Genomic Psychiatry (Genomic Press) 2025 issue-1-1 by genomicpress

2025 CONFERENCE

The Changing Brain

August 18 – 20, 2025 Irvine, California, USA

• The Evolving Brain

• The Learning Brain

• States of the Brain

• The Developing Brain

• The Dynamic Brain

• The Disordered Brain

Paola Arlotta, Harvard University

Xiangmin Xu, University of California, Irvine

Confirmed Speakers: Conference Organizers:

Ishmail Abdus-Saboor, Columbia University

Paola Arlotta, Harvard University

Carlos Brody, Princeton University

Beth Buffalo, University of Washington

Edward Chang, UCSF

Anne Churchland, UCLA

Yang Dan, University of California, Berkeley

Catherine Dulac, Harvard University

Guoping Feng, MIT

Zhigang He, Harvard University

Hailan Hu, Zheijang University, China

Josh Huang, Duke University

Sten Linnarsson, Karolinska Institutet, Sweden

Liqun Luo, Stanford University

Hongkui Zeng, Allen Institute for Brain Science

Guillermina López-Bendito, UMH-CSIC, Spain

Liqun Luo, Stanford University

Michelle Monje, Stanford University

John Ngai, National Institutes of Health

Tom Nowakowski, UCSF

Vanessa Ruta, Rockefeller University

Bernardo Sabatini, Harvard University

Nelson Spruston, Howard Hughes Medical Institute

Karel Svoboda, Allen Institute for Neural Dynamics

Li-Huei Tsai, MIT

Pierre Vanderhaeghen, Leuven Brain Institute of Technology

Hongkui Zeng, Allen Institute of Brain Science

Larry Zipursky, UCLA

Editor-in-Chief

JulioLicinio, StateUniversityofNewYork,UpstateMedicalUniversity,Syracuse,NewYork13210,USA

PublishingManager

Ma-LiWong, StateUniversityofNewYork,UpstateMedicalUniversity,Syracuse,NewYork13210,USA

EditorialBoard

HudaAkil, UniversityofMichigan,AnnArbor,Michigan48109,USA

MauricioArcos-Burgos, UniversidaddeAntioquia,Medellín,Colombia OleA.Andreassen, UniversityofOslo,0318Oslo,Norway

BernhardBaune, UniversityofMünster,48149Münster,Germany

StefanR.Bornstein, TUDDresdenUniversityofTechnology,01307Dresden,Germany

KristenBrennand, YaleUniversitySchoolofMedicine,NewHaven,Connecticut06511,USA

AvshalomCaspi, DukeUniversity,Durham,NorthCarolina27708,USA

MosesChao, NewYorkUniversityLangoneMedicalCenter,NewYork,NewYork10016,USA

SvenCichon, UniversityofBasel,4031Basel,Switzerland

IanDeary, UniversityofEdinburgh,Edinburgh,EH89JZ,Scotland,UK

YogeshDwivedi, UniversityofAlabamaatBirmingham,Birminagm,Alabama35294,USA

StephenFaraone, StateUniversityofNewYork,UpstateMedicalUniversity,Syracuse,NewYork13210,USA

JaniceFullerton, NeuroscienceResearchAustralia&UniversityofNewSouthWales,Randwick,NSW2031,Australia

FredH.Gage, SalkInstituteforBiologicalStudies,LaJolla,California92037,USA

SamuelE.Gandy, IcahnSchoolofMedicineatMountSinai,NewYork,NewYork10029-5674,USA

PatriciaGaspar, INSERMParisBrainInstitute,HôpitalSalpêtrière,75013Paris,France

AnthonyA.Grace, UniversityofPittsburgh,Pittsburgh,Pennsylvania15260,USA

ToddD.Gould, UniversityofMarylandSchoolofMedicine,Baltimore,Maryland21201,USA

RaquelE.Gur, UniversityofPennsylvania,Philadelphia,Pennsylvania19104,USA

Jan-ÅkeGustafsson, UniversityofHouston,Houston,Texas77204,USA

SirJohnHardy, UniversityCollegeLondonDementiaResearchInstitute,London,WC1E6B,UK

NoboruHiroi, UniversityofTexasHealthSanAntonio,SanAntonio,Texas78229,USA.

YasminHurd, IcahnSchoolofMedicineatMountSinai,NewYork,NewYork10029,USA.

SiegfriedKasper, CenterforBrainResearch,MedicalUniversityofVienna,1090Vienna,Austria

KennethS.Kendler, VirginiaCommonwealthUniversity,Richmond,Virginia23298,USA

LorenzoLeggio, NationalInstitutesofHealth,Baltimore,Maryland21224,USA

ChunyuLiu, StateUniversityofNewYork,UpstateMedicalUniversity,Syracuse,NewYork13210,USA

Xin-YunLu, MedicalCollegeofGeorgiaatAugustaUniversity,Augusta,Georgia30912,USA

RobertMalenka, StanfordUniversity,Stanford,California94305,USA

NickMartin, QIMRBerghoferMedicalResearchInstitute,Brisbane,Queensland4029,Australia

AndrewMcIntosh, UniversityofEdinburgh,Edinburgh,EH105HF,Scotland,UK

MariaOquendo, UniversityofPennsylvania,Philadelphia,Pennsylvania19104,USA

SirMichaelOwen, CardiffUniversity,Cardiff,CF244HQ,Wales,UK

AarnoPalotie, InstituteforMolecularMedicine,UniversityofHelsinki,00014Helsinki,Finland

CarlosN.Pato,RutgersUniversity,Piscataway,NewJersey08854,USA

MichelePato, RutgersUniversity,Piscataway,NewJersey08854,USA

MaryL.Phillips, UniversityofPittsburghSchoolofMedicine,Pittsburgh,Pennsylvania15213,USA

RobertPlomin, InstituteofPsychiatryPsychologyandNeuroscienceatKing’sCollege,London,SE58AF,UK

MaurizioPopoli, UniversitàdegliStudidiMilano,20133Milan,MI,Italy

JamesPotash, JohnsHopkinsUniversitySchoolofMedicine,Baltimore,Maryland21287,USA

JohnRubenstein, UniversityofCalifornia,SanFrancisco,California94158,USA

CarloSala, L’IstitutodiNeuroscienzedelCNR,UniversiyofMilan–Bicocca,20854VedanoalLambro,MB,Italy

AlanF.Schatzberg, StanfordUniversity,Stanford,California94305,USA

JairSoares, UniversityofTexasHealthScienceCenter,McGovernSchoolofMedicine,Houston,Texas77054,USA.

ThomasC.Südhof, StanfordUniversity,Stanford,California94305,USA

KristiinaTammimies, KarolinskaInstitutet,17177Stockholm,Sweden

GiuseppeTesta, UniversitàdegliStudidiMilano,HumanTechnopole,20157Milan,MI,Italy

GustavoTurecki, McGillUniversity,Montréal,QuébecH4H1R3,Canada

MonicaUddin, UniversityofSouthFlorida,Tampa,Florida33612,USA

MyrnaWeissman, ColumbiaUniversity,NewYorkStatePsychiatricInstitute,NewYork,NewYork10032,USA

XiangminXu, UniversityofCalifornia,Irvine,California92697,USA

TakeoYoshikawa, RIKENBrainScienceInstitute,Saitama,351-0198,Japan

MoneZaidi, IcahnSchoolofMedicineatMountSinai,NewYork,NewYork10029,USA

GenomicPsychiatry ispublishedbyGenomicPress.

SCOPE: GenomicPsychiatry hasabroadscope.Asourgoalistointerweavegeneticswithotheradvancesincontemporarypsychiatry, wewelcomeinnovativeresearchfromin-depthstudiesofpsychiatricgenomicstobroaderinvestigationsoftheunderpinnings,treatments,outcomes,andconsequencesofmentalhealth.Inadditiontothegeneticaspectsofmentalillness,ourscopeincludesadvances inneuroscienceofpotentialrelevancetomentalillness,imaging,psychology,pharmacology,therapeutics,microbiologyincludingthe microbiome,immunology,endocrinology,brainstimulation,functionalneurosurgery,“bigdata,”computationalapproachesincluding artificialintelligence(AI),epidemiology,andpublichealthinitiatives.

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GenomicPsychiatry TableofContents

Volume1 Number1 January2025

InauguralEditorial–Introducing GenomicPsychiatry:Advancingsciencefromgenestosociety

Real-worldimplicationsoftheprospectsforpreventionofclinicalAlzheimer’sdementia

NataliaAcosta-Baena:Thegeneticgapbetweenneurodevelopmentandneurodegeneration

MariaA.Oquendo:Thetranslationalpathwayfromtheelucidationofthebiologicalcontributionstosuiciderisktothedevelopment ofinterventionsaimedatpreventingmorbidityandmortality

MariaA.Oquendo

GustavoTurecki:Threefundamentalquestions–Howdoesthebrainrespondtosocialandemotionalexperiences?Whydoes psychologicaltraumatriggerdepressivestates?Whatarethemechanismsofantidepressantresponses?

EdoRonalddeKloet:Howdoestheactionofglucocorticoidschangefromprotectivetoharmful?Whatisthecause?Andwhatarethe consequences?

MayanaZatz:Twocriticalquestionstakecenterstage–Whichvariantsmitigatetheimpactoflethalmutationsinsevereconditions withmildphenotype?Whatfactorscontributetothehealthandlongevityofcentenarians?

NoboruHiroi:Exploringthecellularanddevelopmentaloriginsofneuropsychiatricdisorderslinkedtohumancopy-numbervariation

Prepartumbumetanidetreatmentreversesalteredneonatalsocialcommunicationbutnonspecificallyreducespostpubertal socialbehaviorinamousemodeloffragileXsyndrome

Anovelneurodevelopmental-neurodegenerativesyndromethatcosegregateswithahomozygousSPAG9/JIP4stop-codondeletion NataliaAcosta-Baena,JohannaTejada-Moreno…CarlosAndrésVillegas-Lanau

TreatmentwithshRNAtoknockdownthe5-HT2Areceptorimprovesmemoryinvivoanddecreasesexcitabilityinprimarycorticalneurons TroyT.Rohn,DeanRadin…FabioMacciardi

Rethinkingtheconnectionbetweenbipolardisorderandepilepsyfromgeneticperspectives Jin-HuaHuoandMingLi

CoverArt

ThecoverhighlightskeyelementsofthefragileXsyndromeaticlebySakamotoetal.featuredinthisissue.DNAstrandsrepresenttheFMR1gene,which whensilencedleads tofragileXsyndrome,aconditionassociatedwithhighratesofautismspectrumdisorder.Laboratorymice,widelyusedasmodelsforstudyingthiscondition,areshown alongsidethecomputationalstructuresofneonatalsocialcommunication(visualizedascolorfuldotclusters)andthestructureofbumetanidecentraltounderstandingthe disorder.Thisbodyofworkdemonstratesthatprepartumbumetanidetreatmentcanreversealteredneonatalsocialcommunicationpatternsinamouse modeloffragile Xsyndrome.ThestudyprovidesimportantinsightsintohowFmr1deletionaffectsdistinctelementsofearlyvocalizationandlatersocialinteraction,suggestingpotential developmentalwindowsfortherapeuticintervention.ForfurtherinformationonthistopicpleaseseethepaperbySakamotoetalonpages61–72.

Coverdesigncreatedthroughextensiveanditerativehuman-AIcollaborationusingClaudeandGrokAIassistants.ThefinalcoverislicensedunderCreativeCommons Attribution-NonCommercial-NoDerivatives4.0InternationalLicense(CCBY-NC-ND4.0).Thiscovermaybereproducedwithoutpermissionunderthe termsofthislicense, providedappropriatecreditisgiventoGenomicPress,andthecontentisnotmodifiedorusedforcommercialpurposes.

Thisissueisnowavailableat https://genomicpress.kglmeridian.com/view/journals/genpsych/genpsych-overview.xml

EDITORIAL

InauguralEditorial–Introducing

GenomicPsychiatry:Advancingsciencefromgenes tosociety

©TheAuthor(s),2024.ThisarticleisunderexclusiveandpermanentlicensetoGenomicPress

GenomicPsychiatry January2025;1(1):1–2;doi: https://doi.org/10.61373/gp024d.0004

Wearedelightedtointroduce GenomicPsychiatry,anewandgroundbreakingmedicalresearchjournalthataimstorevolutionizethefieldof mentalhealth.Unliketraditionalgeneticsjournals, GenomicPsychiatry willbridgethegapbetweengenesandthevastarrayofinterconnected disciplinesthatcontributetoourunderstandingofmentalhealth,advancingsciencefromgenestosociety.

Inrecentyears,thefieldofgenomicshasmadesignificantstridesin unravelingthegeneticbasisofpsychiatricdisorders.Yet,oureditorial convictionisthatfarmoremonumentaladvanceswillemergefromanuancedexaminationoftheunbrokenspectrumextendingfromgeneticsto ‘omicssciences,neuroscience,cognitivebehaviors,medicalimaging,clinicalpsychiatry,pharmacotherapy,controlledclinicaltrials,andthefarreachingsocietalimplicationsofmentalwell-being(1)gene-enviorment interactions,socialandenvironmentalexposures.

Recognizingthemultifacetednatureofmentalhealth, GenomicPsychiatry willpublisharticlesthatnotonlydelveintogeneticsandgenomics butalsoembraceawiderangeofrelatedtopics.Weencouragesubmissionsthatexploretheinterplaybetweengeneticmarkers,environmental factors,socialdeterminants,andresultantmentalhealthprofiles(2, 3). Fromcutting-edgeresearchontheroleofepigeneticsinpsychiatricdisorderstostudiesinvestigatingthesocialdeterminantsofmentalhealth, wewelcomeadiversityofperspectivesandmethodologies.

Byembracingacomprehensiveapproach, GenomicPsychiatry willprovideaplatformforresearcherstoshowcasetheirworkattheintersection ofvariousdisciplines(4).Thisjournalwillfostercollaborationandinspire novelinsights,therebypropellingthefieldforward.Wefirmlybelievethat thefutureofmentalhealthresearchliesinembracingamultidimensional approachthatintegratesgenetics,genomics,andbeyond.

Toensureinclusivityandfosterinnovation, GenomicPsychiatry invitessubmissionsfromresearchersandcliniciansworkingacrosstheentirespectrumofmentalhealth.Weencourageauthorstoexplorethe applicationofgenomicsandgeneticsinclinicalpsychiatry,pharmacologicalinterventions,andtreatmenttrials(5).Wearealsointerestedin digitalmedicine,e-health,andtheuseofartificialintelligenceincomputationalpsychiatry(3).Additionally,wewelcomepapersthatinvestigatetheneurobiologicalunderpinningsofpsychiatricdisorders,advancementsinbrainimagingtechniques,andbehavioralstudiesthatshedlight onthecomplexitiesofmentalhealth.

Asthefieldofmentalhealthcontinuestoevolve,itisimperativethat wecreateaspacethatencouragestranslationalscience,interdisciplinary collaboration,andknowledgeexchange(6). GenomicPsychiatry endeavorstobethatspace,wherescientists,clinicians,andresearchersfrom variousbackgroundsjoinforcestoexploretheintricatenatureofmentalhealthdisorders.

Oureditorialboard,comprisingsofar50esteemedmembers,stands asacornerstoneof GenomicPsychiatry’s strength.Thesemembersare notjusteminentintheirfieldsbutalsogloballyrecognizedfortheircontributions.AmongthemisNobelLaureateThomasSüdhof,underscoring

Received:19January2024.Accepted:23January2024. Publishedonline:25January2024.

theboard’sprestige.Severalmembersdistinguishthemselveswithaffiliationstoesteemedinstitutions:intheUSNationalAcademyofSciences, wehavethelikesofHudaAkil,MosesChao,FredGage,Jan-AkeGustafsson,YasminHurd,RobertMalenka,JohnRubenstein,andagainThomas Südhof.WithintheUSNationalAcademyofMedicine,ourrosterincludes FredGage,RaquelGur,YasminHurd,KennethKendler,RobertMalenka, MariaOquendo,JohnRubenstein,AlanSchatzberg,ThomasSüdhof,GustavoTurecki,andMyrnaWeissman.TheRoyalSocietyhonorsJonathan FlintandJohnHardyasitsFellows.Additionally,theBritishmonarchyhas knightedtwoofourboardmembers,JohnHardyandMichaelOwen,in recognitionoftheirextraordinaryachievements.Thisdiverseandaccomplishedgroupmirrorstheunparalleledexpertiseandglobalrecognition oureditorialboardenjoys.

Wewouldliketohighlightour InnovatorsandIdeas sectionthatspotlightsindividualswhohavemadenoteworthycontributionstothefield. Fourofoureditorialboardmembershavealreadycontributedtothis excitingsectionasresearchleaders:MariaOquendo(neurobiologyand clinicalapproachestosuicidality)(7),GustavoTurecki(trauma,depression,neuropathology,andgenomics)(8),AnthonyGrace(braincircuits, schizophrenia,anddepression)(9),andNoboruHiroi(neurobiologyof humancopy-numbervariation)(10).

Weinviteyoutoembarkonthisexcitingjourneywithus.Together, letusunravelthemysteriesofthehumanmind,leveragingthepower ofgenomics,genetics,andthewealthofscientificdisciplinesthatconvergeupontherealmofmentalhealth.Joinusinredefiningthelandscape ofpsychiatricresearchandfosteringabetterunderstandingofmental healthforthebenefitofindividualsandsocietyasawhole.

Welcometo GenomicPsychiatry –wherethegenetic,thebehavioral, theenvironmental,andthesocietalmergetodevelopnewpathstowards optimalmentalhealth.

JulioLicinio1

1 Editor-in-Chief,GenomicPsychiatry,GenomicPress, NewYork,NewYork10036,USA e-mail: julio.licinio@genomicpress.com

References

1.MerikangasKR,MerikangasAK.HarnessingProgressinPsychiatricGeneticstoAdvancePopulationMentalHealth.AmJPublicHealth.2019;109(S3):S171-S175.DOI: 10.2105/AJPH.2019.304948 PMC6595514

2.ReubenA,ManczakEM,CabreraLY,AlegriaM,BucherML,FreemanEC,etal.TheInterplayofEnvironmentalExposuresandMentalHealth:SettinganAgenda.Environ HealthPerspect.2022;130(2):25001.DOI: 10.1289/EHP9889 PMC8848757

3.TopolEJ.High-performancemedicine:theconvergenceofhumanandartificialintelligence.Naturemedicine.2019;25(1):44-56.DOI: 10.1038/s41591-018-0300-7

4.InstituteofMedicine(U.S.).CommitteeonBuildingBridgesintheBrainBehavioraland ClinicalSciences.,PellmarTC,EisenbergL.Bridgingdisciplinesinthebrain,behavioral, andclinicalsciences.Washington,D.C.:NationalAcademyPress;2000.xiv,130p.p.

5.GartlehnerG,WagnerG,MatyasN,TitscherV,GreimelJ,LuxL,etal.Pharmacologicalandnon-pharmacologicaltreatmentsformajordepressivedisorder:reviewof

systematicreviews.BMJOpen.2017;7(6):e014912.DOI: 10.1136/bmjopen-2016014912 PMC5623437

6.BornsteinSR,LicinioJ.Improvingtheefficacyoftranslationalmedicinebyoptimally integratinghealthcare,academiaandindustry.Naturemedicine.2011;17(12):15671569.DOI: 10.1038/nm.2583

7.OquendoMA.Thetranslationalpathwayfromtheelucidationofthebiologicalcontributionstosuiciderisktothedevelopmentofinterventionsaimedatpreventingmorbidityandmortality.GenomicPsychiatry.2024.DOI: 10.61373/gp024k.0001

8.TureckiG.Threefundamentalquestions–Howdoesthebrainrespondtosocialand emotionalexperiences?Whydoespsychologicaltraumatriggerdepressivestates? Whatarethemechanismsofantidepressantresponses?GenomicPsychiatry.2024. DOI: 10.61373/gp024k.0007

9.GraceAA.Elucidatingthecircuitriesthatunderlieschizophreniaanddepressionmay revealtheimpactofstressduringdevelopmentandidentifynoveltreatmenttargets. GenomicPsychiatry.2024.DOI: 10.61373/gp024k.0010

10.HiroiN.Exploringthecellularanddevelopmentaloriginsofneuropsychiatricdisorders linkedtohumancopy-numbervariation.GenomicPsychiatry.2024.DOI: 10.61373/ gp024k.0013

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GUESTEDITORIAL

Real-worldimplicationsoftheprospectsforpreventionofclinical Alzheimer’sdementia

©TheAuthor(s),2024.ThisarticleisunderexclusiveandpermanentlicensetoGenomicPress

GenomicPsychiatry January2025;1(1):3–4;doi: https://doi.org/10.61373/gp024g.0034

OnValentine’sDay2024, TheLosAngelesTimes publishedastoryentitled,“InsidetheplantodiagnoseAlzheimer’sinpeoplewithnomemoryproblems—andwhostandstobenefit”(1).Thestoryfocuseson thefinancialimplicationsfordrugcompaniesandpatientadvocates. TheNationalInstituteonAging’sAHEAD3451 study(2)isalegitimate, federallyfunded,randomizedclinicaltrialthatisdesignedtothehighestclinicalresearchstandards.Thisisaprovenmethodfornewdrugs tobeputtothemostrigoroustestusingaplacebo-controlled,doubleblind,state-of-the-artdesign.TheAHEAD345trialwilltestthepossibilitythatclinicalAlzheimer’sdementiamaybepreventableifdiagnosisand interventionaretriggeredbyblood-basedbiomarkerchangesdetectedat midlifedespiteanabsenceofsymptoms.Thereisnowclearevidencethat Alzheimer’spathologydevelops20ormoreyearspriortotheappearance ofclinicalsymptoms.The Times pieceemphasizesthattheefforttoseize uponthispotentialwindowforinterventionisatleastpartiallymotivated bytheprospectthatdrugcompaniesanddementiaadvocacygroupswill befinanciallyenrichediftrialslikeAHEAD345succeed.The Times articleavoidswordslike“breakthrough”and“moonshot”thatarefrequently usedtodescribethemitigationoreliminationofmajorillnessessuchas cancer,diabetes,heartdisease,andAIDS.

Thecostofbringingeachnewprescriptiondrugtomarketisestimated at$350million(3).Usually,generatingprofitstounderwriteongoingresearchhasbeenacceptedasasoundbusinessmodelandviewedasevidenceoftheentrepreneurialspiritofscientistsandclinicians.Whywas thedevelopmentofblockbusterdrugsthatpreventclinicalmanifestationsofatherosclerosiswelcomed,whiletheprospectofpreventingdementiaisviewedinthefirstanalysisasprimarilyprofit-driven?Isthe sufferingofyoungerpersonswithatherosclerosisandcancermoreimportantthanthesufferingofelderpersonslivingwithdementiaandtheir families?Arescientificdiscoveryandfinancialprofitabilitymutuallyexclusive?Iwouldhavepredictedthatanytensionbetweenthesetwooutcomeswouldbeasmallpricetopayifweeliminateanillnessthatcosts 2trilliondollarsperyearintheUSalone(4).

Manybreakthroughsenrichinventors.TheNobelPrizehasamonetary valueof11millionSwedishkronor(5).Rigoroustrialsofdrugsareessential,regardlessofwhostandstobenefiteitherfinanciallyoremotionally.Whileitisentirelyreasonablethatskepticsholdinventors’feetto thefire,theinventorsshouldbeentirelyopentoscrutinytorealizethe commongoalofauthentic,valid,reproducibledata.Skepticismaboutthe outcomedoesnotmeanthataudaciousandpotentiallylucrativehypothesesshouldnotbetested.Theunpredictabilityofscienceistheessenceof whyexperimentsareconducted.

OnegoaloftheRANDCorporationistoelucidatehowsuccessfuldementiatreatmentandpreventionmightmodifytheclinicaland economiclandscapeinarangeofsituations(6).RANDrecognizesthe

1 AcronymforanNIHclinicaltrialofblood-basedbiomarkerguidedtreatmentwith anti-amyloidantibodyorplacebo.

Received:21April2024.Accepted:23April2024. Publishedonline:26April2024.

substantialvariationinthecapacitiesofvarioushealthcaresystems todetect,diagnose,andtreatorpreventearly-stageAlzheimer’swith disease-modifyingtreatments(DMTs).Theestimatedwaittimesandthe numberofpatientstreatedaresensitivetotheuptakeofbriefcognitive assessmentsbythepublicandbyprimarycareproviders.Theestimated averagewaittimesvarybystateandcanbethreetimeslongerinrural areasthaninurbanareas.Caremodelsthatenableprimarycarephysicianstodiagnoseandevaluatepatientsfortreatmenteligibilitywould significantlyreducewaittimesforspecialistsandincreasethenumberofpeopletreatedfrom2025through2044.Improvedtriageofpatientsusingblood-basedbiomarkertestscouldfurtherreducecaseloads forspecialists.WidespreaddeliveryofAlzheimer’sDMTswillrequirea combinationofstrategiesto(1)communicatethevalueofdetectionand treatmenttopatients,(2)integrateprimarycarephysiciansintothedetectionanddiagnosispathway,and(3)addresscapacitydisparitiesacross theUnitedStatesandaroundtheworld.ThesechallengesforimplementationcanonlybeaffordedifDMTsgenerateenoughresourcesto offsetthisincreaseindemand.Criticswithlegitimateconcernsshould allowforthepossibilitythatthepotentialprofitabilityofbreakthroughs doesnotmeanthatweshouldavoidaskingwhetherpreventionis possible.

Thornyquestionsremaintobeanswered.Trialshavenotbeenadequatelyinclusiveanddiverse(7).Standardsforminimumclinicallysignificantbenefitarestillunderdevelopment,bothforpersonslivingwith dementiaandfortheircaregivers(8).Nevertheless,thereisnoreasonnot tobeginsortingthroughtheseimplicationssothatweareappropriately preparedifAlzheimer’spreventionsucceeds.Currentevidencesuggests thatasmanyas76%ofpatientsreceivingsubcutaneouslecanemab(vs 55%ofpatientsreceivingplacebo)haveacompletearrestoftheircognitivedecline(9).Onwhatplanetisthisabadoutcome?

SamGandy,MD,PhD1 , 2

1 MountSinaiAlzheimer’sDiseaseResearchCenter,IcahnSchoolofMedicineat MountSinai,NewYork,NewYork10029,USA; 2 DepartmentofNeurology,JamesJ PetersVAMedicalCenter,BronxNewYork10468,USA e-mail: samuel.gandy@mssm.edu

CompetingInterests

Dr.Gandyisaco-founderofRecuerdoPharmaceuticals.Hehasservedas aconsultantinthepastforJ&J,Diagenic,andPfizer,andhecurrently consultsforCognitoTherapeutics,GLGGroup,SVBSecurities,Guidepoint,ThirdBridge,MEDACORP,Altpep,VigilNeurosciences,andEisai.He hasreceivedresearchsupportinthepastfromWarner-Lambert,Pfizer, Baxter,andAvid.HecurrentlyreceivesresearchsupportfromtheNIHand theCureAlzheimer’sFund.

FundingSources

Theauthorwassupportedasfollows:NIHgrantsU01AG046170, RF1AG058469,RF1AG059319,R01AG061894,P30AG066514toMary Sano,andCureAlzheimer’sFund.

RoleoftheFunders/Sponsors

Thefunders/sponsorshadnoroleinthepreparation,review,orapprovalofthemanuscript,orthedecisiontosubmitthemanuscriptfor publication.

References

1. https://www.latimes.com/science/story/2024-02-14/inside-controversial-plan-todiagnose-alzheimers-in-people-without-symptoms

2. https://www.aheadstudy.org/

3.SchlanderM,Hernandez-VillafuerteK,ChengCY,Mestre-FerrandizJ,BaumannM.How MuchDoesItCosttoResearchandDevelopaNewDrug?ASystematicReviewand Assessment.Pharmacoeconomics.2021;39(11):1243-69.DOI: 10.1007/s40273-02101065-y.PMID:34368939;PMCID: PMC8516790

4.TayLX,OngSC,TayLJ,NgT,ParumasivamT.EconomicBurdenofAlzheimer’sDisease: ASystematicReview.ValueHealthRegIssues.2024;40:1-12.DOI: 10.1016/j.vhri.2023. 09.008.PMID:37972428.

5. https://www.nobelprize.org/prizes/about/the-nobel-prize-amounts/

6. https://www.rand.org/

7.XueD,BlueEE,ConomosMP,FohnerAE.Thepowerofrepresentation:StatisticalanalysisofdiversityinUSAlzheimer’sdiseasegeneticsdata.AlzheimersDement(NY). 2024;10(1):e12462.DOI: 10.1002/trc2.12462

8.HortonMC,OyebodeJ,ClareL,MegsonM,ShearsmithL,BrayneC,etal.MeasuringQualityofLifeinCarersofPeopleWithDementia:DevelopmentandPsychometricEvaluationofScalesmeasuringtheImpactofDEmentiaonCARers(SIDECAR).Gerontologist. 2021;61(3):e1-11.DOI: 10.1093/geront/gnz136

9.vanDyckC,JohnsonK,SperlingR,IrizarryM.LecanemabforearlyAlzheimer’sdisease: Long-termoutcomes,predictivebiomarkers,andnovelsubcutaneousadministration. CTAD23,AbstractS4.JPreventAD2023;10:S1,59-60.

Publisher’snote: GenomicPressmaintainsapositionofimpartialityandneutralityregardingterritorialassertionsrepresentedinpublishedmaterialsandaffiliationsofinstitutionalnature.Assuch,wewillusetheaffiliationsprovidedbytheauthors,withouteditingthem.Suchusesimplyreflectswhattheauthorssubmitted tousanditdoesnotindicatethatGenomicPresssupportsanytypeofterritorial assertions.

OpenAccess. ThisarticleislicensedtoGenomicPressunderthe CreativeCommonsAttribution-NonCommercial-NoDerivatives4.0 InternationalLicense(CCBY-NC-ND4.0).Thelicensemandates:(1)Attribution: Creditmustbegiventotheoriginalwork,withalinktothelicenseandnotification ofanychanges.Theacknowledgmentshouldnotimplylicensorendorsement. (2)NonCommercial:Thematerialcannotbeusedforcommercialpurposes.(3) NoDerivatives:Modifiedversionsoftheworkcannotbedistributed.(4)Noadditionallegalortechnologicalrestrictionsmaybeappliedbeyondthosestipulated inthelicense.Publicdomainmaterialsorthosecoveredbystatutoryexceptions areexemptfromtheseterms.Thislicensedoesnotcoverallpotentialrights,such aspublicityorprivacyrights,whichmayrestrictmaterialuse.Third-partycontent inthisarticlefallsunderthearticle’sCreativeCommonslicenseunlessotherwisestated.Ifuseexceedsthelicensescopeorstatutoryregulation,permission mustbeobtainedfromthecopyrightholder.Forcompletelicensedetails,visit https://creativecommons.org/licenses/by-nc-nd/4.0/.Thelicenseisprovided withoutwarranties.

GenomicPsychiatry

INNOVATORS&IDEAS:RISINGSTAR

NataliaAcosta-Baena:Thegeneticgapbetweenneurodevelopmentand neurodegeneration

©GenomicPress,2024.The“GenomicPressInterview”frameworkisprotectedundercopyright.Individualresponsesarepublishedunderexclusive andpermanentlicensetoGenomicPress.

GenomicPsychiatry January2025;1(1):5–7;doi: https://doi.org/10.61373/gp024k.0082

Keywords: SPAG9,JIP4,retrogradeaxonaltransport,polygenic, pleiotropy,Intellectualdisability,dementia,genetics,epidemiologist, neurodevelopment

Dr.NataliaAcosta-Baenaembodiestherareconfluenceofclinicaland basicscienceexpertisethatmodernneurosciencedemands.Asa physician-scientistattheUniversityofAntioquia’sNeurosciences Group,shecombineshermedicaltrainingwithamaster’sinclinical epidemiologyanddoctoralstudiesinbasicbiomedicalsciences, specializinginGenetics.Hergroundbreakingworkbeganwith contributingtocharacterizetheworld’slargestpopulationaffected byautosomaldominantgeneticAlzheimer’sdisease(mutationE280A inPSEN1),establishingafoundationfornumerousstudieson biomarkers,clinicaltrialdesign,andgeneticmodifiersinthispivotal cohort.Inalandmarkdiscovery,Dr.Acosta-Baena’sresearchrevealed anovelsyndromelinkedtoaSPAG9variant,demonstratinghowa singlegeneinvolvedinneuronalretrogradetransportcandrive neurodevelopmentalproblemsandneurodegenerationinaffected patients.Thisfindingchallengesthetraditionalseparationbetween theseprocessesandsuggestssharedbiologicalpathways.Through hercontinuedworkwithfamiliesaffectedbyneurodevelopmental disorders,shehasuncoveredgeneticnetworksthatreshapeour understandingofrarebraindiseases.Hercurrenttranslational medicineandgeneticepidemiologyresearchfocusesonfurther exploringtheseunexpectedconnectionsbetweenneurodevelopment andneurodegeneration.InaGenomicPressInterview, Dr.Acosta-Baenasharedherlifebeyondthelaboratory–fromher earlyfascinationwiththehumanbraintofindingjoyinColombia’s mountainsunsetswithherhusbandandsonanddrawinginspiration fromLatinAmericanwriterslikeCortázarandGarcíaMárquez.Her dedicationtoscientificrigorandhumanconnectionisreflectedinher philosophythateachfailureteachessomethingessentialasshe workstowardtranslatinggeneticdiscoveriesintomeaningful healthcarepoliciesandpersonalizedmedicineapproaches.

Part1:NataliaAcosta-Baena–LifeandCareer

Couldyougiveusaglimpseintoyourpersonalhistory,emphasizing thepivotalmomentsthatfirstkindledyourpassionforscience? Itmaysoundstrange,buteversinceIcanremember,Ihavewantedto studymedicineandunderstandourbrain.Idonothaveparentswhowere doctorsoranyoneinmyfamilytoinfluenceme.Ithasmotivatedmylife sinceIwaslittle.IrememberthatIdidn’tlikegoingtoschool,butIunderstoodthatitwastheonlywaytogettoworkinwhatIwaspassionate about.

Wewouldliketoknowmoreaboutyourcareertrajectoryleadingupto yourcurrentrole.Whatdefiningmomentschanneledyoutowardthis opportunity?

Amotivation:neuroscienceresearch.Twoopportunities:IstartedbyattendingastudygroupinNeuroanatomywiththeNeuroscienceGroupof

Received:3November2024.Accepted:5November2024. Publishedonline:14November2024.

theUniversityofAntioquia,andIacceptedtheopportunitytodoamaster’sdegreeinepidemiologywiththesameresearchgroupwithProfessor FranciscoLoperaandthencompleteadoctorateingenetics.

Pleasesharewithuswhatinitiallypiquedyourinterestinyour favoriteresearchorprofessionalfocusarea.

Thecomplexityofhumanthoughtandactionandthesearchfortheevolutionaryquestionofwhatthebrainofhomosapienshadgeneticallyand physiologicallymadeusthespeciesthatmanagedtosurviveabovethe otherhominids.

Whatimpactdoyouhopetoachieveinyourfieldbyfocusingon specificresearchtopics?

ThegreatestimpactIhopetoachievewithmyresearchistofostera collaborativeeffortthatreachesindividualswhoaresickandtheircaregivers.Ienvisionafuturewhereourknowledgeempowerspeopleand

Figure1. NataliaAcosta-Baena,MD,MsC,PhD(c),UniversidaddeAntioquia, Colombia.

influenceshealthpoliciestowardstrueprevention.Ourstudies,currently intheformofarticles,holdthepotentialtobeappliedtothegeneralpopulation,shapingdecisionsandresponsesforpatients.

Pleasetellusmoreaboutyourcurrentscholarlyfocalpointswithin yourchosenfieldofscience?

Neurodevelopmentalgeneticsisafieldwhereweonlyseethetipofthe iceberg.Wedonotseewhatwedonotunderstand.Whenwemanageto understandtherelationshipbetweengeneticnetworks,wewillbeableto decipherneurodevelopmentandneurodegeneration.Myfocusisgenetics forpreventionandpersonalizedandcommunitymedicine.

Whathabitsandvaluesdidyoudevelopduringyouracademicstudies orsubsequentpostdoctoralexperiencesthatyouupholdwithinyour researchenvironment?

Continuewiththemedicalconsultation.Eachrareclinicalpresentationof adiseasethatsurprisesiswhatmotivatesnewquestions.

AtGenomicPress,weprioritizefosteringresearchendeavorsbased solelyontheirinherentmerit,uninfluencedbygeographyorthe researchers’personalordemographictraits.Arethereparticular culturalfacetswithinthescientificcommunitythatwarrant transformativescrutiny,oristhereacausewithinsciencethatdeeply stirsyourpassions?

Iampassionateaboutnewquestionsandnewchallenges.Iamboredby absolutecertaintyandthosewhobelievetheyhaveit.

Whatdoyoumostenjoyinyourcapacityasanacademicorresearch risingstar?

ThatIdonotfeellikeone.

Outsideprofessionalconfines,howdoyouprefertoallocateyour leisuremoments,orconversely,inwhatmannerwouldyouenvision spendingthesemomentsgivenachoice?

IalwaysenjoytheviewofthesunsetortheraininthemountainsofColombiawithmyhusbandandson.

Part2:NataliaAcosta-Baena–Selectedquestionsfromthe ProustQuestionnaire1

Whatisyourideaofperfecthappiness? Thereisnotone,bothwordsareapleonasm.

Whatisyourgreatestfear?

“Withoutmusic,lifewouldbeamistake”“OhneMusikwäredasLeben einIrrtum”(FriedrichNietzsche, DieGötzen-Dämmerung–Twilightofthe Idols,section:“SprücheundPfeile”(MaximsandArrows)aphorism#33, 1895).

1 Inthelatenineteenthcentury,variousquestionnaireswereapopulardiversion designedtodiscovernewthingsaboutoldfriends.Whatisnowknownasthe35questionProustQuestionnairebecamefamousafterMarcelProust’sanswersto thesequestionswerefoundandpublishedposthumously.Proustansweredthequestionstwice,atages14and20.In2003Proust’shandwrittenanswerswereauctioned offfor$130,000.Multipleotherhistoricalandcontemporaryfigureshaveanswered theProustQuestionnaire,includingamongothersKarlMarx,OscarWilde,ArthurConanDoyle,FernandoPessoa,StéphaneMallarmé,PaulCézanne,VladimirNabokov, KazuoIshiguro,CatherineDeneuve,SophiaLoren,GinaLollobrigida,GloriaSteinem, Pelé,Valentino,YokoOno,EltonJohn,MartinScorsese,PedroAlmodóvar,Richard Branson,JimmyCarter,DavidChang,SpikeLee,HughJackman,andZendaya.The ProustQuestionnaireisoftenusedtointerviewcelebrities:theideaisthatbyansweringthesequestions,anindividualwillrevealhisorhertruenature.WehavecondensedtheProustQuestionnairebyreducingthenumberofquestionsandslightly rewordingsome.Thesecuratedquestionsprovideinsightsintotheindividual’sinner world,rangingfromnotionsofhappinessandfeartoaspirationsandinspirations.

Figure2. NataliaAcosta-BaenaandhersonembracingthebeautyoftheAntioquiaMountainsduringColombia’sCOVID-19lockdown(25March2020). Againstabackdropoflushtropicalvegetationandbamboogrovescharacteristicoftheregion’smountainside,theystandonaclearedhillsideslope,arms outstretchedinamomentofjoyandfreedomdespitetheglobalpandemicrestrictions.Thecontrastbetweenthecultivatedslopeintheforegroundand thedenseforestcanopyabovecapturesthetypicallandscapemosaicofthe Andeancountryside.

Whichlivingpersondoyoumostadmire?

ToeverywomaninLatinAmericawhoprioritizeshertimefortheintegral educationofherchildrenaboveanyotherneed.

Whatisyourgreatestextravagance? Behappy.

Whatareyoumostproudof? Beingamother.

Whatisyourgreatestregret? Idonotregretanythingsofar.

Whatisthequalityyoumostadmireinpeople? Honestyandgoodhumor.

Whatisthetraityoumostdislikeinpeople? Arroganceandprepotency.

Whatdoyouconsiderthemostoverratedvirtue? None.Eachvirtueisrelevanttohumanity,andresearchonhumansand animalsisanexample.

Whatisyourfavoriteoccupation(oractivity)? Writing–andIcan–dancing.

Wherewouldyoumostliketolive?

InColombia,inmycurrenthouseinaruralareainthemountains.

Whatisyourmosttreasuredpossession? Mymemories.

Whenandwherewereyouhappiest?Andwhyweresohappythen? Whenmysonwasborn,10yearsago,andallthegoodtimessincethen.

Whatisyourcurrentstateofmind? IcurrentlyfeelcalmandgratefulforeverythingIhavereceivedinlife.

Whatisyourmostmarkedcharacteristic? Imagination.

Amongyourtalents,whichone(s)give(s)youacompetitiveedge? Iwasnotbornwithspecialtalents,butIwasbornwithagreatdealof curiosityandadesiretodevelopnewskillseveryday.

Whatdoyouconsideryourgreatestachievement?

Everygoalachievedisthebiggestatthetime.Everyarticleachievedand publishedisthesumoftheeffortsofmanypeople,soitisgreat.

Ifyoucouldchangeonethingaboutyourself,whatwoulditbe? Idonotwanttochangeanything.WhatIwasandwhatIamhaveleftme whereIamnow,andIfeelproudofwhereIam,howIam,andwhoIam with.

Whatdoyoumostvalueinyourfriends?

Appreciationandloyaltydespitethousandsofflawsandmistakes.

Whoareyourfavoritewriters?

JulioCortazar,HoracioQuiroga,JorgeLuisBorgesandGabrielGarcia Marquez.

Whoareyourheroesoffiction?

None.Myfavoriteisanantihero,theJoker,whoremindsusofourdark humanity.

Whoareyourheroesinreallife?

Anonymouspeoplehelpothers,buttheydonotappearinnewspapersor socialmedia.

Whataphorismormottobestencapsulatesyourlifephilosophy?

“Stairsareclimbedfromthefront,sinceclimbingthemfrombehindor thesidewillresultparticularlyuncomfortable.”—JulioCortázar,“InstructionsonHowtoClimbaStaircase”(fromCronopiosandFamas,1962). Trans.PaulBlackburn.

Intheoriginal:“Lasescalerassesubendefrente,pueshaciaatrás odecostadoresultanparticularmenteincómodas.”—JulioCortázar, “Instruccionesparasubirunaescalera”(deHistoriasdecronopiosyde famas,1962).

NataliaAcosta-Baena1

1 UniversidaddeAntioquia,FacultaddeMedicina,GrupodeNeurocienciasde Antioquia(GNA),Medellín,Antioquia050012,Colombia e-mail: natalia.acosta@gna.org.co

Publisher’snote: GenomicPressmaintainsapositionofimpartialityandneutrality regardingterritorialassertionsrepresentedinpublishedmaterialsandaffiliations ofinstitutionalnature.Assuch,wewillusetheaffiliationsprovidedbytheauthors, withouteditingthem.Suchusesimplyreflectswhattheauthorssubmittedtousand itdoesnotindicatethatGenomicPresssupportsanytypeofterritorialassertions.

OpenAccess. The“GenomicPressInterview”frameworkiscopyrightedtoGenomicPress.Theinterviewee’sresponsesarelicensedtoGenomicPressundertheCreativeCommonsAttribution-NonCommercialNoDerivatives4.0InternationalLicense(CCBY-NC-ND4.0).Thelicensemandates: (1)Attribution:Creditmustbegiventotheoriginalwork,withalinktothelicense andnotificationofanychanges.Theacknowledgmentshouldnotimplylicensorendorsement.(2)NonCommercial:Thematerialcannotbeusedforcommercialpurposes.(3)NoDerivatives:Modifiedversionsoftheworkcannotbedistributed.(4) Noadditionallegalortechnologicalrestrictionsmaybeappliedbeyondthosestipulatedinthelicense.Publicdomainmaterialsorthosecoveredbystatutoryexceptionsareexemptfromtheseterms.Thislicensedoesnotcoverallpotential rights,suchaspublicityorprivacyrights,whichmayrestrictmaterialuse.Thirdpartycontentinthisarticlefallsunderthearticle’sCreativeCommonslicenseunless otherwisestated.Ifuseexceedsthelicensescopeorstatutoryregulation,permissionmustbeobtainedfromthecopyrightholder.Forcompletelicensedetails,visit https://creativecommons.org/licenses/by-nc-nd/4.0/.Thelicenseisprovidedwithoutwarranties.

GenomicPsychiatry

INNOVATORS&IDEAS:RESEARCHLEADER

MariaA.Oquendo:Thetranslationalpathwayfromtheelucidationofthebiological contributionstosuiciderisktothedevelopmentofinterventionsaimedat preventingmorbidityandmortality

©GenomicPress,2024.The“GenomicPressInterview”frameworkisprotectedundercopyright.Individualresponsesarepublishedunderexclusive andpermanentlicensetoGenomicPress.

GenomicPsychiatry January2025;1(1):8–10;doi: https://doi.org/10.61373/gp024k.0001

Keywords: Suicide,suicidality,brain,populationhealth,womenin science

Dr.MariaOquendoisRuthMeltzerProfessorandChairmanof PsychiatryatUniversityofPennsylvaniaandPsychiatrist-in-Chiefat theHospitaloftheUniversityofPennsylvania.Asummacumlaude graduateofTuftsUniversity,sheattendedCollegeofPhysiciansand Surgeons,ColumbiaUniversityandcompletedresidencyatPayne WhitneyClinic,NewYorkHospital,Cornell.Sheisamemberofthe NationalAcademyofMedicine,oneofthehighesthonorsinmedicine. Dr.OquendohasusedPositronEmissionTomographyandMagnetic ResonanceImagingtomapbrainabnormalitiesinmooddisordersand suicidalbehavior.Herexpertiserangesfrompsychopharmacologyto GlobalMentalHealth.Shehasover500peer-reviewedpublications, anH-index116and49,472citations(GoogleScholar).Intermsof organizationalleadershippositions,Dr.OquendoisPastPresidentof theAmericanPsychiatricAssociation(APA),theInternational AcademyofSuicideResearch,theAmericanCollegeof Neuropsychopharmacology(ACNP),andtheAmericanFoundationfor SuicidePrevention’sBoardofDirectors.SheisVicePresidentofthe CollegeofInternationalNeuropsychopharmacologyandhasservedon theNationalInstituteofMentalHealth’sAdvisoryCouncil.Dr. OquendoservesonTuftsUniversity’sBoardofTrustees,servesonits ExecutiveCommitteeandchairsTufts’AcademicAffairsCommittee.A recipientofmultipleawardsintheUnitedStates,Europe,andSouth America,mostrecently,shewashonoredwiththeSymondsAward (APA2017),theAPA’sResearchAward(2018),theShockleyAward (ACNP2018),andtheGlassmanAward(ColumbiaUniversity2021). Dr.Oquendohassharedsomeofherthoughtsandperspectivesonher lifeandcareer.

TheGenomicPressInterviewPart1:MariaOquendo:Lifeandcareer Couldyougiveusaglimpseintoyourpersonalhistory,emphasizing thepivotalmomentsthatfirstkindledyourpassionforscience? Ihavelovedmathematicalconceptsandnumberssincemiddleschool,but Ialsocherishedlanguage,art,anddesign.IoriginallythoughtthatArchitecturewouldhelpmemeldtheseinterests,yettheliberalcollegeIattendeddidnotoffersuchstudies.Thus,Ifocusedontheoreticalmathand Romancelanguageliterature.Therestofthetrajectorytomedicineisa yarn,butsufficeittosaythatatnotimeduringmyteensortwentiesdid Iconsiderscientificinquirymycalling.Iviewedscienceaskeytrainingto supportpragmaticapplications:architecture,medicine.Andso,Ifinished residencyinPsychiatryandchoseapositionasateachingfacultymemberinabusyclinicalservice.ItwasnotuntilIhadbeeninthatpositionfor 8yearsthatIbegantothinkaboutotheropportunities.Fortuitously,one ofmyresidencysupervisorswasrecruitedtoColumbiaUniversity,whereI wasonthefaculty.Hewasoneofmyresearchmentorsduringresidency. AkeypointisthatheknewIwouldworkhardandofferedmeafull-time positiononhisteam.IwasnotsureIwouldlikethejob.Infact,Ithought

Received:11January2024.Accepted:12January2024. Publishedonline:25January2024.

therewasagoodchanceIwouldhateit.ButIwaswrong.Theexperience wastransformational.Ilovedthinkingabouthowtointerpretdata,Iloved statistics,Ilovedwritingpapers,carefullyandmethodicallydelineating theapproach,theanalysis,theresults,andtheconclusions.Ievenloved writinggrants.Iwasinheaven.EventhoughIhavehadseveraladministrativepositions,itisundeniablethatthecoreofmyprofessionalidentity isasascientist.

Wewouldliketoknowmoreaboutyourcareertrajectoryleadingup toyourmostrelevantleadershiprole.Whatdefiningmoments channeledyoutowardthatleadershipresponsibility?

ManyofmystudentsandmenteeshaveaskedmehowIforgedthepathto becomingChairmanofamajorDepartmentofPsychiatry.Theyaremostly takenabackwhenItellthemthatIdidnotplanitatallandthatIamas surprisedasthenextpersonthatithappened.Ialsotellthemthatmany leadersatmylevelhavesharedwithmethattheirexperiencewasnotso different.Itwasnotplanned.Itwastheresultofrespondingtoopportunitiesevenwhendoingsowasnotalignedwithapersonalvisionofthe trajectory.Forexample,whenIshiftedfromafacultypositionasaclinical educatortoaresearchpsychiatrist,Ididnothaveanyleadershipresponsibilities.However,withinafewyears,mymentorappointedmeDirectoroftheClinicalLabforwhatwasthentheDivisionofNeurosciencein

Figure1. MariaA.Oquendo,MD,UniversityofPennsylvania,USA.

PsychiatryatColumbia.ThiswasanopportunityIrelished,andIdidthe jobhappilyfor8yearsuntilIwastappedbytheExecutiveVice-Chairof theDepartmentofPsychiatrytobecometheDirectorofAmbulatoryResearchClinics,ofwhichtherewere28(!).Iwasfarfromsurethatthis administrativejobwasforme,butIappliedthesamemethodicalapproachIusedinresearchtounderstandwhateachclinicfocusedonand howitwasorganized.Iwantedtoknowwhethertherewasaneedfora moreuniformstructureorwhethertheclinicsusedrobuststrategiesin theirmanagementofclinicalresearchpatientsintermsofsafety,rigor, andproductivity.Iwantedtounderstandwhattheirscientificoutputwas andhoweachresearchclinicsupporteditsefforts.Weretheysupported solelybystateresources(afterall,thiswastheNewYorkStatePsychiatricInstitutewhichhousestheColumbiaUniversityDepartmentofPsychiatry)ordidtheyhavefoundationorfederalgrants?Didtheyrelyon philanthropyordidtheyuseincomefrompharmacologicaltrialsorconsultations?SufficeittosaythatIlearnedalot,butwithinoneyear,the ChairmanoftheDepartmenttappedmetobecomeVice-ChairmanforEducationandTrainingDirector.Heretoo,Iwasquiteambivalentaboutthe role.Iwasconcernedthatitwouldbeall-consumingandtakemeaway frommyscienceorworse,thatitwouldboreme.Nonetheless,Idecided togiveitawhirl.Thisendedupbeingakeystepinthejourneytowards Chairmanship.TheChairmanoftheDepartmentalsourgedmetorunfor PresidentoftheAmericanPsychiatricAssociation.Nothingcouldhave beenfurtherfrommymind.Tome,suchastepwasmisalignedwithmy goalsandwouldprobablydrivemeinsanebecauseofthepolitics,toboot. Soon,Ihadleadersfromaroundthecountrycallingandemailingasking metorun.MyChairmanwasinsistentandafterall,hewasmyboss.So,I wentahead.Iwasstunnedtoseehowfun,yetdifficultitwas.Itamused menoendthatatinternationalconferences,psychiatristswhomIhad nevermetcameuptometohavetheirpicturetakenwithme.Iwashonoredthatsomanypeopleseemedtotrustmetolead.Itturnedoutthis, too,wasanimportantimprimaturforbeingconsidered“Chairmanmaterial.”Itshouldnothavesurprisedmethathighvisibilitymarriedwith academicchopswasanexcellentcombinationtobeseenasaleader,but itdid.

Pleasesharewithuswhatinitiallypiquedyourinterestinyour favoriteareaofresearchorprofessionalfocus Thatthefocusofmyworkhasbeenonsuicidalbehaviorwashappenstance.Iwasnotlookingtodoresearchperse,butwhenmymentorapproachedmetoworkinaclinicalresearchlab,Isaidmyinterestwasin depressionandcross-culturalissuesrelatedtoit.Istartedoffwiththat, butsoongravitatedtowardsthemainstreamworkofthelabbecausethat iswheremostofthebiologicalfocuswas.Itwascleartomethatbiological workwasthemosthighlyvaluedinthatlab.Ilearnedaboutpositronemissiontomography,cerebrospinalfluidstudies,postmortembrainstudies enoughthatIcouldconductsomeofthestatisticsandinterpretthedata. Itturnedoutthatneurobiologywassomeofthemostinterestingpartof theworktome.

Whatkindofimpactdoyouhopetoachieveinyourfieldthroughyour focusonyourspecificresearchtopics?

Ihopetoraisescientificawarenessofthebiologicalcontributionstosuicideriskwhichcantranslatetointerventionstopreventmorbidityand mortality.Ialsohopetodecreaseclinician’sanxietyaboutmanagingsuicidalpatientsusingimplementationsciencestrategies.

Couldyoutellusmoreaboutyourcurrentscholarlyfocalpointswithin yourchosenfieldofscience?

Ihavebeenworkingondelineatingtheriskforsuicidalideationand behavioramongpersonswhodonotmeetthecriteriaforpsychiatric disorders.Asadepartingpoint,Ihavefocusedonraisingscientificawarenessaboutthefrequencywithwhichsuicidalbehavioroccursunaccompaniedbyotherpsychiatricmorbidities.WhenIfirststartedwritingup thedata,Iwasstunnedbyhowmarriedthefieldwas/istothenotion thatsuicidalbehavioronlyrarelyhappensabsentatleastonepsychiatric disorder.Datadocumentingthecontraryappearedinpublicationsbut wentunmentionedindiscussions,nevermindthetitlesofarticles.The

prevailingclinicalloreisthatifsuicidalbehavioroccurswithoutmental illness,itmustbebecausethedisorderis“masked.”Thedatathatcontradictsthatnotionaboundsandrequiresscientificattentionforwhatit does:defyourcurrentclinicalwisdom.

Whathabitsandvaluesdidyoudevelopduringyouracademicstudies orsubsequentpostdoctoralexperiences,thatyouupholdwithinyour ownresearchenvironment?

Attentiontodetail,internallogicinformulatingresearchstudiesand writingmanuscripts,andlucidityandlinearityinwriting.

AtGenomicPress,weprioritizefosteringresearchendeavorsbased solelyontheirinherentmerit,uninfluencedbygeographyorthe researchers’personalordemographictraits.Arethereparticular culturalfacetswithinthescientificcommunitythatyouthinkwarrant transformativescrutiny,oristhereacausewithinsciencethatdeeply stirsyourpassions?

Manypeopletalkaboutthis,buttome,themetricstomeasureproductivityandqualityinresearchareheavilybiasedtowardwesternscientists’ workandinmedicine,towardbasicscience.Wemustdobetter.

Whatdoyoumostenjoyinyourcapacityasanacademicand researchleader?

Ilovediscussingideasforexperimentsandstudiesaswellaswriting grantsandmanuscripts.AsaChair,Ienjoyencouragingfacultywhodon’t usuallyworktogethertocollaborateonanimportantresearchopportunityandseeingwhattheycomeupwith.

Outsideprofessionalconfines,howdoyouprefertoallocateyour leisuremoments,orconversely,inwhatmannerwouldyouenvision spendingthesemomentsgivenachoice?

IverymuchenjoytravelingandappreciatediversityinwhatIdo.Insome cases,thegoalistoenjoyculturalaspectsofthelocation(history,local culture,art,music,architecture),butatothertimesitismoreaboutthe gastronomyoftheplace,andnotnecessarilyinfancyestablishments.I alsothoroughlyenjoynatureandamanavidhikeralthoughnotamountaineer,byanystretch!Forme,itisaboutbeingoutside,exploringhabitatswiththeirfloraandfauna,andsoakinginvistaswithadaypackand myrecentlyacquired,andimmediatelybeloved,walkingsticksformore challengingtreks.

TheGenomicPressInterviewPart2:MariaOquendo:Selected questionsfromtheProustQuestionnaire1 Whatisyourideaofperfecthappiness?

Adayatthebeach,sittingintheshade,watchingthewavesrollin.

Whatisyourgreatestfear?

Losingmymemory.

Whichlivingpersondoyoumostadmire?

SoniaSotomayor.

1 Inthelate19thcenturyvariousquestionnaireswereapopulardiversiondesigned todiscovernewthingsaboutoldfriends.Whatisnowknownasthe35-question ProustQuestionnairebecamefamousafterMarcelProust’sanswerstothese questionswerefoundandpublishedposthumously.Proustansweredthequestions twice,atages14and20.MultipleotherhistoricalandcontemporaryfigureshaveansweredtheProustQuestionnaire,suchasOscarWilde,KarlMarx,ArthurConanDoyle, StéphaneMallarmé,PaulCézanne,MartinBoucher,HughJackman,DavidBowie,and Zendaya.TheProustQuestionnaireisoftenusedtointerviewcelebrities:theidea isthatbyansweringthesequestionsanindividualwillrevealhisorhertruenature.WehavecondensedtheProustQuestionnairebyreducingthenumberofquestionsandslightlyrewordingsome.Thesecuratedquestionsprovideinsightsintothe individual’sinnerworld,rangingfromnotionsofhappinessandfeartoaspirations andinspirations.

Whatisyourgreatestextravagance?

Ilovejewelry.Ibuyitsparinglyandcarefully.ItisaninterestthatIshared joyfullywithmylatemotherwhoboughtmejewelryasayoungster. Irelishedgoingtojewelryshopswithherassheagedandbuyingherlovely piecesthatcapturedherfancy.

Whatareyoumostproudof?

Mysonsarekind,compassionate,considerate,hardworking,wonderful people.

Whatisyourgreatestregret?

Attheriskofsoundingglib,notdoingajunioryearabroadincollege.

Whatisthequalityyoumostadmireinpeople?

InSpanish,onecandescribeapersonasnoble.Ithasnothingtodowith lineage.Itisaboutkindness,morality,andcompassion.

Whatdoyouconsiderthemostoverratedvirtue? Carefreeness.

Whatisyourfavoriteoccupation? Architecture.

Wherewouldyoumostliketolive?

IamhopingtospendayearlivinginSpain,mycountryoforigin,inthenext years.AlthoughitwouldideallybeinBarcelona,therearemanywonderful placesinSpainIwouldlovetocallhome.

Whatisyourmosttreasuredpossession? Byfar,mysenseofhumor.

Whenandwherewereyouhappiest?Andwhyweresohappythen? Iwouldsaythatwitheachpassingyear,Ifeelhappier.Ithinkthatthewisdomthataccruesbringspeaceandperspective.Eventhoughmanythings declinewithage,theaccrualofwisdomovershadowsthoselosses.

Whatisyourmostmarkedcharacteristic? Apropensityforraucouslaughter.

Amongyourtalents,whichonegivesyouacompetitiveedge? Anaturalinclinationtotellpeoplewhattheydowelloraboutpositive thingsIhaveheardaboutthem.

Whatdoyouconsideryourgreatestachievement? ElectiontotheNationalAcademyofMedicine.

Ifyoucouldchangeonethingaboutyourself,whatwoulditbe? Mytendencytoangstaboutthefuture.

Whatdoyoumostvalueinyourfriends? Integrity,trustworthiness,intelligence,humor,andwarmth.

Whoareyourfavoritewriters? FavoritecurrentwritersincludeJillLeporeandJohnMcPhee.Also,Gabriel GarciaMarquez,AlejoCarpentier,JulioCortazar,andJorgeAmado.

Whoisyourherooffiction? Idon’ttendtothinkthatway.Everyonehasfoibles.

Whoareyourheroesinreallife? Asabove.

Whataphorismormottobestencapsulatesyourlifephilosophy? Dotherightthing.TowhichIwouldadd,“timely.”

MariaA.Oquendo1 1 UniversityofPennsylvania,Philadelphia,Pennsylvania19104,USA e-mail: Maria.Oquendo@pennmedicine.upenn.edu Publisher’snote: GenomicPressmaintainsapositionofimpartialityandneutrality regardingterritorialassertionsrepresentedinpublishedmaterialsandaffiliations ofinstitutionalnature.Assuch,wewillusetheaffiliationsprovidedbytheauthors, withouteditingthem.Suchusesimplyreflectswhattheauthorssubmittedtousand itdoesnotindicatethatGenomicPresssupportsanytypeofterritorialassertions.

GenomicPsychiatry

INNOVATORS&IDEAS:RESEARCHLEADER

GustavoTurecki:Threefundamentalquestions–Howdoesthebrainrespondtosocial andemotionalexperiences?Whydoespsychologicaltraumatriggerdepressive states?Whatarethemechanismsofantidepressantresponses?

©GenomicPress,2024.The“GenomicPressInterview”frameworkisprotectedundercopyright.Individualresponsesarepublishedunderexclusive andpermanentlicensetoGenomicPress.

GenomicPsychiatry January2025;1(1):11–13;doi: https://doi.org/10.61373/gp024k.0007

Keywords: Majordepressivedisorder,treatment-resistantdepression, suicide,trauma,antidepressantresponse

GustavoTureckiMDPhDFRSCisaclinicianscientistwhosework focusesonunderstandingbrainmolecularchangesthatoccurinmajor depressivedisorderandsuicide,aswellasmolecularprocessesthat explainantidepressanttreatmentresponse.Dr.TureckiisFull ProfessorandChairoftheDepartmentofPsychiatryatMcGill University,theScientificDirectorandPsychiatrist-in-Chiefofthe DouglasInstituteinMontreal,Canada,wherehealsoheadsthe DepressiveDisordersProgram.Hehasauthoredover600publications, includingresearcharticlesinleadingpeer-reviewedjournalssuchas NatureNeuroscience,NatureMedicine,and TheLancet andisamong theworld’smosthighlycitedscientistsaccordingtoClarivate,Webof Science.Hehasreceivedseveralnationalandinternationalawards andsitsonseveraladvisoryboards.Dr.Tureckigraciouslyoffersour audienceaglimpseintohispersonalandprofessionaljourney.

TheGenomicPressInterviewPart1:GustavoTurecki–Lifeandcareer Couldyougiveusaglimpseintoyourpersonalhistory,emphasizing thepivotalmomentsthatfirstkindledyourpassionforscience? Sincemychildhood,Ihavebeenfascinatedwithscienceandmedicine. WhenIgotintomedicalschool,Isoonbecameinterestedinphysiologyof exercise.Havingbeenacompetitiveswimmer,thisseemedlikeanatural extensionofmypreviousinterests.Iwasreadytoworkinsportsmedicine untilIdidmyrotationinpsychiatry.Unexpectedly,Ifoundmyselffullyfascinatedbythisfield;therefore,aftersomeinternaldebateandambivalence,Idecidedtopursuethisspecialty.Earlyinmypsychiatryresidency,I wasinvolvedinacaseofdizygotictwinsthatstronglyinfluencedmyprofessionaltrajectoryandresearchcareer.Iwasalsofortunatetocountwith excellentrolemodelsearlyon.Theywereinstrumentalinmyprofessional development,providingmewithexcellentadviceand,aboveall,theyinstilledinmecoreprofessional,scientificandpersonalvaluesthathave beenessential,asIpursuedacareerinacademicmedicine.

Wearekeentoexploreyourcareertrajectoryleadinguptoyourmost relevantleadershiprole.Whatdefiningmomentschanneledyou towardthatleadershipresponsibility?

AlthoughtodayIholdseveralleadershiproles,leadershipdidnotcome naturallytome.Iremainedinacademiabecauseoftheresearchwork andtheintellectualstimulationthatitprovides,nottobeamanager. Ifirsttookaleadershiproleoutofduty,butitwasdifficultasleadershipinvolvesskillsthatIhadtoacquirewitheffort.Inaddition,Iwas veryconcernedaboutthepotentialimpactthatthetimeIhadtodedicate wouldhaveonmylabandresearch.Aftermanyyearsindiverseleadership roles,Inowappreciatetheopportunitythatleadershipprovides,andparticularly,theopportunitytobuildcapacityandcontributetodevelopacademicpsychiatry,researchandclinicalservices.

Received:14January2024.Accepted:22January2024. Publishedonline:25January2024.

Pleasesharewithuswhatinitiallypiquedyourinterestinyour favoriteareaofresearchorprofessionalfocus.

Asaresident,Itreatedoneofadizygotictwinwhohadashareddelusion withherco-twin.Thecasewasfascinatingandledmetoexplore,conceptually,theroleofgeneticsintheetiologyofmentalillness.Ihavebeen workingingeneticsandgenomicseversince.

Whatkindofimpactdoyouhopetoachieveinyourfieldthroughyour focusonyourspecificresearchtopics?

Aboveall,Ihopemyworkwillcontributetoelucidateprocessesandmechanismsunderlyingpsychopathology,andparticularlymajordepressive disorderandsuiciderisk,whicharemyareasofmoredirectinterest.More specifically,Ihopemyworkwillhelpgainsomeinsightintohowthebrain respondstosocialandemotionalexperienceandhowtraumaticexperiencestriggerpathologicaldepressivestates.Ialsohopethatmyworkmay helpelucidatemechanismsofantidepressantresponse.

Ikeepaclinicalpractice,specializinginrefractoryortreatmentresistantmajordepressivedisorder.Itisextremelyrewardingtohelppeoplewhosufferandareunabletofunction.Whilethetreatmentsweuse

Figure1. GustavoTurecki,MD,PhD,McGillUniversity,Canada.

todayaregenerallyeffective,theydonotalwayswork,andsometimes,it takeswaytoolongtoidentifythepropertreatmentorforthetreatment toworkeffectively.IhopetheworkthatIdowilleventuallyhelpthelife ofpeoplelikethepatientsItreat.

Couldyouletusknowyourcurrentscholarlyfocalpointswithinyour chosenfieldofscience?

Currently,Iaminterestedintheunderstandingofmolecularchanges associatedwithdepressionatsingle-cellresolution.Wehaveadapted diversesingle-cellgenomicmethodstostudypostmortemhumanbrain tissueandareexploringdifferentaspectsofmajordepression.Weare alsoveryinterestedintheroleofextra-cellularvesiclesinsystemiccommunicationandhowtheircargomaybemanipulatedtoelicittherapeutic responses.

Whatdoyoumostenjoyinyourcapacityasanacademicand researchleader?

Theintellectualstimulationoftheworkandthepossibilityofcontributing toknowledge.

Whathabitsandvaluesdidyoudevelopduringyouracademicstudies orsubsequentpostdoctoralexperiences,thatyouupholdwithinyour ownresearchenvironment?

Researchisstressfulandcompetitive,butIbelievethatthelabenvironmentshouldbewelcomingandsupportive,andverycollegial.

AtGenomicPress,weprioritizefosteringresearchendeavorsbased solelyontheirinherentmerit,uninfluencedbygeographyorthe researchers’personalordemographictraits.Arethereparticular culturalfacetswithinthescientificcommunitythatyouthinkwarrant transformativescrutiny,oristhereacausewithinsciencethatdeeply stirsyourpassions?

IampassionateaboutscienceandtheworkthatIdo,andthisiswhat drivesmeandhasbeenaconstantmotivationthroughoutmyprofessional trajectory

Outsideprofessionalconfines,howdoyouprefertoallocateyour leisuremoments,orconversely,inwhatmannerwouldyouenvision spendingthesemomentsgivenachoice? Ihavemanypersonalinterests.Besidesmyfamilylife,Ikeepbusyand trytoliveabalancedlife.Iamphysicallyactive,exercisingalmostdaily.I enjoyskiing,biking,cookingandgoodwine.Igrowavegetablegardenin thesummer,andlovepolitics.Iamanavidreaderoftheeconomistand diversenewspapers,andaregularlisteneroftheGoodFightbyYascha Mounkandseveralotherpodcasts.

TheGenomicPressInterviewPart2:GustavoTurecki–Selected questionsfromtheProustQuestionnaire1 Whatisyourideaofperfecthappiness? Allmomentsofhappinessarejustperfect.

Whatisyourgreatestfear? Decline.

Whichlivingpersondoyoumostadmire? Toomanytolist.

1 Inthelatenineteenthcenturyvariousquestionnaireswereapopulardiversiondesignedtodiscovernewthingsaboutoldfriends.Whatisnowknownasthe35questionProustQuestionnairebecamefamousafterMarcelProust’sanswersto thesequestionswerefoundandpublishedposthumously.Proustansweredthequestionstwice,atages14and20.Multipleotherhistoricalandcontemporaryfigures haveansweredtheProustQuestionnaire,suchasOscarWilde,KarlMarx,ArthurConanDoyle,StéphaneMallarmé,PaulCézanne,MartinBoucher,HughJackman,David Bowie,andZendaya.TheProustQuestionnaireisoftenusedtointerviewcelebrities:theideaisthatbyansweringthesequestionsanindividualwillrevealhisorher truenature.WehavecondensedtheProustQuestionnairebyreducingthenumber ofquestionsandslightlyrewordingsome.Thesecuratedquestionsprovideinsights intotheindividual’sinnerworld,rangingfromnotionsofhappinessandfeartoaspirationsandinspirations.

Whatisyourgreatestextravagance? AbottleofGranEnemigoGualtallary.

Whatareyoumostproudof? Mythreekids.

Whatisyourgreatestregret? None.

Whatisthequalityyoumostadmireinpeople? Theirintellect.

Whatdoyouconsiderthemostoverratedvirtue? Virtuesarevirtues,can’tbeoverrated.

Whatisyourfavoriteactivity(physicalorintellectual)? Toomanytolist.

Wherewouldyoumostliketolive? RightwhereIlive,inMontréal.

Whatisyourmosttreasuredpossession? Possessionscomeandgo,soIdonottreasurethem.Theyareworthfor theirtransactionvalue.

Whenandwherewereyouhappiest?Andwhyweresohappythen? Rightnow,yesterdayandtomorrow.

Whatisyourmostmarkedcharacteristic? NotsureIcananswerthis,butprobablypersistence.

Amongyourtalents,whichonedoyouthinkgivesyouacompetitive edge?

PerhapsthefactthatIampatient,butverypersistent.

Whatisapersonality/characteristictraityouwishyouhad? Tobemoreextroverted.

Whatdoyouconsideryourgreatestachievement?

Scientifically,itwasthefirstdescriptionofhowearly-lifeadversityleads tomolecularchangesinthebrainthroughepigeneticchanges(McGowan etal,2009).

Whatdoyoumostvalueinyourfriends?

Theirsenseofhumor.

Whoareyourfavoritewriters?

Chabon,Singer,Borges,Amis,JoshuaCohen,Richler,Cortazar,PhilipRoth, BioyCasares,Beauvoir,Atwood,AmosOz,andseveralothers.

Whoareyourheroesoffiction?

Many,buttocitearecentone,GyuriKövesinFatelessnessbyImreKertész, whichIhavejustread.

Whoareyourheroesinreallife?

Mygrandparents,whoescapedNazioccupiedEastEuropeandestablishedinSouthAmericaafterlosingmanyoftheirfamilymembersand muchhardship.Theyhadnothing,workedveryhard,keptgoing,appreciatedeverythingtheyhadandwerealwaysinagoodmood.Theyhavea beenaconstantsourceofinspiration.

Whataphorismormottobestencapsulatesyourlifephilosophy? Youmakethebestofeverysituation.

GustavoTurecki1 1 McGillUniversity,Montréal,QuébecH4H1R3,Canada e-mail: gustavo.turecki@mcgill.ca

Publisher’snote: GenomicPressmaintainsapositionofimpartialityandneutralityregardingterritorialassertionsrepresentedinpublishedmaterialsandaffiliationsofinstitutionalnature.Assuch,wewillusetheaffiliationsprovidedbythe authors,withouteditingthem.SuchusesimplyreflectswhattheauthorssubmittedtousanditdoesnotindicatethatGenomicPresssupportsanytypeofterritorial assertions.

andnotificationofanychanges.Theacknowledgmentshouldnotimplylicensorendorsement.(2)NonCommercial:Thematerialcannotbeusedforcommercialpurposes.(3)NoDerivatives:Modifiedversionsoftheworkcannotbedistributed.(4) Noadditionallegalortechnologicalrestrictionsmaybeappliedbeyondthosestipulatedinthelicense.Publicdomainmaterialsorthosecoveredbystatutoryexceptionsareexemptfromtheseterms.Thislicensedoesnotcoverallpotential rights,suchaspublicityorprivacyrights,whichmayrestrictmaterialuse.Thirdpartycontentinthisarticlefallsunderthearticle’sCreativeCommonslicenseunless otherwisestated.Ifuseexceedsthelicensescopeorstatutoryregulation,permissionmustbeobtainedfromthecopyrightholder.Forcompletelicensedetails,visit https://creativecommons.org/licenses/by-nc-nd/4.0/.Thelicenseisprovidedwithoutwarranties.

GenomicPsychiatry

INNOVATORS&IDEAS:RESEARCHLEADER

EdoRonalddeKloet:Howdoestheactionofglucocorticoidschangefromprotective toharmful?Whatisthecause?Andwhataretheconsequences?

©GenomicPress,2024.The“GenomicPressInterview”frameworkisprotectedundercopyright.Individualresponsesarepublishedunderexclusive andpermanentlicensetoGenomicPress.

GenomicPsychiatry January2025;1(1):14–17;doi: https://doi.org/10.61373/gp024k.0088

Keywords: Psychoneuroendocrinology,pharmacology,stress,brain, cortisol,stress-relateddisease

Forhalfacentury,EdoRonald(Ron)deKloethaspursueda fundamentalquestioninneuroscience:howdostresshormones switchfromprotectingourbraintopotentiallyharmingit?After receivinghisPhDin1972attheUniversityofUtrechtunderDavidde Wied’smentorship,hespenttwoformativeyearswithBruceMcEwen atRockefellerUniversitybeforereturningtotheRudolfMagnus Institute.In1990,hewasappointedProfessorofMedical PharmacologyatLeidenUniversity,wherehediscoveredhowasingle hormone–cortisol–couldprotectanddamagethebrainthroughtwo distinctreceptorsystems(MRandGR).Thisfindingopenednewpaths forunderstandingandtreatingstress-relatedmentaldisorders.His research,spanningover600publications,hastransformedourgrasp ofhowthebraincopeswithstressandearnedhimnumeroushonours, includingtheGeoffreyHarrisAward(2005),theECNPAward(2007), andtheGoldenEmilKraepelinMedal(2014)foradvancingour understandingofdepression.Thoughofficially“retired”since2009, heremainsactiveasanemeritusprofessoratLeidenUniversity MedicalCentreandacademyprofessorattheRoyalNetherlands AcademyofArtsandSciences.Recognizinghiscontributionsto scienceandsociety,hewasknightedintheOrderoftheDutchLionin 2010.Recently,alongsidehislong-timecollaboratorProfessorMarian Joëls,hereceivedthe2024GlobalStress&ResilienceNetwork PioneerAward.InthisGenomicPressInterview,Dr.deKloetreflects onhisremarkablejourneyandsharesfreshinsightsintothe fascinatingworldofstressneuroscience.

Part1:RondeKloet–LifeandCareer

Couldyougiveusaglimpseintoyourpersonalhistory,emphasizing thepivotalmomentsthatfirstkindledyourpassionforscience?

MytwoolderbrothersobtainedaPhDinbiochemistryandmolecularbiology.Duringtheirthesisresearch,Isometimesjoinedtheminthelab, whichinspiredmetoenterabiochemistryprogramattheUniversityof Utrechtin1961.AfteradullBachelor’s,Ibecameexcitedwhenstarting myMaster’shands-onresearchbyisolatingnovelbioactivepeptidesfrom thesheeppinealgland,evenmoresoduringmyEndocrinologytrainingat OrganonPharmaceuticals.IlearnedfromProfessorMariusTausk,thedirector,that“Endocrinologyisaconcept,anapproach,orevenamethod. Whateverthespecificendocrinesubdiscipline,topic,orsubject,thebindingelementistheobjective:understandinghowsignalscoordinatethe processesincells,tissues,andorgans.”ThissophisticatedviewofendocrinologyintriguedmeandastudentcolleagueatOrganonsomuch thatwebothwantedtoapplyforanavailableneuroendocrinologyPhD positionguidedbythefamousProfessorDaviddeWied.However,since wedidnotwanttocompete,wediditbyflippingacoin.Mycolleague wonandgottheposition,andIwasleftempty-handed!

Received:25November2024.Accepted:29November2024. Publishedonline:10December2024.

Netherlands.

Amonthlater,IarrangedanappointmentwithProfessordeWied.Irelatedthecoin-flippingstoryonThursday,28November1968,at9.00am. Then,aftersomediscussion,ProfessordeWiedsaid:“I’llcalltheDirector ofOrganon.”After5minutes,thecallended,andDeWiedsaid,“Youcan startthiscomingMonday,2December,atOrganonwithaPhDproject.”He alsodefinedthetopicofmythesis.BruceMcEwenhadjustpublishedhis hallmarkpaperontheretentionoftraceramountsof 3 H-corticosterone incellnucleiofthehippocampalpyramidalanddentategyrusneurons.De Wiedsaid,“Wecandothisbetter,Ron!Wewillexaminethecentralaction ofthemuchmorepotentglucocorticoiddexamethasone.”

Aftertwoyears,IwroteBrucealetterstatingthatIcouldnotreproducehisfindingofcorticosteronebindinginthehippocampuswith

Figure1. EdoRonalddeKloet,PhD,LeidenUniversityMedicalCentre,The

gp.genomicpress.com

dexamethasone,andheinvitedmeovertoNewYorktosolvetheissuein hislab.AsapostdocinBruce’slab,weconfirmedtheinabilityofdexamethasonetolabelthecorticosteronereceptorsinthehippocampus.Only 20yearslater,usingmousemutantsobtainedfromPietBorst,wefound outwhy:Dexamethasone,ratherthancorticosterone,isasubstratefor multidrugresistanceP-glycoprotein(mdr-Pgp)intheblood-brainbarrier,whichpumpsthesyntheticsteroidoutofthebrain!Instead,dexamethasoneactsintheanteriorpituitarycorticotrophstosuppressstressinducedACTHrelease,afundamentalfindingforunderstandingthe DexamethasoneSuppressionTest,alaboratorytestassistinginthediagnosisofdepression,furtherdevelopedintotheDex/CRHtestbymycolleagueFlorianHolsboerinMunich.

Ilearnedthat(i)luckrequiresapreparedmind,(ii)partialreinforcementextinctionworks,and(iii)oneneedspatience.

Wewouldliketoknowmoreaboutyourcareertrajectoryleading uptoyourmostrelevantleadershiprole.Whatdefiningmoments channelledyoutowardthatleadershipresponsibility? Thesecondphaseinmycareerstartedwithmytenuredappointmentin 1975asAssociateProfessorattheRudolfMagnusInstituteunderDe Wied’sguidanceuponreturningfromRockefellerUniversity.ThestaffpositionrequiredthedevelopmentofaneuropharmacologyteachingprograminbiomedicalsciencesandparticipationintheInstitute’sresearch onneuropeptides.Theterm’neuropeptides’wascoinedinthelatesixtiesbyDaviddeWiedtodefinethecentraleffectsoffragmentsofvasopressin,oxytocin,andACTHthatweredevoidoftheirclassicalendocrine activity.Forinstance,thefragmentvasopressin(4–9),the“memorypill”, reinforcedmemoryconsolidationoffear-motivatedbehaviour.

Thelate’70swereexcitingtimesforneuropeptides!WithEvaMezey andDanDorsa,weshowedthatpeptidescleavedfrompituitaryhormones couldreachthebrainviaretrogradetransportinthepituitarystalkand theperivascularspace.WithPeterBurbach,weidentifiedthebrainendopeptidasesthatgeneratedvasopressin,oxytocin,andACTH-derived neuropeptidesfromlargerprecursormolecules.MiklosPalkovitstaught meinthemid-70sneuro-anatomyandtheabilitytopunchmorethan100 differentnucleifromfrozenbrainsections(600punches/hour),serving manyotherresearchgroups.

In1984,withAnatBiegon,DoorVoorhuis,andJackElands,wediscoveredthedistributionofoxytocinandvasopressinreceptorsindiscrete ratbrainregionsusinginvitroautoradiography.Thatdiscoveryculminatedinanexcitingtwistinthesongbird:testosterone-inducedvasotocin receptorswereconcentratedaroundasongnucleusinthecanarybrain (n.robustusarchistriatalis).Stimulationofthesereceptorsmodulated thedevelopmentofthestereotypedcanarysong.

Ilearnedthattogrowtowardaleadershiproleinneuroscience,you needtocollaboratewithexpertsinresearchonvariouslayersofbiologicalorganization,frommoleculestocellsandcircuitstobehaviour.While theaboveexperienceswereexciting,therealbreakthroughtowardleadershipwasunderstandinghowglucocorticoidsact,asdetailedinthenext section.

Pleasesharewithuswhatinitiallypiquedyourinterestinyour favouriteresearchorprofessionalfocusarea.

In1985,wehada“Eureka”momentinrecognizingtheidentityoftherodenthippocampalcorticosteronereceptors.Atthattime,RousselUclaf hadsynthesizeda’pure’glucocorticoid,distinguishingbetweenmineralocorticoidreceptors(MR)thatbindcorticosteronewitha10-foldhigher affinitythantheclassicalglucocorticoidreceptors(GR).WithDickVeldhuisandHansReul,werealizedthatthetracerdosesof 3 H-corticosterone providedasufficientamounttooccupytheMRbutnottheGR.ForGRoccupancy,corticosteroneconcentrationsmustincreasetolevelscirculatingaroundthecircadianpeakorafterstress.WithHansReul,Ankevan Eekelen,andWinSutanto,wepublishedthedistributionofMRandGRin theratbrainusinginvitroautoradiography,immunocytochemistry,andin situhybridization.WithChrisEdwardsfromEdinburgh,wedemonstrated thattheenzymaticbreakdownofnaturallyoccurringglucocorticoidswas essentialfortheMRtobecomealdosterone-specificinepithelialcells suchasthekidney.

WithanunderstandingofthecomplementaryMR-andGR-mediated actionsofcorticosteroneandcortisol,weminedgold.Suddenly,weknew howtodesignexperimentsthatmadebiologicalsenseinstressresearchtheEurekamomenttriggeredanavalancheofstudies.Itprovidedtheinroadtothegroup’stransitiontoLeidenUniversityin1990,theDivisionof MedicalPharmacology,withmypromotiontofullProfessorattheLeiden/ AmsterdamCentreofDrugResearch(LeaderDouweBreimer).Inneurophysiology,wehadanintensecollaborationwithProfessorMarianJoëls attheUniversityofAmsterdam,whodiscoveredMRandGR’scomplementaryroleinregulatingtransmitterresponsesandionconductancesina U-shapedrelationship:MRactivationtransientlyincreasedhippocampal excitability,whichwassuppressed,subsequently,bystress-inducedGR activation.MarianJoëlsandHenkKarstdiscoveredin2005thatMRcan alsomediaterapidnon-genomicactionsinthehippocampus.

WithAnnaRatka,weshowedthephysiologicalrole:MRactivation isessentialforthetoneandactivationofthestressresponsesystem, whilesubsequentGRactivationfacilitatessuppressionbynegativefeedback.Inbehaviour,MellyOitzlfoundthatMRisnecessaryforretrievinginformationandselectingacopingstyle,whileGRactivationpromotesmemoryconsolidation.MennoKrukmadeacaseforMR-dependent anxiety/aggression-drivenphenotypes.NicoleDatsonpioneeredgeneexpressionprofilinginlaser-dissectedbrainregionsandidentifiednumerousnovelglucocorticoid-responsivepathwaysinthebrainunder stress,particularlyinepigeneticprocesses.ErnoVreugdenhildiscovereda glucocorticoid-responsiveneuroplasticitygenedoublecortin-likekinase, whichfunctionsinmicrotubulesduringdevelopmentandneurogenesis. WithSeymourLevine,wemadesignificantcontributionstotheroleofglucocorticoidsinprogrammingstress-copingandadaptationinneonates forlaterlife.

Thus,a1968PhDprojectdevelopedintoasuccessfulresearchprogram.Ilearnedthatfocus,collaboration,andmutualrespectaretheingredientsforanexcitingscientificjourney.Melly,Menno,Erno,Nicole, OnnoMeijer,andRoeldeRijkformed,asgroupleaders,adreamteam, guidingtogethermorethan200Master’sandPhDstudents,postdocs, guests,andtechnicalandadministrativestaff,eachdeliveringaunique contributionthatIcannothighlightduetospacelimitations.Seefora summaryofthefirst30yearsofmycareerinDaviddeWied’sFestschrift: deKloetER.Stressinthebrain.EurJPharmacol.2000;405:187–198.

Whatimpactdoyouhopetoachieveinyourfieldbyfocusingon specificresearchtopics

2009Ireachedemeritusstatus,butIhadenoughgrantmoneytocontinue forfiveyearsbeforetheDutchmandatoryretirementat70.

Collectively,wehavecontributedtotheknowledgeofhowcortisolcoordinatesbodyandbrainfunctiontosupportcopingandadaptationand howthehormoneprogramsthisadaptiveresponseforlife.Weformulated theMR:GR(im)balancehypothesis:“UponimbalanceoftheMRandGRmediatedactions,thestressresponse’sinitiationandmanagementbecomescompromised.Atacertainthreshold,thismayleadtoacondition ofneuroendocrinedysregulationandimpairedbehaviouraladaptation, whichpotentiallycanaggravatestress-relateddeteriorationandpromote vulnerability.”

Wecollecteddata(andarestilldoingso)totestthishypothesis.Two outofseveralhighlights.Firstly,RoeldeRijkandLianeKlokdiscoveredan MRhaplotypeassociatedwithoptimismthatprotectsagainstdepression. ThisdiscoveryearnedUSandEUpatentsassignsofasuccessfultranslationalopportunity.Secondly,OnnoMeijerbecamemysuccessorinLeiden andcontinuedtoworkonglucocorticoidsdeveloping,withthesupportof CorceptTherapeutics,novelSelectiveGlucocorticoid/Mineralocorticoid ReceptorModulators(SGRM/SMRM)targetingtissueselectivereceptor co-regulators.Withthisprospect,glucocorticoidtherapywillhavefewer sideeffects.

Pleasetellusmoreaboutyourcurrentscholarlyfocalpointswithin yourchosenfieldofscience.

IwasfortunatetoparticipateintheexcitingprojectsofAlexdeNicola’s group(BuenosAires),whichhasbeenpioneeringthestrikingabilityof SGRMandSMRMtoreverseneuropathologyinanimalmodelsofchronic

Figure2. RondeKloetsailingNordicFolkboatVoiVoi.

stress,hypertension,andneurodegenerativediseases.Someofthese Corceptcompoundsarenowinphases2and3.

IamhappytoparticipateinachallengingprogramledbyMegan Galbally(Melbourne,Australia),whoexploredtheprevalenceofchildhoodanxietydisorderintheoffspringofmothersfromtheMercyPregnancyEmotionalWellbeingStudy,alongitudinalcohortstudyofpregnant womenexploringtheimpactofperinataldepression(fromconceptionto birth)atdelivery,and6months,12months,and4yearspostpartum.The outcomeoftheseexcitingstudiesalignswithdevelopmentalanimalstudies:stressduringearlylifeprogramsviacortisolactionintheamygdala emotionalreactivityforlaterlife.

Ioccasionallywriteacommentaryorreviewonstress.Forinstance, withMarcMolendijk,wewroteaseriesofarticlesonanthropomorphism inneuropharmacology,usingtheforcedswimtestasanexample.

Whathabitsandvaluesdidyoudevelopduringyouracademicstudies orsubsequentpostdoctoralexperiencesthatyouupholdwithinyour researchenvironment?

Examiningafundamentalneuroscientificquestionrequiresamultidisciplinaryapproachinasocial,behavioural,biochemical/molecular,and physiologicalcontext.Keepingupteamspiritrequiresfrequentmeetings todiscussprogress.Teamspiritandsociallablifegotogether:jointcoffee breaks,lunches,sports,and’cabaret’areessentialforshapingcollegial trustandpassion.

AtGenomicPress,weprioritizefosteringresearchendeavoursbased solelyontheirinherentmerit,uninfluencedbygeographyorthe researchers’personalordemographictraits.Arethereparticular culturalfacetswithinthescientificcommunitythatwarrant transformativescrutiny,oristhereacausewithinsciencethatdeeply stirsyourpassions?

IsupporttheEuropean’AgreementonReformingResearchAssessment’. ThisagreementbetweentheEuropeanCommission,ScienceEurope,and

theEuropeanUniversityAssociation(EUA)endorsestheobjectivesof ourNationalRecognition&Rewardsprogram.Theagreementfocuseson recognizingandrewardingacademicsfortheirvarioustasks.Thisfocus impliesthatresearchassessmentprimarilyoccursthroughaqualitative evaluationratherthanbasedonthenumberofpublications,h-indices ofauthors,orjournalimpactfactors.See https://recognitionrewards.nl/ 2022/10/10/dutch-knowledge-institutions-sign-european-agreementon-reforming-research-assessment/.

Whatdoyoumostenjoyinyourcapacityasanacademicorresearch leader?

Toinspirestudentsaboutthebeautyofthebrain,fromgenestobehaviour,inallimaginablecontexts,discussin-depthnewfindings,and guideyoungscientistsinthefirstyearsoftheirscientificlife.

Outsideprofessionalconfines,howdoyouprefertoallocateyour leisuremoments,orconversely,inwhatmannerwouldyouenvision spendingthesemomentsgivenachoice?

Ihaveasemi-professionalonlineweatherstationcalled’TheHippocampus,’inapolder4mbelowsealevelclosetowhereIlive.Justafewhighlights:Icouldmeasuretheairpressurechangesandwindshiftsfollowing theJanuary2022PacificHungaTonga-HungaHa’apaisubmarinevolcano outburst.Ilikeiceskating,swimming,sailing,hiking,andgardening.

Part2:RondeKloet–SelectedquestionsfromtheProust Questionnaire1

Whatisyourideaofperfecthappiness?

Irealizethathappinessiscontext-dependent,andherearesomeingredients.Duringexercise,myendorphinswork;duringsociallife,oxytocin maypeak;ifIcompete,dopaminehelpstopursuesuccess;andserotonin givesasenseofcontrolduringstress.Itisallperfectifitworks.

Whatisyourgreatestfear?

ThefeelingthatIhavenocontrol.

Whichlivingpersondoyoumostadmire?

Iadmiremybrotherforsaying,“Doingnothingisnotanoption,”andhe succeededinextendingthehigh-qualitylifeofoneofhisbelovedsonsfor another8years.

Whatisyourgreatestextravagance?

Tokeepmy65-year-oldmahoganywoodenNordicFolkboatinexcellent shape.

Whatareyoumostproudof?

Inmyscientificlife,Iammostproudofmy57PhDstudents,whoall successfullydefendedtheirthesis.

Whatisyourgreatestregret?

Ishouldhavereservedmoretimeforsocialactivitiesandreading.

Whatisthequalityyoumostadmireinpeople? Senseofhumour,reliability.

Whatisthetraityoumostdislikeinpeople? Narcissism

Whatdoyouconsiderthemostoverratedvirtue?

Inscience,whenanindividual’sh-indexisacriterionforjudgingscientific qualityandapredictoroffutureperformanceandsuccess.

Whatisyourfavouriteactivity?

Adailyhikeofanhourisinspiringandgivesagoodfeeling.

Wherewouldyoumostliketolive?

Ruralenvironment,atalakefront,withDutchweather.

Whatisyourmosttreasuredpossession?

Science-related:my1000+ hippocampusitems.

Whenandwherewereyouhappiest?Andwhyweresohappythen?

Again,context-dependent.Anexample:February1996,Wengen,Switzerland.Werentedacottageandwentskiingandhiking;theweatherwas fantastic,andtherewasasilentandimpressiveredeveningsunonthe 4000mhighmountains.Alternatively,sailingorwanderingwithMarian throughnature,orunderstandingexperimentaldata.

Whatisyourcurrentstateofmind?

Quiet,butwithasenseofurgencyforthingstodo

Whatisyourmostmarkedcharacteristic?

Interestintheotherperson,whattheydo,howtheythink.

Amongyourtalents,whichone(s)give(s)youacompetitiveedge?

Inscience,toidentifytalentthatcansynergizeinamultidisciplinaryfashiontoreachacommongoal.

Whatdoyouconsideryourgreatestachievement?

Withmyassociates’help,wehaveprovidedevidencetosubstantiatethat cortisolactioncontrolsaswitchbetweenresilienceandvulnerabilityin adaptationtochronicstress.

Ifyoucouldchangeonethingaboutyourself,whatwoulditbe?

Bemorecreativeinthinkingoutofthebox.

Whatdoyoumostvalueinyourfriends? Integrity.

Whoareyourfavouritewriters?

JohnGrisham’sdetectives,MartenToonder’s177Bommelstories,Val Howells’s‘SailingintoSolitude,’andMarianJoëls’latestbook,“Finished,” (ResearchgGate),anovelabouttheworldofscience,confrontingand writtenwithcompassionandwit.

Whoareyourheroesoffiction?

Kwetal,asubterraneandwellerandmastermind,ismyherointhe38th storyfromtheBommelsaga,writtenanddrawnbyMartenToonder. Kwetalcoinedtheterm“Denkraam”(noEnglishtranslation)todescribe thebrain.

Whoareyourheroesinreallife?

HermanvanPraag(1929–).Iattendedthepresentationofhislatestbook (November2024)afewdaysbeforethisinterview.Entitled:Gemoedsbewegingen(emotionalmovements).ProfessorHermanvanPraagfounded BiologicalPsychiatryintheNetherlands.Myotherheroespassedaway.

Whataphorismormottobestencapsulatesyourlifephilosophy?

“As‘tnetkinsa’tmoat,danmoat‘tmarsa’tkin”.ItisfromtheFrisian language,wheremyrootsare.InEnglish,itwouldbesomethinglike:“Ifit cannotbedoneasitshouldbe,thenitshouldbedoneasitcan.”

Leiden,TheNetherlands 25November2024

EdoRonalddeKloet,PhD1 1 DivisionofEndocrinology,DepartmentofMedicine,LeidenUniversityMedical Centre,2333ZALeiden,TheNetherlands e-mail: erdekloet@gmail.com

FundingSources

Thisarticlewasnotsupportedbyexternalfunds.

AuthorDisclosures

Theauthordeclaresthatnoconflictofinterestexists.

Publisher’snote: GenomicPressmaintainsapositionofimpartialityandneutrality regardingterritorialassertionsrepresentedinpublishedmaterialsandaffiliations ofinstitutionalnature.Assuch,wewillusetheaffiliationsprovidedbytheauthors, withouteditingthem.Suchusesimplyreflectswhattheauthorssubmittedtousand itdoesnotindicatethatGenomicPresssupportsanytypeofterritorialassertions.

GenomicPsychiatry

INNOVATORS&IDEAS:RESEARCHLEADER

MayanaZatz:Twocriticalquestionstakecenterstage–Whichvariantsmitigatethe impactoflethalmutationsinsevereconditionswithmildphenotype?Whatfactors contributetothehealthandlongevityofcentenarians?

©GenomicPress,2024.The“GenomicPressInterview”frameworkisprotectedundercopyright.Individualresponsesarepublishedunderexclusive andpermanentlicensetoGenomicPress.

GenomicPsychiatry January2025;1(1):18–20;doi: https://doi.org/10.61373/gp024k.0016

Keywords: Neuromusculardisorders,protectivevariants,aging,genome studies,geneticcounseling,ethics

MayanaZatzhasbeenaProfessorofGeneticsattheInstituteof Biosciences,UniversityofSãoPaulo(USP),Brazil,since1982.She becameanassistantprofessorafterapostdocattheUniversityofSão PauloandasecondpostdocattheUniversityofCalifornia,Los Angeles.Hercurrentresearchisfocusedonneuromusculardisorders, aging,genomics,and,morerecently,xenotransplantationandtheuse oftheZikavirusasanoncolytictherapyagainstbraintumors. Functionalstudiesaredoneingeneticallyengineeredmouseandcell models.Sheisparticularlyinterestedininvestigatingprotective mechanismsinrarepatientswithDuchennedystrophyandamilder clinicalcourse,aswellasincentenarians’healthdeterminants.

MayanaZatzisalsoinvolvedinethicalaspectsofgenomicstudiesand governmentpoliticaldecisionsrelatedtoscience.ProfessorZatzis pleasedtoofferourreadersinsightsintoherpersonaland professionalexperiences.

Part1:MayanaZatz–LifeandCareer

Couldyougiveusaglimpseintoyourpersonalhistory,emphasizing thepivotalmomentsthatfirstkindledyourpassionforscience?

IhavebeenfascinatedwithscienceforaslongasIcanremember,andI lovedreadingthebiographiesoffamousscientistssuchasMadameCurie orPasteur.Inhighschool,Ifellinlovewithgenetics.Itwasinthepremolecularera,butIwasintriguedbyhowgenetictraitsweretransmitted acrossgenerations.Idecidedtopursuethesestudiesinmyadultlife.

Wewouldliketoknowmoreaboutyourcareertrajectoryleadingupto yourmostrelevantleadershiprole.Whatdefiningmoments channeledyoutowardthatleadershipresponsibility?

WhenIstartedtostudygenetics,IdidnotimagineIwouldbealeader.My ambitionwastopursueacareerasageneticistattheUniversityofSão Paulo,whichwasandstillisthebestBrazilianUniversity.Afterreturning frommypostdocattheUniversityofCalifornia,LosAngeles,Isubmitted asmallgranttoFAPESP(SãoPauloResearchFoundation,inPortuguese: FundaçãodeAmparoàPesquisadoEstadodeSãoPaulo),ourleading researchfundingagency,tocontinuemyresearchonmusculardystrophies.DNAtechnologywasunavailable;therefore,mystudiesusedserum enzymestoinvestigatedifferentformsofmusculardystrophy.Istarted formingagroupofyoungstudents,primarilyundergraduates,interested inthissubject.Oneday,IwasinvitedtoameetingatFAPESP,andIlearned thatonlyscientificleadershadbeeninvited.ThatwaswhenIrealizedthat IwasconsideredaleaderandthatIhadagreaterresponsibility.

Pleasesharewithuswhatinitiallypiquedyourinterestinyour favoriteresearchorprofessionalfocusarea. Myinitialinterestwasmusculardystrophies.Aturningpointinmycareer waswhenayoungwomanwhohadthreenephewsaffectedbyDuchenne

Received:18February2024.Accepted:19February2024. Publishedonline:21February2024.

dystrophycametomeforadvice.Shewasgettingmarriedandworried aboutthepossibilityofhavingaffectedsons.Atthattime,nobodywas workingwithmusculardystrophiesinBrazil,anditattractedmyinterest.Iwantedtounderstandtheclinicalvariabilityamongdifferentforms ofmusculardystrophiesandtheunderlyinggeneticmechanisms.Ialso aimedtoestimatethegeneticrisksforhealthyfemalerelativestohave affectedsons.Withmycolleagues,MariaRitaPassosBuenoandMariz Vainzof,weidentifiedseveralnovelgenesresponsibleforneuromuscular disorders.Later,wediscoveredthatpatientswiththesamepathogenic mutationcouldhaveahighlyvariablecourse,showingthatotherfactors couldmodulatethephenotype.Sincethen,myresearchhasfocusedon studyingprotectivegeneticvariantsinsporadicpatientswithDuchenne musculardystrophywhoarenotasweakasonewouldexpectfromexaminingtheirgenotype.Wearealsogeneratingmicemodelscarryingcondidatemodifiervariants.Understandingtheunderlying“protective”mechanismscouldopennewavenuesfortreatment.Morerecently,myresearch focushasbeenonhealthycentenarians.Wearedoingfunctionalstudies withIPS-derivedcelllinesfromthesecentenarians.Oneintriguingquestioniswhethertheyhavegeneticvariantssimilartotopathletes.

Figure1. MayanaZatz,PhD,UniversityofSãoPaulo,Brazil.

Whatimpactdoyouhopetoachieveinyourfieldbyfocusingon specificresearchtopics?

Ihopetofindnoveltherapiesformusculardystrophiesifwecanunderstandtheprotectivemechanismsagainsttheeffectsofpathogenicmutations.Inthecaseofcentenarians,wearealsotryingtounderstandthe roleofprotectiveagingvariantsandwhethertheirproductcouldhelp promotehealthyagingforpeoplewhowerenotbornwiththeseprotectivevariants.Idecidedtofocusoncentenariansbecauseitisknownthat geneticsplaysasignificantroleinolderpeople’sresilience,particularly aftertheageof90.

Pleasetellusmoreaboutyourcurrentscholarlyfocalpointswithin yourchosenfieldofscience.

IamcoordinatingseveralprojectsonthesubjectsIjustdescribed: Duchennemusculardystrophy,centenarians,genomics,andethics.Iam alsoinvolvedintwootherprojects:xenotransplantation(aimingtouse geneticallymodifiedpigsasorgandonors),andusingtheZikavirusasan oncolyticvectoragainstbraintumors.

Whathabitsandvaluesdidyoudevelopduringyouracademicstudies orsubsequentpostdoctoralexperiencesthatyouupholdwithinyour researchenvironment?

InmyresearchenvironmentinBrazil,onemustbepreparedtodealwith muchbureaucracy;therefore,youneedtomanagefrustrationandberesilient.However,inSãoPaulo,whereIlive,wehaveanexcellentresearch fundingagency,FAPESP.Therefore,weareinamuchbettersituationthan scientistsfromotherBrazilianstates.Ibelievethatcontactwithpatients ispreciousforenhancingresearchmotivation.Knowingthestorybehind thesampleyouareworkingwithandthehopepatientsputintoyourresearchgivesyouatremendoussenseofresponsibility.Youknowthatyou havetotryyourbest.

InBrazil,themainchallengeistoincreasefundingforscience.Another significantchallengeistohaveadramaticcostreductioninnewlydevelopedtreatmentsforrarediseases,suchasspinalmuscularatrophy, hemophilia,orsicklecelldisease,inordertomakethemavailabletoall patients.

Whatdoyoumostenjoyinyourcapacityasanacademicand researchleader?

Beingascientistisfascinating.Whenyouunderstandaquestion,youopen manyothers,anditislikeplayinganendlesspuzzle.Younevergetbored becausewhatdrivesourmotivationarethequestions.Whatmovesusas scientistsisourtremendouscuriosity.Ilovetodiscussideaswithmystudentsortrytosolveproblemswhilejogginginthemorning.Also,Iloveit whenyoungstudentsapproachmeandsaythattheydecidedtobescientistsbecauseofmyinfluence.

Outsideprofessionalconfines,howdoyouprefertoallocateyour leisuremoments,orconversely,inwhatmannerwouldyouenvision spendingthesemomentsgivenachoice?

Iliketoreadprimarilybiographiesofinterestingpeople;Ilovetraveling andgoodmovies.Ilovetobewithmyfamilyandfriends.

Part2:MayanaZatz–Selectedquestionsfromthe ProustQuestionnaire1

Whatisyourideaofperfecthappiness?

Idonotbelieveinperfecthappiness.Wehavemomentswhenwearedelightedandotherswhenwemaybesad.

Whatisyourgreatestfear?

Tolosemyindependenceorcognitivecapacitywithaging.

MayanaZatzandLaura,a104-year-oldswimmingchampion.

Whichlivingpersondoyoumostadmire?

Iholdgreatadmirationforseveralpioneersingenetics,notablyShinya Yamanaka,EmmanuelleCharpentier,andJenniferDoudna.

Whatisyourgreatestextravagance? Spendingontraveling.

Whatareyoumostproudof? Mychildrenandgrandchildrenandalsosomeofmyformerstudentswho becamegreatscientists.

Whatisyourgreatestregret? Nothavingmorechildren.

Whatisthequalityyoumostadmireinpeople? Honestyandcourage.

Whatdoyouconsiderthemostoverratedvirtue? Modesty.

Whatisyourfavoriteoccupation(oractivity)? Scientificresearch.

1 Inthelatenineteenthcentury,variousquestionnaireswereapopulardiversion designedtodiscovernewthingsaboutoldfriends.Whatisnowknownasthe35questionProustQuestionnairebecamefamousafterMarcelProust’sanswersto thesequestionswerefoundandpublishedposthumously.Proustansweredthequestionstwice,atages14and20.Multipleotherhistoricalandcontemporaryfigures haveansweredtheProustQuestionnaire,suchasOscarWilde,KarlMarx,ArthurConanDoyle,StéphaneMallarmé,PaulCézanne,MartinBoucher,HughJackman,David Bowie,andZendaya.TheProustQuestionnaireisoftenusedtointerviewcelebrities: theideaisthatbyansweringthesequestions,anindividualwillrevealhisorher truenature.WehavecondensedtheProustQuestionnairebyreducingthenumber ofquestionsandslightlyrewordingsome.Thesecuratedquestionsprovideinsights intotheindividual’sinnerworld,rangingfromnotionsofhappinessandfeartoaspirationsandinspirations.

Figure2.

Wherewouldyoumostliketolive? IlovetheplacewhereIlivenow.

Whatisyourmosttreasuredpossession? Myhouse.

Whenandwherewereyouhappiest?Andwhywereyousohappythen? Whenmydaughterwasborn.IwantedtohaveagirlasIalreadyhadason. Andrightbeforeshewasborn,IfinishedwritingmyPhDthesisandmoved tothehousewhereInowlive.Irememberthatcomingfromthehospital withherintomynewhousewasthehappiestmomentinmylife.

Whatisyourmostmarkedcharacteristic? Iamincrediblydriven.

Amongyourtalents,whichone(s)givesyouacompetitiveedge? Creativity,notgivingupquickly,andnotbeingafraidtotestnewideas.

Whatdoyouconsideryourgreatestachievement? Myscientificcareer.

Ifyoucouldchangeonethingaboutyourself,whatwoulditbe? Myage.

Whatdoyoumostvalueinyourfriends? Sincerity.

Whoareyourfavoritewriters?

GeorgeOrwell(1984)andWalterIsaacson(biographies).

Whoareyourheroesoffiction? ForrestGump.

Whoareyourheroesinreallife?

MygreatestherowasNobellaureateRitaLevi-Montalcini,whodiedat age103whilestillactive.

Whataphorismormottobestencapsulatesyourlifephilosophy? Neverbelievethatyouhaveachievedthebest.Thereisalwaysroomfor improvement.

MayanaZatz1

1 UniversityofSãoPaulo,05508-090SãoPaulo,SãoPaulo,Brazil e-mail: mayazatz@usp.br

Publisher’snote: GenomicPressmaintainsapositionofimpartialityandneutrality regardingterritorialassertionsrepresentedinpublishedmaterialsandaffiliations ofinstitutionalnature.Assuch,wewillusetheaffiliationsprovidedbytheauthors, withouteditingthem.Suchusesimplyreflectswhattheauthorssubmittedtousand itdoesnotindicatethatGenomicPresssupportsanytypeofterritorialassertions.

GenomicPsychiatry

INNOVATORS&IDEAS:RESEARCHLEADER

NoboruHiroi:Exploringthecellularanddevelopmentaloriginsofneuropsychiatric disorderslinkedtohumancopy-numbervariation

©GenomicPress,2024.The“GenomicPressInterview”frameworkisprotectedundercopyright.Individualresponsesarepublishedunderexclusive andpermanentlicensetoGenomicPress.

GenomicPsychiatry January2025;1(1):21–23;doi: https://doi.org/10.61373/gp024k.0013

Keywords: 22q11.2,autism,cellmodel,copynumbervariation, intellectualdisability,mousemodel,schizophrenia

ProfessorNoboruHiroiisafacultymemberintheDepartmentsof Pharmacology,CellularandIntegrativePhysiology,andCellSystems& AnatomyattheUniversityofTexasHealthScienceCenteratSan Antonio(UTHealthSanAntonio),USA.Hejoinedhiscurrent institutionin2019afterworkingatAlbertEinsteinCollegeof MedicineinNewYorkfor21years.Hiscurrentworkisfocusedonthe cellularanddevelopmentaloriginsofthedimensionsof neuropsychiatricdisordersingeneticallyengineeredmouseandcell models.ProfessorHiroiishappytoprovideourreaderswith reflectionsonhislifeandcareer.

Part1:NoboruHiroi–LifeandCareer

Couldyougiveusaglimpseintoyourpersonalhistory,emphasizing thepivotalmomentsthatfirstkindledyourpassionforscience?

IwasborninasmalltownneartheportcityofYokohama,Japan.Itissurroundedbymountainsandtheocean,andthereisrichanimallife.Iwas alwaysfascinatedbythenormalandabnormalwaysanimalsbehave.As anundergraduatestudentatWasedaUniversityinTokyo,alecturerwho spenthissabbaticalyearatMcGillUniversity(Montreal,Quebec,Canada) taughtmeaboutthediscoveriesbyJamesOldsandPeterMilnerofarewardcenterintherodentbrain,D.O.Hebb’sconceptualizationofsynaptic plasticity,andmultiplememorysystemsbyBrendaMilnerandNormanM. WhiteatMcGill.IwasfortunatetoreceiveafullscholarshipfromanewspapercompanyinJapanand,later,anotherscholarshipfromtheGovernmentofCanadatocompletemyPhDatMcGill.Thesefirstpivotalevents setthestageformycareerinscience.

Wewouldliketoknowmoreaboutyourcareertrajectoryleadingupto yourmostrelevantleadershiprole.Whatdefiningmoments channeledyoutowardthatleadershipresponsibility?

MyPhDthesisworkwasfocusedontheanatomicallocicriticalforamphetamine’saddictiveproperties.AftercompletingmyPhDthesisin 1991,Iwasofferedpostdoctoralpositionsinlabsthatwereworkingon anatomicalsubstratesofaddictivesubstancesattheUniversityofBritish Columbia(Vancouver,BritishColumbia,Canada),CambridgeUniversity (Cambridge,UnitedKingdom),andtheMassachusettsInstituteofTechnology(MIT,Cambridge,MA).IchosethelaboratoryofProfessorAnn M.GraybielatMITtoexpandmygraduatetraininginbehavioralneurosciencetothecompartmentalizationofthestriatum,afieldpioneeredby Dr.Graybielandothers.AtMIT,Iwasinvolvedinacollaborativeproject withDr.SusumuTonegawa,oneofthepioneerswhointroducedgeneticallyengineeredmiceinstudiesoftheroleofgenesinbehavior.IcompletedasecondpostdoctoralprograminthelaboratoryofDr.EricNestler atYaleUniversity(NewHaven,Connecticut,USA)tofurtherdevelopmy skillsinthemolecularanalysesofaddictivebehaviorsingeneticallyengineeredmousemodels.Myworkthereinvolveddeterminingtherolesof twogenes,FBJmurineosteosarcomaviraloncogenehomologB(FosB) anddopamine-andcyclicadenosinemonophosphate–regulatedphosphoprotein,Mr32kDa(DARPP-32),ingeneticknockoutmousemodels,in

Received:8February2024.Accepted:9February2024. Publishedonline:16February2024.

Antonio,USA.

collaborationwithDr.MichaelE.GreenbergatHarvardandDr.PaulGreengardatRockefellerUniversity.

IwasrecruitedtoAlbertEinsteinCollegeofMedicineinNewYork in1998asanindependentjuniorfacultymembertofurtherstrengthen itsaddictionresearchprogram.However,Iwassoonfascinatedbymy colleagues’workonpatientswhocarriedcopy-numbervariants(CNVs)at humanchromosome22q11.2.PatientswiththeseCNVsexhibit schizophrenia,intellectualdisability,andautismspectrumdisorder atratesfarabovewhatisexpectedinthegeneralpopulation.Itbecame apparentthatthesepatientsrepresentedgeneticallyidentifiablecases ofmentaldisorders,whichwastoointerestingatopictopasson,andI startedanewprojectexploringthisassociationin1999.Mypostdoctoral trainingintheintegrativeuseofgeneticallyengineeredmiceenabled metocontributetotheworkofEinstein’s22q11researchteam.Iwas fortunatetocollaboratewithmany22q11pioneers,includingDrs.Raju Kucherlapati,BerniceMorrow,andmanyothers.

Asmymouseworkdeveloped,Istartedexpandingthelevelofanalysis byformingateamofinvestigatorsspecializingintheimagingofmouse brains,computationalmodeling,andcellmodels.Thiscollaborativeteam grewtoinvolvemanyinvestigatorsoutsideEinstein,includinggroupsin IrelandandJapan.Icurrentlyorganizeaninternationalteamwiththose investigators.

Figure1. NoboruHiroi,PhD,UniversityofTexasHealthScienceCenteratSan

Whatkindofimpactdoyouhopetoachieveinyourfieldbyfocusing onyourspecificresearchtopics?

Ihopetoincreasetheknowledgeofcellularandmolecularsubstratesfor 22q11.2CNV-linkedpsychiatricdisorderssothattheimplementationof precisionmedicineinpsychiatrycanbecomeareality.

Couldyoutellusmoreaboutyourcurrentscholarlyfocalpointswithin yourchosenfieldofscience?

Mycurrentpointoffocusistoelucidateandfinelydefinethecellularand developmentaloriginsofcognitivedeficitscommonlyaffectedinCNVassociatedcasesofschizophrenia,autism,andintellectualdisabilities. Theultimatevalidationofourfindingsincellandmousemodelswould comewhentherapeuticoptionsdevelopedfromthemechanisticunderstandingderivedinthesemodelsystemsprovetobeeffectivefortreating highlyspecificdimensionsofmentaldisorders,whichwouldbemydream. Evenifpotentialtherapeuticoptionsturnouttobenoteffective,thenegativeoutcomeswouldfurthermotivatemetoexploreotherpotentialcellularanddevelopmentalmechanismsofmentalillnessinmodelsystems.

Whathabitsandvaluesdidyoudevelopduringyouracademicstudies orsubsequentpostdoctoralexperiencesthatyouupholdwithinyour ownresearchenvironment?

Frommythesismentor,NormanM.White,Ilearnedtosticktomyown ideas,evenwhentheyarenotwellaccepted.Ilearnedscientificrigorfrom AnnM.Graybielandtheimportanceofvisionsfrommycollaborationwith SusumuTonegawaatMIT.Ilearnedcutting-edgemolecularapproaches fromEricNestleratYale.Fromallofmymajorscientificmentors,Ilearned theimportanceofcontinuouslyincorporatingnewtechniquesandideas intomyproject.

Thecurrenttrend,whichIdonotparticularlyappreciate,isthatarticlesconsistentwithprevailingdogmastendtopopulatemajorhighimpactjournalsandthoseinconsistentwiththeseprevailingdogmasare notpublishedinprominentjournals.Thistrendisexacerbatedbythe geographiclocationsoftheauthors.Authorswhodonotresideincountrieswheredogmasarepopularmightbepublishedlessfrequently,ifthe reviewersoftheirworkarefromcountrieswherethedogmasarewidely held.Iampassionateaboutpromotingworkthatdoesnotsupportthe prevailingconcepts.

Whatdoyoumostenjoyinyourcapacityasanacademicand researchleader?

ThemostenjoyablemomentsarethetimeswhenIdiscussnewideaswith mycolleagues.

Outsideprofessionalconfines,howdoyouprefertoallocateyour leisuremoments,orconversely,inwhatmannerwouldyouenvision spendingthesemomentsgivenachoice?

MymotherishousedinacarefacilityinJapanthatspecializesin Alzheimer’sdisease.WheneverIhaveachance,Itrytoflytoseeher.

Part2:NoboruHiroi–SelectedquestionsfromtheProust Questionnaire1

Whatisyourideaofperfecthappiness? Forme,perfecthappinessinvolvesseeingthatkindofhappinessinmy familyandtwodogs.

Whatisyourgreatestfear?

MymotheriscurrentlyafflictedwithAlzheimer’sdisease.Theprospect ofdevelopingthesameconditionatsomepointinmylifeiscurrentlymy greatestfear.

Whichlivingpersondoyoumostadmire? Therearetoomanytopickafew.

Whatisyourgreatestextravagance?

MygreatestextravaganceinvolvessharingsuperbJapanesefoodwithmy colleaguesandcollaborators.

Whatareyoumostproudof?

IamtryingtoachievethisstateofmindaboutmyworkbythetimeIdie orceasetofunctionintellectually.

Whatisyourgreatestregret? ItendtoforgetwhatIregret.

Whatisthequalityyoumostadmireinpeople?

Oneofthemostadmirablequalitiesinpeopleistheirabilitytoachieve goalsdespiteadversity.IgavemysonthemiddlenameMoses.Youget theideaofwhatqualitiesIadmireinpeople.

Whatdoyouconsiderthemostoverratedvirtue?

Iamnotsure.Itdependsoncertainperspectives,andtheydifferindividually.

Whatisyourfavoriteoccupation?

Mycurrentoccupationismyfavorite.ThisjobiswhatIdreamedofasa child.

Wherewouldyoumostliketolive?

IwouldliveanywhereIcancollaboratewithgoodpeopleuntilIdevelop dementiaorretire.Afterretirementordevelopingdementia,Iwould mostlikelyliveinJapantoenjoythegreatfoodandhotsprings.Moreover,Japanoffersgood,affordablemedicalcareandcarefacilities.Major surgeriesandgoodcarefacilitiesintheUnitedStateswouldcostmea fortune.

Whatisyourmosttreasuredpossession?

Idonottreasurephysicalpossessions.Seeingmyfather’sbelongingsafterherecentlypassedawaymademerealizethatphysicalpossessionsdo notmeanmuch.Icannot“possess”nonphysicalthings.Myfamilyisnot my“possession.”Therefore,Icannotthinkofanytreasuredpossessions.

Whenandwherewereyouhappiest?Andwhyweresohappythen? Iamhappiestnow.Ihaveneverbeenthishappy.Relativelyspeaking,my lifewasnotsogreatearlier.

Whatisyourmostmarkedcharacteristic?

Itendtospeakhonestlyandfrankly,eventotheextentthatmyspeech maybebluntandabrasiveattimes.

Amongyourtalents,whichonegivesyouacompetitiveedge?

ItendtosaywhatIthinkistrue,evenifitmakesothersuncomfortableor infuriated.ThatistheonlythingIcanthinkof,ifyoucountitasatalent.

1 Inthelatenineteenthcenturyvariousquestionnaireswereapopulardiversiondesignedtodiscovernewthingsaboutoldfriends.Whatisnowknownasthe35questionProustQuestionnairebecamefamousafterMarcelProust’sanswersto thesequestionswerefoundandpublishedposthumously.Proustansweredthequestionstwice,atages14and20.Multipleotherhistoricalandcontemporaryfigures haveansweredtheProustQuestionnaire,suchasOscarWilde,KarlMarx,ArthurConanDoyle,StéphaneMallarmé,PaulCézanne,MartinBoucher,HughJackman,David Bowie,andZendaya.TheProustQuestionnaireisoftenusedtointerviewcelebrities:theideaisthatbyansweringthesequestionsanindividualwillrevealhisorher truenature.WehavecondensedtheProustQuestionnairebyreducingthenumber ofquestionsandslightlyrewordingsome.Thesecuratedquestionsaimtoprovide insightsintotheindividual’sinnerworld,rangingfromnotionsofhappinessandfear toaspirationsandinspirations.

Whatisapersonality/characteristictraityouwishyouhad? IdonotwishtohavesomethingIdonothaveoramincapableofhaving.

Whatdoyouconsideryourgreatestachievement?

MygreatestachievementsofaristhatIhavesurvivedasaresearcher.

Whatdoyoumostvalueinyourfriends?

TheattributesImostvalueinmyfriendsarethattheyareforgivingand notjudgmental.

Whoareyourfavoritewriters?

Ienjoymanynonfictionwriters.

Whoareyourheroesoffiction?

Idon’tlikeheroesinfictionorfictionalworlds.IdonotlikeDisneyorany otherthemeparksthatincludeheroes.Ifanything,Iprefervillains.They aretough.

Whoareyourheroesinreallife?

Heroesinreallifearethosewhosacrificeeverythingforthebenefitand well-beingofothers.

Whataphorismormottobestencapsulatesyourlifephilosophy?

TheessenceoflifedescribedinEcclesiastesbestencapsulatesmylifephilosophy.Despiteitsseeminglypessimisticviewoflife,itsconclusionis

thatoneshouldneverthelesspursuewisdombecausethatiswhatprovidencedictates.

NoboruHiroi1

1 DepartmentsofPharmacology,CellularandIntegrativePhysiology,andCell Systems&Anatomy,UTHealthSanAntonio,SanAntonio,Texas78229,USA e-mail: hiroi@uthscsa.edu

Publisher’snote: GenomicPressmaintainsapositionofimpartialityandneutrality regardingterritorialassertionsrepresentedinpublishedmaterialsandaffiliations ofinstitutionalnature.Assuch,wewillusetheaffiliationsprovidedbytheauthors, withouteditingthem.Suchusesimplyreflectswhattheauthorssubmittedtousand itdoesnotindicatethatGenomicPresssupportsanytypeofterritorialassertions.

VIEWPOINT

Whystatinghypothesesingrantwritingisusuallynecessary

©TheAuthor(s),2024.ThisarticleisunderexclusiveandpermanentlicensetoGenomicPress

GenomicPsychiatry January2025;1(1):24–25;doi: https://doi.org/10.61373/gp024v.0031

Keywords: Hypothesisevaluation,researchmethodology,philosophical perspectivesinscience,scientificdiscourse,knowledgeevolution

Inthisviewpoint,weexploretheprovocativeargumentbyHernánand Greenland,presentedinJAMA,regardingthetraditionalnecessityof statinghypothesesingrantapplications.Theyproposethatthis conventionmayhindertheexplorativenatureofresearch,callingfor areevaluationthatcouldimpactglobalresearchpracticesand methodologies.Hypothesesprovideastructuredframeworkcrucial forclarifyingresearchquestionsandfacilitatingsuccessfulfunding. However,HernánandGreenlandmergegrantwritingwithresearch execution,potentiallyundervaluingthestrategicroleofhypotheses. WediscusstheperspectivesofphilosophersKarlPopperandThomas Kuhn,emphasizingtheessentialroleofhypothesesinfostering scientificprogressthroughcriticalscrutinyandparadigmshifts. WhileacknowledgingthevalueinHernánandGreenland’sflexibility fordata-drivenresearch,weassertthathypothesesremain fundamentalinguidingscientificinquiry,balancinginnovationwith traditionalrigor.Ourdiscussionaimstocontributetotheevolutionof researchmethodologies,ensuringtheyarebothinnovativeand groundedinsystematic,hypothesis-drivenapproaches.

Intheirthought-provokingcommentarypublishedin JAMA (1),MiguelA. HernánandSanderGreenlandproposeareevaluationofthetraditional necessitytostatehypothesesingrantapplications,suggestingthatthis practicemightbeunnecessaryandevendetrimentaltotheessenceofresearchtoexploreeffectswithprecisionandopenness.Ourmotivationto engagewithHernánandGreenland’sdiscourse,particularlygivenitspublicationinaprestigiousplatformlike JAMA,stemsfromanunderstandingoftheprofoundimpactthisdebatecanhaveonclinicalpracticesand researchmethodologies.Theconversationextendsbeyondacademicdiscourse,affectinghowresearchisconceptualized,funded,andexecuted globally.Engaginginthisdialogueisessentialfordevelopingresearch methodologiesthatcombineinnovationwiththerigornecessaryforsignificantadvancementsinmedicalscienceandbeyond.

TheoriginalpurposeoftheHernánandGreenlandarticle,asinferred fromitstitle,appearstofocusontheroleofhypothesesingrantwriting.However,thecontentextendsbeyondthistoencompasstheimplementationofresearch,blurringthelinesbetweenthesedistinctphases. Ingrantwriting,hypothesesarecrucialastheyencapsulatetheresearch question,direction,andrationale,providingaclearandstructuredframeworkforthestudy(2–4).Theyserveasfoundationalelementsthatguide theresearch’sconceptualandanalyticaltrajectory,facilitatingsuccessful grantacquisition.

However,HernánandGreenland’sblendofthegrant-writingprocess withresearchexecutionoverlooksthefoundationalrolehypothesesplay intheformer.Whiletheircallforflexibilityanddata-drivenapproachesin researchexecutionisvalidandvaluable,itsomewhatdiminishestheimportanceofawell-articulatedhypothesisinsecuringgrantfunding.This overlookcanleadtounderestimationofthestrategicimportanceofhypothesesinguidingtheresearchjourney,accommodatingnewdata,and fosteringunanticipateddiscoveries.

Received:11March2024.Revised:20April2024.Accepted:21April2024. Publishedonline:2May2024.

ExpandinguponthephilosophicalperspectivesofKarlPopperand ThomasKuhnprovidesaricherunderstandingofthisdebate.KarlPopper andThomasKuhnaretwoofthe20thcentury’smostinfluentialphilosophersofscience.Eachoffersdistinctperspectivesontheroleofhypothesesinscientificprogressandthedynamicsofparadigmshifts.Popper, knownforhistheoryoffalsifiability(5),arguesthatscientifictheories shouldbeframedinsuchawaythattheycanberigorouslytestedandpotentiallydisproven.AccordingtoPopper,thegrowthofscientificknowledgeisanevolutionaryprocessdrivenbythecycleofconjecturesand refutations.Heproposesthatscientistsputforwardboldhypothesesand thenattempttofalsifythem.Inthisview,hypothesesarecrucialasthey offerclear,testablepropositionsthatchallengethestatusquo.Popper contendsthattheinabilitytofalsifyahypothesisdoesnotconfirmitasaccuratebutmerelyupholdsitasthebestapproximationoftruthcurrently available.Thus,forPopper,thehypothesis-drivenapproachiscentralto scientificdiscovery,asitencouragesrobusttestingandcriticalscrutiny, leadingtotheeliminationoferrorsandtheadvancementofknowledge.

Ontheotherhand,Kuhnintroducestheconceptofscientific paradigms(6, 7)whichhedefinesasuniversallyrecognizedscientific achievementsthat,foratime,providemodelproblemsandsolutionsto acommunityofpractitioners.AccordingtoKuhn,normalscienceoperateswithintheconfinesofthecurrentparadigm,focusingonsolving puzzlesthattheparadigmdelineates.However,whentheparadigmencountersanomalies,itcannotbeexplained,thismayleadtoascientificcrisisandtheeventualemergenceofanewparadigm—aparadigm shift.ForKuhn,hypothesesareembeddedwithintheprevailingscientific paradigms,guidingwhatquestionsscientistsaskandhowtheyinterpret data.Hesuggeststhatsignificantscientificprogress—paradigmshifts— occursnotjustbyaccumulatingfactsordisprovinghypotheseswithinthe currentparadigm,butbyfundamentallychangingtheconceptualframeworkthroughwhichscientistsviewtheworld.

Thus,frombothPopper’sandKuhn’sperspectives,hypothesis-driven approachesarefundamentaltothedynamicsofscientificprogress.They supportthesystematicandcriticalexaminationofourtheoriesandpractices,promotingcontinuousimprovementandadaptationinourquestto understandtheuniverse.Theseapproachesencouragenotonlytherefinementofexistingknowledgewithincurrentparadigmsbutalsothe revolutionaryshiftsthatredefinescientificunderstanding.Inessence,by fosteringarigorous,question-drivenapproachtoresearch,hypotheses playavitalroleinboththeevolutionaryandrevolutionaryaspectsofscientificadvancement.

Incontemporaryscientificresearch,acleardistinctionemergesbetweentraditionalhypothesis-drivenstudiesandhypothesis-freeinvestigationstypicalof‘bigdata’approaches,suchasgenome-wideassociationstudies(GWAS)(8).Traditionalmethods,deeplyrootedinspecific, testablehypotheses,remainessentialfortargetedscientificinquiries. Conversely,GWASandsimilarbigdatamethodologiesanalyzeextensivedatasetstoidentifypotentialcorrelationswithoutinitialhypotheses.Theseexplorations,whilenotimmediatelygroundedinhypothesis testing,oftengeneratefindingsthatnecessitatesubsequenthypothesisdrivenresearch.Suchsequentialapproachesensurethatstatisticallysignificantresultsfromlarge-scaledataanalysisarerigorouslytestedfor

theirbiologicalsignificance,therebybridgingthegapbetweenstatisticaldiscoveryandbiomedicalinsight.Thisiterativecycleofdiscoveryand validationembodiesthedynamismandadaptabilityofmodernscientific practice.

WhileHernánandGreenlandraisesignificantpointsthatwarrantseriousconsideration,itisessentialtoreflectonthebroaderimplications oftheirarguments,particularlyinthecontextoftheirpublicationina high-impactjournallike JAMA.Thediscoursesurroundingtheroleofhypothesesinscientificresearchisvital,asitshapesthefutureofhowwe approach,understand,andsolvethecomplexproblemsfacingthemedicalandscientificcommunities.Itisourhopethatbyaddingourvoiceto thisconversation,wecancontributetotheongoingevolutionofresearch methodologiesthatarebothinnovativeandgroundedintherobusttraditionsofscientificinquiry.

YunyuXiao,PhD1 ,andMyrnaM.Weissman,PhD2 , 3

1 DepartmentofPopulationHealthSciences,DepartmentofPsychiatry,Weill CornellMedicine,NewYork,NewYork,10065,USA; 2 DepartmentofPsychiatry, VagelosCollegeofPhysiciansandSurgeons,ColumbiaUniversity,NewYork, NewYork10032,USA; 3 NewYorkStatePsychiatricInstitute,NewYork, NewYork10032,USA

e-mail: yux4008@med.cornell.edu

tion,review,orapprovalofthemanuscript,orthedecisiontosubmitthe manuscriptforpublication.

References

1.HernánMA,GreenlandS.WhyStatingHypothesesinGrantApplicationsIsUnnecessary. JAMA.2024;331(4):285-6.DOI: 10.1001/jama.2023.27163

2.ArdehaliH.HowtoWriteaSuccessfulGrantApplicationandResearchPaper.CircRes. 2014;114(8):1231-4.DOI: 10.1161/CIRCRESAHA.114.303695

3.MonteAA,LibbyAM.IntroductiontotheSpecificAimsPageofaGrantProposal.Acad EmergMed.2018;25(9):1042-7.DOI: 10.1111/acem.13419

4.LockeLF,SpirdusoWW,SilvermanSJ.ProposalsThatWork:AGuideforPlanningDissertationsandGrantProposals.SagePublications;2013.

5.PopperK,HansenTE,PickelA,KinoryJ.TheTwoFundamentalProblemsoftheTheory ofKnowledge.Routledge;2014.

6.KuhnTS.TheStructureofScientificRevolutions.UniversityofChicagopress;2012.

7.KuhnTS.Secondthoughtsonparadigms.StructSciTheor.1974;2:459-82.

8.UffelmannE,HuangQQ,MunungNS,etal.Genome-wideassociationstudies.NatRev MethodsPrimer.2021;1(1):1-21.DOI: 10.1038/s43586-021-00056-9

FundingSources

ThisworkwassupportedinpartbygrantsfromtheNationalInstitutesof Health(NIH)R01MH121921andRF1MH134649Y.X.andR01MH121922 M.M.W.

ConflictsofInterest

Dr.WeissmanreceivesbookroyaltiesfromPerseusPress,OxfordPress, andAPAPublishingPress.Noneofthempresentsaconflictofinterest.

RoleoftheFunder/Sponsor

Thesponsorhadnoroleinthedesignandconductofthestudy,thecollection,management,analysis,andinterpretationofthedata,theprepara-

OpenAccess. ThisarticleislicensedtoGenomicPressundertheCreativeCommonsAttribution-NonCommercial-NoDerivatives4.0InternationalLicense(CCBY-NC-ND4.0).Thelicensemandates:(1)Attribution:Credit mustbegiventotheoriginalwork,withalinktothelicenseandnotificationofany changes.Theacknowledgmentshouldnotimplylicensorendorsement.(2)NonCommercial:Thematerialcannotbeusedforcommercialpurposes.(3)NoDerivatives: Modifiedversionsoftheworkcannotbedistributed.(4)Noadditionallegalortechnologicalrestrictionsmaybeappliedbeyondthosestipulatedinthelicense.Public domainmaterialsorthosecoveredbystatutoryexceptionsareexemptfromthese terms.Thislicensedoesnotcoverallpotentialrights,suchaspublicityorprivacy rights,whichmayrestrictmaterialuse.Third-partycontentinthisarticlefallsunderthearticle’sCreativeCommonslicenseunlessotherwisestated.Ifuseexceeds thelicensescopeorstatutoryregulation,permissionmustbeobtainedfromthe copyrightholder.Forcompletelicensedetails,visit https://creativecommons.org/ licenses/by-nc-nd/4.0/.Thelicenseisprovidedwithoutwarranties.

BENCHTOBEDSIDE

Theimportanceofelderlygenomes

MayanaZatz1

Thedifficultyinclassifyingararegeneticvariantas“likelypathogenic,”“likelybenign,”orVUS(variantofunknownsignificance)representsa significantchallengeingeneticcounseling(GC)whentryingtoestablishadiagnosisorasaresultofincidentalfindings.Thisclassificationmay impactthecommunicationofprognosisinlate-onsetconditions,suchasneuromusculardisorders,andtheconsultants’reproductivedecisions regardingfutureoffspring.Here,wereporttwounrelatedfamilies,oneBrazilianandoneofEastAsianancestry,wherearareandpreviously unreporteddeletioninthedystrophingenewasidentified.Inthesetwofamilies,theanalysisofoldermalerelatives(from56to89yearsold) whowerefullyasymptomaticprovidedrelevantinformationtotheirfamiliesaboutthepotentialpathogenicityofthisdystrophinvariant. Thesecasessupportourprevioussuggestionhighlightingtherelevanceofgenomesequencingofolderhealthyindividualsorfamilymembers, abovetheageof50andgoingintothe80’sad90’s,andtheimportanceofsharingnewrelevantinformationfordecision-makingwithfamilies whopreviouslyunderwentgeneticcounseling.Inaddition,thesecasereportscontributetotheclassificationofVUS,enhancingourknowledge oftheimpactofspecificmutationsinfunctionalstudies.

GenomicPsychiatry January2025;1(1):26–27;doi: https://doi.org/10.61373/gp024b.0019

Keywords: Beckermusculardystrophy,Duchennemusculardystrophy,exomesequencing,geneticcounselling,genomics,wholegenomesequencing

Next-generationsequencing(NGS)hasallowedimmenseimprovement indiagnosinggeneticdisorders,facilitatingprecisionmedicine.Furthermore,thecurrentcostofwholeexomesequencing(WES)andwhole genomesequencing(WGS)makesthemincreasinglyaccessiblediagnostictools.However,wefrequentlyhavetodealwithvariantsofunknown significance(VUS),whichcouldcauseamajorillnessorjustbeararegeneticvariantnotyetdepositedintheinternationalgenomicdatabanks.

Anotherethicalchallengegeneticistsfacewhensequencingagenome isaccidentalfindingsthatcouldbeutterlyunrelatedtothediseaseof theproblem.Forexample,amutationinthe BRCA1 gene,responsiblefor breastcancer,inan8-year-oldboywithanundiagnosedmyopathy.Should theprobandorthefamilybeinformed?TheAmericanCollegeofMedical GeneticsandGenomics(ACMG)publishedalistofgenesandgeneticvariantsthatshouldbereportedasincidentalfindings(orsecondaryfindings) whentheyarediscoveredduringgenomictesting,eveniftheyareunrelatedtothesuspecteddiagnosis(1, 2).

OurstrategyhasbeentosequencethegenomeofhealthyelderlyindividualsinBrazil,asthosesequencescould(1)contributetodatabanks ofouradmixedBrazilianpopulation,(2)helptoclassifythepathogenicityofrareunknownvariants,and(3)provideessentialinsightsonconditionsthatareprevalentlaterinlife,suchashypertension,type2diabetes, Parkinson’s,andcancer,amongothers.Topursuethisstrategy,in2008, welaunchedthe80+ project,aimingtosequencethegenomesofolder Brazilians.Afirstdraft,with609exomes,waspublishedin2017(3),and asecondstudy,includingWGSof1171individuals,waspublishedin2022 (4),representingthemostextensivegenomicdatabankofolderindividualsinLatinAmerica.

Acommentin Cell publishedseveralyearsafterourfirststudywasinitiatedcalledattentiontotheimportanceofstudyingthegenomesofadmixedpopulations,asavailabledatabankshavebeenconstructedmainly withindividualsofEuropeanancestry(5).Indeed,inourrecentWGSstudy ofmorethan1000individuals,weidentified2milliongeneticvariantsnot reportedpreviously.Morerecently,theAllofUsResearchProgram(6),a longitudinalcohortstudyaimingtoenrolladiversegroupofatleastone millionindividualsacrosstheUnitedStates,involved77%ofparticipants fromcommunitiesthatarehistoricallyunder-representedinbiomedical researchand46%individualsfromunder-representedracialandethnic

minorities.TheAllofUsResearchProgramidentifiedmorethan1billion geneticvariants,includingmorethan275millionpreviouslyunreported ones.Thisreinforcesthevalueofstudyingthegenomesofadmixedpopulations.Inadditiontopopulationgeneticdatabanks,thegenomestudyof olderprobands’relativescanbeextremelyvaluableinreal-lifedecisionmaking,asillustratedbytwoexamplesbelow.

Case1

In2012,a44-year-oldmanwasreferredtoourcenterbecausehehada mutationinthedystrophingene,whichwasidentifiedinageneticcenter intheUnitedStates.Hewasperfectlyhealthyandrobust,buthewasinvestigatedasaresultofthataccidentalfindingbecausehis10-year-old daughterhadadiagnosisofcolobomaandsomehearingdifficulties(7). Thegenomestudyoftheyounggirldidnotuncoveranyvariantthatcould explainhercondition.However,itrevealedthatshecarriedanunrelated mutationinthedystrophingene,encompassingexons38–44,inherited fromher44-year-oldfather.Mostmutationsinthedystrophingeneare responsibleforDuchennemusculardystrophy(DMD),aseverelethalconditionthataffectsabout1in5000malenewborns(8).Thosearedisruptivemutationsthatresultintheabsenceofmuscledystrophin.Affected boysusuallyloseambulationbyage10–12andareentirelydependenton allactivitiesintheirseconddecade.However,somemutationscanresult inapartiallyfunctionaldystrophinandamilderbuthighlyvariablephenotype,asseeninBeckermusculardystrophy(BMD).Dependingonthe typeandsiteofthemutationalongthegene,BMDpatientscanbeconfinedtoawheelchairaroundage16orremainambulantintheirsixtiesor seventies.Forexample,itisknownthatsomemutationsintheroddomain (centralpartofthegene)thatmaintaintheRNAreadingframe(in-frame deletions)cancauseonlycardiopathylaterinlifebutnomuscularweakness.Therefore,mutationsinthedystrophingeneshouldbeclassifiedas dystrophinopathiesandnotDuchennemutations,astheyareresponsible forawiderangeofclinicalvariability.

Theprobleminthiscaseisthatthedystrophinmutationfoundinour probandhadneverbeenreportedbefore.Coulditberesponsiblefora late-onsetdisorder,orwasitjustalikelybenignrarevariant?Although hewashealthyandstrongatage44,hewantedtoknowwhetherhe mightdevelopmuscularweaknesslaterinlife.Ifhehadcarriedanovel

1 HumanGenomeandStem-cellResearchCenter,InstituteofBiosciences,UniversityofSãoPaulo,05508-090SãoPaulo,Brazil

CorrespondingAuthor: MayanaZatz,HumanGenomeandStem-cellResearchCenter,InstituteofBiosciences,UniversityofSãoPaulo,05508-090SãoPaulo,SP,Brazil. E-mail: mayazatz@usp.br

Received:10March2024.Revised:12March2024and14March2024.Accepted:14March2024. Publishedonline:15March2024.

mutation,itwouldnothavebeenpossibletoanticipatehisclinicalstatuslaterinlife.Theonlyalternativewastoinvestigatehisolderrelatives,hopingtheirgenomicdatamightbeinformative.Inotherwords,we neededtoinvestigatewhetherthoseelderlyrelativescarriedthesame dystrophinmutation.Thatwasthecase:westudiedseveralfamilymembersandfoundoutthattheproband’smotherandonematernaluncle, whowas56yearsoldatthattime,alsocarriedthesamemutation,and theywereasymptomatic.Itwasgoodnews.Wepublishedthiscasereport withatake-homemessage:ifyouwanttosequenceyourgenome,keep yourolderrelatives’DNA.Theycanbringimportantinformation(7).

Case2

Morerecently,IreceivedanemailfromayoungwomanofEastAsian ancestrywhowrotetomebecauseshediscoveredthatshecarriesa DMDmutationencompassingexons38–44,thesameraredeletionof theBrazilianfamilyincase1.Hermutationhadbeeninheritedfromher 60-year-oldmother.Becauseofthelackofinformationingenomedata banks,themutationwasclassifiedaslikelytobepathogenic,andsheunderwentanabortionat27weeksofpregnancy.Shewroteinheremail thatthis“ledhertobedevastated,butalsotoconducttonsofresearch.” Searchingreferences,shediscoveredthathermutationwasthesamein ourpreviouslyreportedfamily,andshewantedmoreinformationabout ourcase.Herquestionswere:(1)couldyoupleaseprovidemorecontext onwhat“asymptomatic”meansforthatfamily?(2)Dotheynotshowany signsofDMD/BMD,includingnosignsofCKincrease/cardiomyopathy?(3) Ifthisresearchshowsexondeletion38–44isasymptomaticforthisBrazilianfamily,canIsafelyassumeitwillalsobeasymptomaticformyfamily?

(4)Whatisthecurrentstatusofyourpatients12yearsafteryourreport?

(5)HowmuchshouldI(she)beworriedaboutthismutationinmy(her) futureoffspring?

Ourreportreinforcingtheimportanceoftestingolderrelatives promptedhertostudyhergrandparents.Hermaternalgrandmother wasalreadydeceased,andshehadthreebrotherswhorefusedtobe tested.However,hermaternalgrandfatherunderwentgenetictesting, whichrevealedthathecarriesthe38–44mutation.Itcouldnotbebetternewssinceheiscurrently89yearsold,fullyambulant,andhasno cardiomyopathy.

Asymptomaticgeneticvariantsinpatientsofdifferentethnic backgrounds:“VUSorlikelybenign?

Followingtheselastgeneticresults,IcontactedtheBrazilianfamilyto sharetheexcellentnewsaboutthehealthy89-year-oldmancarrying the38–44deletion,andtheyinformedmethattheyalsocontinuetobe healthyandstrong.Ourprobandandhismaternalunclecarryingthedystrophindeletionarecurrently56and68,respectively.Thenewobservationthatthissamevariantisnotassociatedwithanymuscularweakness intwofamilieswithdifferentethnicbackgroundssupportsthehypothesisthatitisa“likelybenign”variant.However,somegeneticistswould stillclassifyitasaVUS.Mostimportantly,itreinforcestherelevanceof genomicscreeningofolderpopulationsandprobands’familymembers.

ThepathogenicityofVUSscanalsobestudiedusingin silico strategiesthatincludecomputationalstructuralbiologyorin vivo experiments inwhichanewvariantiscreatedviaCRISPRandinsertedinalivingorganism.However,webelievethatsuchmodelswilllacktheinputfromother putativeprotectivevariants;moreover,theoutcomesofgene-geneinteractionsmaybemissed.Therefore,weadvocateforthestudyofelderly genomesasakeytooltodeterminetheclinicalsignificanceofVUSs.

Inarecentreviewoftheliteratureanddatabase,Fortunateetal.reported22casesofpatientswhocarried in-frame deletionsinthedystrophingeneandwerefullyasymptomatic(9).Theywereolderthan43, whilethethreeindividualsreportedwereolderthan55.Accordingto Fortunateetal.,somedeletionsshouldbecarefullyconsideredwhen identifiedasincidentalfindings,andgeneticcounselingmustalwaysbe offeredtohelpinterprettheseraredystrophingenotypes.Indeed,inthe currentcase,sharingnewdatawithourfamilywasveryhelpfulintheir decisionaboutfutureoffspring,whichalsoreinforcestheimportanceof re-visitingpreviouslycounseledfamilieswithnew,relevantinformation. FowlerandRehamrecentlyquestionedwhetherVUSwouldstillbe presentby2030(10).TheysuggestthatinvestingineliminatingVUSsis

worthwhilebecausetheirpredominanceremainsoneofthebiggestchallengestoprecisiongenomicmedicine.Sharingcasereportssuchasthose synthesizedherecannotonlybringrelieftofamilieswithsuchgenetic variantsbutcanalsocontributetoclassifyingthepathogenicityofVUS andrarevariants.

Acknowledgments

Iwouldliketothankourfundingagenciesforthefinancialsupport (FAPESP,grant2013/08028-1andCNPq,grant465355/2014-5),ourcolleagueswhoperformedgenomicanalysis,andthetwofamiliescitedhere fortheirpermissiontosharetheirhistories.

References

1.KaliaSS,AdelmanK,BaleSJ,ChungWK,EngC,EvansJP,etal.Recommendationsforreportingofsecondaryfindingsinclinicalexomeandgenomesequencing,2016update(ACMGSFv2.0):apolicystatementoftheAmerican CollegeofMedicalGeneticsandGenomics.GenetMed.2017;19(2):249–55. DOI: 10.1038/gim.2016.190

2.RiggsER,AndersenEF,CherryAM,KantarciS,KearneyH,PatelA,etal.Technicalstandardsfortheinterpretationandreportingofconstitutionalcopynumbervariants:ajointconsensusrecommendationoftheAmericanCollege ofMedicalGeneticsandGenomics(ACMG)andtheClinicalGenomeResource (ClinGen).GenetMed.2020;22(2):245–57.DOI: 10.1038/s41436-019-0686-8 PMC7313390

3.NaslavskyMS,YamamotoGL,deAlmeidaTF,EzquinaSAM,SunagaDY,PhoN,et al.ExomicvariantsofanelderlycohortofBraziliansintheABraOMdatabase. HumMutat.2017;38(7):751–63.DOI: 10.1002/humu.23220

4.NaslavskyMS,ScliarMO,YamamotoGL,WangJYT,ZverinovaS,KarpT, etal.Whole-genomesequencingof1,171elderlyadmixedindividualsfrom SaoPaulo,Brazil.NatCommun.2022;13(1):1004.DOI: 10.1038/s41467-02228648-3 PMC8897431

5.SirugoG,WilliamsSM,TishkoffSA.Themissingdiversityinhumangeneticstudies.Cell.2019;177(1):26–31.DOI: 10.1016/j.cell.2019.02.048 PMC7380073

6.AllofUsResearchProgramInvestigators,DennyJC,RutterJL,GoldsteinDB, PhilippakisA,SmollerJW,etal.The“AllofUs”ResearchProgram.NEnglJMed. 2019;381(7):668–76.DOI: 10.1056/NEJMsr1809937 PMC8291101

7.ZatzM,PavanelloRdeC,LourencoNC,CerqueiraA,LazarM,VainzofM.Assessing pathogenicityfornovelmutation/sequencevariants:thevalueofhealthyolder individuals.NeuromolecularMed.2012;14(4):281–4.DOI: 10.1007/s12017012-8186-x PMC3505535

8.CrisafulliS,SultanaJ,FontanaA,SalvoF,MessinaS,TrifiroG.GlobalepidemiologyofDuchennemusculardystrophy:anupdatedsystematicreviewand meta-analysis.OrphanetJRareDis.2020;15(1):141.DOI: 10.1186/s13023020-01430-8 PMC7275323

9.FortunatoF,TonelliL,FarneM,SelvaticiR,FerliniA.DMDdeletionsunderliningmilddystrophinopathies:literaturereviewhighlightsphenotyperelatedmutationclustersandprovidesinsightsaboutgeneticmechanismsand prognosis.FrontNeurol.2023;14:1288721.DOI: 10.3389/fneur.2023.1288721 PMC10823016

10.FowlerDM,RehmHL.Willvariantsofuncertainsignificancestillexistin 2030?AmJHumGenet.2024;111(1):5–10.DOI: 10.1016/j.ajhg.2023.11.005 PMC10806733

Publisher’snote: GenomicPressmaintainsapositionofimpartialityandneutrality regardingterritorialassertionsrepresentedinpublishedmaterialsandaffiliations ofinstitutionalnature.Assuch,wewillusetheaffiliationsprovidedbytheauthors, withouteditingthem.Suchusesimplyreflectswhattheauthorssubmittedtousand itdoesnotindicatethatGenomicPresssupportsanytypeofterritorialassertions.

THOUGHTLEADERSINVITEDREVIEW

Thegeneticsofcognitioninschizophrenia

MichaelJ.Owen1 ,andMichaelC.O’Donovan1

Thisconceptualreviewfocusesonrecentinsightsintothenatureoftherelationshipbetweengeneticpredispositionandcognitiveimpairment asriskfactorsforschizophrenia,andthefactorsthatinfluencethedegreeofcognitiveimpairmentinthosewiththedisorder.Thereisclear evidencethatpremorbidcognitiveimpairmentisfrequentlypresentinthosewhodevelopschizophrenia,and,acrosstherangeofabilities, poorerpremorbidcognitionisassociatedwithhigherliabilitytothedisorder.Evidencefromgeneticandpopulationstudiesstronglysupports thehypothesisthatpremorbidcognitiveimpairmentisamarkerforunderlyingneurodevelopmentalriskfactorsforthedisorder,ratherthana prodromalmanifestation.Thepremorbidcognitivedeficitseemstobelargelyexplainedbynon-familialfactorsratherthanbyfamilialfactors thatjointlyinfluenceliabilitytoschizophreniaandcognitiveability,andthesenon-familialriskfactorsappearacttosensitizeindividualsto familialrisk.Thereisalsoevidencethatneurodevelopmentalriskmaybebetterindexedbythedegreetowhichpremorbidcognitiveability deviatesfromfamilialexpectationsthanbycognitiveability perse.Premorbidcognitiveimpairmentthusdoesnotitselflieonthecausal pathwaytoschizophrenia,ratheritisamarkerofaneurodevelopmentalabnormalitythatissubstantiallynon-familial,andwhichincreases riskforschizophrenia.Geneticriskfactors,includingbothcommonandrarealleles,thatinfluenceIQinthegeneralpopulationalsocontribute bothtoliabilityforschizophreniaandtothedegreeofcognitiveimpairmentinthosewiththedisorder.Thereisalsoevidenceforfurther declineincognitivefunctionafterdiagnosisinsomeindividualsaswellasanincreasedriskofdementia.Thisdoesnotappeartoreflect substantialsharedheritabilitywithneurodegenerativedisorders,butthecausesofpostonsetcognitivedeclineanditsrelationshipto schizophreniapathophysiologyremainuncertain.

GenomicPsychiatry January2025;1(1):28–35;doi: https://doi.org/10.61373/gp024i.0040

Keywords: Cognition,genetics,genomics,neurodevelopment,schizophrenia

Introduction:CognitiveImpairmentinSchizophrenia

Schizophreniaisdiagnosedbasedonthepresenceofcorepositive(psychotic),negativeanddisorganizedsymptoms(1).Individualsmeetingdiagnosticcriteriashowconsiderableheterogeneityintheseandotherclinicalfeatures,aswellasincourseandoutcome(2).Comparisonsbetween peoplewithschizophreniaandcontrolsrevealgroupwiseimpairmentsin mostaspectsofcognitivefunction(3, 4)includingIQ,whichisonaverage reducedbyapproximately1SDincases(5).Again,thereisenormousvariationbetweenindividualsintheextentoftheimpairmentpresent,anda diagnosisofschizophreniadoesnotprecludeestimatesofcognitiveabilitythatareabove,andsometimesmarkedlyabove,average(6, 7).CognitiveimpairmentisnotacoresymptomofschizophreniainDSM5orICD11, butasitisstronglyassociatedwithpoorerfunctionaloutcomesinareas suchaswork,independentliving,andsocialintegration(3),itisofgreat importancetothosewiththedisorder,andtothosewhoareinvolvedin theircareandmanagement,Arecentreviewhasbroadlyconsideredevidenceconcerningtheetiology,pathogenesis,andtreatmentofcognitive impairmentinschizophrenia(4).Thecurrentconceptualreviewismore circumscribedandfocusesonadditionalimportantinsightsprovidedby recentstudiesintothenatureoftherelationshipbetweengeneticrisk andcognitiveimpairmentasriskfactorsforschizophrenia,andthefactorsthatinfluencethedegreeofcognitiveimpairmentinthosewiththe disorder.

Manyofthestudieswereviewhavebeenbaseduponderivedmeasures ofgeneralcognitivefunctionsuchasIQratherthanuponperformanceon specifictestsmeasuringspecificdomainsofcognitivefunction.Giventhat impairmentsinmostaspectsofcognitivefunctionareassociatedwith schizophrenia(3),webelievethatsuchglobalmeasuresareinformative forthespecificquestionsweaimtoaddress.Accordingly,weusetheterm cognitiveimpairmentinterchangeablywithlowIQratherthantoindicate impairmentinspecificcognitivedomains.

Peoplewhoreceiveadiagnosisofschizophreniafrequentlyexhibit “premorbid”cognitiveimpairmentsbeforepsychosisismanifest(8), includinganaverageIQthatisapproximately0.5SDbelowthatofcontrols(5).Thereisalsoevidencethattheseverityofpremorbidimpairmentisassociatedwithearlieronsetofschizophrenia(9).Impairments areevidentinchildhood(5, 10–12)andappeartorepresentafailureof typicaldevelopmentalacquisitionoffunctionratherthanadeterioration perse asmightbeexpectedofadegenerativeprocess(12).Whilethe evidencesuggeststhatthedevelopmentaltrajectoriesofthosewhodevelopschizophreniadivergefromcontrolsmanyyearsbeforepsychosis emerges,itremainsunclearwhetherthisisprecededbyaperiodofnormaldevelopmentand,ifso,exactlywhendivergencebegins(10, 12). Alongsideotherpremorbiddevelopmentalandenvironmentalriskfactors,thefindingspointtoschizophreniahavingoriginsindisturbances ofneurodevelopment,onemanifestationofwhichisimpairedcognition priortoonsetofpsychosis(13–15).

Whiletheweightofevidencesupportsaneurodevelopmentalexplanationforpremorbidcognitiveimpairmentinschizophrenia,thenatureof therelationshipbetweenthetwoisuncertain.Inprinciple,therearethree possibilities.First,premorbidcognitiveimpairmentcouldbeaprodromal manifestationofaninsidiousonsetofschizophrenia.Secondly,itcould beacausalriskfactormediatingtheeffectsofgeneticorenvironmental riskonthedevelopmentofschizophrenia,aso-calledintermediatephenotypeorendophenotype.Thirdly,itmightbeariskindicatorthatresults fromthepleiotropiceffectsofanunderlyingneurodevelopmentalabnormalitythatindependentlyincreasesriskofthesubsequentemergence ofschizophrenia.Inthislatterinstance,premorbidcognitiveimpairment wouldbeamarkerofthepresenceorextentofaneurodevelopmentalabnormalitybutnotitselflieonthecausalpathwaytoschizophrenia.

Somestudiescomparingcognitivefunctioninthesameindividuals beforeandafteronsetofschizophrenia(5, 16–18)suggestthat,aswell

1 TheCentreforNeuropsychiatricGeneticsandGenomics,DivisionofPsychologicalMedicineandClinicalNeurosciences,andNeuroscienceandMentalHealthInnovation Institute,HadynEllisBuilding,MaindyRoad,CardiffUniversity,CardiffCF144HQ,UK.

CorrespondingAuthor: MichaelJ.Owen,HadynEllisBuilding,MaindyRoad,CardiffUniversity,Cardiff,UK,CF144HQ.E-mail: owenmj@cardiff.ac.uk Received:26April2024.Revised:19June2024.Accepted:19June2024. Publishedonline:16July2024.

aspremorbiddeficits,thereisfurtherdeclineinfunctionafterdiagnosisinsomeindividuals.However,thisremainsacontroversialareaand twometa-analysesoflongitudinalstudiesfoundnoevidenceforpostonsetdeclineinthefirst5yearsafteronset(19, 20).Moreover,arecent umbrellareviewconcludedthatmostofthereviewsassessedpointto nodeclineofcognitivefunctionovershorttomediumtimeframes(21). Nevertheless,studiesoverlongerfollow-upperiodshavefoundevidence foracceleratedcognitivedecline(18)andsubstantiallyincreasedrisks ofdementiahavebeenreportedinthosewithschizophrenia(22, 23). Somehavearguedthatthislaterdeclinepointstotheoperationofaprimaryneurodegenerativeprocessinschizophrenia(24),whereasothers havearguedthatmanypeoplewithschizophreniadonotprogressively deteriorateandpointedtothepossibilitythat,wheredeclinedoesoccur,itreflectsnon-specificfactorssecondarytoschizophreniasuchasantipsychoticexposure,metabolicsyndrome,smokingandothersubstance abuseandvarioussocialconfounders(25, 26).

Finally,theconsiderablevariationincognitiveimpairmentseenin peoplewithschizophreniaraisesthequestionastowhatextentthismight beassociatedwiththesamegeneticfactorsthatinfluencevariationin cognitiveabilityinthegeneralpopulation,orwhetherfactorsthatare relativelyspecifictoschizophreniaoperate.

GeneticArchitecturesofSchizophreniaandIntelligence Schizophreniaishighlyheritableandpolygenic,withriskconferredbyallelesacrossthefrequencyspectrum(27).Genome-wideassociationstudieshavesofaridentified287locithatcontaincommonriskallelesof smalleffect(28),butmanymoreexist,likelythousands,thatarecollectivelyresponsibleforaroundathirdofgeneticliability(29, 30).In addition,atleasteightrarecopy-numbervariants(CNVs)havebeenidentifiedthatconfersubstantialindividualrisk(31, 32).Morerecently,exomesequencingstudieshaveshownrareproteintruncatingvariantsand damagingmissensemutations(33–39)canconferlargeeffectsonriskof schizophrenia.Thelargestexomewidesequencingstudyofthedisorder todatereportedbytheSchizophreniaExomeMeta-AnalysisConsortium (SCHEMA)(38)identified10geneswithanexome-widesignificantexcess burdenoftheseclassesofmutationincases.Anadditionaltwogenesmet thissignificancethresholdinafocusedsequencingstudytargetinggenes thathadshownsomeevidenceforassociationinapreliminaryanalysisof theSCHEMAexomewidestudy(40).

Genomicstudiesofcommonvariantshavefoundevidenceforgenetic correlationbetweenschizophreniaandotherpsychiatricconditionsand personalitytraits,withthestrongestrelationshipseenwithbipolardisorderwherethesharedheritabilityisapproximately0.7(41).Incontrast, forrarevariants,whilethereisevidenceforoverlapswithbipolardisorder (42),sofar,thegeneticoverlapsaremoreprominentbetweenschizophreniaandchildhoodonsetneurodevelopmentaldisorders(NDDs)which,like schizophrenia,arealsoassociatedwithcognitiveimpairment:allknown schizophreniaassociatedCNVshavebeenimplicatedinintellectualdisability,andsomehavealsobeenimplicatedinautism(43).Peoplewith schizophreniaarealsoenrichedforultra-raredamagingmutationsin NDD-associatedgenes(33, 36–39),includingspecificmutationsthatare pathogenicforNDDs(44).Finally,commonschizophreniariskallelesare enrichedingenesimplicatedbyrarevariantstudiesofNDDs(28).

Intelligenceismoderatelyheritableinthegeneralpopulation(45). Genomicstudieshaveshownthat,likeschizophreniaandaspredicted (45),itishighlypolygenic,andimpactedbyallelesacrossthefrequency spectrumincludingmanycommonvariantsofsmalleffect(46),aswellas rareallelesincludingchromosomalabnormalities,CNVs,andrarecoding variants,someofwhicharealsoassociatedwithschizophreniaandother NDDs(47–50).

TheRelationshipBetweenGeneticRiskandCognitiveImpairmentas RiskFactorsforSchizophrenia

FamilyandPopulationStudies

Thereisevidencefrommanyindividualstudies,supportedbymetaanalyses,thatunaffectedfirst-degreerelatives(FDRs)ofthosewith schizophreniashowgeneralizedimpairmentsofcognitivefunctionbutto alesserdegreethanthoseseeninprobands(51, 52).However,suchfindingsarenotuniversal.Onestudyofschizophreniacasesandtheirrela-

tivesfromrelativelyhighlyfunctioningfamiliesfoundthatthesiblingsof probandsdidnotdifferincognitiveperformancefromacommunitycontrolsample(53).Inaddition,averylargestudyoftheSwedishpopulation foundnoevidenceforadolescentcognitiveimpairmentinthesiblingsof peoplewithschizophrenia(54).Thesefindingsraisethepossibilitythat theroleoffamilialriskfactorsforlowIQinschizophreniamayhavebeen influencedbyascertainmentbiasinstudiesofprobandsandtheirrelatives.Anotherpossibleconfounderisthatfamiliesascertainedforhaving aschizophreniaprobandmayhavehigherratesofexposuretoenvironmentswithanimpactoncognitiveability,forexamplecannabisabuse (55).Finally,thereisevidenceforassortativematingsuchthatpeople withschizophreniawhohavechildrenonaveragehavepartnersoflower cognitiveabilitythancontrols(56).Asaresult,assortativematingcould contributeto,orevenaccountfor,deficitsinFDRsratherthanthesereflectingasubstantialoverlapbetweenthegeneticriskforschizophrenia andlowIQ.Thus,whilemanystudieshavefoundevidenceforimpaired cognitioninFDRs,questionsremainabouthowthesefindingsshouldbe interpreted.Genomicstudiesofparent-probandtriosmightthrowlight onthisissue.

Populationstudieshavefurtherilluminatedthecausalrelationship betweenpremorbidIQandgeneticrisk.InalargestudyoftheSwedish population(57),riskofschizophreniaincreasedby3.8%witheachonepointdecreaseinpremorbidIQ.Theeffectswerestrongerinthelower IQrange,butimportantly,theassociationwasmonotonic,meaningatno pointinthedistributionwashigherIQassociatedwithhigherrisk,althoughsuchaneffectinpeoplewithexceptionallyhighIQcouldnotbe excludedduetotherarityofthoseindividuals.Overall,thesefindings,and themagnitudeoftheeffect,werealmostidenticaltothoseobtainedina meta-analysisofearlierpopulationstudies(9).TheSwedishstudy(57) notedthattheassociationbetweenpremorbidIQandriskofschizophreniawasofsimilarmagnitudewhenonsetswithin5yearsoftestingwere excluded,suggestingthatitdoesnotreflectpotentialprodromaleffects ofdecliningIQassociatedwithinsidiousonset.Again,thesefindingsare inaccordwiththosefromearlierpopulationstudieswhichexcludedonsetsimmediatelypriortotesting(51).Finally,theSwedishstudyfound thatthatriskofschizophreniainpeoplewithhighfamilialliabilityto thedisorderwassubstantiallymodifiedbypremorbidIQ,familialsusceptibilityhavingamuchstrongerimpactonriskofillnessinthosewith lowIQ.

Sinceintelligenceandschizophreniaarebothfamilialandsubstantiallyheritable,theauthorsoftheSwedishstudy(57)testedthehypothesisthatthepremorbidIQ-schizophreniaassociationmightbetheresultof genetic(andfamilyenvironment)factorsthatpredisposetobothtraits. Theyundertookco-relativeanalyses,comparingthestrengthoftheassociationbetweenintelligenceandschizophreniawithinvariousclasses ofpairsofrelativeswhoseIQsweredifferent,thatisnotinthesame decile.Siblingssharemorefamilialfactors(geneticandenvironmental) thandomoredistalpairsofrelatives,andthereforemoreofthedifferenceinintelligencebetweenthemislikelytobeattributabletononfamilialfactorsthanisthecasefordifferenceinintelligencebetween moredistallyrelatedpairs.Accordingly,iftheassociationbetweenlowIQ andschizophreniaissubstantiallyduetogenesorfamilialenvironments thatinfluencebothtraits,thestrengthofthisassociationwithinsiblings willbelessthaninthemoredistantlyrelatedrelativepairs.However,the findingswereinconsistentwiththis;associationbetweenpremorbidIQ andschizophreniawasasstrongwithinsiblingsaswithinmoredistant relatives,andevenbetweenpeopleinthegeneralpopulationwhoare effectivelyunrelated,suggestingthelinkbetweenthetraitsisnottheresultofheritablegeneticorfamilialenvironmentalriskfactorsthatjointly influencebothtraits.Thesefindingshavebeensupportedbytworecent population-basedsiblingstudies(58, 59).

Evidencethatnon-familialfactorsmayhaveanimportantinfluence onpremorbidIQdeficitsinschizophreniaalsocomesfromstudiesshowingthatriskofschizophreniamaybebetterindexedbyameasureofthe extenttowhichanindividual’spremorbidcognitiveperformanceislower thanexpectedbasedonestimatesoffamilialcognitiveaptitudethanby theirabsolutepremorbidcognitiveperformance perse (54, 60, 61).Itis conceivablethatsuchdeviationsfromfamilialexpectationmightoccur

inindividualswho,byanunluckyrollofthegeneticdice,inheritanexcessofpoorercognitionallelesthathavepleiotropiceffectsonliability toschizophrenia.However,thisinterpretationwasnotsupportedbythe findingthat,whenschizophreniaandcontrolprobandswerematchedfor cognitiveperformance,thesiblingsoftheschizophreniaprobandshad scholasticaptitudesandIQsthatdidnotdifferfrompopulationmeans andwhichweresignificantlyhigherthanthoseofthesiblingsofcontrol probands(54).Instead,thisseriesofstudiesfromSweden(54, 57, 60) suggestthatanimportantcontributiontoriskforschizophreniacomes fromneurodevelopmentalperturbationsthatimpactcognitivedevelopmentinpeoplewhodevelopschizophreniathatarenotcausedbyfamilialfactorsthattypicallyinfluencecognitionwithinfamilies.Thisgeneral conclusionalsoisindirectlysupportedbyevidencethattheheritabilities ofarangeofcognitiveabilitiesinschizophreniaseemtobelowerthanin thegeneralpopulation(61).

OverlapinCommonRiskAllelesforSchizophreniaandIQ Thereisevidencethatsomeofthecommonallelesthatinfluencecognitiveabilityinthegeneraladultpopulationalsoinfluenceliabilityto schizophrenia,butthegeneticcorrelationof 0.21betweenthetwois modest(46).Asimilargeneticcorrelationof 0.22hasalsobeenreported betweenliabilitytoschizophreniaandintelligenceestimatedin16year olds,anagepriortothetypicalonsetofschizophrenia(62).Thesecorrelationssuggestthat,onaverage,commonvariantgeneticeffectsassociatedwithlowerintelligenceinthegeneralpopulationexplainaround 5%ofliabilitytoschizophrenia,anestimateconsistentwithalongitudinalpopulation-basedtwinstudywhichestimatedthisfigureat7%(63). Thesefindings,basedonverydifferentstudydesigns,convergeonthe conclusionthat,whilethereissomesharedgeneticliabilitybetweenIQ andriskofschizophrenia,theoverlapissmall,andinlinewiththeconclusionsfromtheSwedishfamilystudiesreviewedabove.Itshouldbe notedthatgeneticcorrelationsofthismagnitudecouldpotentiallybe theresultofthetypeofcross-traitassortativemating(64)discussed above(56),aswellasothersourcesofconfounding(65),possibilities thatwarrantfurtherstudy.Moreover,sharedliabilitydoesnotimplythat riskofschizophreniaismediatedbytheeffectsoncognitiveability,it mayinsteadindicatetheexistenceofpleiotropywherebycommonalleles independentlyinfluencecognitiveabilityandliabilitytoschizophrenia, perhapsbycontributingtothepleiotropicneurodevelopmentalperturbationsdiscussedintheprevioussection.

Astudy(66)basedonmodelingtherelationshipsbetweenschizophreniapolygenicliability[indexedbypolygenicriskscore(PRS)],schizophreniaandcognition(expressedaslatenttraits)withinfamiliesfoundthat thebestmodelsolutionssuggestedaroundathirdofgeneticriskof schizophreniacouldbeexplainedthroughcausaleffectsoncognition. However,themodelingdoesnotappeartohaveaccountedforthepossibilityofpleiotropy,and,astheauthorsnoted,thelimitationsandassumptionsmeanthattheirapproachcannotprovecausality,andlongitudinal designsand/orMendelianrandomizationapproachesareneeded.However,todate,therequiredlongitudinalstudieshavenotbeenconducted andMendelianrandomizationmethodshavenotbeenabletoclearlydistinguishbetweencausalandpleiotropichypotheses(67).Moreover,arecentreportthatalmostallvariantsitesthatinfluenceIQinthegeneral populationalsoinfluenceliabilitytoschizophrenia,butthatthespecific allelesthatincreaseschizophreniariskareamixoflowerandhigherIQ alleles,suggeststhatsharedliabilitycannotindicateasimplecausallink betweenlowerIQ perse,andschizophrenia(68).

GenomicStudiesofVariationinCognitiveFunctioninSchizophrenia CommonAlleles

Separatetothequestionabouttherelationshipbetweenallelesthatinfluencecognitioninthegeneralpopulationandthosethatinfluenceliabilitytoschizophreniaiswhatistherelationshipofthosesetsofalleles tocognitiveabilityinpeoplewiththedisorder?

ThereisstrongevidencethatallelesinfluencingIQinthegeneral populationalsohaveeffectsoncognitiveabilityinindividualswith schizophrenia(69–71).Incontrast,theevidenceforarelationshipbetweencognitivefunctioninschizophreniaandcommonvariantliability forthedisorderisinconsistent(69, 70, 72–75).Thisinconsistencymayre-

flecttherelativelymodestsamplesizesandpowerofsomeofthestudies aswellasdifferencesindurationofillnessatthetimeofcognitivetesting andthenatureofthecognitivetestsemployed.Nevertheless,theresults suggestthattheeffectsofcommonschizophreniariskalleles,asindexed byapolygenicriskscore(PRS),oncognitioninpeoplewithschizophrenia areatbestsmall,andwheredirectcomparisonshavebeenmade(69–71), considerablysmallerthantheeffectsofcommonallelesthatinfluence IQinthegeneralpopulation(asindexedbyanIQPRS).TheIQPRSexplainsaround9%ofvarianceinIQ(76)inpeoplewithschizophrenia.This issimilartoestimatesinthegeneralpopulation(62),althoughonestudy suggestedthevarianceexplainedincasesislessthaninUKBiobankcontrols,albeitusingdifferentcognitivemeasuresincasesandcontrols(70). Thesefindings,togetherwiththoseindicatingthatthegeneticcorrelationbetweenliabilitytoschizophreniaandIQismodest,andthefindingsfromthelargepopulationfamilystudiesreviewedabove,suggest thatvariationinpremorbidcognitivefunctioninpeoplewholaterdevelop schizophreniaisnotsubstantiallytheresultofcommongeneticriskfactorsforschizophrenia.Thereisalsoclearevidencefromgenomicstudiesthatcommonallelesthatinfluencevarianceincognitiveabilityinthe generalpopulationalsodosoinpeoplewiththedisorder.However,inthe absenceofdirectcomparisonsofcasesandcontrolsbasedonidentical cognitiveassessments,itisuncleartowhatextentthoseallelescanexplaintheaveragepremorbidcognitivedeficitseeninpeoplewhodevelop schizophrenia.

RareVariants

PeoplewithschizophreniawhocarryschizophreniaassociatedCNVstend tohaveworsecognitivefunctionthanthosedonot,theaveragedifferencebetweenthetwogroupsinmeasuresofcognitiveperformancebeingaround0.5–1.0SD(77).Thegreatestreductionsincognitiveability areseeninthosewithCNVsspanningloss-of-functionintolerant(LoFI) genes,thatisgenesinwhichloss-of-functionmutationsarehighlydisadvantageousforreproductivefitness.Thereisalsoevidencethatinpeople withschizophrenia,rarecodingriskvariants(RCVs),particularlyproteintruncatingvariantsinLoFIgenes,areassociatedwithreducedcognitivefunction(71, 76),poorereducationalattainment(33, 78)andanincreasedriskofcomorbidintellectualdisability(37, 78).Theeffectsofrare variantsoncognitionarelargelymanifestbeforeillnessonset(seebelow),suggestingthattheireffectsimpactneurodevelopmentalprocesses. FurthersupportforneurodevelopmentaleffectscomesfromtheobservationthattheeffectsofRCVsoncognitivefunctioninschizophreniaareon averagegreaterforthosewithingenesthathavebeenimplicatedinchildhoodNDDsthanforRCVsinothermutationintolerantgenes(37, 76, 78). Givenevidencereviewedabovethatnon-familialeffectsareimportant,it isalsonotablethattheeffectsizesofmutationsoccurring denovo (which bydefinitionarenon-familial)arelargerthanthoseoftransmittedmutations(38, 76, 78).

PremorbidversusPostonsetEffects

Fewgenomicstudieshaveincludedmeasuresofbothpremorbidandcurrentcognitioninpeoplewithschizophrenia,butasmallnumberofrecent studiesthathavedonesohavebeguntothrowsomelightonthelinksbetweengeneticriskfactorsandthetimingofcognitiveimpairment.Most oftheimpactsofCNVs,RCVs,andIQPRSoncognitionarepremorbid(69, 71, 76, 77).Incontrast,thereissomeevidencethattherelativelysmall effectsofcommonvariantschizophrenialiabilityoncognitionoccurafter onset(69)butdonotinfluencepremorbidcognitiveability(69, 71, 76). Thefindingsthatschizophreniacommonvariantliability,whilenotassociatedwithpremorbidimpairment,maybeassociatedwithlaterimpairmentrequiresreplication,butifconfirmed,mighteitherreflecteffectson cognitivedeclinearoundthetimeofonsetofpsychosisoratsomepoint thereafter.ThefindingsfromalongitudinalstudythatassociationbetweenschizophreniaPRSandpoorercognitionisstableacrossa20-year follow-upperiod(75)wouldtendtosuggestthattheeffectsoncognition aremostlikelytooccuraroundthetimeofonset.

Regardingthesubstantialincreasesinriskofdementiareportedin schizophreniamentionedabove,itisnotablethatthereisnoevidence thatschizophreniasharesheritabilitywithAlzheimer’sorParkinson’s

diseases,althoughthereisweakevidenceforsomesharingwithfrontotemporaldementia(79–81).

ConclusionsandImplications

Thefindingsfromgeneticstudiesthatwehavedescribedarerelevant tothreebroadsetsofissues.Thefirstconcernsthequestionofwhat accountsforthelowerpremorbidIQseeninschizophreniaandthenatureoftherelationshipbetweenthisimpairmentandotherriskfactors forschizophrenia.Thesecondrelatestowhatgeneticfactorsinfluence theextentofcognitiveimpairmentamongthosewithschizophrenia.The thirdconcernsthepossibleinfluenceofgeneticeffectsoncognitivedeclineinlaterlife.

PremorbidCognitiveImpairmentandRiskofSchizophrenia

EvidencereviewedabovesuggeststhatlowpremorbidIQisariskfactor forschizophreniaandthatthisisnotexplainedbyprodromaleffectsofan insidiousonsetofthedisorder.Moreover,ratherthanan“U”-shapedrelationshipbetweenIQandriskofthedisorder,highestriskoccursinpeople withlowestIQ,lowestriskinthosewiththehighestIQ,andonaverage, peoplewhosubsequentlydevelopschizophreniahavea0.5SDdeficitin premorbidIQcomparedwiththegeneralpopulationaverage.

Inprinciple,thestrongevidencethatschizophreniariskallelescan alsoinfluencecognitiveabilityinthegeneralpopulationprovidesaplausibleexplanationforlowpremorbidIQ.Intuitivelyattractivethoughthis hypothesismaybe,itisnotsupportedbytheevidencewehavediscussed. Commonvariantgeneticliabilitytoschizophrenia,whichcurrentlyexplainsbyfarmostoftheattributableheritabilityofthedisorder(82), doesnotappeartobeassociatedwithpremorbidIQ(69, 71),afinding broadlyconsistentwiththesmallgeneticcorrelationsobservedbetween liabilitytopsychosisandpremorbidIQintwinsandbetweenschizophreniaandIQinthegeneralpopulation.Thus,thegenomicevidencetodate isconsistentwiththatoutlinedabovefromgeneticepidemiologyinsuggestingthatthepremorbidcognitivedeficitinschizophreniaislargely explainedbynon-familialratherthanfamilialfactorsthatjointlyinfluenceliabilitytoschizophreniaandcognitiveability.Thereisstrongevidencethesenon-familialfactorsinclude denovo mutations(SNVsand CNVs),butthesehavebeenimplicatedinfewerthan5%ofcases(82)and thereforeothernon-familialriskfactorsmustcontribute,includingasyet unidentified denovo mutations,suchasrarestructuralvariantsandnoncodingvariants,non-familialenvironmentalfactors,forexample inutero orperinatalbirthtraumaandinfections,andstochasticevents(83)that contributetovariationinneurodevelopment.

Somecaveatsshouldbenoted.First,itisconceivablethatstudiesof theeffectsofschizophreniaPRSoncognitionincasesmayunderestimate effectsduetoBerkson’sparadox(84),alsosometimesknownascollider bias(84).AssumingPRSandlowIQtobeatleastpartlyindependent riskfactors,peoplewithexposuretohigherPRSwillrequirelessexposuretotheriskconferredbylowIQinordertomanifestthedisorder,and viceversa.Thiscanresultinaspuriousnegativecorrelationbetweenthe tworiskfactorsincaseonlystudies,or,whereatruepopulationassociationexists,areductionintheestimatedeffectsize.Secondly,onlya minorityofschizophreniaheritabilityiscurrentlyattributabletoknown typesofvariant(82)and,inprinciple,classesofvariantsresponsiblefor theunexplainedheritabilitycouldshowstrongerassociationswithcognition,althoughthefindingsfromstudiesofsiblingsandotherrelativesreviewedabove(57–59)suggestthatthisisunlikelytobethecase.Thirdly, thefindingsofthekeygeneticepidemiologicalstudiesrequirefurther replication.

HavingestablishedthatlowpremorbidIQisariskfactorfor schizophreniathenextquestioniswhetherlowIQis perse causaloris insteadariskindicatorofapleiotropicneurodevelopmentalabnormalitythatcan(largely)independentlymanifestaslowIQinchildhoodand theemergenceofschizophreniainlaterlife.Thelatterinterpretationis supportedbytheobservationthatriskofschizophreniaisbetterindexed bythedeviationofcognitiveperformancefromthatexpected,thanby absolutepremorbidcognitiveperformance,whichiscontrarytotheexpectationiflowIQ perse isdirectlycausal.Moleculargeneticstudiesof commonandrarevariationarealsoinconsistentwiththeideathatlowIQ perse isonthecausalpathwaytoschizophrenia.Thus,whileallknown

schizophrenia-associatedCNVs,andcertainschizophrenia—associated RCVsareassociatedwithlowIQ,theirimpactsonriskinschizophreniais notcontingentonthepresenceoflowIQ(41).Theobservationthatcommonschizophreniasusceptibilityallelesincludethoseassociatedwith higherIQaswellaslowerIQsimilarlysuggeststhatthereisnorobust causallinkbetweenlowerIQandschizophrenia.Thereis,however,evidencethatlowpremorbidIQmaysensitizeindividualstofamilialriskfactors(57)(Figure1A)andthereisaneedtosubstantiatethisfindingand, ifconfirmed,explorepossiblemechanisms.

Afurtherissuethatwarrantsdiscussioniswhetherthereisaneurodevelopmentalsubtypeofschizophreniacharacterizedbypremorbid cognitiveimpairment.Wehavediscussedthiselsewhere(44)andour viewisthattheevidencebettersupportsthehypothesisthatthereisa spectrumofneurodevelopmentalimpairmentinthosewithschizophrenia,ratherthanacleardistinctionbetweenaformofthedisorderwith cognitiveimpairmentandonewithout(15).Themonotonicchangeinrisk ofschizophreniaacrossthefullIQrange,ratherthantherebeinganIQ thresholdthatisassociatedwithastepchangeinliability,supportsthis (9, 57)asdoestheobservationthatpeoplewithlowercognitiveability inschizophreniadonotsubstantiallydifferfromthosewithhighercognitiveabilitywithrespecttocommonallelesthatconferrisktoschizophreniagenerally(70).Finally,inpeoplewithCNVsknowntoaffectneurodevelopment,schizophreniaistheresultofboththeCNVandthecommon variantliabilitythatissharedwithgeneralformsofthedisorder(85, 86). Whilewedonotbelievethatthedatasupporttheexistenceofadistinct neurodevelopmentalsubtypeofschizophrenia,theextentofpremorbid cognitiveimpairment,andmoreparticularlytheextentthatthisdeviates fromfamilialexpectations,seemstoindexthedegreeofunderlyingneurodevelopmentalimpairmentandmaybeanimportantclinicalandprognosticmarkerwithinthedisorder(Figure1B).

Weadditionallynotethatwhileourfocushereisschizophrenia,comparisonsofthedegreeofpremorbiddeviationfromfamilialcognitiveaptitude(60)indifferentpsychiatricdisorderssupporttheviewthatthe neurodevelopmentalcontinuumextendsacrossanumberofconditions, aspreviouslyproposed(15, 87).Thus,theeffectsizeofdeviationfrom familialcognitiveaptitudewasgreatestforASD,followedbyschizophreniaandothernon-affectivepsychoses,andleastinbipolardisorder(60) supportingthesuggestion(15, 87)thatthereisagradientofneurodevelopmentalpathologyacrossneurodevelopmentalandpsychiatricdisorders.Accordingtothisviewschizophreniaoccupiesanintermediate positionbetweenchildhoodneurodevelopmentalconditionsandbipolar disorder.Thishelpsexplainwhycognitiveimpairmentisassociatedwith schizophreniabuttoamuchlesserextentbipolardisorderdespitethe highcommonvariantgeneticcorrelationbetweenthetwoconditions.

Itisnowimportanttoidentifytheriskfactorsfor,andthenature of,theneurodevelopmentalabnormalityunderlyingpremorbidcognitive impairments.

Itwillalsobeimportanttodeterminehowandwhenneurodevelopmentalimpairmentmoderatestheimpactoffamilialgeneticriskfor schizophrenia.Onehypothesiswithpotentialimplicationsforinterventionsisthattheeffectsofdivergencefromfamilialcognitiveexpectationsonpsychopathologymightbemediatedbytheevocationofdisruptedfamilydynamicsand/orimpairmentofindividual’sself-esteem (88)(Figure1).

GeneticFactorsInfluencingtheDegreeofCognitiveImpairment inSchizophrenia

Aswehaveseen,recentgenomicstudiessuggestthatallclassesofallelethatinfluenceliabilitytoschizophreniaortovarianceinIQcontribute tovariationinpremorbidcognitionandtocross-sectionaldegreeofcognitiveimpairmentinpeoplewithestablishedschizophrenia.Thesetypes ofvariantcurrentlyexplainaround10%ofvarianceinpremorbidIQ,of which,asnotedabove,90%isexplainedbyIQPRSandtheremainderby rareCNVsandraredamagingcodingvariantsinconstrainedgenes.They explainlessvarianceincognition,around6%,inthosewithestablished schizophrenia(76),butafterallowingfortheeffectsofpremorbidcognition,thisdropstoaround1.6%,equallysplitbetweenIQandschizophreniaPRS.Thus,mostofthegeneticcontributiontovariationincognitionin

Figure1. (A)Amodeloftherelationshipbetweenpremorbidcognitiveimpairmentandriskofschizophrenia.ThedegreetowhichpremorbidIQdeviatesfrom familialexpectations,ratherthanIQ perse, isakeyriskindicatorforschizophrenia.Thisdeviationindexesanunderlyingneurodevelopmentalimpairment(NDI) thatincreasestheriskofschizophrenia,andwhichsensitizestheindividualtofamilialriskfactorsincludingcommonvariantliabilityindexedbyschizophrenia PRSaswellassomeotherformsoftransmittedgeneticvariant(1).TheNDIreflectspredominantlynon-familialfactorsincludingenvironmentalriskfactors, stochasticfactorsandraredamagingdenovomutations.However,familialriskofschizophrenialikelycontributestoNDI(2)givenevidencethatgenesimplicated bycommonriskvariantsoverlapthoseassociatedwithraredisruptivecodingvariantsinschizophrenia,areenrichedforgenesimplicatedbysuchvariantsin NDDs(28)andevidencethattheyareenrichedforgeneswithhighexpressionspecificityindevelopingfetalneuronalpopulationsindependentlyofthose expressedinadulthood(89).Anindividual’sdeviationfromfamilialcognitiveaptitudeexpectationmightimpactontheirpsychopathologyviainfluenceson theirself-esteemandfamilyrelationships(3).(B)Severityofpremorbidcognitiveimpairmentinschizophrenia.Theseverityofpremorbidcognitiveimpairment inindividualswithschizophreniareflectsboththecontributionofcommonallelesthatareassociatedwithIQinthegeneralpopulationindexedbyIQPRSand theseverityoftheunderlyingNDIwhichisindexedbytheextentofpremorbidcognitiveimpairmentrelativetofamilialexpectations.Thelatterislikelytobean importantmarkerofstratificationwithinthedisorderindicatingapropensitytopoorfunctionaloutcomes.ThedirectarrowfromNDItooutcomesacknowledges thatmoreresearchisneededtodeterminetheextenttowhichdifferentoutcomesaremediatedbycognitiveimpairment.

schizophreniaismediatedbyeffectsthatwehavearguedaboveareneurodevelopmental.Alsonotethat,whileraremutationscontributeonlya smallamounttovarianceinpremorbidcognition,theyareassociatedwith relativelylargeimpairmentsofcognitivefunction(76).

Acaveattoourconclusionthatmostofthegeneticinfluencesoncognitioninschizophreniaarepremorbidandlikelyneurodevelopmentalis that,givenourincompleteunderstandingofgeneticarchitecture,wecannotexcludetheexistenceofasyetunknowntypesofriskvariantthatshow adifferentbalanceofmorbidandpremorbideffectsoncognition.Theexistenceofsuchvariantsisplausiblegivenevidencewehavediscussedthat anyeffectsoncognitionofcommonriskallelesforschizophreniaseemto manifestpostmorbidlyratherthanpremorbidly(69).Anothercaveatis theremaybespecificgeneticcontributionstocognitioninschizophrenia thatareindependentofthosethatconferliabilitytothedisorder,orto intelligenceinthegeneralpopulation.

GeneticInfluencesonPostonsetCognitiveDecline

Aswehaveseen,uncertaintiesremainconcerningtheextenttowhich thereiscognitivedeclineafteronsetandifsowhatproportionofcases

areaffectedandwhenthedeclineoccurs.Thereisemergingevidence ofdeclineincognitivefunctionafterschizophreniaonsetrelativetoage matchedcontrols,whichappearstobeprogressiveovermanyyears(18) andforsubstantiallyincreasedrisksofdementia(22, 23).Thepresenceof latelifecognitivedeclineandincreasedriskofdementiainpeoplewith schizophreniaintheabsenceofincreasedgeneticliabilitytodementia suggeststhatschizophreniamayleadtohigherexposure,orgreatervulnerability,tothesameenvironmentalriskfactorsthatoperateinthegeneralpopulationtoincreaseriskofdementia,forexamplesmoking,poor cardiovascularhealth,andlowercognitivereserve,thelatterbeingaconsequenceoflowerpremorbidIQ,socialisolation,andlowratesofemployment.Asecondexplanationisthattheincreasedriskofdementia reflectsintrinsicpathogenicmechanismsrelatedtoschizophrenia,orenvironmentalexposures,thatarerelativelyspecifictothosewithsevere mentalillnessforexamplemedicationeffects.Thisisanareathatneeds furtherresearchthatincludesmeasuresofgeneticandpotentialenvironmentalriskfactorsandrobustmeasuresofcognitionideallyovermultiple timepoints(24, 26).

Limitations

Animportantlimitationofmanyofthecitedstudiesisthatdifferentmeasuresofcognitionhavebeenusedandanalyseshavetypicallybeenbased uponderivedmeasuresofgeneralcognitivefunctionsuchasIQrather thanuponperformanceonspecifictestsmeasuringspecificdomainsof cognitivefunction.Indefenseofthisapproach,theevidencesuggests thatreductionsinmostaspectsofcognitivefunctionareassociatedwith schizophrenia(3)inlinewithanunderlyingneurodevelopmentalimpairmentthatimpactsbroadlyoncognitiveperformance.However,itispossiblethattherearespecificcognitiveimpairmentsthatareimportantmediatorsofriskthatwillbeidentifiedbyfutureresearch.Additionally,the measuresofpremorbidcognitivefunctionusedinmanygenomicstudieshavebeenindirect.Concernismitigatedtosomeextentbystudies showingthatsuchmeasuresarestronglycorrelatedwithdirectmeasures ofpremorbidIQ(90).However,weacknowledgethatlargelongitudinal cohortswithdirectmeasuresofcognitivefunctionwouldofferabetter meansofinvestigatinghowgeneticandotherriskfactorsinfluencecognitivefunctionoverthelifespaninthosewithschizophrenia.Finally,as wehavenoted,onlyaround10%ofvarianceincognitioninschizophrenia iscurrentlyattributabletoallelesoftheclassessofarstudied,andeven then,thevastmajorityofthisisattributabletopolygenicscoresrather thanspecificcausalalleles.Ourinferencesarethereforebasedonanincompleteunderstandingofthegeneticarchitectureofcognitioninthe generalpopulationaswellasinschizophrenia.

Conclusions

Despitetheselimitationsandthecaveatsanduncertaintieswehave notedthroughout,theavailableevidencefromgeneticstudieshasallowedustoidentifysomeimportantconclusionsandtoproposeamodel oftherelationshipbetweenpremorbidcognitiveimpairmentandriskof schizophreniawhichbestfitsthecurrentdata(Figure1).Thismodelwill nodoubtneedtoberevisedasfurtherfindingsaccumulate.Inparticular, itwillbeimportanttofurtherreplicatethefindingthatdeviationfrom familialcognitiveaptitudeisabetterriskindicatorthanIQ perse andto understandthegeneticandenvironmentalfactorsthatunderliethisdeviationandhowitinteractswithgeneticriskforschizophrenia.However, asitstandsourmodelandthedatauponwhichitisbasedhaveimportantimplicationsforinterpretingbothendophenotypeandanimalmodel studiesaswellasforinterventionsaimedatimprovingcognitivefunction inschizophrenia.

Thefactorsunderlyingcognitivedeclineafteronsetremainunclear anditisnotapparenttowhatextenttheseareintrinsictothedisease processorsecondarytoschizophreniasuchasantipsychoticexposure, metabolicsyndrome,smokingandothersubstanceabuseandvarioussocialconfounders.Understandinghowandwhentheeffectsoncognition, premorbid,andpostonset,arisearekeyquestionsforresearchgiventhe potentialforpreventionandearlyintervention.

Acknowledgments

TheworkwassupportedbyaMedicalResearchCouncilCentregrant MR/L010305/1andprogrammegrantMR/P005748/1.Thecontentisthe responsibilityoftheauthorsanddoesnotnecessarilyrepresenttheofficialviewsofthefundingbodies.WearegratefultoVictoriaHirstforassistancewithmanuscriptpreparation,andCatrinHopkinsandEllieShort forhelpwiththefigures.

AuthorContributions

M.J.O.andM.C.O.reportedreceivinggrantsfromAkriviaHealthandthe TakedaPharmaceuticalCompanyLtdoutsidethesubmittedwork.Takeda andAkriviaplayednopartintheconception,design,implementation,or interpretationofthisstudy.

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THOUGHTLEADERSINVITEDREVIEW

LiverXandthyroidhormonereceptorsinneurodegeneration

MargaretWarner1 ,XiaoyuSong1 ,andJan-ÅkeGustafsson1 , 2

Theroleofthyroidhormone(TH)inthedevelopmentandfunctionofthecentralnervoussystem(CNS)hasbeenknownformanyyears. However,theroleofliverXreceptors(LXRs)inTHfunctionandprotectionagainstneuronaldegenerationwasnotrecognizeduntilrecently.The relationshipbetweenthyroidhormonereceptors(TRs)andLXRsbecameapparentwiththecloningofsteroidhormonereceptors,leadingtothe discoveryofthenuclearreceptorsuperfamily.Thisfamilyincludesnotonlyreceptorsforclassicalsteroidhormonesbutalsomanynewly discoveredligand-activatednuclearreceptors.LXRsandTRsregulateoverlappingpathwaysinlipidandcarbohydratemetabolism,aswellasin overallCNSdevelopmentandfunction.TheseCNSpathwaysincludeneuronalmigrationduringcorticalandcerebellarlayering,myelination, oligodendrocytematuration,microglialactivation,andastrocytefunctions.Furthermore,LXRslikelyhaveuniquefunctions,asevidencedby theinabilityofTHtocompensateformicroglialactivation,oligodendrocytematuration,spinalmotorneurondeath,anddegenerationof retinalandcochlearneuronsinLXRβ knockoutmice.Thecommonanduniquefunctionsofthesetworeceptorsarethesubjectofthisreview.We analyzedsomeofthemostrelevantliteratureontheregulationandfunctionofLXRsandTRsandinvestigatedwhybothreceptorsarerequired inthehumanbody.WeconcludethatLXRsandTRsdonotrepresentparallelpathwaysbutratherconstituteasinglepathwaythroughwhichthe THendocrinesystemregulatescholesterolhomeostasis.Subsequently,LXRs,activatedbycholesterolmetabolites,functionasa paracrine/autocrinesystemthatmodulatesthetargetcellresponsetoTH.

GenomicPsychiatry January2025;1(1):36–46;doi: https://doi.org/10.61373/gp024i.0073

Keywords: LiverXreceptors,thyroidhormonereceptors,steroidhormonereceptors,cholesterolhomeostasis,neurodegenerativediseases,paracrine/autocrinesystem

HistoricalPerspective:LXRs

LiverXreceptors(LXRs,LXRα ,andLXRβ )belongtoasubfamilyofthe nuclearreceptorsuperfamilyofligand-activatedtranscriptionfactors, whichcomprises48membersinthehumangenome(1).Nuclearreceptors playcrucialrolesinregulatingmetabolism,endocrinesystems,andthe developmentandfunctionofthecentralnervoussystem(CNS).Although thefunctionsofthyroidhormone(TH)havebeenstudiedformanyyears, LXRswereonlydiscoveredinthe1990s.Thyroidhormonereceptors(TRs), TRα andTRβ ,aredifferentiallyexpressedinvarioustissuesandhavedistinctrolesinTHsignaling(2).LXRβ (genename NR1H2)wasindependentlydiscoveredbyseverallaboratories(3–6)in1996andwasinitially designatedasOR1,UR,NER,andRIP-15.ItwaslaterrenamedLXRβ due toitshomologywithLXRα (alsoknownas NR1H3),areceptordiscovered in1994(7, 8).

LXRα hastwomajorfunctionsinthebody:lipidmetabolisminorgans suchastheliver,intestine,andadiposetissue,andregulationoftheimmunesystem,notablyinmacrophages(9).LXRβ hasabroadertissuedistributionthanLXRα ;whileitsexpressionintheliverislow,LXRβ iswellexpressedinimmunesystemcells,glialcellsintheCNS,colon,gallbladder, pancreaticislets,retina,andinnerear(10–16).Althoughitisexpressedin veryfewneuronsintheadultmousebrain(17),LXRβ iswidelyexpressed inneuronsofthefetalbrain(18, 19).BothLXRα andLXRβ areexpressed intheovary,testis,prostateepithelium,andepididymis,wheretheyplay significantroles(20–23).

Whilethemostwell-studiedfunctionofLXRsistheirroleincholesterolhomeostasis(24),afunctionsharedwithTRs,cholesteroltransport isjustoneofmanytransportfunctionsofLXRs.LikeTRs,LXRsregulate thetransportofwaterbymodulatingaquaporins(25–29)andglucose throughGLUT4regulation(30–32).Inaddition,LXRsregulatethetransportofTHsandlactatethroughmonocarboxylatetransportersMCT8and MCT10(33).Transportoflactateintoneuronsisessentialforneuronal

nutrition,anditsregulationbyLXRβ (viaMCT1)mayexplainthelossof neuronsinLXRβ / mice.

Classicalhormonessuchasandrogens,estrogens,progesterone,glucocorticoids,andthyroidhormonefunctioninendocrinepathwayswhere glands(suchasthetestis,ovaries,adrenalglands,andthyroidgland)secretehormonesintothebloodstream,whichtargetorgansreceivevia thevascularsystem.WiththeexceptionofthevitaminDreceptor,more recentlydiscoveredmembersofthenuclearreceptorsuperfamilyare activatedbyligandsnotsecretedfromendocrineglandsbutrathersynthesizedinvariouscellsthroughoutthebody.Insomecases,ligandsare acquiredfromthedietorarepharmaceuticalagents.Thenaturalligands ofLXRsareoxygenatedmetabolitesofcholesterol(oxysterols).Some cellsthatsynthesizeoxysterolsalsoexpressLXRs,makingtheLXRsystem anautocrineandparacrinesystemratherthanapurelyendocrineone.

ThetwomajordifferencesbetweenTHandLXRsignalingare:1)TH governstheregulationandintegrationofmetabolichomeostasisatthe hypothalamic-pituitarylevel,butLXRdoesnot;and2)sinceoxysterolsare notcirculatinghormones,LXRactivationisnotnecessarilydeterminedby plasmalevelsofoxysterols(34).

Itisimportanttonotethatevenclassicalsteroidreceptorscanactina paracrinemanner.Forexample,dihydrotestosterone(DHT),aligandfor theandrogenreceptor,isnotacirculatinghormonebutissynthesized fromtestosteroneincellsexpressingtheenzymesteroid5α -reductases. Similarly,3β -Adiol(5α -androstane-3β ,17β -diol),aligandforestrogen receptorbeta(35),issynthesizedincellsexpressingsteroid5α -reductase and17β -hydroxysteroiddehydrogenasetype6(36).IfTHandLXRhavea relationshipsimilartothatoftestosteroneandDHT,theeffectsofTHin cellsmayvarydependingonLXRexpression.

AlthoughLXRsignalingisnotregulatedbythehypothalamic–pituitary–thyroidaxis,LXRdoesregulatethyrotropin-releasinghormone(TRH).BymediatingTH’sactiononTRHrelease,LXRinfluences

1 CenterforNuclearReceptorsandCellSignaling,DepartmentofBiologyandBiochemistry,UniversityofHouston,Houston,TX77204,USA; 2 DepartmentofBiosciences andNutrition,KarolinskaInstitutet,Huddinge14186,Sweden

CorrespondingAuthors: Jan-ÅkeGustafsson,CenterforNuclearReceptorsandCellSignaling,3517CullenBlvd,Bldg545,Houston,TX77204,USA.E-mail: gustafsson@uh.edu;MargaretWarner,e-mail: mwarner@central.uh.edu andXiaoyuSong,e-mail: xsong7@central.uh.edu

Received:6August2024.Revised:20September2024and9October2024.Accepted:13October2024. Publishedonline:24October2024.

thyroid-stimulatinghormone(TSH)levels.IntheabsenceofLXR,thereis excessiveTSHrelease,whichstimulatesthyroxine(T4)releasefromthe thyroidgland.Inaddition,becauseLXRrepressesdeiodinases,thelossof LXRcancreateahyperthyroidstate,whichmayhelpexplainwhyLXRβ / miceareresistanttoobesityinducedbyahigh-fatdiet(33).

LXRsandTRs

ThefunctionofTHsismainlymediatedthroughtheirbindingtoTRsat specificTREs(thyroidresponseelements)onDNA.BothTRsandLXRs bindtotheseresponseelements,whichconsistofdirectrepeatsofthe half-sitesequence5 -G/AGGTCA-3 ,separatedbyfournucleotides(DR4). Intheabsenceoftheirligands,bothTRsandLXRsbindtoDR4,recruiting corepressorsandinhibitingthetranscriptionofresponsivegenes.When ligandsbind,theyrelievethisrepressionbycausingthereleaseofcorepressorsandsubsequentbindingofcoactivators,leadingtotheactivation oftranscriptionofresponsivegenes(37).

TRscanbindtoDNAeitherashomodimersorasheterodimerswith retinoidXreceptors(RXRs),whileLXRsformobligatoryheterodimers withRXR(38–40).RXRsareasubgroupofthenuclearreceptorsuperfamily,comprisingisotypes α , β ,and γ ,whichcanformhomodimericandheterodimericcomplexeswithothernuclearreceptors(41).Theendogenous ligandforRXRis9-cisretinoicacid(42).Thus,vitaminAalsoplaysasignificantroleintheregulationoftheimmunesystembyTHandLXR(43).

T3,T4,andDeiodinases

T4isaprohormonethatisconvertedtotheactivehormonetriiodothyronine(T3)throughtheactionofdeiodinases.Thelocalactivationof T4toactiveT3bydeiodinasesisakeymechanismofTHregulationof metabolism.Therearetwoactivatingdeiodinases,DIO1andDIO2,and oneinactivatingdeiodinase,DIO3(44, 45).Inhumans,DIO1ishighlyexpressedintheliver,whileDIO2isexpressedinthehypothalamus,white fat,skeletalmuscle,andbrownadiposetissue,whereitisessentialfor adaptivethermogenesis(46).OnekeymechanismbywhichLXRregulates THfunctioninbothrodents(47)andhumans(48)isthroughthedownregulationofdeiodinases.

SomeComplexitiesofLXRsandTRsSignalingintheBrain

Sincecholesterolcannotcrosstheblood–brainbarrier(BBB),itmust besynthesizedwithinthebrain.Astrocytesareresponsibleforcholesterolsynthesis,whichisthentransportedtoothercellsviathetransportproteinapolipoproteinE(ApoE)(49, 50).Additionanally,thebrain synthesizestwooxysterols:25-hydroxycholesterol(25-HC),producedin microglia(51),and24-hydroxycholesterol(24-HC),whichiscatalyzedby theenzymeCYP46A1(cholesterol24-hydroxylase)andexpressedinneuronsofthehippocampus,cortex,Purkinjecellsofthecerebellum,and interneuronsinthehippocampusandcerebellum(52).24-HCisamajor metaboliteofcholesterolinthebrainandservesastherouteforexcreting excesscholesterol(53, 54).Furthermore,thebraincaninactivateoxysterolsthroughCYP7B1,whichcatalyzeshydroxylationofoxysterolsatthe 6and7positions(55).AlthoughthecellulardistributionofCYP7B1has notbeenwellinvestigated,itisoneofthemostactivecytochromeP450 enzymesinthebrain(56),makingitveryunlikelythatthecellsharboring thisenzymewillrespondtooxysterols.

T3doesnotcrosstheBBB,butT4does.Therefore,deiodinasesareextremelyimportantfortheTHfunctioninthebrain,anddefectivedeiodinasescanleadtobrainTHdeficiency.THentersthebraineitherdirectlyviatheBBBorindirectlyviatheblood–cerebrospinalfluid(CSF) barrier,withtheBBBservingastheprimaryentrypathforT4(57).TH entersthechoroidplexusthroughtransmembranetransportersMCT8 andorganicanion-transportingpolypeptide1C1(OATP1C1)andexitsthe choroidplexustoentertheCSFviaTHtransmembranetransportersor throughchoroidplexus-derivedtransthyretinsecretedintotheCSF(58). DIO2isexpressedinthechoroidplexus(59).LXRsregulateCSFdynamics atboththechoroidplexusandtheastrocyticendfeet,andinactivation ofLXRresultsindegenerationofthechoroidplexusandlackofCSFin thelateralventricles(28).Thisdegeneration,alongwiththelossofDIO2, leadstoreducedTHlevelsinthebrain.Consequently,somephenotypicaspectsofLXRknockoutmiceresembleTHdeficiency.OnceTHshavepassed BBB,theirlocalavailabilitydependsontheactivityoftheastrocyticDIO2

toconvertT4toT3.T3issubsequentlyinactivatedinneuronsbyDIO3, whichremovesthe3’iodine,producing3,5-diiodothyronine(T2).

Inadditiontoregulatingdeiodinases,LXRsalsoregulateT4transporters.Inhumans,asinotherprimates,theBBBcontainsMCT8butlacks OATP1C1(60, 61).MCT8isahighlyspecifictransmembraneTHtransporterresponsibleforthecellularinfluxandeffluxofT4andT3(62). ItisindespensiblefordrivingTH-dependentoligodendrocytedifferentiationand,consequently,myelination(63, 64).Inhumans,mutations in SLC16A2,thegeneencodingMCT8,leadtoanX-linkedsyndromecharacterizedbysevereneurologicalimpairmentandalteredT3concetrations duetoimpairedTHuptakeinthedevelopingbrain.Inmicelackingboth MCT8andOATP1C1,THconcentrationsinthebrainaresignificantlyaffected(65).

BothTRsandLXRsbindtoDR4onDNAintheabsenceoftheirrespectiveligands,repreesinggenesregulatedbyDR4responseelements.The knockoutofLXRrelievesrepressiononDR4-responsivegenes;however, whatcannotoccurinLXRknockoutmiceistheactivationofLXRbyligandsandtherecruitmentofcoactivatorstoenhancethetranscriptionof LXR-responsivegenes.TounderstandingthephenotypeofLXRknocknout mice,wemustconsiderboththederepressionofcertaingenesandthe absenceofactivationofothersbyLXRligands.

THregulatesmetabolicrate,bodytemperature,cholesterolhomeostasis,andadrenergicfunction.Ofthese,onlyadrenergicstimulation isnotsharedbyLXRs.THregulatescholesterolhomeostasisattwomajorpoints:itincreasesthelow-densitylipoprotein(LDL)receptortofacilitatecholesterolremovalfromcirculationandstimulatescholesterol 7alpha-hydroxylase(CYP7A)topromotecholesterolremovalfromthe bodyintheformofbileacids.LXRsactascholesterolsensors,activated bycholesterolmetabolites(1).Uponactivation,theyassistTHineliminatingcholesterolfromthebodybyinducingcholesteroltransporters ABCA1andABCG1,whichtransportcholesteroloutofcells.However,LXRs alsoactatmultiplelevelstoreduceTHfunction:1)LXRreducesdeiodinases,preventingtheconversionofT4toT3;2)LXRlowersTHlevelsby facilitatingnegativefeedbackatthehypothalamiclevel;and3)LXRinducestheexpressionoftheinducibledegraderoftheLDLreceptor(IDOL), whichdecreasesLDLreceptorexpressiononthecellsurfaceandlimits LDL/cholesteroluptake(66).

LXRsandTRsinNeurodegeneration

BothLXRandTHareessentialfornormalbraindevelopment,influencingneurogenesis,neuronalandglialcelldifferentiationandmigration, synaptogenesis,andmyelinationduringearlyfetallife(67, 68).DysregulationofcholesterolmetabolismintheCNShasbeenlinkedtoseveral neurologicaldisorders(49, 69–74).Preclinicalstudieshaveindicatedthat LXRsandTRscanbeusedastargetsforthetreatmentofneurodegenerativediseases(Figure1),suchasAlzheimer’sdisease(AD),Parkinson’s disease(75, 76),amyotrophiclateralsclerosis(ALS)(77),Huntington’s disease,andmultiplesclerosis(MS)(78).

Althoughthesecommonanddevastatingneurodegenerativediseases areassociatedwithaging,neurodegenerationlikelybeginsmuchearlier, asdiseasesymptomsemergeonlyafterasignificantnumberofneurons havealreadybeenlost.Ourstudieshaveshownmarkedexpressionof LXRβ incorticalneuronsinthefetalmousebrainduringlaterembryonicstages(19).LXRβ expressionfirstappearsinthecerebralcortexas earlyasE14.5andisstronglyexpressedinthecortexplatefromE16.5 untilE18.5.Afterbirth,LXRβ ismainlylocalizedincorticallayersII/III.In LXRβ / mice,thereisnodefectinneuronalproliferation;however,laterbornneuronsfailtomigratetocorticallayersII/IIIastheydoinwild-type (WT)littermates(19).Thismigrationdefectisthoughttoresultfromadefectinradialgliaandreducedexpressionofthereninreceptor,ApoER2 (79).ThedefectiscorrectedwhenTHlevelsincrease,andbypostnatal day14,thereisnodetectabledifferenceinthecortexbetweenWTand LXRβ / mice(79).Theseobservationssuggestthatintheabsenceof LXRβ ,thereisinsufficientTHinthefetalbrain,leadingtoaprolonged repressiveroleofTR-onTH-responsivegenes.

Despitethewell-knowneffectsofTHinthedevelopingbrainandthe clearroleoffetalhypothyroidisminmentalretardation,THisnotimplicatedinlate-onsetneurodegenerativediseases.However,thelossof

Figure1. LXRandrelatedCNSneurologicaldisorders.

LXRβ inmicedoesleadtoage-relatedneurodegeneration.InLXRβ / mice,thereisalossofdopaminergic(DA)neuronsinthesubstantianigra(80),largemotorneuronsintheventralhornofthespinalcord(81), epithelialcellsofthechoroidplexus(28),retinalganglioncells(15),and spiralganglionneurons(Figure2)(16).Alloftheseconditionsdevelop withageafterthemiceare6monthsofage.

Oneperplexingobservation,inviewofthelossofDAandmotorneurons,istheabsenceofLXRβ expressionintheseneuronsinadultmice. ThishasledtotheconclusionthatLXRβ incellsotherthanDAandmotor neuronsprotectstheseneuronsfromage-relatedloss.Thesespecificcell typesinvolvedremaintobeidentified.Todate,LXRβ hasbeenspecifically deletedfromastrocytes(82)andmicroglia,andtherewasnoobserved lossofDAormotorneuronsinthesemice.ItremainspossiblethatdegenerationofthechoroidplexusanddefectsinCSF,whichoccurintheabsenceofLXR,aremajorcontributorstotheneurodegenerationobserved inLXRβ / mice.

LXRsandTRsinALS

ALSisalate-onset,fatalneurodegenerativedisordercharacterizedbythe specificlossofbothupperandlowermotorneurons(83).Themajority ofcasesareclassifiedassporadic,withtheetiologyremainingunknown. Lessthan10%ofALScasesarefamilialandassociatedwithdefectsinthe SOD1, C9ORF72, FUS,and TARDBP genes.Althoughnoneofthesegenes areregulatedbyLXR,aproteomicanalysisofserumfromALSpatients revealedthattheLXR/RXRpathwayisoneofthemostsignificantlyregulatedpathways,withbothLXRα andLXRβ identifiedasgeneticmodulatorsoftheALSphenotype(84, 85).InmicelackingLXRβ ,thereisprogressiveimpairmentofmotorperformanceleadingtohindlimbparalysis,loss ofmotorneuronsintheventralhornofthespinalcord(Figure3),andloss ofneuromuscularjunctions(80, 81, 86).

AstudyonthepathogenesisofALSindicatedthat25-HC,anendogenousligandforLXR,mayactivelymediateneuronalapoptosis,particularly intheearlysymptomaticstageofthedisease(87).ThefailureoftheCNS toremoveexcesscholesterolcanleadtoneurodegeneration,astheaccumulationofcholesterolmaybetoxictoneuronalcells.However,choles-

terolaccumulationisnottheonlybraindefectcausedbydefectiveLXR; thereisalsoareductionin3β ,7α -dihydroxycholest-5-en-26-oicacid,a neuroprotectivecholesterolmetabolite(88, 89).

AnothercommondefectobservedinbothALSandLXRβ / miceisthe structuralandfunctionaldisruptionoftheblood–CSFbarrier.InALS,there isdisruptionofjunctionsbetweenchoroidplexusepithelialcells,activationofplatelets,immuneinfiltrationintothechoroidplexus,anddegenerationofmajorvasculatureassociatedwiththedisease(90).Thechoroid plexusofLXRknockoutmiceisseverelyaffected(28),withdegeneration andabsenceofCSFinthelateralventriclesbeingprominentcharacteristicsoftheLXR / mousebrain.

Todate,studieshavenotprovidedstrongevidencetosupportarole forTHinALS.InacohortofPortuguesepatientswithALS,thyroiddysfunctionwasnotassociatedwiththedisease(91),andinacohortfromSouthwestChina,thyroiddysfunctiondidnotassociatedwithsurvivalorserve asaprognosticfactorforALS(92).DespitethelackofeffectofTHonALS, asimilarmovementdisorderisobservedinbothTHandLXRdeficiencies: pronounced,spontaneous,asymmetricalcirclingbehavior.Thisbehavior wasfirstreportedbyKincaid(93)ingeneticallyhypothyroidmice,which doesnotdevelopathyroidglandduetoadefectiveTSHgene.Thecircling behaviorappearedinbothmaleandfemalemicearoundpostnatalday35 andpersistedthroughouttheirlifespan.Thecirclingwasunidirectional, eitherclockwiseorcounterclockwise.ThisbehaviorwasnotedinallfemalebutnotmaleLXRβ / mice.IntheKincaidstudy,thecyclingwas linkedtothelossofDAneuronsinthesubstantianigra,butitremainsunclearwhythebehavioronlyemergedinadultmice.IntheLXRβ / mice, asimilarcyclingbehaviorwasobserved,thoughitsetiologyhasnotbeen thoroughlyinvestigated.

LXRsandTRsinDopaminergicNeurons

InthedevelopingmousebrainatE11.5,theLXRagonist24(S),25epoxycholesterolincreasedmidbrainDAneurogenesisfromprecursor cellsbyabout40% invitro and invivo (94–96).TheLXR-regulatedtranscriptionfactorresponsibleforthisincreaseinthedifferentiationofradialgliaintoDAneuronswasidentifiedasthebasichelix-loop-helix

Figure2. ThenumberofspiralganglionneuronsinthecochleaofLXRβ / miceislessthanthatofWTmice.12monthsofage.Scalebars:AandC,100 μm; BandD,50 μm.

transcriptionfactorSREBP1(sterolregulatoryelementbindingprotein1; genename Srebf1)(97).DespitethisroleofLXRβ inthedifferentiationof DAneurons,thereisnoapparentreductioninthenumberofDAneurons inthesubstantianigrain5-month-oldLXRβ / mice,andtheirperformanceontherotarodtestwascomparabletothatofWTmice(80).Thus, thereisadisconnectbetweenLXR’sactionsinthefetaldevelopmentof DAneurons invitro anditsroleintheadultbrain.

KnockingoutLXRaffectsthesurvivalofDAneurons.Inthesubstantia nigraofLXRβ / mice,thelossofDAneuronsbeginstobenoticeableafterthemicereach6monthsofage,andby16months,thereisamarked reductioninthenumberofDAneurons.Thesemiceperformpoorlyon therotarod.LXRβ knockoutmicealsoshowincreasedsensitivitytochallengeswithMPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)(17) or β -sitosterol(80).AconfoundingfactorintheseeffectsisthatLXRβ is notexpressedinDAneurons.Thus,LXRappearstoinfluenceDAneurons attwolevels:survivalwithageandneurogenesisatE11.5.Inbothcases, itisnotLXRβ intheDAneuronsthemselves,butinothercellsthatinfluencethedifferentiationofDAneurons.Thecellsinvolvedduringembryonicdevelopmentarelikelyradialglia,butthespecificcellsresponsible forthelossofDAneuronsinadultmicewithageremaintobedetermined. ItmaybethatmultipleLXR-regulatedcellsandfactors,includingcholesterolaccumulation,microglialactivation,astrogliosis,oradysfunctionof thechoroidplexus,influencethesurvivalofDAneurons.

THisalsoessentialforthedifferentiationofDAneurons(98),butin thiscontext,itisevidentthatTRα 1inprecursorcells,ratherthaninDA neurons,isresponsible(99).Thetranscriptionfactorrequiredforembryonicventralmidbrainneuralstemcells(NSCs)todifferentiateinto DAneuronsisOtx2.TRα 1iscoexpressedwithOtx2inculturedventral midbrainNSCs.Otx2,inturn,inducesanumberofotherfactors,includingNeurogenin2(Ngn2)andNurr1(alsoknownasnuclearreceptor4A2, NR4A2).

Currently,thedistinctrolesofLXRandTHhavenotbeenfullydefined. PublisheddataindicatethatthefunctionsofTHandLXRhavebeeninvestigatedatdifferentstagesofdifferentiationbetweenE11.5andE13.5. ThisisacriticalperiodforthedifferentiationofDAneurons(100),and manystepsinDAneuronaldifferentiationoccurbeforeE13.5.Untilmore detailedtimedstudiesareconducted,itisnotpossibletoseparatethe rolesofTHandLXRinthedifferentiationofDAneurons.

Ofkeyinteresttohumandiseaseisthelate-onsetoflossofDAneurons inLXRβ / mice.SinceParkinson’sdiseaseisalate-onsetcondition,the LXRβ / micemayprovidevaluableinsightsintothisdisease.

TRsinCerebellum

Ishii etal.summarizedthatvariousmousemodelshavebeenusedto evaluatetheeffectsofTHoncerebellardevelopment,revelingextensiveabnormalitiesthatresultinanataxicphenotype(101).Thepostnatal

Figure3. ThenumberofmotorneuronsintheventralhornofthespinalcordofLXRβ / miceislessthanthatofWTmice.Neurofilamentstaining.11months ofage.Scalebars:AandC,50 μm;BandD,20 μm.

defectsobservedinthecerebellumofhypothyroidmicearerecapitulated inmiceheterozygousforadominant-negativemutationintheTRα 1receptor(102, 103).Thismutationprimarilyaffectsthedifferentiationof PurkinjecellsandBergmannglia.

LXRsandTRsinDevelopmentoftheDentategyrus

Thedentategyrus(DG)ofthehippocampusplaysaprominentrole inlearning,memory,andemotion.Thesubgranularzone(SGZ)ofthe hippocampalDGisoneofthestemcell–containingnichesintheadult mammalianbrain(104).ThepermissivemilieuoftheSGZallowsNSCs toproliferatewhilepromotingthespecificationanddifferentiation ofdentategranuleneurons.IntheDGofLXRβ / mice,thereishypoplasiaandabnormalitiesinprogenitorcellformationandgranule celldifferentiation,resultinginautistic-likebehavior(105).InGW3965treated3xTg-ADmice,thenumberofstemcellsandproliferatingcells increasedintheSGZ(106).Furthermore,LXRactivationameliorated learningandmemoryimpairmentsbypromotingneuronalsurvival,NSC proliferation,andneurogenesisintheDGindifferentanimalmodels (107, 108).

Hypothyroidismresultsinreducedhippocampalvolumeinadults (109).THsaffectneurogenesisintheDGofadultrats(110)andareessentialforpreservingnonproliferativecellsinvolvedinadultneurogenesis(111).In2024,Valcárcel-Hernández etal.providedanexcellent

summaryofTHsintheSVZ(subventricularzone)liningthelateralventricles,thehippocampalSGZ,andthehypothalamus,controllingthegenerationofnewneuronalandglialprogenitorsfromNSCs,aswellastheir finaldifferentiationandmaturationprograms(112).

LXRsandTRsinAlzheimer’sDisease

AD,themostcommoncauseofdementiaglobally,isaprogressiveneurodegenerativediseasecharacterizedbyinitialmemoryimpairmentand cognitivedecline,withthepresenceofamyloidplaquesandneurofibrillarytanglesbeingcrucialforapathologicaldiagnosis(113).BothTHand LXRsignalinghavebeenimplicatedinAD(Figure4).Asdiscussedabove, LXRsignalingisintricatelylinkedtoTHlevels.BecauseLXRinhibitsdeiodinases,areductioninLXRsignalingshouldbeassociatedwithhigherlevelsofTH.Therefore,thereducedsignalingofbothTHandLXRinADis puzzling.

Severalstudieshaveinvestigatedtheassociationbetweenthyroid dysfunctionanddementiarisk(114–116).Meta-analysesrevealeda higherprevalenceofhypothyroidisminAD,buttheauthorscautionedthat thefindingcouldnotdistinguishwhetherhypothyroidismisariskfactor fororaconsequenceofAD(117).Oneofthemostbeneficialeffectsof THinADisitseffectsinrepressionofmicroglialimmuneresponses(118). However,nodefinitivelinkbetweenthyroiddysfunctionandADhasbeen established(119–121).

Figure4. UnravelingthecomplexrolesofLXRsandTRsinneurodegenerativediseases.ThisdiagramdepictstheintricatebiologyofLXRsandTRsandtheir rolesinneurodegenerativediseases.Attheheartofourconceptualframeworkishowthesereceptorsinfluencekeyprocessesinthebrain—frommanaging cholesterollevelstoshapingbraindevelopment.Wecanseehowtheiractionsrippleouttoaffectvariousneurodegenerativeconditions,includingAlzheimer’s andParkinson’sdiseases,aswellasALSandmultiplesclerosis.AninterestingtwistrevealedbythediagramisthatLXRsactuallyhelpregulatethyroidhormone function,addinganotherlayerofcomplexity.WehaveuseddifferentcolorstohighlightwhichprocessesarespecifictoLXRs(inpink)orTRs(inlightblue),while sharedpathwaysareshowninorange.Lookingtothefuture,wehaveincludedpromisingtherapeuticapproachesandexcitingnewresearchdirectionsinlight green.Thisvisualframeworkcapturesourcurrentunderstandingandalsopointstowherethenextchaptersinthisemergingscientificnarrativemightleadus.

InthecontextofLXRandAD,activationofLXRhasbeenconsidereda therapeuticstrategy(11, 71, 73, 122–124)forseveralreasons:1)ApoE, anLXR-inducedgene,promotestheproteolyticdegradationofAβ invariousADanimalmodels(106, 125–128),therebyreducingbrainAβ burden;2)Inhibitionofneuroinflammation(129, 130),includingtheactivationofmicrogliaandastrocytes(131, 132);3)LXRligandsameliorate theimpairmentsinsynapticplasticity(133, 134);4)GeneticlossofLXRs inAPP/PS1transgenicmiceresultsinincreasedamyloidplaqueburden (135);5)T0901317hasbeneficialeffectsonmemorybyenhancingbrain cholesterolturnoverinAPPSLxPS1mutmice(136);6)InAPP/PS1mice, LXRagonistsexertbeneficialeffectsinamelioratingmemoryimpairment byelevatinglevelsofApoEandABCA1,reducingtheexpressionofproinflammatorygenes,anddecreasingAβ aggregation(137–139);7)ActivationofLXRwiththeagonistT0901317decreasedBACE1expression andactivitybyloweringmembranecholesterollevels(140);8)DMHCA, apartialLXRagonist,preventedmemorydeclineandsignificantly decreasedhippocampalAβ oligomerswithoutaffectingplasmalipid levels(141).

OnegenethatisupregulatedbybothLXRandTRβ istheseladin-1 (selectiveADindicator-1),encodedbythe3beta-hydroxysterol-Delta24 reductase(DHCR24).DHCR24isacrucialenzymeincholesterolsynthesis, catalyzingtheconversionofdesmosterolintocholesterolandlanosterol to24,25-dihydrolanosterol.BothLXRα andTRβ upregulatethetranscriptionofDHCR24(142–144),suggestingitmaybeacommongenelinking TRandLXRtoAD.

LXRsandTRsinDemyelinatingDiseases

Sincecholesterolisanessentialcomponentofallcellmembranesand isparticularlyenrichedinmyelinmembranes,itisnotsurprisingthat cholesterolmetabolismisinvolvedintheprocessesofdemyelinationand remyelination(145).Oligodendrocytesarethecellsinthebrainresponsibleformyelination(146).BothLXRsandTRsarecriticalforpromoting andmaintainingmyelination(147, 148).Evenbeforemyelinsynthesisoccurs,bothreceptorsareneededforthedifferentiationofoligodendrocytes.LXRβ regulatesthenumberofoligodendrocytebydrivingradial glialcellsinthedorsalcortextobecomeoligodendrocyteprogenitorcells (149).Meanwhile,THisrequiredfortheterminaldifferentiationofoligodendrocyteprecursorcellsintomyelinatingoligodendrocytesbyinducing rapidcell-cyclearrestandtranscriptionofprodifferentiationgenes(150, 151).

Therefore,itisnotsurprisingthattheknockoutofLXRsinmiceresults inabnormalmyelinationandareductioninthesizeofmyelinatedaxonin themousebrain(70, 152, 153).Asdescribedabove,LXRhaswidespread functionsinthebody,andinactivationofLXRleadstomultipleorgandysfunctioninmice.IfLXRshavesimilarrolesinhumansandmice,itisdiffi-

culttoimagineahumansurvivingwithadefectiveLXRgenewithoutseveredefectsinlipidhomeostasis,vasculardisease,andimmuneandneuronaldysfunction.AmutationinLXRα (p.Arg415Gln)hasbeenreported toberesponsibleforfamilialdevelopingprogressiveMS(154),butthe associationbetweentheLXRα mutationandMScouldnotbeconfirmed bytheInternationalMSGeneticsConsortium(IMSGC)patientcollection (155).Beforethisissuecanbefullyresolved,itisessentialtoexamine thefunctionoftheLXRα withthe(p.Arg415Gln)mutationtodetermine whetheritfunctionsasanormalLXRα andwhethertheLXRmutationsimplysegregateswithanothergeneresponsiblefortheMSphenotype.

Martin-Gutierrez etal.reportedthatLXR-mediatedlipidmetabolism pathwaysweredysregulatedinTcellsfrompatientswithrelapsingremittingMS(RRMS)pathology,potentiallycontributingtoRRMSpathogenesis(156).ThestudyshowsthatLXRregulatesTcellfunctionby regulatingglycosphingolipidandcholesterolmetabolism,althoughthe specificdefectinLXRinTcellsthatcouldcauseRRMSremainsundefined.

MSisanautoimmunedisease(78, 157, 158)thoughttobeduetoT-cell reactionstoantigensassociatedwithmyelin,suchasmyelinbasicproteinandmyelinoligodendrocyteglycoprotein.Inchronicdemyelinating inflammatorydiseasemodels,THrestoresnormallevelsofmyelinbasic proteinmRNAandprotein(159, 160)andpromotesthedifferentiationof oligodendrocyteprogenitorcells,improvingremyelinationthroughTRβmediatedT3effects(161).

T4activatesoligodendrocyteprecursorsandincreasesthecontent ofmyelin-formingproteinsandNGFinthespinalcordduringexperimentalallergicencephalomyelitis(162).StudiesusingtheTRβ -selective agonistSobetirome(GC-1)havefoundthatitpromotesremyelination, enhancesoligodendrocyteproliferation,andprotectsagainstoligodendrocytedeath(163–166).

Inadditiontoitseffectsonoligodendrocytes,anothermechanism throughwhichLXRsignalingrepairsdemyelinationdamageisbyactingonmicroglia/macrophages,inhibitingtheinflammatoryresponseand providingasupportiveenvironmentforoligodendrocytedifferentiation andmyelination(167),whilealsopromotingthephagocyticclearanceof myelindebrisandcholesterol(168).LXRagonistsmaybeusefulinhealing whitematterinjury,asLXRligandshavebeenshowntoinduceoligodendrogenesisinrodentinjurymodels(169, 170).However,thechallengeof limitingLXRactiontothetargetedareamustfirstbeaddressed.

ConcludingRemarks

TheaimofthisreviewwastoanalyzetherolesofLXRsandTRsinneurodegenerativediseases(Figure4).Areviewoftheliteratureclearly showsthatthesetworeceptorsworktogethertoregulatecholesterol homeostasis,anddysregulationofcholesterolhomeostasisisacommon factorinneurodegenerativediseases.Duetotheirwidespreadeffects

AuthorDisclosures

Theauthorsdeclarenoconflictsofinterest.

AuthorContributions

J-ÅG,MW,andXSconceivedthemanuscripttopic,editedandorganized thefinaldraft.XSwrotethefirstdraftofthemanuscriptandpreparedthe figures.Allauthorsrevisedthefinalmanuscriptandapprovedthefinal version.

Acknowledgments

J-ÅGacknowledgesRobertA.WelchFoundationgrantE-0004andthe SwedishResearchCouncil.

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GenomicPsychiatry

THOUGHTLEADERSINVITEDREVIEW

LessonswelearnedfromtheLothianBirthCohortsof1921and1936

IanJ.Deary1 ,andSimonR.Cox1

Theauthorsare,respectively,thefoundingandcurrentDirectorsoftheLothianBirthCohortsof1921and1936.Inthisinvitedand,admittedly, self-regardingandnecessarilyself-citingpiece,weenumerateandexplicatesomethingswelearnedfromworkingwiththecohortsandtheir data.Someofthelessonsarescientificresults,somearetodowithscientificpractice,andsomearemoregeneralreflections.Wehopethe paperprovidesausefulsummaryofsomeofthemainfindingsfromthesetoo-many-papers-to-readcohortsandanenjoyableaccountofour buildingaresearchteamandanetworkofcollaborators.Theoriginalaimofassemblingthecohortswastofashionatooltodiscoverwhysome people’sthinkingskillsagedbetterthanothers’.Thattool,wediscovered,hadmanyadditionaluses.

GenomicPsychiatry January2025;1(1):47–60;doi: https://doi.org/10.61373/gp024i.0076

Keywords: Cognitiveageing,cognitiveepidemiology,longitudinalstudies,LothianBirthCohorts,ScottishMentalSurveys,intelligence.

Introduction

Itisnice(weareBritish,afterall)tohavebeenaskedbytheeditortoreflectonwhatwehavelearnedfromtheLothianBirthCohorts(LBCs).We arehappytodoso.Oneofthebenefitsthisreflectionaffordsisthatit cancollectafractionofthelargenumberoftheLBCs’widely-dispersed scientificarticlesinoneplaceandprovideashopwindowforthem(see PublicationsfromtheLothianBirthCohorts);itcanpointthewaytomany more.Therearesomedrawbacks,too,ofthisexercise.WeshallnecessarilyfocusontheLBCs’contributionstoscientificquestionswhereaswe knowthatothercohortsandsamplesoftenhavemademoreandbettercontributions.Weshallhavetoengageinthefrowned-uponactivityofself-citation.Wetrytoavoidduplicatingothersynopticpieceson theLBCs.Thesecoyworriesnotwithstanding,herearesomelessonsfrom 25yearsofworkontheLBCs.

NoteveryoneknowswhattheLBCsare,sothisenumeratedparagraph isacribsheet.Herearesomekeyfactsthatshouldmaketherestofthe articlemorecomprehensible.

1.OnMondayJune1,1932,theScottishCouncilforResearchinEducationtestedalmosteverychildbornin1921andattendingschoolsin ScotlandontheMorayHouseTestNo.12(atestofintelligencethat correlatedabout0.8withtheStanfordBinettestin1000ofthepupils inavalidationexercise).The N was87,498andthisrepresentedabout 94%ofthewholeScottishpopulationofthatyearofbirth.Thiswas theScottishMentalSurvey1932(SMS1932)(1, 2).

2.OnWednesdayJune4,1947,theScottishCouncilforResearchinEducationtestedalmosteverychildbornin1936andattendingschools inScotlandontheMorayHouseTestNo.12.The N was70,805and thisrepresentedabout94%ofthewholepopulationofthatyearof birth.ThiswastheScottishMentalSurvey1947(SMS1947)(2, 3).

3.Beginningin1999,atameanageof79years,werecruited550 largely-healthycommunity-dwellingScottishpeoplebornin1921to formtheLothianBirthCohort1921(4).Mosthadtakenpartinthe SMS1932;therefore,formostofthem,MorayHouseTestscoreswere availablefromage11.Theyprovideddemographicandhealthinformation;theyweretestedoncognitivefunctions,sensoryfunctions, psychosocialfactors,andfitness;theyprovidedbloodsamplesfora widerangeofbiomarkers,genetics,andother‘omicstests;theywere linkedtodeathrecords;aminorityhadsomestructuralmagneticresonanceimaging(MRI)ofthebrain.Theyweretestedatages79,82, 87,90,and92years(5, 6).

4.Beginningin2004,werecruited1091largely-healthycommunitydwellingScottishpeoplebornin1936toformtheLBC1936(7).Most hadtakenpartintheSMS1947;therefore,formostofthem,Moray HouseTestscoreswereavailablefromage11.Theyprovidedallthe informationthathasbeencollectedintheLBC1921,butinmoredetailandwithmanyextras.Forexample,theircognitivetestbattery wasmuchlonger,theyunderwentlongitudinalstructuralmagnetic resonancebrainimaging,theywerelinkedtomedicalrecordsaswell asdeathrecords,theyprovidedwhitebloodcellsforstemcellcreation,andtheyconsentedtoprovidebraintissueafterdeath.They weretestedatages70,73,76,79,82,86and,aswewrite,theyare beingtestedforwhatwillcompriseWave7atmeanage88(5, 6). Figure1 illustratesthetimelineoftheLBC1936studyandsomeof themajortypesofdatathathavebeencollected.

5.WehavewrittentheprotocolsoftheLBC1921andLBC1936baseline Waves(4, 7),andwehavewrittencohortprofiles(5)andcohortprofileupdates(6)thatgivedetailsofthevariablescollectedinthese twostudies.Werecommendthesearticlestothosewhowouldliketo requestdatatotesttheirhypothesesontheLBCs.

6.WewroteasummaryofwhatwehadfoundoutabouthealthycognitiveageingintheLBC1921andLBC1936upto2018(8).

7.ForthoseinterestedinthebackgroundtotheLBCs,theScottishMentalSurveys,andthesmallerbutslightlyearlier-conductedAberdeen BirthCohortsof1921(ABC1921)and1936(ABC1936)thereisthe book, ALifetimeofIntelligence (2).

8.AkeyvariablethatisavailableintheLBC1921andtheLBC1936was theretestinginoldageoftheMorayHouseTestNo.12.Thisisthe intelligencetestthattheyhadtakenatmeanage11years,whichwas theageoftransitionfromprimarytosecondaryschool(atthetime, compulsoryeducationcontinueduntiltheageof14).

9.IanDearyfoundedanddirectedtheLBCsfromJanuary1999to November2020whenheretired(justbriefly,toberehired,afew monthslater,part-timetocontinueworkingontheLBCs).SimonCox hasDirectedtheLBCssinceDecember2020,havingworkedwiththe LBCssince2009(forhisPhDwhereIanDearywasoneofhissupervisors,thenasStudyCo-ordinator,thenpostdoctoralfellow,andthen LBCCo-Investigatorandleadinghisownfundedworkontheneuroimagingaspectsofthestudy).

10.ItwasafumblingsetofeventsthatledLawrenceWhalley(whodied in2024)andIanDearytodiscoverthattheSMSshadbeenconductedandthattheirdatastillexisted(describedinref. 2).Professor

1 LothianBirthCohorts,DepartmentofPsychology,UniversityofEdinburgh,Edinburgh,UK

CorrespondingAuthor: IanDeary,DepartmentofPsychology,UniversityofEdinburgh,7GeorgeSquare,EdinburghEH89JZ,UK.E-mail: i.deary@ed.ac.uk

Received:19September2024.Revised:23October2024and24October2024.Accepted:25October2024. Publishedonline:7November2024.

Figure1. DatacollectedintheLothianBirthCohort1936.Thecentralrowofwhiteboxesdenoteseachinstanceofmeasurement,startingwithallage11Scottish childrenwhosattheMorayHouseTestNumber12,andproceedingtothe“baseline”(Wave1)andsubsequentassessmentvisitsoftheLBC1936participants. Solidarrowsindicatedata(topandbottomrows)collectedatagivenwave;dottedlinesandboxesdenoteongoing(Wave7)andplanned(Wave8)datacollection.

Whalley(apsychiatrist)ledtheABC1921andABC1936,collaboratingwithIanDearyandgeriatricphysicianProfessorJohnStarr(9). JohnStarrwasthemedicalleadontheLBCsfrom1999untilhis deathin2018,afterwhichDrTomRusstookuptherole.Professor JoannaWardlaw(aneuroradiologist)isthebrainimagingleadonthe LBC1936(10).

ScientificDiscoveryLessons

WhenpeopleaskabouttheaimsoftheLBCs,wesaysomethinglike,“we aretryingtodiscoverwhysomepeople’sthinkingskillsandbrainsage betterthanothers”.However,theLBCshaveprovedtobevaluablefar beyondthatremit.Theyoftenthenask,“whathaveyoudiscovered?”. Afterweprovideananswer,sometimesitismetwithundertakingsto makelifestylechanges(e.g.,stopsmoking),butitisalsonotunusualto hearthefollow-onquestion,“isn’tthatobvious?”.Seewhatyouthink… SomeoftheBigFindingsAppearedEarlyon

Thisisnotascientificdiscoveryperse;rather,itisametacomment.Toarticulatethis,althoughithasaPareto-likequality(andsomeregressionto-the-meanquality),weshallusethecomparisonofpop/rockbands. Mostbandshavemanysongs,onlyafewofwhicharelargehitsandoftenthosehitsappearearlyintheircareers(havealookatnumbersof playsonSpotify).WiththeAberdeenBirthCohorts(ABCs)andLBCssome oftherelativelybiggerdiscoverieshappenedearlyonaswepickedsome low-hangingfruits.Perhaps,withmostcohortstudiesmoregenerally,investigatorswillhave,probably,onlyafewbighitsandmanyworthyalbum tracks(havealookatnumbersofcitationsonGoogleScholar).Slightlyto argueagainstthat,isthatlongitudinalcohortsgainvaluefromhaving morewavesand,therefore,somelargerfindingscanonlyappearafter severalwavesoftesting,notearlyon.Inwhatfollowsweshallprovide anas-pithy-as-we-can-managestatementofsomeofwhatwefound,followedbyabitofexplanationandcontext,andrelevantreferences.There aremanyhundredsofpeer-reviewedarticlesthatanalyzeLBCs’dataand weshallcite,intotal,asmallminorityofthem.

HigherIntelligenceTestScoresatAge11areRelatedtoaBetterChance ofSurvivaltoOlderAge,andtoLowerRiskofDeathFromManyMajor CausesofMortality

These—theassociationsbetweenhigherchildhood(sometimesyoung adulthood)intelligencetestscoresandlongerlifeandbetterhealth— havebeenwidelyreplicated,includinginverylargestudies(somehaving sixorsevenfiguresamplesizes).ThediscoveryfromtheSMSs,thathigher intelligenceinchildhoodisassociatedwithlivinglonger(11, 12),properly beganthefieldofcognitiveepidemiologywhichaimstoreplicate,extend, andexplainthissetoffindings(13). Figure2 showstheresultsoflinking

theMorayHouseTestscoresatage11fromtheScottishMentalSurvey 1947tomajorcausesofdeathseveraldecadeslater.Thisnewfieldtook wepsychometrically-orientedpsychologistsintothestatisticalanalysis worldandtoolsofepidemiologists.Theassociationbetweenchildhood cognitivetestscoresandsurvivalwasanalyzedmostlyusingCoxproportionalhazardregressionandtheresultsexpressedashazardratios.To giveaguidetothesizeofthetypicaleffects,aone-standarddeviation advantageinMorayHouseTestscoreatage11wasassociatedonaverage withabout20%to25%lowerchanceofdyingfrommostmajorcausesof deathuptothelate70s(12).Partofthisworkhasbeenthepicking-apart ofthecontributions(confounders?,mediators?)ofeducationandsocial class(whicharecorrelatedwithintelligencetestscoresandarethemselvesrelatedtohealthandmortalityinequalities),andtheemployingof moleculargenetictechniques.Wehavereviewedthisfieldand,briefly,it appearsthatchildhood(parental)socialclassdoesnotcontributemuchif atalltotheintelligence-longevity/healthassociation,butthattheassociationmightbemediatedsomewhatbyaperson’sownadultsocialclass (14).Withregardtoeducation(or,e.g.,healthliteracy)thisishardtocall, notleastbecauseintelligenceandeducationandhealthliteracyarequite stronglycorrelated(15).

AboutHalftheVarianceinIntelligenceTestScoresinOlderAgeis theSameasThatFoundatAge11 NotlongafterwediscoveredthattheSMSs’datawereextant,weknew thatitwouldbevaluableandunusualtobeabletofindouthowstrongly childhoodintelligencetestscorescorrelatedwiththesametesttaken inolderage.Thisprovidestwousefulpiecesofinformation:theobverse isthestabilityofintelligencedifferencesacrossmostofthehumanlife course(testedusingthePearson[usually]correlationbetweenthetest scoreatage11versusthescoreonthesametestinolderage);andthe reverseofthatcoinisthatitcantellusaboutthechangeswithrespectto individualdifferencesoverthatsameperiod.Fortheformer,we’vepublishedseveralpapersthatdescribethecorrelationbetweentheMoray HouseTestNo.12atage11andolder-ageagesinthe60s,70s,80s,and 90s(4, 16–18).Thebroadresultisthateventherawcorrelationfromage 11tothe70sisnotfarfrom0.7which,whensquared,tellsusthatjust underhalfofthevarianceinintelligenceinolderagewasthereatage11. Thisisalower-boundestimateofthelong-termstabilityofintelligence differences.Itisnotcorrectedformeasurementerrororfortherestriction ofrangeinthesesamplescomparedwiththeirbackgroundpopulations (whichareknownbecauseofthecomprehensivenessoftheScottishMentalSurveys)(18).Forthelatter(i.e.,theremaining ∼50%notexplainedby earlylifedifferences,someofwhichwillbemeasurementerror,ofcourse), understandingwhatsortsoffactors(betheygenetic,health,behavioral,

Figure2. AssociationbetweenMorayHouseTestNo.12scoreatage11andmajorcausesofdeathuptoage79intheScottishMentalSurvey1947.Forvisualization,theMorayHouseTestscoresweredividedintodecilesorquarters.Thepointsinthefiguresareage-andsex-adjustedhazardratiosand95%confidence intervals;thelowestscoringgroupissetto1.0.Theanalyticsample N was67,765ofwhom25,979haddied.Meantimetofollowupwas57(SD = 18)years.This isFigure2fromCalvinetal.(2017)intheBritishMedicalJournal,357,j2708;thisarticleisanopenaccessarticledistributedunderthetermsoftheCreative CommonsCCBY4.0licenseandthefigureisreproducedhere,withthanks,underthatlicense.

social,etc.,thoughsomewillbestochastic/randomerror)perturbpeoplefromtheirchildhoodrankinghasbeenthebasisofmanyoftheearlier LBCdiscoveries.Anditturnsoutthatthosewhoareperturbedlessfrom their11–70scorerankingarealsothosethattend—abit—toalsoageless steeplyintomucholderage(19).In Figure3,weshowthescattergramsof theMorayHouseTestNo.12scoresfortheLothianBirthCohortsatage11 andage79.Wehavepreviouslypublishedaversionofthisscattergramfor theLBC1921butnotLBC1936andwehavenotpublishedthemtogether before.Wenotethat,whereasage79wasthesecondtestingoccasionfor theLBC1921,itwasthefourthtestingoccasionfortheLBC1936whoalso tookthetestatages70and76.

TheGeneticInfluencesonIntelligenceDifferencesarenotAlltheSame inChildhoodandOlderAge

Mostoftheindividualgeneticcontributionstointelligencedifferences aretiny(reallytiny,liketootinytoworkon).Don’tdocandidategenestud-

ies(apartfrom APOE).So,threelessonsthere.Thefirstlessonwasbased onanearlyfindingwiththeLBC1921inwhichwefoundthatpossession ofthe APOE e4allele(assessedbytestingforthetwosingle-nucleotide polymorphisms[SNP]thatdetermine APOE e4status)wasnotassociated withMorayHouseTestNo.12scoreatage11,butwasassociatedsignificantlywiththesametesttakenbythesamepeopleatage79(onaverage,thosewiththee4allelescoredlower)(20).Thesecondlessonbecame obviousasweconductedgenome-wideassociationstudies(GWAS)which grewinsamplesizesfromfourtofivetosixfigures.InGWAS,oneexaminestheassociationbetweentheoutcome(inthiscasethecognitivetest scores)andhundredsofthousandsofSNPsthatcapturegeneticvariationinhumans(seeRef. 21)foradescriptionofthisandothergenetic methods).TheLBCsandABCsformedthemajorityoftheparticipants originally(22)andstillcontributedtothelargerconsortiastudies(23, 24).Onethingthatdidnotchangehugelyasthestudiesgrewinsizewas theestimatedheritabilityofintelligencedifferencesbasedonSNPs—it

Figure3. Stabilityofindividualdifferencesinintelligencefromchildhoodtooldage.AssociationsbetweenMorayHouseTestNumber12scorestakenatage 11andage79intheLothianBirthCohortof1921(left; N = 483)andLothianBirthCohort1936(right; N = 468).Pearson’s r aredisplayedinthetopleftof bothpanels(p < 2.2 × 10 16 ).Scoreshavebeencorrectedforageindaysattesting.Outliers ±3.5SDswereremovedfromthepairwisecorrelationsbasedon fullavailablesamples(N = 3forLBC1921, N = 7forLBC1936)forvisualizationpurposes.Correlationswithoutliersincludedare: r = 0.66(LBC1921, N = 486; p < 2.2 × 10 16 )and r = 0.61(LBC1936 N = 475; p < 2.2 × 10 16 ).

remainedatabouthalf,orabitless,ofthatestimatedfromtwinstudies. Thatheritability,todate,ismadeupofatleasthundredsoftinyindividual associationsbetweenSNPsandintelligencetestscores.Wesummarized thisfield,withconsiderationofwhatthismeansforunderstandingthe biologicalmechanismsthatfoundpartofintelligencedifferences(21). ThethirdlessonwaslearnedfromourearlyexperiencewiththeLBCsand ABCsandtheworkofotherswhichconcludedthat,apartfromvariationin APOE (25, 26),associationsbetweenvariationincandidategenesandintelligencetestscoresinmodestly-sizedstudieshavenotreplicated.With largeerrorsaroundthepointestimates,weestimatedthatgeneticfactorsaccountedforabouttwo-thirdsofthestabilityinintelligencefrom childhoodtoolderagebutaboutonlyaquarterofthechangesinintelligencerankingsacrossthesameperiodofthelifecourse(27).

SomeoftheExpected“Exposures”(IndependentVariables)toLater-life CognitiveAbilityTurnOuttobe“Outcomes”(DependentVariables)of Early-lifeCognitiveAbility(ReverseCausationorConfounding) WhenwesetuptheLBCswewantedtoincludeaswidearangeofpotentialcontributorstopeople’sdifferencesincognitiveageingaswasfeasible/tolerable.Intestingthecognitiveoutcomes,weselectedabroadbatteryofcognitiveteststocoverthemaindomainsofcognitivefunction;in assessingtheexposures,wetriedtobeinclusiveaswaspracticableand includedgenetic,health,fitness,sensory,biomarker,brainimaging,psychological,demographic,andsocialvariables(6).Webegantofindthat someoftheselatter,supposedlyexposure/independent-variablefactors, althoughtheydidassociatewithcognitivefunctioninolderage,alsocorrelatedwithintelligencetestedatage11,manydecadespreviously.Thus dissolvedthesometimes-falseseparationwehadmadebetweenourcognitiveageingandcognitiveepidemiologyinvestigations.Amongtheputativevariablesthatwereinvolvedinourrealizingthiswere,forexample, C-reactiveprotein(28),physicalfitness,TypicalIntellectualEngagement, socialandotheractivities(29),alcoholintake,tendencytotype2diabetes (30)andallostaticload;therewereothers;someofthesearelistedand discussedbyref.

8.Tospellthisout,wefoundthatchildrenwithahigher intelligencetestscoreatage11tendedtobefitter,healthierandmore sociallyandintellectuallyengagedinolderage,andtodrinkaweebit(not alot—nottoexcess)ofalcohol;thatis,sometimes,butnotalways,the associationbetweenthegivenfactorandage-11intelligencetestcould

reducetheassociationbetweenthefactorandolder-ageintelligenceto nonsignificance.Thuswediscovered“reversecausation”/confoundingby early-lifeintelligencetestscore.Thisdoesnotnecessarilyruleoutthe causalnatureofagivenfactorwhoseassociationwithlaterlifefunctioningisattenuated,sinceitcouldalsobethatpeople’sdifferentialexposurestocognitive-ageing-inducingfactorscanbepredicted,atleastin part,byearlierlifefactors.Whatitdoesdo,though,iscastthosefactors whicharenotattenuatedbyage-11intelligenceintomuchsharperfocus asfactorsofinterest.Thelife-coursetimingoffactorsthatmightormight notinfluencepeople’sdifferencesincognitiveaging—includingcontributionsmadebytheLBCs—isdiscussedbyothersalso(31).

PeopleHaveVeryDifferentExperiencesofBrainandCognitiveAgeing Whenanalyzingthethingsthatmightexplaindifferencesinbrainand cognitiveageing,oneneedstohavevariabilityinthoseoutcomes-ofinterest.However,quitehowmuchofadifferencethereisbetweenpeoplehasbeenoneofthestrikingfindingsofthework.TheLBCscanoffera valuablewindowintothisbecauseallparticipantsarethesameage.The brainscansthatweretakenduringthesecondwaveofLBC1936testing areastarkillustrationofjusthowvariablesame-agepeople’sbrainsare intermsofkeyfeaturesofbiologicalageing. Figure4 showsaselection of73-year-oldLBC1936brainMRIs(over700werebrain-scannedatthis age),showingatrophy(wherethebrainshrinksawayfromtheintracranialvaultandalsothecerebrospinalfluid-filledventriclesatthecenterof thebrainenlargetoreplacespacevacatedbythediminishingcerebraltissue;PanelA)andwhitematterhyperintensities(ageing-relateddamage tothebrain’sconnectingfibers;PanelB).Theyarebothorderedfromtopleft(leastaffected)tobottomright(mostaffected).Weandothershave indicatedthattheseandotherimportantaspectsofbrainstructureare importantforcognitiveageingdifferences(seebelow).Wehaveofcourse alsoshownthiswidevariabilityintheageingexperienceelsewhere,with statisticalfiguresandanalysesforbothbrainandcognitiveageing,and fortheirsubsequentchangesintoolderage(whichalsoshowwidevariability)(32, 33).Nevertheless,thisfigureremainsoneofthemostengagingwaystocommunicatetootherssomeofourcentralresearchaims;how canonearriveatolderagewithabrainthatlookslikethoseinthetopleft, andwhatcanonedotoavoidhavingonethatlookslikethosetowardthe bottomright?Andhowcanwemaintainthatforaslongaspossibleaswe

Figure4. Brainstructural(MRI)scansfromaselectionofindividualsfromtheLothianBirthCohort1936takenduringWave2(whenallparticipantswereabout 73yearsold). PanelA showsglobalatrophy(brainvolumetricshrinkage)orderedfromleast(topleft)tomost(bottomright). PanelB showstotalwhitematter hyperintensityvolume(increasingfromtoplefttobottomright).PanelsAandBarereproducedfromCoxandDeary(2022)inBrainAging,2,100032(74);this articleisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsCCBY4.0licenseandthefigureisreproducedhere,withthanks,underthat license. PanelC showswhitematterpathwaysofamiddle-agedmaleadult,identifiedusingdiffusionMRI.Viewsfromlefttoright:superior,lateral,anterior, inferior.

continuetoage?Italsooffersarayofhopetothoseofusonthejourney toour70s(whereIJDhasjustarrived)thatadversebrainandcognitive ageingoutcomesarenotaninevitability.

BrainSizeReallyis(Modestly)RelatedtoIntelligence.WhetherMore IntelligentPeopleTendtoHaveaLargerBrainwasaDebatedIssue OverManyYears ThenadirofrespectabilityforthisquestionmighthavebeenwithStephen J.Gould’sbook TheMismeasureofMan.ThearrivalofMRItoassessbrain sizesettledtheissue.Earlymeta-analyses,anLBC1936studythatwas thelargeststudyatthetimeitwaspublished(34),anddatafromtheUK Biobankstudy(35)agreethattheassociation(correlation)betweengeneralcognitiveabilityandtotalbrainvolumeasassessedinMRIisabout 0.27.Wecautionthat,inolder-agesamplessuchastheLBC1936andUK Biobank,theremightbesourcesofvariationintotalbrainvolumethatare associatedwithintelligencetestscoresthatarenotpresentorasmarked inyounger-agesamples.Therefore,itisimportanttostudytheassociationatdifferentages.Whydoesonedothiswork?:becauseit’sthebrain thatthinksandwewanttoknowhowvariationsinitsbiologicalparametersassociatewiththinkingskillsthroughthelifecourse.Werecognize (seefollowingsections)theimportanceinunderstandingwhatitisabout alargerbrainthatmakes,onaverage,formoreefficientthinking.But,of course,thereismuchmoretothinkingskillsthanjusthavingalargebrain, includingotherbrainvariables(34, 35),andwecoversomeofthatinthe followingsection.

BrainWhiteMatterMattersforIntelligence

Aswejustsaid,thereismuchmoretothinkingskillsthanjusthavinga largebrain.AroundthetimethattheLBC1936began,therewasincreasingrealizationof,andinterestin,measurementsofthebrain’swhitematterandtheirimportanceforstudyingageing.Ourteam’sdecisiontomeasureparticipants’whitemattermicrostructureusingnewdiffusionMRI (see Figure4,PanelC)wasinresponsetothis,andourintentiontoaddressthiswaswritlargeinourapplicationtothecharityAgeUKforfundingas“TheDisconnectedMindproject”(10).WiththeLBCs’brainimagingdata,wediscoveredthatthehealthofthebrain’sconnections—the whitematter—inthemainbraintractswereallpositivelycorrelated,that is,healthybrainwhitematterinonetractwasstronglyrelatedtohaving healthywhitematterelsewhereinthebrain(36).Wesubsequentlyalso replicatedthisimportantfindinginotherhealthyadultsampleswitha wideragerangesuchasUKBiobank(37),andalsofounditinneonatesand amongpsychiatricpatientswithschizophrenia(38, 39).Moreover,having computedageneralcomponentofthiswhitematterhealth(usingprincipalcomponentsanalysis),wefoundthatpeople’sdifferencesinbrain whitematterhealthweremodestlyassociatedwithcognitivefunctioning(40).Thereafter,wefoundthatthesetwovariableschangetogether inasynchronizedfashionovertime:onaverage,thosewithsteeperageingoftheirbrainwhitematterpathwaysarethosewhosegeneralcognitivefunctioningdeclinesmoresteeply(41).Thisisanotherresultthathas beenreplicatedelsewhere.Moreover,theso-calledwhitematterlesions

thataccumulateinsomepeoplemorethanothersastheyagearealso relatedtointelligencedifferencesand…

BrainGreyMatterMattersforIntelligence,Too(andCarefullyPutting ManyBrainImagingMeasuresTogetherisAdvantageous)

Thereareotherbrainvariables—includinggreymatterparametersfor thecortexandsubcortex,andaspectsofbrainvascularhealth—toconsiderwithrespecttointelligence/cognitiveageingassociations(34).MRIderivedvariablesdon’tstandstill;wehaveexpandedthesetoincludedetailedpropertiesofthecortex,brainconnectomics,“brainage,”andother aspectsofthehealthofthebrain’swhitematter(33, 42–44).Onelesson fromthisaccumulationofbrainimaging–derivedvariablesisthatsome arestronglycorrelatedwithothersuchvariablesandthatoneneedsto ascertaintheindependenceofbrainimagingvariablesfromeachother whenfiringthematintelligencedifferences.Thisavoidsold-wine-innew-bottlesscenarios,buthasalsoallowedusto:i)identifymorepreciselyhowfareveryoneexperiencesthesameaspectsofbrainageing;and ii)maptheextenttowhichinformationgleanedfromthesemanyaspects ofbraingreyandwhitematterarealluniquelyrelevantfordifferencesin cognitiveageing.Wehavelearnedthattheyoftenaccountforsomesmall uniqueproportionofcognitivedifferencesinolderage.Thatis,whereas someclassesofbrainfeaturesarepartlyoverlapping,havinglotsofinformationaboutdifferentfacets,regions,andtissueshelpsustoimprove ourunderstandingofdifferencesincognitiveageing(44, 45).

ThegenerosityoftheLBC1936participantsinprovidingtheirbrain tissueaftertheydie(moreaboutthisimportantandstrikinglegacyin thesectionsbelow)hasalsoenabledustolookdeeperstillintothehallmarksofbetterandpoorercognitiveandbrainageing,identifyingfeatureslikesynapticresilienceandneurograninasimportantaspects(46, 47).WearealsousingnewmethodstoputLBCdatatogetherwithlargescalepostmortemdatafrommanyothersourcestolearnmoreabout theregionsofthebrainthataremostimportantforcognitiveandcognitiveageingdifferences,suchasgeneexpressionpatternsacrossthe cerebralcortex(45).Weareoptimisticabouttheopportunitiesthatthese approachespromise,andwegratefullyrecognizethattheycan’thappen withoutthesadaggregationofmunificentbraindonationsbymembers oftheLBC1936.

IntelligenceisFarFromAllThatMatters(atAnyandEvery AgeasaHuman)

Itmightbepossible—giventhestatementofLBCs’aimsgivenabove—to imaginetheLBCsashaving,asoutcome(dependentvariable),abullseye labelledcognitivefunctioningand,asexposures(independentvariables), hundredsofarrows(genetic,health,lifestyle,biomarker,psychosocial, etc.)firedtowardsit.Thatwouldbewrong.Fromthebeginningofthe LBCs,thenoncognitivevariablesweincludedsometimesbecameoutcomesadditionaltothecognitiveones.Webecameinterestedinhealth, fitness,personality,mood,lifesatisfaction,socialposition,socialengagementetc.aspartofhealthyageingandstudiedtheassociationsofthese outcomestoo(48–50).

TheAgeofPeople’sDNA(byComparisonwithTheirChronologicalAge) Predicts(toaWeeExtent)HowLongPeopleWillLive ThisprovidesausefullessonregardingtheLBCs’expansionwithrespect tobothexposuresandoutcomes.Bythisstage,wehadalreadygenomewidescannedtheLBCs’DNAsamplesforSNPs.Butone’sDNAnucleotide sequenceisnotthewholestorywithrespecttohowtheDNAworks(i.e., eventuallyleadstoproteinproduction).AlongDNAstrandsthereare, attached,methyl(CH3)groupswhichhaveeffectsongeneexpression; theseareoneformofepigenetic(inthiscaseDNAmethylation:DNAm) marks.Peopleshowdifferencesinthesemarkswhichare,inpart,due togeneticdifferences((51))and,inpart,toenvironmentalcauses(e.g., smoking(52)).Weundertookmethylome-widescanningintheLBCs.We thoughtthatindividualdifferencesinDNAmethylationmightbeinformativeaboutcognitivedifferencesandage-relatedcognitivechanges,which theyweretoanextent(53).MethylationmarksonDNAchangewithage, andwewereawareoftheconceptofepigeneticage,thatis,thatsome people’sDNAhadmethylationpatternsthatlookedolderoryoungerthan istypicalfortheirchronologicalage.Wefoundthatyoungermethylation

ageatbaseline(age70fortheLBC1936andage79fortheLBC1921)was associatedwithhowlongpeoplelived.Thisreplicatedinothersamples. Thiswasoutsideofourfieldbutisoneofourcitationhits(51).Pursuant tosomeoftheScientificStrategypointslistedbelow,DNAmresearch hasbeenasuccessfulandinterestingcollaboration.Itemergedthathow methylatedyourgenesarecorrelatedquitestrongly(accordingtoFunder andOzer(54)ratherthanCohen[seethesubsectiondirectlybelow])with smoking,BMI,andinflammation,tonameafew,andthattheseinturnare alsorelatedtobrainandcognitivedifferences(53, 55–57).

GetReadytoEnjoySmallEffectSizes,MovingFromthePsychologists’ Crud(Meehl,1990(58))ValueofAbout0.3DowntotheEpidemiologists’ 0.1andBelow

WeandotherssawanddiscoveredintheLBCsforourselvesearlyonthat effectsizesincognitiveageingaretypicallysmall;afitnessvariable,or possessionofthe APOE e4allele,orsmokingorjustpickyourfavoritecandidatevariablethatcontributestoindividualdifferencesincognitiveageingandaboutthebestyoucanexpectfromanyoftheseisthat,netofcognitivecapabilityinyouth,theywill,ifyouarelucky,contributeabout1%of thevariancetocognitivecapabilityinolderage.Wesummarizedthisrealityinourpaperentitled Marginalgainsnotmagicbullet (8).Mindyou, andthisseemstooobvioustohavetowrite(butweseepapersandstatementsthatrefutethat),itisimportanttokeepinmindthat,incognitive ageing,whatoneisseekingisfactorsthatareassociatedwith change in cognitivecapability,whetherthatchangeisfromyouthtomiddletoolder age,orjustchangewithinolderageitselfwhenmoredeclinetakesplace. Tounderlineevenmore,anassociationbetweenaputative“cognitiveageing”factorandacognitivetestscoreassessedononeoccasion—whether itiscross-sectionalorwhethertheputativepredictorwasassessedsome timepreviously—isnotinformativeaboutcognitiveageing.Thereisless varianceincognitivechangethanthereisincognitivestatus,andcognitivechangeisnoisier,andsuchchangesareaccordinglyhardertoaccount forwithpredictors.Here’sanexample.Ina12-cohortsconsortiumthat includedLBC1936,therewasasignificantcross-sectionalassociationbetweentelomerelengthandvariouscognitivetestscores(59).However, inastudythatincludedonlytheLBC1936,therewasnoassociationbetweenchangeintelomerelengthandchangeincognitivetestscores(or withchangeinphysicalabilities)acrossthreewavesoftestingatages70, 73,and76(60).Itisalsoworthnotingthatmeasuredchangeisalsorarer (sinceitisharderandmorecostlytomeasure/fund),whichalsodetracts fromtherelativepoweroflongitudinalstudiesonwithin-persondifferencesascomparedtocross-sectionalstudiesofbetween-persondifferences(asillustratedbythe12:1ratiointhetelomereexampleabove).

Beyondgenetics—thegiven—whatcananindividualdoiftheywantto agewell,includingcognitively?:playthenumbers;maybebygettingoneselfontherightsideofthemany(manyofwhicharenotconfirmed)possiblecognitiveageingvariablesonemightbeleaningintherightdirection towardhealthierageing.Somehaveexplainedthatsmallassociations, thoughtheycanmeanalotforlargepopulations(e.g.,bloodpressure controlfortheavoidanceofstroke)arenotpracticallyinformative—when consideredinisolation—forindividuals(61);however,wewouldargue againthatstayingonthecorrect/sunnysideofthemanysmallputative effectsisthebestchoiceforimprovingone’shealthycognitiveandbrain ageing(andgeneralhealth)odds.Withregardtothepointswemadein thissection,andelsewhere,Walhovdetal.(31)—sometimescitingLBCs’ results—madeastrongcasefor,“sobrietyregardingthetimingandquantity”ofinfluencesonbrainandcognitiveageing;weagree—wehavetried tostaysobertoo,andweencourageotherstodoso.

MultivariateAnalysesofCognitiveAgeingareMoreBracingThan UnivariateAnalyses

Yes,itgetsworse.Justaswediscussedforthevarietyofbrainimaging variables(youhavetoaskwhateachbrainmeasureistellingyouabout cognitivedifferencesthatisunique),onehastoaskthesameaboutthe otherlifestyle,health,genetic,andothercandidatepredictorsofcognitiveageing;thatis,dotheysurvivewhenenteredtogether?Therearefew reliableassociationswithcognitivechanges(usuallydeclines,onaverage)inolderage.Andtheireffectsizesaresmall.Itisnotunusualforresearchreportstoinclude(inadditiontosomesensible,basiccovariates)

asinglepredictorofcognitiveageing.Indeed,wehavehadexperiencesof findingsuchreportseasiertopublishthanwhenwehaveincludedmultiplepredictors/exposures.Butlifeisnotlikethat;wedon’texperienceinfluencesonourageinginisolationfromeachother.Wehaveconducted, withtheLBC1936’slongitudinaldata,twostudiesinwhichwethrewin acoupleofhandfulsofpopular(fromthescientificliterature)determinantsofcognitiveageing(32, 62).First,welookedatthemaspredictors ofcognitivechangeoneatatime.Then,wepoppedthemtogetherina multivariateanalysis.Whathappened?:positivefindingsfelllikesnowoff adyke(aswesayinScotlandaboutsuchephemera)asmostofthesignificantunivariatefindingsleftpossessionof APOE e4andtheoccasional othervariablelookingratherlonelyintheircontinuedsignificance.Thatis nottosaythattheuniquecontributionsofthosemanyfactorsmightn’tbe additivelyimportant(weexaminethisinthispaper—(62)),butthattheir uniqueeffectsarelikelyevensmaller;accuratelyquantifyinghowmuch smallerwillrequireevenbiggersamplesandconsortiaeffort(towhich wearecontributing)withcomparablydeepphenotyping.

YouWillBetonSomeDuds,ButNull(-ish)ResultsareValuableToo WiththeLBCswehavetriedtoscanthehorizonforpossiblecontributors tocognitiveageingdifferencesfrommanyfieldsofstudy.Amongthese, wehavekeptaneyeonbiomarkersofageingbecausethatseemedlikea likelysourceoftractablecontributors.Lookingback,onecouldsaythat earlierworkwasconductedinthetimeofcandidatebiomarkersandthat wearenowinatimewhenmulti‘omicsplatformsprovidethecapability toexaminehundredsandeventhousandsofproteins/peptides,lipids,glycans,othermetabolitesetc.Andthesewillbeusedinhypothesis-free(ish) studiesandwillprobablydeliversomereplicableandprobablysmalleffects.Butthepointhereisthatwesoughtexpertcollaboratorsinlikely biologicalvariablesrelatedtocognitiveageing,foundtheresourcesto assessthemandthensometimesfoundnotverymuchwhenitcameto lookingattheresults.Thisappliesto,forexample,retinalvesseltopography(63),andseetelomerelength,above.Itshouldquicklybesaid,by wayofbeingpositive,thatthesevariablesprovedusefulinotherstudies withothervariablesandthatnullresults—knowingwhatisprobablynot associatedwithdifferencesincognitiveageing—provideknowledgetoo. Oneshouldnothaveanemotionalreactiontoascientificresult,butwe confesstomildpleasureatfindinganullassociationbetweenchildhood intelligenceandlifesatisfactioninoldage(64).

ItHelpstoHavea“theory”butTheoryDoesnotAlwaysHelpScientific ProgressinThisField

Wehaveputtheory,there,insneerquotesforthereasonsthatoneofus hasalreadywrittenatlengthregardingtheassessmentofthequalityof theoriesinthepsychologyofcognitivecapabilityandmostofthatcritique applieshere(65).Thislessonwaslearnedfromsomerefereesandeditors whohavefromtimetotimeenjoyedourmanuscriptsbuthavewanted forsome“moretheory.”Andsometimesanonharmfulsprinklingofthat condimentwillsuffice;wedonotwishtoappearcynical,butithelpsto haveatheorytobepublished,althoughtheoriesinourfieldareoften skyhooksratherthancranes(66).Someoftheso-calledtheoriesthatcirculateinthefieldofcognitiveageingincludebrain/cognitivereserve(67, 68),brainmaintenance(whereothershavesimilarreservationstoours aboutwhetherthesetwoaforementioned“theories”constituteexplanationsornot(69)),commoncause(70),andprocessingspeed(71).The lattertwoarecircumscriptionsofinterestingempiricalregularitiesand thefirsttwovarybetweenausefultrellisonwhichtohangcognitiveageingstudiestoadiversionarysoupstone(72).Theonesuggestionfromour teamthatothershavetakentobeatheoreticalarticulationwasthenotionof“systemintegrity”whichwaspositedinourfirstcognitiveepidemiologystudyusingdatafromtheSMS1932(11).Wetooktheopportunity thereaftertoclarifywhatitmightmeanandmightnot,andwhatitsweaknessesandpredictionswere.Ifitdeservesanameitisprobablyhypothesisratherthantheory(73);isitatrellisorasoupstone?—neitherperhaps, beingmorelikeastickynotetoremindustoexplorethispossibility(both theoreticallyandempirically)abitmore.Inthefieldofcognitiveandbrain ageingwewouldpreferavery-large-Ndatasetwithwell-measured,relevantvariablesratherthananapparently“well-aimed”so-called“theory” (cf.GWASversuscandidategenestudies).Wetrustthatthisshortmen-

tionoftheoryisnottooglib,andwerefertheinterestedreadertoour longerdiscussionsoftheory(8, 65, 74, 75)andtoahandbookthathasa sectionon“modelsofcognitiveaging”(76).

IrrespectiveoftheLargeAmountofDatayoudoHave,PeopleWill (Rightly)AskAbouttheDatayoudon’tHave

Runningalongitudinalstudyofolderadultsinevitablyresultsinthesad truthofdropoutandmissingdata.SincethebaselineofbothLBCstudies, attritionistypicallyabout20%perevery3-yearcyclebetweenwavesof assessment.Abouthalfoftheattritionisduetomortality,withtheremainderbeing—anecdotally—amixtureofpeoplenotwishingtocome backbecausetheyhave“doneenough,”becauseofthedevelopmentof illnesses,orbeingunavailableduetocaringdutiesforgrandchildrenor— increasingly—foraspouse/significantother.Understandinghowandwhy participants“dropout”ofthestudyisimportant.Ithasimportantstatisticalimplicationsforourcoreaimofcharacterizingcognitiveandbrain ageing,andaskingwhatcorrelateswithdifferencesinthosetrajectories. Weknowthat,onaverage,peoplewhodropoutarelikelydoinglesswell intermsoftheirbrainandcognitiveageing,andgeneralhealth,than thosewhokeepcomingback(“completers”;e.g.,ref. 62).Wealso,therefore,knowthat,whenweplottheaveragechangesinjustcompleters, wemostlyunderestimatetheamountofcognitivedeclineinoursample(e.g.,ref. 77).Toensurethatwedon’tbiasourestimatesagainstthe leasthealthyparticipants(whoarejustasimportantandinformative), wewilloftenusefullinformationmaximumlikelihood(FIML)toinclude allavailabledatatoestimatethosedeclines.However,reviewersoften askwhetherwearedoingthecorrectthinghere,sinceFIMLassumesthat thepatternsofmissingnessareeitherrandomormostlyaccountedforby variablesincludedinourmodels.Whereasweareunabletoaccountcompletelyforthepatternsofdropoutweobserve(e.g.,refs. 32, 78),wehave previouslyindicatedthatthefurtherreductioninvariance/greaterrange restrictioninanalreadyself-selectingsamplewouldlikelyyieldaslight underestimationofeffectsizes(e.g.,ref. 79)aswellassubstantiallylower statisticalpower.

ScientificStrategyLessons

SomeofthelessonsthatwelearnedfromtheLBCspertainmoretohow togoabouttheprocessofscientificenquiryratherthanresultsfromanalyzingthedata.

“MaximumStrategicIntransigence,MaximalTacticalFlexibility”(with ThankstoS.Reicher)

Ouroriginalstatedaimwastoinvestigatenonpathologicalcognitiveageing.We’vestucktothat.Notwithstandingthatcontinuedfocus,itsoonbecameclearthatwehadusefuldatawithregardtootheraspectsofhealthy ageingandweinvestigatedthose—thoughneverasamainstreamofthe work.Also,astheparticipantsgrewolder,someofthemdevelopeddementia,andwebegantoascertainthatandtousetheinformationsometimesasanexclusioncriterionandsometimesasanoutcome(80).

SometimesOneFindsSomethingThatisTooGoodnottoDevelop Indeed,that’swhathappenedwhenwediscoveredthattheSMSs’data wereextant.BothLawrenceWhalleyandIanDearywerebusydoingresearchbuttheopportunitiesseemedtooimportantnottodevelop,that is,thepossibilitytostudylifetimecognitiveageingwithachildhoodbaselinecognitivetest,andthechancetoconductlinkagestudiesandfind outwhetherchildhoodcognitiveabilitywasrelatedtosurvival(and,ifso, why).Changeofstrategy?AndthegoodluckoffindingtheSMSs’datawas followedbythediscoveryofotherdataandthedecisionsaboutwhether timespentinpursuingthosewasworthwhile.Forexample,wediscoveredbirthrecords(includingbirthweight)ofsomeofthepeopleborn inEdinburghin1921(81).AndwefoundoutthatsomeoftheSMS1947 participantshadhadmoreinformationcollectedfromthematage11 andsomeintotheir20s.Wefollowedupbothofthesewithadd-onstudiesof,forexample,cognitiveageing,cognitiveepidemiology,personality, andlife-longwellbeing(82–85).AndwealsofoundoutthattheScottish “Midspan”studieshadhadmanypeoplebornin1921andweobtained permissiontolinkthemtotheScottishMentalSurvey1932.Thisresulted inseveralcontributionstoourcognitiveepidemiologywork(86–90)and toourworkonsocialmobility(91).

OnlySetUpaCohortifithasSomethingThatitCandoThat OthersCannot

Leadingacohorttakesoverone’slife.TheLBCsneverhadguaranteed fundingbeyonda3-or5-yeargrant-fundingperiod.Yet,theyhavebeen fundedcontinuouslysinceJanuary1999.ThismeansthattheDirectorand co-investigatorsgohomeeacheveningwiththeresponsibilitytoretain thecohortandtheresearchteam.Therefore,especiallywiththeeaseof accessingandanalyzingsecondarydatafromexistingcohorts(withUK Biobankprovidingacurrentapogeeforsomeinvestigations),onemust askwhyoneistakingthetroubletosetupacohort,whichwillthenput otherpeopletothetroubleoftakingpart.Thewhyisthatthecohort shouldbeabletoaddressanimportantsetofscientificquestionsina waythatisvaluable,thatis,bybeingtheonlysamplethatcanaddress thequestionsoratleastbyaddingusefullytowhatcanalreadybedone inothersamples.WhenwebegantheABCsandLBCs,weknewofnoother cohortsthatcouldadjustforsuchawell-validatedcognitivetestinyouth. Aboutthebestwewereawareofwasthecognitivesurrogatesemployed intheNunStudy,whichwefoundimpressive.

MaketheCohortaHubThatConcentratestheTeam’sScientificExpertise andThenAttachHigh-qualitySpokestoEnhancetheCohort’sScope TheLBCsbeganwithfourpeopleinvolvedinthehubthatdesigned,ran, andanalyzedtheLBC1921study:IanDeary(trainedinmedicineandpsychiatryandaPhDindifferentialpsychology),JohnStarr(geriatricphysician),MarthaWhiteman(PhDinpsychology),andAlisonPattie(nurse andresearchassistant).Thus,thehubhadexpertiseincognitivetesting, multivariatestatistics(includingstructuralequationmodelling),other aspectsofpsychologicaldifferences,gerontology,andgeriatrics.With time,thehub/coreteamenlarged—especiallywiththebeginningofthe LBC1936,atwhichtimeweaddedfull-timeindividualstolookafterthe growingdatabases.Evenearlyonthough,werealizedthatweneeded additionalexpertise,someofwhomcamefromourDepartmentofPsychology,someofwhomwerealsofromourUniversityofEdinburgh,and someofwhomwerefromotherUKandoverseasuniversities.Let’scall theseexpertsandtheirteamsspokestoourhubs.(Weshallseebelow thatsomespokesbecomepartofthehub[yes,there,themetaphorbreaks downabit]).Whatspokeexpertisedidweadd?:webroughtinexperts inmoleculargenetics,statisticalgenetics,brainimaging,ophthalmology, biomarkers,medicaldatabaselinkage,telomerebiology,neuropsychology,epidemiology,education,environmentalgeography,music,qualitativemethods,physicalactivity,stemcellbiology,postmortempathology, molecularneuroscience,psychiatry,hematology,epigenetics,immunology,transcriptomics,lipidomics,proteomics…Thereareprobablymore andmorewillcome,andweapologizetoanywhomwehaveforgotten tolist.Oh,andwe’vetendedtoworkwithverygoodexperts.Itwouldbe invidioustopickoutafew,sothereadercanspotthemasco-authorson ourarticles.Finally,workingwithsuperbexpertsalsomeansthatsome ofthatknow-howinanewfieldrubsoffonyou—notahugeamount andwewouldneverclaimtobeexpertsinournon-nativefields—but enoughthatyouareinabetterpositiontospotnewopportunitiestooccasionallycontributetonewdiscoveriesorperspectivesinunexpected fields.

ConsiderWhethertoAddanExpertisetotheCoreTeamortoOutsource ittoaSpoke LookingattheLBCs’hub/coreteamasitisnow,thatis,thoselocated inthesameplaceinPsychologyin7GeorgeSquareattheUniversityof Edinburgh,therearein-housegeneticistsandbrain-imagingexperts,for example,whowouldpreviouslyhavebeeninspokes.Earlieron,asolegeneticistorbrainimagerwouldnothavehadanenvironmentthatwould havenourishedtheirexpertise.Therefore,itwasaswewereabletoattractmoreofsuchexpertsthatwehadacommunitythatcouldhelpeach other.Now,with >1numbersofpsychologists,brainimagers,geneticists (allofwhomarealsoexpertinmultivariateanalyses)inoneplacetheycan notonlyhelptheircolleaguesinthesamefield,theycanconductcrossdisciplinarystudieseasilybecausetheyarecolocated.

ForEachProposedAdditionalVariable,AskWhetheritisofUse inThisCohort

Generally,whenwehavedecidedtoconductananalysisormeasureavariableintheLBCswehaveaskedourselvestheserelatedquestions:does theLBCmakeavaluablecontributiontothisliterature?;andcouldthisbe doneinanycohortorsample?Insummary,wehavetriedtoplaytothe strengthsoftheLBCs’informationwhichmeans,alotofthetime,having childhoodintelligencetestscoresinolderpeople.However,astheLBCs’ databasesgrow,othervaluableopportunitiesemerge.Forexample,havinglongitudinaldataonclonalhematopoiesisofindeterminatepotential(92, 93)andDNAmethylation,theirlinkagewithexistingLBCdata washighlyvaluableandhardtoreplicateelsewhere.So,themoredata thathavebeencollected,themoreopportunitiesthereareforpossiblyunique/at-least-valuablecollectionofstillmore(tosomeextent,and withinwhatispracticalandissometimesdrivenbyopportunityand/or serendipity)becausetherearesomanydataalreadycollectedtowhich theymightbetestedforassociation.Alsoworthsayingisthatthevalue incollectingnewdataduringthelaterwavesassamplesizessadlydwindlealsomeansthatpowerisaffectedandthereislessreasontoaddnew data;tocounterbalancethat,therearelotsofinnovativewaysinwhich onecancontinuetocollectnewdataonthefullsamplebycapitalizingon emergingmethodsandpossibilities—forexample,retrospectivegeocodingbasedonlinkedlifetimeaddressesinthefullsample(e.g.,ref. 94), medicalrecorddatalinkage,analyzingbloodsamplesstoredfromprior waves,andsoforth.Thus,peoplebecomemoreratherthanlessinterestingastheygrowolder.

CovertheBasesWhenTestingtheCohort

Fromourinterestincontributorstononpathologicalcognitiveageing,we knewthosevariablescouldcomefromawiderangeofdomains.Therefore,wehadtogatherawiderangeofdata.Ofcourse,welookedaround atothercohortsforguidance.Weneededdatafromcognitivefunctioning,otheraspectsofpsychology,socialanddemographicfactors,lifestyle andhealthbehaviors,demographics,biologicalandgeneticfactors,and medicalinformationandfitness.ThesecanbeseeninourLBCs’study protocolsandprofilearticles(4–7).Oneisinhibitedfromcollectingwhat seemsliketoomuch,wishingnottofatiguetheparticipantsordiscouragingthemfromreturning.Ourlessonwasthanolderpeoplecanandwilldo morethanyouthink.Initially,wewerecautiouswithLBC1921andmore detailedandwide-rangingwiththeLBC1936(whostartedwithuswhen theywereyounger).

EvenifTheyarenotPerfect,RetaintheSameVariablesinthe NextWaveofTesting

Inpsychologyandinmedicineandbeyond,thewaysofmeasuringthings donotstaystill.Onechooses,say,cognitiveandpersonalityandmood andfitnesstests(topluckoutafewfrommanytypesofdata)forthebaselinestudy.Sometimes,anewerandseeminglybettertestwillappearafter onehascollectedthedata.Whatshouldonedo?Holdyournerve:unless thereisabigproblemwiththeoriginaltest,collectthesamethingagain. Thereisvalueinlongitudinaldatawiththesamemeasures.Ofcourse, ifwehavehadtime,wehaveincludedthebettermeasureandtheolder measureatthenextwave,butwehavetendednottodropvariablesand wehavebeengladofthatwhenitcomestolongitudinalanalyses.

IfYouHaveOnly2MintoTestaPersoninaCognitiveAgeingStudy,do theWechslerDigitSymbolTest(Other,EquivalentTestsareAvailable) Scoresonthistestagebadly,thatis,itdeclinesmoresteeplythanother cognitivetestsanddomains,andsoitprovideswhatoneislookingforin acognitiveageingstudy.Individualdifferencesintheageingofitscognitivedomain—processingspeed—correlatestronglywiththeindividual differencesintheageingofothercognitivedomainssuchasreasoning andmemory.

AddtheNationalAdultReadingTestifyouhaveanothertwominutes (other,equivalenttestsareavailable).Thistestwillgiveyouadecentestimateofthepersons’peakpriorcognitiveability,evenwhentheyhave mildcognitiveimpairmentorearlydementia(82, 95).Toanextent,itwill

makeupfornothavingtheearlylifecognitivetestscoresthattheABCs andLBCshave,thoughittookthesestudiestoestablishthat.

Addgripstrengthifyouhaveanother2min.Itisahandy(punintended)indexoffitnessand,inlargesamples,ispredictiveormortality (tightergripperslivelonger)(96, 97).

DoWhatYouCanwithSufficientPoweronYourOwnandJoinaBigger GangWhenYouCan’t

Wehavedoneunder-poweredstudies.Manypeoplehave.Wetrynotto. WesetuptheLBC1936,especially,with N > 1000,tobepowerfulinphenotypicstudieslookingatdeterminantsofcognitiveageing.Withover 700oftheLBC1936havingbrainimagingdata,itwasoneofthelarger single-cohortstudiesofcognitiveageingwithsuchdataatthetime.For APOE geneticstudies,itwasadequateinpower.However,assoonas webegandoingGWAS,withoneexception(seebelow)weknewwehad toolittlepowertodoanythingthatwouldberobustandsowecollaborated.First,wehadaUK-basedgangthatincludedus,theABCsandthe Manchester-Newcastlestudies(22).Itsoonbecameclearthatthatwas toosmallandwejoinedtheCHARGENeurologyconsortium,whichhad acognitivegroup.ThattooktheNstofiveandthensixfiguresandonly thendiditseemthatreplicableresultswereappearing(24).Anotherexampleisthedebateaboutreplicablebrain-behaviorassociationsrequiringthousandsofsamples(98).WealsojoinedENIGMAandotherconsortiabecausewerecognizeasimilarsituationinthebrainimagingdomain, thoughofcoursethereremainthingsthatcanbedoneinLBCthataren’t easytofindappropriatedatasetsforreplication.Byadoptingmulticohort approachesinthebrain-imaginganalyses,weleadwehaveshownthat, actually,theLBC1936soloresultsdon’tstackuptoobadly(45).

HavingaCohortThatisSuccessfulatSomeThingsMakesOneallthe MoreAppreciativeofOtherCohortsThatCandoSomeThingsBetter Therearelotsofstudiesouttherewiththeirdifferentstrengths.Inthe fieldofcognitiveageing,wherewecouldpickoutmanygoodstudies,we haveastrong“we’renotworthy”responsetotheROSMAPstudieswhich haveanastonishingrangeofvariedandgoodvariables,awonderfulretentionrate,andaterrificsign-uprateforpostmortemdonation.Wehave asimilarresponsetoUKBiobank,whichiswhywehavespentsomuchtime analyzingtheirdataontopicsrelevanttotheLBCs.Thevisiontocollect suchalarge N (500,000)—withaveryboldaimtocollectwithbrainimagingdatafrom100,000ofthem—meansthattheUKBiobankdataisused world-wide.

FeelFreetoMoonlightwithOtherCohortstoTestHypothesesThatYou CareAbout Bearinginmindthatweareinterestedinvariationinbrainandcognitive variablesandtheirageing,wehavefelttheneedtobeunfaithfultothe LBCswhenwecananswerquestionsmorepowerfullyelsewhere.Thelist istoolongtonamethemall,butwehaveanalyzeddataandpublishedresultsfromUKBiobank,GenerationScotland,NLSY1979,ThethreeBritish birthcohorts(1946,1958,1970),theWestofScotlandTwenty-07Study, andothers.

YourSampleCanbeaControlSamplefortheIllnessesTheyDon’tHave MembersoftheLothianBirthCohorthavebeenproudtoappearas healthycontrolsinvariousmedicalstudies.Topickoutjusttwoexamples, theyhavebeencontrolsingeneticstudiesofcolorectalcancer(99)and motorneurondisease(100).

YouWillRegrettheThingsYouDidNotTest Onecan’tgobackandtestatbaselineagain.Onehastolivewiththe decisionsthatweremade.TheLBC1936willneverhavebaselinebrain imagingdataatage70,thoughtheydohavethosedataateverywave afterthat.So,thinkcarefullyaboutthatinitialtestingwave.Relatedto that,youwillbemoan,often,whatthecohortdoesnothaveandperhapsenvyothercohortsforhavingthosedata.Forexample,thereareno contemporaneously-collecteddataintheLBCsbetweenage11andolder age(thoughwemanagedtofind—vialinkagetotheScottishMidspan study—datafrommiddleageinsomeparticipantsoftheScottishMental Survey1922inourcognitiveepidemiologywork(86–90, 101)).Itwould

havebeenhelpfultohavemoreearly-andmid-adultvariablescollected intheLBCs.Wehavedoneourbesttofillthesegapsbyretrospective self-reportsandothertechniquessuchasgeo-linkagesforpasthomeaddresses.OnepracticalexamplefromLBC1936isthatwedidnotaskfor linkagetoparticipant’smedicalrecordsatthefirstwaveoftheirtesting atage70;wecorrectedthat,butwekickedourselvesforhavingomitted torequestthatfromparticipantsfromthebeginning.

ConsiderWhatSizeofCohortandTeamisOptimalforPurposes(toRetain Focus)andQualityofLife(ThoughtheCohortwillTakeOverYourLife) WehavekepttheLBCteamtoamoderatesize.IfonecountsthePIs,the teamthatrunsthestudy,theemployedandad-hominem/feminampostdoctoralfellowsandresearchassistants—thatis,mostlykeepingittothe hub—thenumbersvaryaroundascore.Allmembersoftheteamareencouragedtocontributetoanalysisandwrite-ups.Wehavenopurelyclericalstaff—allaretrainedinscience,usuallypsychologyand/orgenetics and/orbrainimaging.Mostarelocatedinthesamecorridorornearby.

IfSomethingisExciting,doit,EvenifitisnotinYourField WegavetheexampleaboveoffindingthatDNA-methylationagewas relatedtolongevity.Itwastooexcitingnottodo.Evenlessrelatedto ourcoremissionthanthatwasatthetimewhenwehadrecentlyobtainedthegenome-widescanningofSNPsintheLBCs.Asanexercise forournewishly-appointedstatisticalmoleculargeneticist,weransome biomarkervariablesthroughaGWASprocedure.WefoundthreeSNPsthat accountedfor18%ofthevarianceinactivatedpartialthromboplastin time,andimportantmeasureinhematology(102).Therewasthenthe searchforwhohadreportedthisalready.Noonehad.Weranwithit,with addedhematologicalexpertise.Itwasaninterestingresultanditwasa goodexerciseintheanalyzingandwriting-upofGWAS.Perhapsevenfurtherfromthiswasourinvolvementwithclonalhematopoiesis(92, 93) which,again,seemedbothtooexcitingandimportantnottobecomeinvolvedwith.

WeLearnedThatThereAreMultiple,PartlyOverlapping-Campsof CognitiveAgeingResearch

Let’scalltheseindividual-differencespsychology,experimentalpsychology,andmedically-oriented.Theindividualdifferencesapproachmight beexemplifiedby,sayTimothySalthouseorWarnerSchaie,usinglarge community-dwellingcohortstoexaminepatternsofcognitiveageingin differentcognitivedomainsandwhattheyshare.Thesampleisallone group.Sometimesthesestudiesarecross-sectionalandsometimeslongitudinalandsometimescross-sequential.Theexperimentalpsychology approachmightbeexemplifiedby,sayMichaelRugg,andismorelikely tousesmaller,separatesamplesofolderandyoungerindividualsand comparethemoncognitivetestscores.Therearethenmoremedicallyorientedstudiesthatfocusonmildcognitiveimpairmentanddementia. Sometimesthesearecase-controlstudiesandsometimescohortsthat arefollowedintoandthroughcognitiveimpairments.

MuchofourworkwiththeLBCshasbeendonewithintheindividual differencesframework.Inpart,thiscaninvolvedatareductionstatisticaltechniques(principalcomponentsanalysis,factoranalysis,sometimes inastructuralequationmodellingframework)—thebreadandbutterof differentialpsychologymethods.However,thenecessityofdoingmultivariatelongitudinalmodellingtaughtusthat,withtheLBCs,wewerein oneofthemoretechnicalanalyticalfieldsofpsychologyandalsothatthe measurementofanddeterminationofdifferencesincognitive(andother) changewas,tosaytheleast,much-discussedandsometimesfraught.Our teambecamefamiliarwith,forexample,growthcurvemodeling(32, 41, 62, 103).Ifonewantstostudycognitivechangeanditsdeterminants,one hastobepreparedtolearntodrivesomeheavyandcomplicatedstatisticalmachinery.

TakeBiologicalSamples,EvenifTheydonotHaveanImmediateUse FromearlyoninourworkwiththeLBCswestoredblood,plasma,and serum.Weknewweneededtheseforbasichealthbiomarkers(e.g.,blood chemistry,hematology,glycatedhemoglobin,etc.)andforgenetics.We knewthatmorebiomarkerswouldappearandthatsomewouldneedto

bemeasuredlongitudinally.Itishardtoexaggeratenowhowusefulthese sampleshavebeen.Anindicationoftheirusescanbeseeninourcohort profilearticles.

Future-prooftheCohortinTheirAfterlife

ThedatafromtheLBCswillbeanalyzedaftertheyarenolongerwithus and,wehope,afterwearelong-gone,too.However,thereisamorebiologicalmeaningtothislesson.Havingdrawncellswhichcanbetransformedtostemcellsthatcanthenbedifferentiatedtomanycelltypes meansthattheLBC1936participantshaveprovidedmaterialthatcan beused,invitro,totesthypothesesaboutneuralandothercellageing (104).Relatedtothat,thepostmortembraintissuedonatedbysomeof theLBC1936participantswhohavediedhasalreadybeenusedtoinvestigatethebiologyofcognitiveageing,thoughthenumberstodate arestillverysmall(cf.theROSMAPstudies)(47, 105).Suchdiscussion ofattemptedfuture-proofingoftheLBCsremindsustomentionthat this—and,indeed,thepanoplyoftheLBCs—takesplacewithinthestricturesofconsentandethicsandthatthese,duringourwork,havebeen movingtargets.Theconsentrequiredfordifferentaspectsofthestudieshasbecomemoredetailedaswaveshavepassed,andpostmortem braintissuecollectionandstorageandstem-cellcreationandstorage haveeachneededtheirowndetailedconsentproceduresandethicalapprovals.Otheraspectsofthestudy,suchaslinkagetomedicalrecordsrequiredadditional(additional,i.e.,totheethical/consentworkforthecollectionofthedatawithineachwave)ethical/consentapplications.From thebeginningoftheLBCsuntilthepresentwehaveproceededviathe ethicalcommitteesofthenationalHealthServiceinScotland;thatis,we havetakenamedicalratherthanapsychologicalroutetoethicalapproval andconsent,whichreflectsthebroadandhealth-relatedcontentofthe studies.

RealizetheResponsibilityofAssemblingaLongitudinalCohortand InvolveThem

Oneliveswithacohort.Theyarenotlikeaconveniencesamplethat onewillthankandneverseethereafter.Onemustformrelationships withthecohort.Onemustlistentothemandmakechannelsforthatto happen.WiththeLBCs,wehave:newsletters(readsomeofthemhere: https://edin.ac/4dN8unc)attheendsofwavesandatChristmasandat someothernotabletimes;reunionsattheendsofwavesandatnotable anniversaries(therearetalksonthenewresultsandquestionandanswersessionsandinformationaboutthefutureplans;seeour20-year anniversarybooklethere: https://edin.ac/3VnQiLi);andtheLBCs’participantshavemademanynationaltelevision,radioandnewspaperappearances.Therehavebeenhistoricalandartexhibitions(portraitssomeof theLBCs’participantsandtheresearchteam)abouttheLBCs.Therewas aplayabouttheLBCsperformedattheEdinburghInternationalFringe Festival.AfilmwasmadeabouttheLBCs.Abookwaswrittenrecountingthepersonalhistoriesofsomeoftheparticipantsandsomeofthe team.TheLBCs’participantshavefeaturedinumpteensciencefestivals, andknowledgeexchangeeventsforschoolchildrenandmembersofthe public.There’sasummaryhere: https://edin.ac/4dTXQee.LBCs’participantsandteammembershavetwicebeentotheUKHouseofLordstodescribewhattheirfindingsweretoexpertgroups.Whenwewrote,above, thatonemustlistentothecohorts’members,thatwasnotemptyvirtue signaling.Here’sanexample.AtoneofthereunionsoftheLBC1936,a participantaskedwhy,giventhatwehadcollectedsomuchinformation onthem,wehadnotaskedfortheirbrainafterdeath.Thatbeganalong processofobtainingpermissionforandsettinguptheLBC1936BrainTissueBank(whichhasmultiplesmallsamplesfrombrains,andnotwhole brains).

BeinginanObservationalStudyCanbeanUnintendedTreatment

Thisprobablyhasnothappenedoftenortoanylargeextent.However, beingaparticipantintheLBCs,itwouldbeimpossiblenottobealerted toaspectsofcognitiveandbrainandmoregeneralageing.Togivejust oneexample,oneLBC1936participantenrolledforandsuccessfullycompletedadegreeinphilosophywiththeUK’sOpenUniversitybecauseshe thoughtsheshoulduseherbrainmore.

RefereesandJournalEditorsWantLongitudinalDatainCognitiveand BrainAgeing,butFundersDon’tWanttoFundThem(UnlessYouHave NewHypothesesatEachWave)

Aswesaidabove,theLBCshaveneverhadguaranteedfunding.However, wehavehad,forexample,consecutivegrantsfromAgeUKthatspanned theyearsbetween2004and2020fortheLBC1936.WehavealsohadmultiplegrantsfromtheUKResearchandInnovationbodies,especiallyBBSRCandMRC.Here,wearereferringmostlytograntsforcoreaspectsof thestudy;therearemanyothergrantsforspecificprojectsandforfellowships.But,aswenoteinparenthesesabove,ithasneverbeensufficient tostate,whenapplyingforfunding,thatwewerecollectinganothervaluablewaveofdatafromtheLBCs.Almostalways,wehavehadtodevelop freshhypothesesforeachwave.Althoughwehavebeenablesuccessfully todothis,andkeeptheshowontheroad(anddeliverthespecifiedwork), theprocessoffocusingonsomespecifichypotheses—fromcohortsthat haveasolidtrackrecordofbeingarichsubstrateonwhichtotestsomany hypotheses—wasnoteasy.

HaveaGoodSuccession,EvenifitHappensDuringaPandemic Ourcohortshavelastedalongtime,beyondthefull-timecareerofIan Deary,forexample.WewereabletokeeptheLBC1936cohortandresearchteamgoingthroughtheCovid-19pandemic(106–108)andthey arenow(thesecondhalfof2024)passingthroughWave7atageabout88. TheDeary-to-Coxsuccessionisbuiltnotjustonthatonepositiveworking relationship,butalsoupongreatloyaltyandcontinuityofteammembers andparticipantsandcollaborators.Westillhave,workingintheteam,AlisonPattie,whowasfirstemployedatthestartoftheLBC1921in1999, andJanieCorley,whowasfirstemployedatthestartoftheLBC1936in 2004.Directingacohortstudyisanintricatebusiness,andwebothcount ourselvesluckytohavebenefittedfromasuperbanddedicatedteam, withoutwhosecontinuitythewholeoperationwouldhavebeenimpossibletokeepontheroad.

AppreciateOne’sHistory

ThehistoryoftheSMSsandtheresearchenvironmentinScotlandand nationallyandinternationallythatbroughtthemabouthasbeenasource ofinterestingstudyinitself.Centraltothatwastheinterestingfigure ofProfessorSirGodfreyThomson,agiantineducation,intelligence,and statisticsandwhoisunfairlyrelativelyunknown(109–113).Hisportrait hangsinthecurrentdirector’soffice(alongwithanappreciationof in umerisgigantumstamus)asitdidinthefoundingdirector’s.Hereisalink toavideocoveringtheexhibitiondevotedtoThomsonthatweproduced in2016: https://www.youtube.com/watch?v=ZObidTDX4lI

And,nowthattheLBCsare25yearsold,theyfoldintoandbecome partofthathistory.Thenextfewyearswillseetheage-90/Wave8testing oftheLBC1936.Alliedwiththatwillbeanefforttotakeourverylarge, securely-storedpaperrecordsanddigitizethem,sothateverymarkmade foreverytestforeveryparticipantateverywavecanbemadeavailable tofutureresearchers.Also,thepresentauthorshave(it’samug’sgame, though)triedtopredictwhatmighthappen,scientifically,inthebrainand cognitiveageingfieldoverthenextwhile(74).

EnjoyWhatYoudo

TheLBCcohorts,theLBCresearchteam,ourcollaborators,ourfunders (here,wemakeaspecialmentionofAgeUK,withwhomwehadalong andrewardingrelationship),andtheUniversityofEdinburgh(withaspecialmentionfortheSchoolofPhilosophy,PsychologyandLanguageSciencesanditsvariousHeadsfortheirsupport)areandhavebeenenjoyable toworkwith.Wevalueandhumblyappreciatepersonal,social,andscientificpremiumsthatreturnfromthatpositiveenvironment.TheLBCshave openeddoorsthatothertypesofresearchinvolvementwouldn’thave:we andtheymetseveralmembersoftheBritishRoyalFamily(includingthe lateQueenElizabethII),LordsandMPs,andstarsofstageandscreen.FuelledbytheLBCs,wehaveseenjuniorresearchersrisetoprofessorships andtoothervaluedvocationaldestinations;theLBCshaveseededcognitiveandbrainageingresearchersinotherplaces.

ConcludingThoughts

TheresearchwiththeLothianBirthCohortswasoftensummarizedas somethinglike,“todiscoversecretsofhealthycognitiveageing.”In

variouswaysthathandy-but-crudestatementdissolved:first,wewere sometimesconfirming/incrementingothers’findingsratherthandiscovering(i.e.,forthefirsttime);second,weacceptedthatweshouldhaveto studypathologicalaswellashealthycognitiveageing,astheparticipants experienceddementiainlargernumbers;andweexpandedouroutcomes remitbeyondcognitivefunctioning.Thosebelt-looseningchangestoour originalmethodologicalpuritynotwithstanding,wehopethatdiscoveriesfromtheLBCs,asenumeratedinthe“ScientificDiscoveryLessons” willbeusefultoscientistsincognatefields.Wealsohopethatourdiscoveriesandincrementalcontributionstothefieldsinwhichwework willhelppeopletomakebetterchoicesregardinghealthylifestylesand provideunderstandingregardingcontributionstoindividualdifferences incognitiveandbrainageingandageingmorebroadly(alongsideother teams’findings);wehopethatscientistsandlaypeoplewillappreciate thatwhattheythinkareoutcomescanbeexposuresandviceversa(reversecausation/confounding);thiswasallsummarizedinour“marginal gains”approach(8).Wealsoreferthereadertoourvariouspolicyinfluencingattemptsandcontributions(here https://edin.ac/4dTXQee andhere https://edin.ac/48te7G9)andalsomentionthatwehaveundertakenhundredsofmediaandin-personappearances/activitiestospread thewordaboutgoodscienceandhealthyageing.Theseactivitiescoverall agesfromprimaryschoolstoolder-people’sgroupsanduseeducational programs,games,andart.Finally,wehopetohaveencouragedreaders tofindandreadmoreofourpublicationsandtokeepupwiththosethat appearinthecomingyears;theyarelistedhere: https://edin.ac/3UixD26.

DataAvailabilityStatement

Nooriginaldataweregeneratedinthisworkthatrequirespublicdissemination.InformationaboutdataaccessandcollaborationfortheLothian BirthCohortsof1921and1936,theLBCs’datadictionaries,theLBCs’ datasummarytables,theLBCs’cohortprofilearticles,theLBCs’datarequestform,anddatarequestcontactinformationareallavailablehere: https://edin.ac/3YkR3Ev

Acknowledgments

WegratefullyacknowledgethecontributionsoftheLBC1921and LBC1936participants,thepresentandpastmembersofourresearch teamwhocollect(ed),manage(d),andanalyze(d)theLothianBirthCohorts’dataandwrotemanyofthepaperswecitehere,andourvalued collaboratorswithwhomwepartneredbecausetheyknewthingsthat wedidn’tandcoulddothingsthatwecouldn’t.WethankDrSarahMcGroryforassistancewith Figure3.Thepresentpaperisdedicatedtoour lateandmuch-missedcolleaguesProfessorsJohnM.StarrandLawrence J.Whalley.

AuthorContributions

Bothauthorscontributedequallytoallaspectsofthiswork.

FundingSources

IJDissupportedbyaNationalInstitutesofHealth(NIH)researchgrant (R01AG054628)andbyBBSRCandESRC(BB/W008793/1).SRCissupportedbyaSirHenryWellcomeFellowshipjointlyfundedbyWellcomeand theRoyalSociety(221890/Z/20/Z),andreceivesfundingfromtheBBSRC andESRC(BB/W008793/1),NIH(R01AG054628),MRC(MR/X003434/1), MiltonDamerelTrust,andtheUniversityofEdinburgh.

AuthorDisclosures

Theauthorshaveconfirmedthatnoconflictofinterestexists.

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HIGHPRIORITYRESEARCHCOMMUNICATION

Prepartumbumetanidetreatmentreversesalteredneonatalsocialcommunication butnonspecificallyreducespostpubertalsocialbehaviorinamousemodelof fragileXsyndrome

YuiSakamoto1 , # ,TakeshiTakano2 , 3 , # ,ShujiShimoyama4 , # ,TakeshiHiramoto2 ,NoboruHiroi2 , 5 , 6 ,andKazuhikoNakamura1

FragileXsyndromeiscausedbymonogenicsilencingofthe FMR1 geneandischaracterizedbyhighratesofautismspectrumdisorder.A previousstudydemonstratedthatprepartumadministrationofbumetanide,achloridetransporterblocker,normalizedneonatalvocalization innon-congenic Fmr1 knockout(KO)pups.However,thegenuinecontributionof Fmr1 deletiontothisphenotypeinacongenic Fmr1 KOmouse modelandthelong-lastingeffectofprepartumbumetanideadministrationonpostpubertalsocialinteractionremainsunclear.Thecurrent studyaimedtodeterminetheimpactofprepartumbumetanideadministrationonvocalizationatpostnatalday7andsocialinteractionat 6and8weeksofageinacongenic Fmr1 KOmousemodelinwhichthegeneticbackgroundswerehomogeneousbetweenKOandwild-type(WT) littermates.Moreover,weappliedacomputationalanalyticalalgorithmanddeterminedpredictivevariablesofneonatalvocalizationfor postpubertalsocialinteraction.Ourdatashowedthat(1)KOmiceexhibitedalterednumbersandsequencesofdistinctcalltypesduring neonatalvocalizationandreducedsocialinteractionat6weeks,(2)selectsetsofneonatalvocalizationvariablespredictedpostpubertalsocial interactionlevels,and(3)bumetaniderestoredneonatalvocalizationinKOpupsbutnonspecificallyreducedsocialinteractioninWTandKO miceat6weeks.Thesedataindicatethat Fmr1 deletionselectivelyimpactsdistinctelementsofneonatalvocalizationandpostpubertalsocial interaction.Additionally,bumetanideselectivelyrestoresneonatalvocalizationbuthasatransientnonspecificnegativeimpactonsubsequent postpubertalsocialinteraction.

GenomicPsychiatry January2025;1(1):61–72;doi: https://doi.org/10.61373/gp024h.0094

Keywords: FragileX,Fmr1,FMRP,criticalperiod,GABA,socialcommunication,socialinteraction,machinelearning,predictors

Introduction

Precisionmedicinehasnotbeenutilizedinpsychiatrybecausetheprecisemechanistictargetsofpsychiatricdisordersarenotwellestablished. Geneandgenomicvariantsprovideareliableentrypointforamechanistic understandingofpsychiatricdisordersandmechanism-basedtherapeuticoptions(1).

FragileXsyndromeisaneurodevelopmentaldisordercausedbymonogenicmutationandtranscriptionalsilencingofthefragileXmessenger ribonucleoprotein1(FMR1)gene,resultinginlossofitsproteinproduct, fragileXmessengerribonucleoprotein(FMRP).Thesyndromeincludes theclinicaldiagnosesofautismspectrumdisorder(ASD)andintellectualdisability.Although FMR1 silencingbeginsintheembryonicperiod inhumans(2),thephenotypesarenotreliablyidentifieduntillaterinthe postnatalperiod,partlybecauseclinicaldiagnosesarenotfeasibleuntilformaltestscanbereliablyapplied.However,manysocial,cognitive, affective,motor,andsensoryphenotypesappearduringearlypostnatal periods(3).

Inthemousebrain,FMRPexpressionishighonembryonicday11.5 (E11.5),E18.5,andduringthefirstpostnatalweekbutsteadilydeclinesthereafter(4, 5).The Fmr1 peaksduringtheperinatalperiodmirrorthoseinhumanbrains(5).Thedevelopmentalphasefromtheembryonicperiodtothefirstneonatalweek,exceptforthetimeofbirth (i.e.,postnatalday0,P0),isauniqueperiodinwhichtheinhibitory neurotransmittergamma-aminobutyricacid(GABA)exertsanexcitatory actiononneuronsbecauseoftheirhighintracellularchlorideconcentration(6, 7).Remarkably,aseminalstudybyBen-Ariandcolleagues

reportedthatthistransientinhibitoryroleofGABAatandnearP0isattenuatedinamousemodeloffragileXsyndrome;whenpregnantmothers weretreatedwithbumetanide,anNKCC1chloridetransporterinhibitor, onedaybeforedelivery,theincreasedprobabilitiesoftwoneonatalcall types(i.e.,chevronanddownward)werenormalizedinnon-congenic Fmr1 knockout(KO)pupsatP8(8).Humanstudieshavenotaddressed thecriticalperiodforbumetanidetreatmentduetotechnicalandethicalissues.ThetherapeuticeffectsofbumetanideonpatientswithidiopathicASDarelargelynegativewhentreatmentstartsat2yearsofage orlater(9).

Theimpetusofthepresentstudywas3-fold.First,anon-congenic mousemodelposesaninterpretativeissue.Althoughnon-congenic mousemodelshaverandomlyshuffledallelicdistributionsthroughout thegenomesofKOandwild-type(WT)littermates,theyhaveasystematic,consistentbiasnearthetargetedgene.Becauseofthelowrecombinationratesbetweenthetargetedgeneandgeneslocatednearby,the allelesofnearbygenesofthetargetedgenetendtobeinheritedtogether.Becausegenetargetingisinducedinembryonicstem(ES)cells ofthe129/Svsubstrainsandmicearebredwithanotherstrain(e.g.,FVB orC57BL/6J),KOoffspringaccumulateallelesofneighboringgenesderivedfromEScells,andWToffspringaccumulateallelesofbreeders.Becausethesestrainsdifferintheirmolecular,cellular,electrophysiological,anatomical,andbehavioralphenotypes,anyphenotypicdifferences betweennon-congenicKOandWTlittermatescannotbeunequivocallyattributedtothetargetedgene(10).Weaddressedthisissueusingacongenic Fmr1 KOmousemodel.

1 DepartmentofNeuropsychiatry,HirosakiUniversityGraduateSchoolofMedicine,Hirosaki,Aomori,Japan036-8562; 2 DepartmentofPharmacology,UniversityofTexas HealthScienceCenteratSanAntonio,SanAntonio,TX,USA78229; 3 TokyoDenkiUniversity,Ishizuka,Hatoyama-machi,Hiki-gun,Saitama350-0394,Japan; 4 Department ofNeurophysiology,HirosakiUniversityGraduateSchoolofMedicine,Hirosaki,Aomori,Japan036-8562; 5 DepartmentofCellularandIntegrativePhysiology,Universityof TexasHealthScienceCenteratSanAntonio,SanAntonio,TX,USA78229; 6 DepartmentofCellSystemsAnatomy,UniversityofTexasHealthScienceCenteratSanAntonio, SanAntonio,TX,USA78229

# Theseauthorscontributedequallytothiswork.

CorrespondingAuthors: KazuhikoNakamura,HirosakiUniversityGraduateSchoolofMedicine,5Zaifu-cho,Hirosaki,Aomori,036-8562,Japan.Phone:81-172-395066;E-mail: nakakazu@hirosaki-u.ac.jp;andNoboruHiroi,UniversityofTexasHealthScienceCenteratSanAntonio,7703FloydCurlDr,SanAntonio,TX78229,USA. Phone:210-567-4169;E-mail: hiroi@uthscsa.edu

Received:23October2024.Revised:16November2024and4December2024.Accepted:9December2024. Publishedonline:24December2024.

Figure1. Numberofneonatalvocalizationcalltypes.Theaveragenumber(±SEM)ofeachcalltypeemittedbymaleWTandKOmiceisshown.Asterisks (∗ , ∗∗ ,and ∗∗∗ )indicatestatisticallysignificantdifferences(1%,0.5%,and0.1%,respectively)betweenWTandKOpups.Inset:WTandKOmicediffered (F(1,41) = 18.839, p < 0.0001)andbumetanideincreasedcallsinKOmice(Treatment,F(1,41) = 23.080, p < 0.0001;Interaction,F(1,41) = 6.7576, p = 0.0129). Student t-testsshowedthatWTandKOmicedifferedinthetotalnumberofallcallswithoutbumetanidetreatment(p = 0.0013),butnotwithbumetanide(p = 0.0850).Mainfigure:WTandKOmicediffereddependingoncalltypesandtreatment(GenotypexCalltypesxTreatment,F(11,451) = 8.8573, p < 0.0001). Vehicle-treatedWTandKOpupsdifferedinthenumbersofharmonic(p = 0.0007)andflat(p = 0.0072).Thesesignificantdifferencesof t-testssurvived Benjamini–Hochberg’scorrectionsformultiplecomparisonsat5%FDR.Bumetanide-treatedWTpupsdidnotdifferinanycalltypefrombumetanide-treated KOpups(p > 0.05).Vehicle:WT, n = 9;KO, n = 12.Bumetanide:WT, n = 12;KO, n = 12.Ha,harmonic;Ch,chevron;Co,complex;Df,down-frequencymodulation; Fl,flat;Ms,multi-step;Rc,reversechevron;Sh,short;Sd,step-down;Su,step-up;Ts,two-step;Uf,up-frequencymodulation.

Second,phenotypicvariabilityisanorm,andthuscomparisonsbased ongroupaveragesdonotfullycapturethenuancednatureoftheimpactsofgenomicvariations. Fmr1 mutationdoesnotcausecompletepenetrance,andapproximately60%ofmalecarriersarediagnosedwithASD. ElementsofASDalsoshowphenotypicvariability;socialcommunicationis weakamong Fmr1 mutationcarrierswithanASDdiagnosis(3).Morethan 10distinctcalltypesandtheirspecifictemporalsequencesareuniquely impactedbydosealterationsofgenesimplicatedinneurodevelopmental andpsychiatricdisordersinmousemodels(11–13).Therefore,tocapture thevariablenatureofthissyndromeanditsdimensionalelements,we appliedcomputationalapproaches(11, 12, 14, 15).

Third,specificrolesofGABAduringtheperinatalperiodinthedevelopmentofpostpubertalsocialinteractionhavenotbeenexplored.This issueispertinenttothetheoreticalquestionofwhethernormalneonatalsocialcommunicationisaprerequisitefornormalsocialinteraction atlatertimesorwhetherthesetwoprocessesindependentlydevelop. Weaddressedthisquestionbycomputationallyevaluatingthepredictivepowerofvariablesofneonatalvocalizationforpostpubertalsocial interactionwithoutandwithbumetanide.Differentialalterationofphenotypesofthetwodevelopmentalstagesbythistreatmentwouldsupportmechanisticallyindependentprocesses;incontrast,thepresenceof predictiveneonatalvariablesforpostpubertalsocialinteractionandimprovementofbothbehaviorsbybumetanideatthetwodevelopmental stageswouldbeconsistentwiththehypothesisthatthetwodevelopmentalstagesshareacommonmechanisticbasis.

TotestthehypothesisthataGABAergictoneduringtheperinatalperiodisadeterminantforneonatalsocialcommunicationandpostpubertal socialinteraction,weadministeredbumetanideprepartumandevaluated itsimpactsonneonatalvocalizationatP7andonpostpubertalsocialinteractionat6and8weeksofage.AsfragileXsyndromeisassociatedwith variableexpressivityanddevelopmentaltrajectoriesamongcarriers,we appliedacomputationalanalyticalalgorithm(12)topredictthevariabilityofpostpubertalsocialinteractionscoresfromthevariationinneonatal vocalizations.

Results

Fmr1 DeletionAlterstheNumberofCallswithSpecificGeometricShapes Evidenceindicatesthattheperinatal/neonatalperiodiscriticalforthe developmentofsocialbehavior(16),andneonatalvocalizationistheearliestexpressionofsocialcommunicationinrodentsandhumans(11, 12, 17, 18).Itwasreportedthattheprobabilitiesofcallswithtwospecific geometricshapes,termedchevronanddownward,wereincreasedand theseincreaseswerenormalizedbybumetanideinanon-congenicmouse modeloffragileXsyndrome(8).However,mousepupsgenerallyexhibit morethan10distinctcalltypes,andtheyaredistinctlyimpactedbygenes linkedtoneurodevelopmentaldisorders(11–13, 19, 20).Inparticular,the calltypesthatarealteredinvariousmousemodelsoffragileXsyndrome varydependingongeneticbackgrounds,sex,age,callclassifications,and otherfactors.Whenneonatalcalltypeswereanalyzedinmousemodels offragileXsyndromeinwhichthegeneticbackgroundsweremadehomogenousbetweenWTandKOpups,variouscalltypeswerefoundtobe altered,resultingingreaternumbersof“frequencyjump”calls,whichincludedtwo-syllableandfrequencystepcalls,atP7(13),increasedpercentagesofchevronandfrequencystepcalltypesanddecreasedpercentagesofcomplex,composite,downward,harmonic,two-syllable,andshort calltypesatP8(21).

Wecomprehensivelycharacterizedcalltypesthatwerealteredby Fmr1 deletionandbumetanidetreatmentincongenic Fmr1 KOpupsand theirWTlittermates.KOpupsemittedfewertotalcallsthanWTpups, andthiseffectwasamelioratedbybumetanideatP7(Figure1 inset).The numbersofthe12calltypeswereseparatelycomparedbetweenWTand KOmice(Figure1;SupplementalTableS2).ComparedwithWTpups,KO pupsemittedfewerharmonicandflatcalltypes.Bumetanideincreased thenumberofcallsinKOpupstotheextentthatKOandWTpupsdidnot differforanycalltypes.

Weadditionallyexaminedtheproportionofeachcalltypewithinthe totalnumberforeachpup.Inthismeasure,WTandKOmicedidnotdifferforcalltypesandbumetanidehadnosignificanteffect(Supplemental FigureS1;SupplementalTableS2).

Figure2. Three-dimensionalUMAPofVocalMatparameters.UMAPwasusedtoconvertthequantitativeacousticparametersintolowerdimensionswhilemaintainingtheircharacter.TheVocalMatparametersincluded“duration,”“min_freq_main,”“max_freq_main,”“mean_freq_main,”“bandwidth,”“min_freq_total,” “max_freq_total,””mean_freq_total,”“min_intens_total,”“max_intens_total,”and“mean_intens_total”.Callswereseparatedintothreeclusters.Calltypesare indicatedbydistinctcolorsinthethreeclusters.

Thelengthofallcalls(SupplementalFigureS2inset)oreachcalltype (SupplementalFigureS2)didnotdifferbetweenWTandKOpups.While thelengthsofallcallswereprolongedbybumetanide(Figure2 inset), theydidnotreachsignificancewheneachcallwasseparatelyanalyzed (SupplementalFigureS2).

Fmr1 DeletiondoesnotAltertheQuantitativeAcousticParametersof CallTypes

Theseanalyseswerebasedonthecategoricalclassificationofgeometric callshapes.However,eachcalltypealsohasquantitativeacousticparameters.Wethusadditionallyanalyzedthe(1)bandwidthinHz,(2)maximum,mean,andminimumfrequenciesinHzofacalloritsmaincomponents,and(3)maximum,mean,andminimumintensitiesofeachcall. Vehicle-treatedWTandKOpupsdidnotdifferinanyoftheseacousticparametersforallcalltypes(SupplementalTableS3A–S3J).Thus,theacousticparametersdidnotdiscriminategenotype.

Fmr1 DeletionImpactstheQuantitativeParametersofCallsina DimensionalSpace

Havingestablishedthatthesimpleacousticparametersdidnotdifferentiategenotypeordrugtreatment,wenextaimedtoevaluatecallsinan independentdimension.Wevisualizedandevaluatedthecomplexnature

ofVocalMat’squantitativeacousticparametersinthreeandtwodimensionsusingUniformManifoldApproximationandProjection(UMAP).

WefirstusedthreecommonlyusedUMAPmetrics:Manhattan,Euclidean,andChebyshev(SupplementalFigureS3).ToevaluatetherelationshipbetweentheseUMAPclustersandcalltypes,wecolor-coded eachdatapointbasedonthecallclassificationofourmodifiedVocalMat. BecausetheEuclideanmetricsprovidedbetterclusterseparationthan theManhattanorChebyshevmetrics,weusedtheEuclideanmetricfor allthesubsequentanalyses.

Thisanalysisreducedallcallsintothreemainspatialclustersina three-dimensionalUMAPspace(Figure2;SupplementalFiguresS4and S5).Thedecreasednumbersofcallsareapparentinallthreeclusters inKOpups;allthesespatialclusterswererestoredbybumetanide.HarmoniccallswerepredominantlyrepresentedinspatialCluster1andCluster3(SupplementalFiguresS5andS6);chevronwasthemostpredominantcalltypeinCluster2(SupplementalFiguresS5andS6).Thisanalysis showedquantitativedifferencesandsimilaritiesamongVocalMat-based calltypes.Callsclassifiedas“harmonic”havethemostvariablequantitativeprofilethanothercalltypes.

Thecombinedanalyticalapproachesforcategoricalcalltypes andquantitativeacousticparametersshowedthat Fmr1 deletionand

Figure3. Probabilitiesofintercallintervals.Theobservedandexpectedprobabilitiesofintercallintervalsofvehicle-treatedWT(A),vehicle-treatedKO(B), bumetanide-treatedWT(C),andbumetanide-treatedKOmice(D).Vehicle-treatedKOpupshadlongerintercallintervals,comparedwithvehicle-treatedWT pups(p < 0.0001).BumetanidereducedintercallintervalsinKOpups(p < 0.0001)butbumetanide-treatedKOpupsstillmaintainedlongerintercallintervals thanbumetanide-treatedWTpups(p < 0.0001).Theactualintercallintervals(ms)atcross-pointsoftheobservedandexpectedprobabilitiesareshown.Intercall intervalsshorterthanthehighercross-pointsbetweentheobservedandexpecteddistributionshadprobabilitieshigherthantheexpecteddistributionsandthus areconsideredintervalsbetweencallswithinsequences(Vehicle-WT,306.62ms;Vehicle-KO,473.18ms;Bumetanide-WT,306.12ms;Bumetanide-KO, 334.63 ms);intercallintervalslongerthanthecross-pointswereconsideredintervalsfromtheendofasequencetothebeginningofthenextsequence.

bumetanideimpactcallswithdistinctcategoricalandquantitative parameters.

Fmr1 KOAlterstheTemporalDistributionofCallSequences Callswereemittedwithvariousintercallintervals.Toobjectivelyidentify atemporalclusterofcalls,wefirstdeterminedthePoissondistribution, atheoreticallyexpecteddistributionofintercallintervalswithagiven numberofcallswithina300-stesttime(Figure3,redlines).BecauseKO pupsemittedfewercallsthanWTpups,thetheoreticallyexpectedintercallintervalsofKOpupswereshiftedtotheright(Figure3B,redlines) comparedwiththoseofWTpups(Figure3A,redlines).Thesedataindicatethat Fmr1 deletionalterstheintercallintervalswithoutchangingthe lengthofeachcall(seeSupplementalFigureS2).

Thelargestpeaksofobservedintercallintervals,representingthe mostfrequentintercallintervals,werefoundaround200ms(Figure3, blacklines).Therewerealsomuchsmallerpeaksthatoccurredmore frequentlythanexpectedfromthePoissondistributionsbelowapproximately30ms(Figure3,blacklines).However,theseshortintercallintervalsoccurredinlessthan5%ofallcallsofallpups.

Wedefinedacallsequenceasaseriesofcallsthatwereemittedwith intercallintervalsshorterthanthelargervalueofthetwocrossesbetweenthehighestprobabilitypeakoftheobservedcurveandtheexpectedprobabilitycurve(see Figure3A,WT,Vehicle,306.62ms;B,KO, Vehicle,473.18ms;C,WT,Bumetanide,306.12ms;D,KO,Bumetanide, 334.63ms).

Figure4. Markovmodel.Therelativeprobabilitiesofeachtwo-callsequenceforeachofthe12calltypesidentifiedbytheMarkovmodelsareshown.Thesum ofallproportionsofeachstartingcallis1,andthesumofallMarkovprobabilitiesis100% × 12calls.Thelinethicknessrepresentstherelativeprobabilityofa callsequence.

Whencallsequencesweresodefined,shortandlongsequenceswere identifiedaround100msand1s,respectively(SupplementalFigureS7). BumetanidelengthenedthelongsequencedurationinKOpupscompared withthatinvehicle-treatedKOpups.

Fmr1 KOAltersCallSequences

Havingdefinedcallsequences,wenextdeterminedhowvariouscalltypes wereorderedwithinsequences.Wedeterminedtheprobabilitiesoftwo consecutivecallsforagivenstartingcallusingMarkovmodeling.This modelisbasedontheMarkovproperty,wherefuturestatesdependonly onthecurrentstate.Theprobabilitiesofcallsequencesweredetermined basedontwoconsecutivecalls.Inotherwords,theprobabilityofthenext calltypewascomputedwithineachcalltype;thesumofprobabilitiesof alltwo-callsequencesstartingfromagivencalltypewasalways1.0.Thus, thisanalysiswasnotinfluencedbytheprobabilitiesofthefirstcalloftwo consecutivecallsemittedbyeachsubject.Thisanalysisrevealeddistinct frequentlyemittedtwoconsecutivecallsofvehicle-treatedWTandKO pups(Figure4).Bumetaniderestoredthealteredtwo-callsequencesof KOpups.

BecauseoftheMarkovproperty,thetwo-callsequencesstartingfrom lessemittedcalltypestendtobeoverestimated.Toevaluatecallsequenceswithineachmousewithallstartingcalltypes,weincorporated aseparateanalysisofhowfrequentlyeachtwo-callpairwasemitted.In thisanalysis,thesumofprobabilitiesofalltwo-callsequencesstarting fromallcalltypeswasalways1.0.Two-callpairsstartingandendingin harmonicwerepredominantinallgroups(SupplementalFigureS8).

PrepartumBumetanideTreatmentReducesPostpubertalSocial InteractioninKOMice

Whenthepupsreached6and8weeksofage,theyweresequentially testedforsocialinteraction.Wetestednaturalisticsocialinteractionin ahomecagesettingbecauseitisabettervalidationthanotherprocedures(22),andmolecularmechanismsunderlyingdirectsocialcontactin suchaset-upandindirectcontactwithabarrierdiffer(23).

Therewereafewtestorstimuluspartnermiceineachgroupthatexhibitedaggressivebehavior.Suchcaseswereeliminatedfromtheanalysis(24)forthreereasons.First,geneticbasesofaffiliativesocialinteractionandaggressivebehaviorarenonidentical(25).Second,aggressive

Figure5. Socialinteraction.Thetime(mean±SEM)spentinactivesocialinteractionat6weeksofage(A)and8weeksofage(B)isshown.(A)KOmicespent lesstimeinsocialinteractionthanWTmice(Genotype,F(1,36) = 4.1160, p = 0.0499)andbumetanidetreatmentequallyreducedsocialinteractioninboth genotypegroups(Treatment,F(1,36) = 37.1341, p < 0.0001;Interaction,F(1,36) = 1.1674, p = 0.2871).WT/Vehicle, n = 8;KO/Vehicle, n = 11;WT/Bumetanide, n = 11,KO/Bumetanide, n = 10.(B)WTandKOmicedidnotdiffer(Genotype,F(1,33) = 0.0399, p = 0.8429)andtheeffectofbumetanidetreatmentwasnot significant(F(1,33) = 3.4975, p = 0.0704)withoutaninteractioneffect(F(1,33) = 0.1661, p = 0.6862).WT/Vehicle, n = 7;KO/Vehicle, n = 11;WT/Bumetanide, n = 9,KO/Bumetanide, n = 10.

behaviorisnotaprominentelementoffragileXsyndrome,butalteredaffiliativesocialinteractionisanelement.Third,aggressivebehaviorindirectlysuppressestheoccurrenceofaffiliativesocialinteraction,thereby artificiallyunderestimatingit.Thefollowingtestmicewereeliminated ineachgroup:6weeks:VehicleWT,1mouse;BumetanideWT,1mouse; BumetanideKO,2mice.8weeks:VehicleWT,1mouse;BumetanideWT, 3mice;BumetanideKO,1mouse.Wealsoeliminatedcaseswherethe stimulusmicewereagitatedandhyperactive(6weeks,VehicleKO,1case. 8weeks:VehicleWT,1case;BumetanideKO,1case),assuchbehaviorof thestimulusmousemakesitphysicallyimpossibleforatestmousetoengageinaffiliativesocialinteraction.

KOmiceshowedlowersocialinteractionlevelsthanWTmice;prepartumbumetanidetreatmentequallyloweredsocialinteractionlevelsin bothWTandKOmiceat6weeksofage(Figure5A).

At8weeksofage,vehicle-treatedWTandKOmicedidnotdiffer,and bumetanidehadnostatisticallysignificanteffectonsocialinteractionin WTandKOmice(Figure5B).

PredictiveVariablesofNeonatalVocalizationforPostpubertalAffiliative SocialInteraction

Ifamechanisticlinkexistsbetweenneonatalvocalizationsandthedevelopmentofpostpubertalsocialbehavior,theformershouldpredictthelatter.Toidentifysuchpredictivevariablesofneonatalvocalizationsamong thenumbersandprobabilitiesofcalltypesandcallsequences,wedevelopedLeastAbsoluteShrinkageandSelectionOperator(Lasso)regressionmodelsforeachgroup.Theacousticvariableswerenotincludedin thisanalysis,astherewasnodifferenceintheiraveragesbetweengenotypesortreatments,andsomecalltypeswerenotemittedinsomemice, thusprovidingnoacousticparameter(e.g.,bandwidthandamplitude) (SupplementalTableS3).

Thisanalysisidentifiedauniquesetofspecificvariableswithineach groupthatpredictaffiliativesocialinteractionlevelsat6weeksofage (Figure6).Themostrobustpredictorsofvehicle-treatedWTmicewere thenumberofflat->chevronandtheproportionofchevron->chevron; thoseofvehicle-treatedKOmiceweretheMarkovprobabilityofchevron>short,andthenon-Markovproportionsofharmonic->chevronand chevron->two-stepcalls.Thesevariableswerealsosignificantlycorrelatedwiththescoresofsocialinteraction(see Figure6 inset;SupplementalTableS2;SupplementalFigureS6).

Inbumetanide-treatedmice,severalvariablesofneonatalvocalizationwereidentifiedaspredictorsbyLassomodels(Figure6).Themost robustpredictorsweretheproportionoftwosteps->flatandMarkov probabilityofharmonic->step-upinWTmice;theMarkovprobabilities ofstep-down->harmonic,harmonic->chevron,complex->upwardfrequencymodulation,andchevron->twosteps,andnon-Markovproportionsofdownwardfrequencymodulation->flatinKOmice.Remarkably, noneoftheLasso-selectedvariablesweresignificantlycorrelatedwith socialinteractionscoresinbumetanide-treatedWTorKOmice(Figure6 inset;SupplementalTableS2;SupplementalFigureS6).Thisresultis likelyduetothenonspecificeffectsofbumetanideonsocialinteraction inbothWTandKOmice(see Figure5).

Intheanalysisabove,weidentifiedneonatalcallparametersthatbest predictedpostpubertalsocialinteractionwithin6-weekvehicle-treated WTorKOmice.Wenextidentifiedneonatalcallparametersthatbest differentiatedsocialinteractionscoresandgenotypesinpooleddataof bothvehicle-treatedWTandKOmiceat6weeks.First,aLassoregressionmodelwithsocialscoresasthecoefficientidentifiedsequencesand callnumbersaspredictorsofsocialinteraction(SupplementalFigure S9A,e.g.,Fl->Ch,Sh->Fl).Onlytwoofthemweresignificantlycorrelatedwithsocialinteractionscores(Fl->ChandCh->Sh;Supplemental TableS2;SupplementalFigureS9A).Second,aLassoregressionmodel identifiedthenumberofFlat->Chevronasapredictorofthegenotype (SupplementalFigureS9B).However,thispredictorfailedtodiscriminatethegenotype(seeSupplementalFigureS9BandSupplemental TableS2).

Althoughtherewasnoeffectofgenotypeorbumetanideonsocialinteractionat8weeksofage(Figure5),weappliedtheLassomethodtodeterminewhichneonatalvocalizationvariablespredictindividualvariation insocialinteraction(SupplementalFigureS10).Distinctsetsoftwo-call sequencespredictedindividualvariationwithineachgenotype,including theMarkovprobabilityofcomplex->chevronandthenon-Markovproportionofchevron->complexinvehicle-treatedWTmice.Invehicle-treated KOmice,theMarkovprobabilityofupwardfrequencymodulation->two stepsandthenon-Markovproportionofharmonic->chevronwererobust predictors.

Inbumetanide-treatedWTmice,Lassomodelsidentifiedsomesequences(e.g.,complex->upwardfrequencymodulationandtwo-step>upwardfrequencymodulation;SupplementalFigureS10),allofwhich

Figure6. Neonatalvocalizationpredictorsforsocialinteractionat6weeksofage.Thefractionofdevianceexplainedandthecoefficientsofvariableswere determinedbytheLassoregressionmodel.Allparameters(calltypenumberandproportions,callsequencenumber,Markovprobabilities,andnon-Markov proportions)wereusedforselection.Lassomodelsidentifiedseveralpredictorsforsocialinteractionscoreswithineachgroup.Eachinsetshowscallsequences thatweresignificantlycorrelatedwiththeindividualpost-pubertalsocialinteractionscores(seeSupplementalTableS2and Figure6).TheMarkovprobabilities, non-Markovproportions,andnumbersoftwo-callsequenceswereselected.Thesmallvaluesalongthefractionofdevianceexplainedaxisarerobustpredictors ofsocialinteraction.Ha,harmonic;Ch,chevron;Co,complex;Df,down-frequencymodulation;Fl,flat;Ms,multi-step;Rc,reversechevron;Sh,short;Sd,stepdown;Su,step-up;Ts,two-step;Uf,up-frequencymodulation,MP,Markovprobability;P,non-Markovproportion;#,number.

weresignificantlycorrelatedwiththepostpubertalsocialinteraction scores(SupplementalFigureS10inset;SupplementalTableS2).

Inbumetanide-treatedKOmice,aLassomodelselectedtheMarkov probabilityofflat->upwardfrequencymodulationandupwardfrequency modulation->upwardfrequencymodulationandnumberofshort->short asthemostrobustpredictorsforsocialinteraction(SupplementalFigure S10),noneofwhichweresignificantlycorrelatedwiththesocialscores (SupplementalFigureS10inset;SupplementalTableS2).

Discussion

Wehypothesizedthattheintracellularconcentrationofchlorideionsin neuronsaroundthetimeofbirthisacriticaldeterminantforthecausal sequentialdevelopmentofneonatalandpostpubertalsocialbehaviors.

Thishypothesiswasbasedonthepioneeringreportthatbumetanide, anNKCC1(Na+ -K+ -2Cl cotransporter)inhibitorthatcontrolsintracellularchlorideionconcentration,restoredneonatalvocalizationsinnoncongenic Fmr1 KOpups(8).Weusedacongenic Fmr1 KOmouseto determinethepredictivevariablesofneonatalvocalizationforthepostpubertalsocialinteractionscoresaswellastheimpactofbumetanide onthesedevelopmentalvariables.Ourdatashowedthat(1) Fmr1 deletionreducedthenumberofspecificneonatalcalltypesandprobabilities ofcallsequences;(2)bumetaniderestoredtheseneonatalphenotypes; (3) Fmr1 deletionreduceddirectsocialinteractionat6weeks,butnotat 8weeks,ofage;(4)bumetanidenonspecificallyreducedthepostpubertal socialinteractionlevelinWTandKOmice,resultinginindistinguishable

socialinteractionlevelsat6weeks;(5)distinctneonatalvocalizationcall sequencespredictedthepostpubertalsocialinteractionlevelinvehicletreatedWTandKOmiceat6weeks;and(6)suchpredictorsdidnotexist inbumetanide-treatedWTandKOmicebecausebumetanidenonspecificallyreducedsocialinteractionscorestothelevelwherethetwogenotypegroupswereindistinguishable.

Unlessunavoidable,wewilldiscussonlythosestudiesthatusedcongenicorcoisogenic Fmr1 KOmicelinesoranF1hybridlinewherethehomogeneousgeneticbackgroundsofWTandKOmicearemaintained.Noncongenicmicewith“mixed”geneticbackgroundsconsistentlycarrymore breederlineallelesinWTmiceandmoreEScellalleles(e.g.,129/Sv)inKO micenearthedeletedgene(10).Suchmodelsdonotidentifyphenotypes thatgenuinelyreflecttheimpactof Fmr1 deletionalone.Moreover,we limitourdiscussiontostudiesthatmanuallyclassifiedcallsorusedautomaticcallclassificationsystemswithlowfalsenegativeandfalsepositive ratesbecausehighfalsenegativeandpositiveratescompromisetheaccurateevaluationofneonatalcalls(26).

Weobservedthatmalecongenic Fmr1 KOmiceemittedfewercallsof specificcalltypes(harmonicandflat)butexhibitednormalpercentages ofallcalltypes.Previouswell-controlledstudiesidentifiedalterationsof variousneonatalvocalizationparametersincongenic Fmr1 KOpups.Congenic Fmr1 KOpupswithanFVBbackgroundemittedmoretwo-syllable andfrequencystepsatP7thanWTpups;however,sexwasnotspecified inthisstudy(13).Inanotherstudy,malecongenic Fmr1 KOpupswith anFVBbackgroundemittedfewercallsatP9andP13andmorecallsat P12thanWTpups(27).Ourresultsareconsistentwiththisobservationin thatKOpupsemittedfewercallsthanWTpups(see Figure1 inset).While poweranalysesindicatethatstatisticallysignificantdifferencesmight beachievedwithlargersamplesizesforchevron(n = 22),step-up(N = 13),up-frequencymodulation(N = 15),alltheothercalltypesareestimatedtorequiremuchlargesamplesizes(seeSupplementalTableS2and Figure1,Poweranalysis).Thus, Fmr1 KOaffectsharmonicandflatcall typesmorerobustlythanothercalls.

Whencallswereanalyzedbyproportions,wefounddecreasedproportionsofharmonicandtwo-stepcallsandhigherproportionsofchevron inKOpups.However,noneofthesealterationsreachedstatisticalsignificance.Inapreviousstudy,male Fmr1 KOpupswithanFVBbackground producedproportionallymorechevronandfrequencystepcalltypesand decreasedproportionsofcomplex,composite,downward,harmonic,twosyllable,andshortcalltypesatP8(21).Alargersamplesizeoftheirstudy (WT, n = 17;KO, n = 13)isonelikelyfactorforsignificanceinmorecall typesintheirstudythaninourstudy(WT, n = 9;KO, n = 12).Poweranalysesshowedthatwithlargersamplesizes,vehicle-treatedWTandKO micearelikelytoachieveastatisticallysignificantreductioninproportionsofharmonic(N = 13)andstep-up(similartotwo-syllable, N = 45) callsandincreasesindown-frequencymodulation(similartodownward) (N = 39)andshort(N = 48)calls(SupplementalTableS2;SupplementalFigureS1).Theothercalltypesrequiremuchlargersamplesizesto achievestatisticalsignificance:chevron(N = 69),frequencystep(multiplesteps, N = 830;twosteps, N = 704),complex(N = 638)calltypes (SupplementalTableS2;SupplementalFigureS1).Togetherwiththese poweranalyses,ourdataclearlyindicatethat Fmr1 deletionimpactsthe proportionsofharmonic,down-frequencymodulation,step-up,andshort callsinthisorder.

Apreviousstudydemonstratedthatnon-congenic Fmr1 KOmiceexhibited higher probabilitiesofchevronanddownwardcalltypesthanWT pupsatP8(8).Ourstudyshowedthatthepercentagesofchevronand down-frequencymodulationcallswerehigherinmaleKOpupsthanin maleWTpupsatP7(SupplementalFigureS1),andbumetanidetended tocorrectthesetrendswhenusingsamplesizes(n = 9–12)similarto thoseinthepreviousstudy(8)(n = 9–13).However,noneofthesetrends achievedstatisticalsignificanceinourstudy,althoughapoweranalysisindicatesthatanincreasedproportionofdown-frequencymodulation (N = 39)andchevron(N = 69)callsinKOmicemayachievesignificance withmuchlargersamplesizes(seeSupplementalTableS2andSupplementalFigureS1).Severalfactorsarelikely.First,thestudybyTyzioand colleagueseliminatedpupsthatemittedlessthan50callsduringa3minperiod,butwedidnoteliminatesuchcasesbecauseweconsidered

lowcallnumbersasaphenotype.Second,ourandtheirrecordingtime durationswere5minand3min,respectively.Thephenotypetheydetectedmightoccurinthefirst3minofourtesting.However,thiswas notthecase;whendataofthefirst3minwereanalyzed,westilldidnot findstatisticallysignificantincreasesinchevronordownwardfrequency modulation(SupplementalTableS2,Figure1_Number_3min;SupplementalTableS2,Figure1_Proportion_3min).Third,thestudybyTyzio andcolleaguesdidnotdeterminethesexofmiceused,whereasourstudy usedmalesonly.Fourth,theirstudyanalyzedandpresentedonlychevron anddownwardcalltypes.Despitethesemethodologicaldifferences,our dataareconsistentwiththeirfindingthat Fmr1 deletionalterstheproportionsofspecificneonatalcalltypes,andtheeffectisnormalizedby bumetanide.OurstudyfurthershowedthatbumetaniderestoreddefectivecallsequencesofKOpupstolevelssimilartothoseofWTpups.While alargersamplesizemightidentifymorecalltypesthatdifferbetween thegenotypes,itisclearthatsomecalltypes(e.g.,harmoniccalltypes) aremoreeasilyaffectedby Fmr1 deletionthanothers.

OurobservationsfurtherextendedthestudyofTyzioandcolleagues byincludingananalysisofpostpubertalsocialinteractionandtheeffects ofbumetanideonthisphenotype. Fmr1 deletionimpairedpostpubertal directsocialinteractionat6butnot8weeksofage.Previousstudiesof Fmr1 KOmicedidnotconsistentlyfindrobustsocialinteractiondeficits. Congenic Fmr1 KOmiceshowedhigher,indistinguishable,orlowerlevels ofactivedirectsocialinteractionthanthoseofWTmice(28–34).These studiesused8-to24-week-oldmice.Ourandothers’datafrom8-weekoldmiceindicatethat Fmr1 deletionhaslittleornoeffectonsocialapproachandsociabilityatthisage(32–34).Ourobservationssuggestthat detectabledeficitsinaffiliativesocialinteractionappearat6weeksof age.Moreover,wedetectedastatisticallysignificantgenotypedifference afterexcludingcaseswhereeitherstimulusortestmiceexhibitedaggressivebehaviorsorhyperactivity(see Results).Itmightbedifficulttodetectasubtledifferenceinaffiliativesocialinteractionin Fmr1 KOmiceif suchconfoundingfactorsarenoteliminatedornotdetectableinathreechamberapparatuswhereaggressiveandaffiliativesocialapproachcannotbeseparatedandareequallyrecordedasmoretimeinthevicinityof acagedstimulusmouse.Ingeneral,theratherweakdefectsinsocialbehavioronagroupbasisin Fmr1 KOmicearecongruentwithclinicalobservationsthatindividualswith Fmr1 deletionsshowincompletepenetrance forthefullcriteriaofASDdiagnosis(35).

Anovelaspectofthepresentstudyisthatweidentifiedneonatal vocalizationsequencesthatbestpredictpostpubertalsocialinteraction scores.Thenumberofflat->chevronandMarkovprobabilityofchevron>shortsequenceweresignificantlycorrelatedwiththepostpubertalsocialinteractionscoresinapooleddataofvehicle-treatedWTandKO mice(SupplementalFigureS9A).Theseparameterswerealsoidentifiedasthemostrobustpredictorswhenthebestpredictorswereexploredwithineachgenotype(see Figure6).Inotherwords,thelevelof thesecallsequencescanprovideinsightsintothefuturedevelopmentaltrajectoryofsocialinteraction.Thisobservationisnotinconsistent withthehypothesisthatacommondevelopmentalmechanismexistsbetweenneonatalsocialcommunicationandpostpubertalsocialinteractionandthatthenormalpostpubertalsocialinteractionrequiresnormal neonatalsocialcommunication,includingthenumberofflat->chevron sequence.Whilethebiologicalsignificanceofthesecallsequencesis notclear,wepreviouslydemonstratedthatalteredcallsequencesina mousemutantforanothergeneimplicatedinneurodevelopmentaldisorderslostthecapacitytoelicitmaternalapproach(11).Moreworkis neededtocriticallyevaluatewhetherthecallsequencealterationof Fmr1 KOpupscontributestoacausalchainfromthegenotypeofpups,impairedmaternalcare,andimpaireddevelopmentofsocialandcognitive capacities(17, 18).

Whilethenumberofflat->chevronwasidentifiedbyaLassomodelas apredictorforthegenotype(SupplementalFigureS9B),thisvariabledid notclearlydiscriminateWTandKOgenotypes(seeSupplementalTable S2andSupplementalFigureS9B).Thisweakdiscriminatingpowerofthe Lasso-identifiedvariableislikelyduetotheoverlappingnatureofsocial interactionscoresbetweenWTandKOmiceandaveryweakdifference insocialscoresbetweenthetwogenotypes(Figure5A).Inmoregeneral

terms,theneonatalvariablesmightbemoresuitableinpredictingthe continuousnatureofpostpubertalsocialscoresthanthecategoricalclassificationofthegenotype.

AlthoughtheLassomodelsextractedpredictiveneonatalcallvariablesforthesocialinteractionscoresofeachgenotypewithbumetanide treatment,nomodelsachievedstatisticallysignificantcorrelationcoefficientswiththesocialinteractionscoresinbumetanide-treatedWTandKO mice(Figure6).Onereasonforthelackofcorrelationsisthatbumetanide treatmenteliminatedtheeffectsofgenotypeonpostpubertalsocialinteractionbynonspecificallyreducingthesocialinteractionlevelsinWT andKOmice(see Figure5).Thus,theeffectsofbumetanideonneonatal andpostpubertalsocialbehaviorsaredissociated;bumetaniderestored neonatalsocialcommunicationbuthadnonspecificnegativeeffectson postpubertalsocialinteractioninWTandKOmice.Thisdissociationcould beinterpretedassuggestingthatdistinctmechanismsexistforneonatalsocialcommunicationandpostpubertalsocialinteraction.Morework isneededtocriticallyevaluatethemechanisticoriginsofneonatalsocial communicationandpostpubertalsocialinteraction.

Thisresultdoesnotsupportthehypothesisthatbumetanide,when givenaroundthetimeofbirth,hasbeneficialeffectsonpostpubertalsocialimpairmentsinpatientswithfragileXsyndrome.However,possibilitiesremainthatlowerdosesofbumetanidehavemorespecificameliorativeeffectsonthelatersocialinteractionorthatthisdrughasbeneficial effectsonothertypesofsocialbehaviorsuchassocialincentivelearning (36)ormaternalsocialbehavior(11, 37)ornonsocialbehaviors,includingsensoryhypersensitivity(38)andtheircellularcorrelates(38, 39).Alternatively,distinctmechanisticoriginsanddifferentialdependenceon peripartumGABAsignalingmightexistforneonatalsocialcommunicationandpostpubertalsocialinteraction.Moreworkisneededtoexplore thepossibleeffectsofbumetanideadministeredatpostnatalorearlier embryonicperiods.

AlthoughbumetanideislargelyineffectiveinalleviatingASDsymptomsinhumans,thistreatmentisgenerallystartedafteradiagnosisof ASDat2–3yearsofage.Iftheperinatalperiodisthecriticalperiodforthe therapeuticeffectsofbumetanideonneonatalsocialcommunication,as suggestedbyourobservations,itstherapeuticeffectswouldbeexpected tobemostrobustwhenitisappliedperinatallyanditsoutcomeisevaluatedmuchearlier.Ourobservationsandcomputationalapproachesprovideatemplateforfutureworktoexplorethecausallydistinctneuronal substratesthatsubserveneonatalandlaterASD-linkedbehaviors.

Methods

Mice

WeusedmaleFVB.129P2-Pde6+ Tyr c-ch Fmr1tm1Cgr /Jmice(Fmr1 /y , #004624,JacksonLaboratory,BarHarbor,ME,USA)andtheirWTlittermates.Wechosemalemice,asthesymptomsofFragileXsyndrome inhumansaremoresevereinmalesthanfemalesandfemalepatients tendtoexhibitagreaterdegreeofinterindividualvariability(40).These miceweregeneratedbycrossingmale Fmr1 /y mice(12–24weeksold) withfemale Fmr1+/ mice(8–20weeksold)asbreeders.TheirgenotypesweredeterminedbyPCRusingthefollowingprimers:5’-TGTGATA GAATATGCAGCATGTGA-3’,WTforward;5’-CACGAGACTAGTGAGACGTG-3’, homozygousforward;5’-CTTCTGGCACCTCCAGCTT-3’,reverseforboth genotypes.

This Fmr1 mutantstrainoriginallycontained129P2/OlaHsdalleles derivedfromE14EScells,butwasconvertedtoacongeniclinethrough 11generationsofbackcrossingofmutantmicetotheFVBstrain.This backcrossingeliminatedbothcopiesofthe Pde6b mutantallele,agene responsibleforretinaldegeneration;mutantmicedonotsufferfrom retinaldegenerationorblindness.Becausethisisacongenicstrain,the confoundingeffectsofunequallyenriched129P2/OlaHsdallelessurroundingthe Fmr1 geneinmutantsandthoseofFVBallelesintheirWT littermatesareminimized(10).

Treatment

Femalebreederswereexaminedforaplugeveryday;whenitwaspresent, thiswasdefinedas0.5dayspostcoitum(dpc).Pregnantfemaleswere thenseparatedfromtheirmalepartners.Inthelastweekofpregnancy, cagebeddingwasnotchangedtominimizestress.Micewererandomly

assignedtoeitherthevehicleorbumetanidetreatmentgroup.At18.5 dpc,bumetanide(#14630,CaymanChemicalCompany,AnnArbor,MI, USA)orvehiclewasgiveninthedrinkingwater,basedontheexpected volumeconsumptionandthedam’sbodyweight.Thistimepointwaschosenbasedonapublishedstudy(8).Ourpilotstudyshowedthatmiceconsumeatleast5mlin24h,andthesolutionconcentrationwasadjusted, basedonthedam’sweight,togreaterthan2mg/kgbumetanide.Thisregimenachievedthetargetdosewhenpartumwasconfirmedatandafter 19.5dpc.Theconsumeddoseofbumetaniderangedfrom2.928to 4.12mg/kg(SupplementalTableS1).

Behavior

Malecongenic Fmr1+ /y (WT)and Fmr1 /y (KO)micewereusedfor behavioralanalyses.

UltrasonicVocalization

WhenmalepupsreachedP7,thecagecontainingthemotherandthelitterwastransferredtothetestroom30minbeforetesting.Thisdevelopmentaltimepointwaschosen,asapreviousstudyshowedthat Fmr1 KO pupsdifferedfromWTpupsinvocalizationsatP7,butnotatP4orP10 (13).Moreover,anotherstudytestedtheeffectsofbumetanideonvocalizationsatP8(8).Eachpupwasthenmovedtoatestchamber(18cm long × 18cmwide × 30cmhigh).Ultrasonicvocalizationswererecorded for5minusinganUltraSoundGate(Avisoft,Germany)connectedtoa computerequippedwithAvisoft-RECORDERsoftware(Avisoft).Thesamplingratewassetto250kHz(format,16bit).Thelowcut-offfrequency wassetat10kHztoreducebackgroundnoiseoutsidetherelevantfrequencyband.Thefrequencywindowforanalysisrangedfrom15to150 kHz.Calldetectionwasperformedusinganautomaticthreshold-based algorithmandaholdtimemechanism(holdtime:10ms).

Theweightsofdamsweremeasuredtocalculatethetotaldoseof bumetanidetheyconsumed.However,wedidnotmeasurethebody weightsofpups.Thepups’bodyweightsarenotalteredinthisspecific mousemodeloffragileXsyndrome(Jax#004624)comparedwiththose ofWTlittermates,andnocorrelationofbodyweightswithchangesincall typeswasreported(41, 42).

AffiliativeSocialInteraction

Malemicethatweretestedforneonatalvocalizationweresequentially testedforsocialinteractionat6and8weeksofage.Thetestsubjectsand age-matchedmale Fmr1+/y non-littermates,usedasstimulusmice,were habituatedtothetestroomfor30min.Thetestandstimulusmicewere simultaneouslyplacedinatestapparatus(20cmlong × 28cmwide × 15cmhigh;50lux),andtheirbehaviorwasrecordedfor5min.Underthis experimentalcondition,micegenerallyexhibitlowlevelsofaggressive behaviors(12, 43–47).Weusedthisnaturalistictestinsteadofthethreechambersociabilitytestapparatusbecausetheformerisrecommended forultimatevalidationandthelatterhasmanytechnicalandinterpretive issues(22, 48).Raterswereblindedtothegenotypeandtreatmentuntil testingandscoringwerecompleted.

ComputationalAnalysis

CallTypeClassification. Sonogramswereinspected,andgenuinenoises wereeliminatedfromtheanalysis.VocalMatsoftware(26)wasusedto determinecalltypes.Thissoftwarehasthelowestfalsepositiveandfalse negativeratesamongallcalltypeclassifiers(26).Onemodificationwas applied.VocalMatdetectsonlysalientelementsinsonogramsofwhatwas classifiedas“harmonic”inourpreviousstudies(11, 12, 45)andclassifies suchelements(e.g.,step-upandstep-down)ascalltypes.Toavoidthese falsenegativecases,wemanuallyinspectedallcalltypesandreclassified suchcasesasharmonic.

UMAP. WeusedtheUMAPmethod(49)toindependentlyclassifycall typesandevaluatetheimpactof Fmr1 /y andbumetanidetreatmenton calltypes.UMAPisadimensionalityreductiontechniquebasedonRiemanniangeometryandalgebraictopologythathelpsclusterdatawith similarfeatures.WeutilizedthePythonlibrary“umap-learn”(version 0.5.5),andthequantitativeparametersofVocalMatwereusedasinputs. Thequantitativeparametersincludedthelength(duration)andbandwidthofeachcall,theminimumandmaximumfrequencyvaluesinkHz

(min_freq_main,max_freq_main,mean_freq_main,min_freq_total,max_ freq_total,mean_freq_total),andsoundintensityindB(min_intens_total, max_intens_total,andmean_intens_total)ofvariouscomponentsofeach call,where“main”and“total”designatethemostintensewavecomponentandallwavecomponents,respectively,ofeachcall.Additionally,the followingparametersweresetforUMAPcalculation:random_state,0and n_neighbors,30.Theremainingparameterswereleftattheirdefaultvalues.AftercallclusterswereidentifiedbyUMAP,welabeledeachdata pointbasedonthemodifiedVocalMatcalltypeclassification.

Totestthevalidityandrobustnessofthisapproach,wecreatedbootstrappedclustersfromrandomlychosendatapointswithineachcalltype 2000times.Eachrandomselectiongeneratedthemediandatapointfor eachcalltype.These2000medianvaluespercalltypewereplottedand comparedwiththepositionsofthedatapointdistributionofeachcallin UMAPs.Thepositionsofthesemedianvaluesclusteredatthecenterof eachcalltypeofUMAP,therebyvalidatingtheUMAPdata.

CallSequenceAnalysis. Aswereportedpreviously(11, 12),wequantitativelydefinedacallsequenceasaseriesofcallswithintercallintervals belowtheintersectionbetweenthetheoreticalandobserveddistribution curves.Twocallswithanintercallintervallongerthanthecross-pointof thetwocurveswereconsideredtobelongtothelastandfirstcalloftwo distinctcallsequences.Two-callpairswithinso-definedsequenceswere thenusedforMarkovmodeling,usingourpublishedprocedure(11, 12). Therewere0countsofsomecallpairsinsomeanimals.Acountof1was addedtoallcallpairsofeachanimaltoavoid0probabilities.

LassoModel. WeappliedtheLassoregressionmodel,followingourpreviousmethod(12),toextractpredictivevariablesofthenumberandproportionofeachcalltypeandtwo-callpairswithinsequencesforindividual socialinteractionscores.

StatisticalAnalysis

Allcomputerprogramsanddataareavailableuponrequest.Analysisof variance(ANOVA)wasusedtocomparemorethantwogroups,andStudent’sunpairedandpaired t-testswereusedforcomparisonsoftwo groups.ThenormalityandhomogeneityofvarianceofdatawereevaluatedusingtheShapiro–WilkandLevenetests,respectively.Wheneither assumptionwasviolated,datawereanalyzedusingtheMann–Whitney testforunpaireddataandtheWilcoxonnonparametrictestforpaired data.However,whenasamplesizewastoosmallforthenormalitytest (n < 10),thehomogeneityofvariancealonewasusedtodecidewhether toanalyzedatawithparametricornonparametrictests.Theminimumsignificancelevelwassetat p < 0.05.Whenmorethantwotestswereappliedtoadataset,thesignificancelevelwasadjustedusingBenjamini–Hochbergcorrectionata5%falsediscoveryrate(FDR).Allstatistical valuesareprovidedinSupplementalTableS2;theoriginal p valuesthat remainedsignificantafterthisadjustmentareshowninfigurelegends. Excludedcasesaredetailedinthe Results section.

StudyApproval

AnimalhandlingandusefollowedprotocolsapprovedbytheAnimalCare andUseCommitteeofHirosakiUniversityGraduateSchoolofMedicine andwereinaccordancewiththeRulesforAnimalExperimentationofHirosakiUniversity.

DataAvailability

Allrawdataandsupportinganalyticalcodeareavailableuponrequest. AllstatisticaldataareprovidedinSupplementalTableS2.Allreagents andthemousemodelarepubliclyavailable.

Acknowledgments

WethankMs.SachikoKamikawa,Mr.DaikiTsushima,Ms.YurikoTakagi, andMs.TakakoTakehanafortechnicalassistance.

AuthorContributions

YSconductedanddesignedallexperimentsandwrotethemanuscript.TT analyzedalldata,madeallfigures,andwrotethemanuscript.SScollected data.THanalyzedalldata.KNoversawtheentirework,designedallexperiments,andsupervisedYSandSS.NHoversawtheentireanalysisof

alldata,supervisedTTandTH,analyzedalldata,madefigures,andwrote theentiremanuscript.

Themanuscripthasbeenreadandapprovedbyallauthors.Allauthors takefullresponsibilityforalldata,figures,andtextandapprovethecontentandsubmissionofthestudy.Norelatedworkisunderconsideration elsewhere.Allauthorsstatethatallunprocesseddataareavailable,and allfiguresprovideaccuratepresentationsoftheoriginaldata.

Correspondingauthors:ProfessorKazuhikoNakamuraforanyaspect oftheworkexceptfordataanalysesandProfessorNoboruHiroifordata analyses.Thesecorrespondingauthorstakefullresponsibilityforthesubmissionprocess.

FundingSources

Theresearchreportedinthispublicationwaspartlysupportedby theNationalInstitutesofHealth(R01MH099660;R01DC015776; R03HD108551;R21HD105287toNH),theJSPSKAKENHI(JP19K17103 toYS),andtheHirosakiInstituteofNeuroscience,Japan(KN).Thecontentissolelytheresponsibilityoftheauthorsanddoesnotnecessarily representtheofficialviewsoftheNationalInstitutesofHealth.An open-accesslicensehasbeenselected.

AuthorDisclosures

Theauthorshaveconfirmedthatnoconflictofinterestexists.Thecorrespondingauthorshadfullaccesstoallthedatainthestudyandhadfinal responsibilityforthedecisiontosubmitforpublication.

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GenomicPsychiatry

RESEARCHARTICLE

Anovelneurodevelopmental-neurodegenerativesyndromethatcosegregateswitha homozygousSPAG9/JIP4stop-codondeletion

NataliaAcosta-Baena1 , 2 ,JohannaTejada-Moreno1 ,AlejandroSoto-Ospina1 , 2 , 3 ,AlejandroMejía-García1 ,MauricioPreciado1 , JessicaNanclares-Torres1 , 2 ,MaríaAntonietaCaro1 ,WinstonRojas1 ,GloriaP.Cardona-Gómez2 ,LucíaMadrigal2 ,MauricioArcos-Burgos4 , andCarlosAndrésVillegas-Lanau1 , 2

Thisreportoutlinestheclinicalfeaturesofacomplexneurologicalphenotypesharedbythreesiblingsfromaconsanguineousfamily, characterizedbyintellectualdisabilities,speechdevelopmentaldelay,gaitdisturbance,cerebellarsyndromesigns,cataracts,anddysmorphic features(squareandcoarsefacialfeatures,thicklips,deeppalate,smallandspacedteeth,low-setears,strabismus,eyelidptosis,andblond hair).Seizuresandbrainatrophywerelaterevident.Inthecosegregationanalysis,fivefamilymembersand12familycontrolswerestudiedby whole-exomeandSangersequencing.Thestructuralandfunctionaleffectsoftheproteinwereexploredtodefinethemutatedvariant’s potentialdeleteriousimpairment.Neurologicalandneuropsychologicalfollow-upsandbrainmagneticresonanceimaging(MRI)were performed.WeidentifiedasingleframeshifthomozygousnucleotidedeletionintheSPAG9/JIP4gene(NM_001130528.3):c.2742del(p. Tyr914Ter),causingaprematurestopcodonandtruncatingtheproteinandoriginatingapossiblelossoffunction.Thevariantcosegregatedin affectedindividualsasanautosomalrecessivetrait.Theinsilicoproteinfunctionalanalysesindicateapotentiallossof66phosphorylationand 29posttranslationalmodificationsites.Additionally,amutatedproteinstructuremodelshowsasignificantmodificationofthefoldingthat verylikelywillcompromisefunctionalinteractions.SPAG9/JIP4isadynein-dynactinmotoradapterforretrogradeaxonaltransport,regulating theconstitutivemovementofneurotrophicfactorsignalingandautophagy-lysosomalproducts.Understressconditions,itcanpotentiatethis transportbythep38mitogen-activatedproteinkinases(p38MAPK)signalingcascade.Bothfunctionscouldbeassociatedwiththedisease mechanism,alteringtheaxon’sdevelopmentandgrowth,neuronalspecification,dendriteformation,synaptogenesis,neuronalpruning, recyclingneurotransmittersandfinally,neuronalhomeostasis—promisingcommonmechanismstobeusedwithinvestigationalmoleculesfor neurodevelopmentaldiseasesandneurodegeneration.

GenomicPsychiatry January2025;1(1):73–84;doi: https://doi.org/10.61373/gp024a.0052

Keywords: Intellectualdisability,neurodevelopment,neurodegeneration,dementia,syndrome,axonaltransport,retrogradesignaling,signalingendosomes, MAPKp38signalingpathway,dynein-dynactinmotoradapter

Introduction

Themoleculartransportofmoleculesattheintracellularlevelisessential toacell’sdevelopmentandsurvival(1).Thischallengeforneuronsispermanentduetotheeverlastinganddistantpolarizationbetweentheaxons andtheneuronalbody.However,distanceisnottheonlychallenge;localizeddeliveryofpresynapticcomponentsmustbesuccessfullyovercome tomaintainsynaptictransmission(2).Tocarryoutthisprocess,neurons use“axonaltransport”toshipmultiplesubstancesthatmovealongthe microtubulesoftheaxoninabidirectionalway(3, 4).

Thekinesincomplexdrivesanterogrademovementtransport(from thesomatotheaxontip)andshipstransportsubstancessuchas RNA,proteins,andorganellestogrowthconesandsynapses(5). Theopposite,retrogrademovement(fromtheaxontotheneuronalbody),isdyneindependentandimportantforneurotrophicfactorsignaling(6),autophagy-lysosomal-autophagy,degradation,and nerveregeneration.Themachineryforthisaxonaltransportincludes motorandmicrotubuleproteinsandessentialadapters(7).Furthermore,proteinkinasesignalingpathwaysandposttranslationalmicrotubulemodificationsarerequiredtoensureefficienttransportinto neurons(2).

Alterationsinaxonaltransportcanemergethroughseveralmechanisms:(1)defectsintheorganizationofthecytoskeleton,(2)alterations inthebindingofmotorproteinstomicrotubules,(3)abnormalkinase ordyneinactivities,(4)destabilizationofmotorcargobinding,and(5)

alterationsinmitochondrialdysfunctionenergy(8).Thusfar,inadequate andnonprogressiveretrogrademovementscandisruptsynapses,axonal growth,plasticity,andneuronalhomeostasis.Multipleneurologicaldiseasesareassociatedwithaxonaltransportdisorders(7, 9).

TheJIP4protein,encodedbythe SPAG9 gene,isadynein-dynactinmotoradapterthatfavorsaxonalretrogradeflow.JIP4isubiquitouslyexpressed,includingthecentralandtheperipheralnervoussystem(10). Proteinexpressionstudiesinbrain-derivedneurodevelopmentaxons haveshownhighlevelsofJIP4,whicharedetectedinlysosomalfractions andautophagicvacuoles(11).JIP4promotesandstabilizestheassociationwithdynactinwhileantagonizingkinesinbinding(12).Thus,inamutuallyexclusivemanner,retrogradetransportisactivated.JIP4isinvolved inpostnatalbraindevelopment(13)andneuronalhomeostasisbyintracellularmetabolitesrecyclingtomaintainneuronalhomeostasis(14).In humans,SPAG9/JIP4hasbeenassociatedwiththeprognosisofdifferent typesofcancer(15)butneverlinkedtointellectualdisabilityand/orcomplexneurodevelopmentalphenotypes.

Thisarticledescribesthreehomozygoussiblingswithamutantvariantofthe SPAG9 gene,affectedbyacomplexphenotypecharacterized bydevelopmentalandlanguagedelay,severelearningdifficulties,and motorcompromiseimpairment.Thefollow-upofthisfamilyformore than10yearshasalsosuggestedacognitivedeteriorationprogressive impairment,suggestingasubsequentneurodegenerativeprocess.Preliminaryfindingswerepresentedonaposter(16).

1 GrupodeGenéticaMolecular(GENMOL),UniversidaddeAntioquia,Medellín,Colombia; 2 GrupodeNeurocienciasdeAntioquia(GNA),FacultaddeMedicina,Universidad deAntioquia,Medellín,Colombia; 3 GrupodeInvestigaciónenAlimentos(GRIAL),Facultaddeingeniería,CorporaciónUniversitariaLasallista,Caldas,Colombia; 4 Grupode InvestigaciónenPsiquiatría(GIPSI),DepartamentodePsiquiatría,InstitutodeInvestigacionesMédicas,Facultaddemedicina,UniversidaddeAntioquia,Medellín,Colombia. CorrespondingAuthor: NataliaAcosta-Baena,UniversidaddeAntioquia,FacultaddeMedicina,GrupodeNeurocienciasdeAntioquia(GNA),SIU,SededeInvestigación Universitaria,AA1226,Calle62Número52-59,Medellín,Colombia.Phone/Fax:57-4-2196444.E-mail: natalia.acosta@gna.org.co Received:6March2024.Revised:6June2024.Accepted:19June2024. Publishedonline:5August2024.

Methods

StudySiteandFamily

Thisstudywascarriedoutatthe“UniversidaddeAntioquia,”Medellin, Colombia,incollaborationbetweenTheGrupodeNeurocienciasdeAntioquia(GNA)andGenéticaMolecular(GENMOL)researchgroups.The bioethicscommitteeoftheuniversity(ComitédeBioética,SedeInvestigaciónUniversitariaCBE-SIU)approvedtheprotocolstudy.Thesubjects whowerestudiedsignedtheinformedconsentformafteradetailedexplanationoftheobjectivesandproceduresofthestudy.Incaseswherethe subjectdidnotknowhowtosign,parentsorrepresentativesgaveconsent andsignedtheform.ThefamilywasidentifiedinAntioquia,Colombia,in 2011andclinicallyfollowedupwithsubsequentfollow-upsuntil2021.

CharacterizationofthePhenotype

Thephysician’steamfromtheGNAcarriedoutmedical,neurological,and psychiatricfollow-ups.Laboratorytests,brainmagneticresonanceimaging(MRI),andneuropsychologicalevaluationswereperformedonthe cases.Affectedindividualswereevaluatedatamedicalschoolpediatricneurologymeetingstaffattheuniversityhospitaltodiscussthecomplexphenotypeandtoconsidersomepotentialdifferentialdiagnoses. TheneuropsychologicalevaluationswerecarriedoutusingtheGNAprotocol,andtheWechslerIntelligenceScale(17)wasadditionallyincluded. Theseverityofthecaseswasassessedusingacomprehensivebatteryof structuredandsemi-structuredclinicaltoolsbasedontheDSM-5criteria, accordingtoIQmeasuresandfunctionalperformancescaleassessment indailylife.Acquireddementiaorcognitiveimpairmentwasdiagnosed accordingtoestablishedstandardcriteriausingneurologicalandneuropsychologicalevaluationtoolsandfamilyreports.Amoredetailed descriptionoftheseinstrumentsispresentedelsewhere(18).

GeneticAnalysis

DNAextractionfollowedastandardextractionprotocolwiththeperipheralbloodsamplesusingthesaltingoutmethod(19),andthesamples werestoredat 20°Cuntilsequencing.Macrogenperformedthewholeexomesequencing(WES).Thecodingregionsofthegenomeweresequencedbynext-generationsequencingusingtheIlluminaplatformwith anaveragecoverageof100X.TheSureSelectXTLibraryPrepKit(Target EnrichmentSystemforIlluminaVersionB.2,April2015)enrichedthelibrary(20).SequencingwasperformedonaHiSeq4000instrumentfollowingthestandardprotocoltoreacha100Xdeepreadaverage.Thedata wereprocessedusingtheHCSsoftware[HiSeqControlSoftware(HCS3.3) version3.3].SequencingdatawereconvertedtotheFASTQformatusingtheIlluminapackagebcl2fastqmodule[version2.16.0.10fromIllumina(21)].Thebioinformaticanalysiswascarriedoutwiththesedata afterward.

BioinformaticAnalysis

Thequalityofthereadswasevaluatedwiththefastqcv0.11.5tool fromtheBabrahamInstitute, http://www.bioinformatics.babraham.ac. uk/projects/fastqc (22).Subsequently,thesequencesweremappedtothe hg19referencegenomeavailableattheUniversityofCalifornia,Santa Cruz(UCSC)website, http://hgdownload.cse.ucsc.edu/goldenpath/hg19/ chromosomes/ usingtheBurrows-WheelerAligner,asimplementedin thebwa-0.7.12software, http://bio-bwa.sourceforge.net/ (23).VariantcallingwasperformedusingtheBroadInstitute’sGenomeAnalysisToolKitGATKtool(GATK)v3.8-1BestPracticesforGermline SNP&IndelDiscoveryintheWholeGenomeandExomeSequence https://software.broadinstitute.org/gatk/ (24).Fortheprocessofmarkingduplicates,thePicardv1.119tool https://broadinstitute.github.io/ picard/ wasused.Baserecalibrationprocesses(BQSR–basequalityscorerecalibration),thesearchforsingle-nucleotidepolymorphism (SNP)-typevariantsandINDELS(variantcalling)andvariantfiltering (hardfiltering)werecarriedoutconsideringtheprotocolsuggested bythetoolGATK(GenomeAnalysisToolKit)fromtheBroadInstitute (BestPracticesforGermlineSNP&IndelDiscoveryinWholeGenome andExomeSequence) https://software.broadinstitute.org/gatk/bestpractices/bp3step.php?case=GermShortWGS.Oncethevariantswere identified,theannotationwascarriedoutwiththewANNOVAR(25)and EnsemblVariantEffectPredictorprograms(26),whichmakeuseofthe

informationcollectedindifferentdatabasesandbioinformatictoolsfor thesearchoftheallelicfrequenciesofthevariantsinthedifferentcontinentalpopulationssuchas1000Genomes,ExAC,ESP6500andgnomeAD.

Theclinicalinterpretationofgeneticvariantswasconsideredbythe guidelinesproposedbytheAmericanCollegeofMedicalGeneticsand GenomicsandtheMolecularPathologyAssociation(ACMG)(27).The toolsInterVar(28) http://wintervar.wglab.org/ andVarSome(29) https:// varsome.com/ wereusedtoclassifyeachoftheidentifiedcandidate variants.

Thevariantswereprioritizedconsideringthefollowingcriteria:(1) Qualityofthesequences:Depthacrosssamples(DP>30).(2)Modeof inheritance:autosomalrecessive.(3)Allelicfrequency(MAF <0.01)ina populationdatabase(1000Genomes,ExAC,ESP6500,gnomAD).Wefilteredonlyvariantswithafrequencylessthan0.01withahigherprobabilityofcausingmajoreffectsandrareMendelianconditions.(4)Exomic variantsorvariantsatsplicingsiteswereincluded.(5)Pathogenicvariants,probablypathogenicorvariantsofuncertainsignificance—VUS— werealsoincluded.(6)Pathogenicitypredictors:variantscatalogedas deleteriousorpossiblydeleteriousbymorethanthreepathogenicitypredictors,including,thatis,SIFT,andPolyphem2Polyphen2,andpresenting withvalueshigherthan14bytheCADDpredictor,and(7)Thepotential relationshipwiththephenotypebyconsideringHumanPhenotypeOntologytermswereevaluated.

SangerSequencing

ThevariantidentifiedwasreplicatedusingSangersequencinginfour samples:twoaffectedindividualswithDNAavailableandbothparents.FinchTVversion1.4.0(Geospiza,Inc.;Seattle,WA,USA), http:// www.geospiza.com/Products/finchtv.shtml wasusedtoevaluatethe qualityofthechromatograms.WeusedAliviewversion1.18 http:// www.ormbunkar.se/aliview/ (30)andnovoSNPversion3.0.1 http://www. molgen.ua.ac.be/bioinfo/novosnp (31)toidentifyandanalyzecandidate variant.

FunctionalandStructuralAnalysis

Thebioinformaticsstudybeganwiththeanalysisoftheprimarysequence inFASTAformattocharacterizethesystemfromthepotentialperspective ofposttranslationalproteinmodificationsconsideringthecalculationof N-glycosylationofaminoacidswithamidegroupsintheirsidechain,from theprobabilityasestimatedbymeasuredwiththeNetGlyc1.0server software.

Similarly,theinteractionofO-glycosylationsitesbytheOHfunctional groupwasmeasuredwiththeNetOglyc4.0serversoftware.Likewise,the phosphorylationpatternsweremeasuredwiththeNetPhosK3.1server softwarefortheaminoacidswhosesidechainisserine,threonine,ortyrosineispresentinthesequenceoftheevaluationof17kinases:ataxiatelangiectasia(ATM),creatinekinaseI(CKI),creatinekinaseII(CKII), Ca2+/calmodulin-dependentproteinkinaseII(CaM-II),DNA-dependent proteinkinase(DNA-PK),epidermalgrowthfactorreceptor(EGFR),epidermalgrowthfactorreceptor-3(GSK3),insulinreceptor(INSR),protein kinaseA(PKA),proteinkinaseB(PKB),proteinkinaseC(PKC),proteinkinaseG(PKG),ribosomalS6kinase(RSK),Srcfamilykinases(SRC),celldivisioncyclegeneinSchizosaccharomycespombe,namedcdk1inmammals (cdc2),cyclin-dependentkinase5(cdk5),andp38mitogen-activatedproteinkinases(p38MAPK)(32–34).

Toidentifythethree-dimensionalstructureofSPAG9,portionsofthe proteinsequenceweresearchedforintheProteinDataBank.TheanalyzedstructurebyX-raydiffractionwitharesolutionof1.80Åwasconstitutedby70aminoacids(ID:2W83).Thisstructuralfragmentwasusedas atemplateforthecompletemodel(35).

TomodeltheproteinstructuresoftheSPAG9andtheSPAG9Tyr914Ter variants,three-dimensionalSPAG9/JIP4wild-type,aswellasotherlikely structures,wereobtainedfromtheAlphafoldrepositorybuiltbythe DeepMind-Evoformermodule(36).Fivemodelswithasignificantspatial arrangementconvergenceasmeasuredbythelocaldistancedifference test(37)werechosen.ThemachinelearningmodelingallowedthereconstructionofthetruncatedproteinencodedbytheSPAG9Tyr914Ter variant.Fromthefivemodels,weselectedtheonewiththebestmolecularscore(38).Theglobalalignmentbetweenthewild-typeandthe

Table1. Clinicalcharacteristicsofaffectedindividuals Case1Case2Case3

Ageatexam,years 363432

Gender MFF

Height(cm) 165168172

BodyMassIndex 27.529.124

Headcircumference(cm) 575858

Faciesandgeneralcharacteristics

Blondhair,thicklips,coarsefacialfeatures,squareface,deeppalate,low-setears,spacedteeth. Singlepalmarcrease,pescavus.

Skin Hyperpigmentedmaculesinthe periorbitalregionandface

Hyperpigmentationinperiorbital region

Reporteddyslipidemia ++−

Otherpathologies Asthma,rhinitis

Delayedpsychomotordevelopment/ Intellectualdisability +++

Languagedisturbance +++

Cataracts +++

Strabismus +++

Eyelidptosis BilateralUnilateral(left)

Seizures

Seizuresfrom34yearsofage

Gaitdisturbance Scoliosis.Walkwithbothfeet apartandwiththeballsofthe feetwithexternaldeviation

Parkinsonism

Cerebellarsyndrome (intention tremor,dysdiadochokinesia,ataxia, dysmetria)

Impairedtandemgait,slight outwarddeviationoftheballsof thefeet

Impairedtandemgait

AtaxiaDysmetriaDysmetriaanddysdiadochokinesis

Dystonia +−−

Dysarthria

Impairedcoordination +++

Pathologicalreflexes Suckingreflex Suckingreflex

Plantarreflexes

Urinaryincontinence +−−

Supranuclearpalsy

Nystagmus ++−

Psychiatricsymptoms/behavior Hepullsouthisnailsandteeth. Aggressiveness.

Apathy,abulia,compulsiontoeatIrritabilityanddisinhibition

Superiormember

Spasticity

Weakness

Hyperreflexia

Sensorydisability

Lowermember

Spasticity

Weakness

Hyperreflexia

Sensorydisability

mutantproteinmodelswasachievedwiththeNeedlemanWunschalgorithmappliedtotheBLOSUM62matrixandquantifyingtheRMSDstandarddeviationbetweenthemodels.Thethree-dimensionalmodelswere picturedwiththeChimeraU.C.S.Fvisualizerv1.1.1(39).

Results

ClinicalDescriptionoftheAffectedSiblings Twoparentsandthreeaffectedchildrenconstitutethenuclearfamily. Table1 detailsthesummaryofclinicalfindingsforeachindividual.

Case1. Male,firstborn,40weeks,normalpregnancy.Spontaneousvertexdeliverywithoutanycomplications.Averageweightatbirth.At1year oflife,significantmotordelaydevelopmentofbilateraleyecataracts. Headsupportisat4yearsold,butwalkingisdifficult.Hecouldnotchew andswallowfooduntilhewas6yearsold,forwhichhewasfedonlywith blendedfood.Thefirsttwowords,“mama”and“papa,”areat4yearsold. Attheageof30,thedevelopmentofcompleteoralsentencesbegins.At

ResearchArticle Acosta-Baenaetal.

34yearsold,developmentofgeneralizedtonic-clonicseizures.Entirely dependentonalldailylivingactivitiesbutwithbehaviorchangesinrecentyears.Hepulledouthisnailsandhisteethaggressivity.Functional scaleshavedeterioratedovertime.

Case2. Female,secondpregnancyproduct,withoutsignificantprenatal orperinatalhistory.Spontaneousvertexdeliverywithoutanycomplications.Holdtheheadandtrunkupat8monthsold.Walkingandpronunciationoffirstwordsaround16monthsold.Severelearningandmental disability(onlycansign).Currently,shepronouncesonlythreewords.She isindependentindailylifefunctionsandhelpshermotherwithsimple householdtasks.

Case3. Thethirdaveragepregnancyfemaleproduct.Spontaneousvertexdeliverywithoutanycomplications.Sittingat10monthsold,walking ageat15monthsold,andfirstwordsat24monthsold.Developedbilateralcataractsat4yearsold.Mildmotorandlearningdelaydevelopment.

Table2. Summaryofneuropsychologicalassessmentofaffectedindividuals

CognitivefunctionCase1Case2Case3

Mini-MentalStateExamination(MMSE)/30 61319

Orientation

FarbelowaverageFarbelowaverageBelowaverage

Verbalfluency/denomination 07Intheaverageageandschooling

Wordlistmemory/10 015

Evocationmemory/10 013

Visualmemory

CannottakethetestCannottakethetestFarbelowaverage

Attention CannottakethetestCannottakethetestCannottakethetest

Executivefunction

CannottakethetestCannottakethetestCannottakethetest

Visuoconstructivefunction CannottakethetestCannottakethetestCannottakethetest

Apraxia +++

FullscaleIQ

VerbalIQ

PerformanceIQ

Sheworksasalaundressinadairy—recentyearswithmarkeddisinhibition.

NeuropsychologicalAssessment. Thethreesiblingswereclassifiedwith moderateintellectualdisabilityaccordingtotheDSM-5criteria,andall threeobtainedanIQscoreof45(Table2).

BrainImaging. BrainMRIwasperformedforallaffectedindividuals.The MRIresultsshowheterogeneitybetweenallofthem(Figure1).Therewas nocorticaldysplasiaorpolymicrogyriainanyofthecases.Tractography bydiffusiontensorimagingarenormal.Cochlea,vestibule,andbrainstem arenormal.Inallthreesiblings,thereismildmicroangiopathy(Fazekas scalegrade1fordeepwhitematter).Ventriculomegalyispredominantly left,withslightprominenceofthesulcioftheuppercerebellarvermis. Itdrawsattentiontodecreasedirondepositsintheglobuspallidusand aslightincreaseintheputamen,beingmoresignificantincase3.Case 1attractsattentionformicrocephaly,decreasedfronto-occipitaldiameteraccordingtobiometricparametersbyageandsex(Figure1A)(40). Thehypoplasticcorpuscallosum(CC)withoutdysplasia,withadecreased thicknessofthegenu(40).Bilateralhippocampalmalrotationwithboth collateralsulciarevertical(Figure1B).Slightirondepositintheputamen.Case2withlowerlimitfrontal-occipitaldiameteranddecreased thicknessofthegenuofCC,lefthippocampalmalrotation.Incase3,we additionallyseemalrotationofthelefthippocampus.Theputamennucleusisseenwithmoresignificantirondeposits,andunliketheother cases,thereisagenesisoftheseptumpellucidum(Figure1B).Also,there isaverythinCC,mainlythesplenium.Lossofsubcorticalvolume,witha moredilatedfourthventricleandincreasedirondepositsinthedentate nucleus.

Identification,ClinicalInterpretation,andValidationof CandidateVariants

Thecompletefamilyincluded36members.Threeaffectedsiblingsand theirtwoparentswereavailableforWES.Weextendtheanalysesto theremainingfamilymembers,availablein17individualexomes.AhomozygousdeletionintheSPAG9gene (NM_001130528.3):c.2742del (p.Tyr914Ter) wasidentifiedinthethreeaffectedsiblings-heterozygous intheirparentsandamaternaluncle.Thisvarianthasnotbeenfound inotherfamilymembersanddisparatepopulationsaccordingto1000 Genomes,ExACandGnomADdatabases.Ithasnotbeenfoundinexomes (coverage:86.8)andgenomes(coverage:31.7),accordingtoVarSome (29). Figure2 showsthecompletegenealogyandtheresultsoftheSanger sequencingofthefourindividuals.

StructuralandFunctionalAnalysisoftheSPAG9/JIP4ModelProteinand ItsRelationshipwiththeSPAG9Tyr914

TheSPAG9/JIP4proteinisexpressedatthecytoplasmiclevelandinthe celllysosome,belongstochromosome17andhasamassof146,205(Da). Ithassixisoforms,andtheisoformwiththehighestfrequencyrelated totheinteractionwithkinesinistheisoformwithacompositionof1321

aminoacids(41–43).Itbindstodyneinandkinesin-1intheleucinezipper II(JIPLZII)domain(Figure3),allowingbidirectionalvesiculartransport alongthemicrotubulesandtheirdynamics(44).

Themutationfoundatposition914isassociatedwithanucleotide changethatproducesaterminationcodonduetoguanine(G)deletion.Thisstopcodonpreventstheproteinfromextendingfurtherand causinglossofstructuralinformation,butitdirectlyaffectsthethreedimensionalarrangement.Therefore,theproteinpresentedchangesin lengthandaminoacidcompositionduetothelackofassemblyof407 aminoacids.Inthecharacterizationofthesystem,theeffectontheposttranslationalmodificationsthattheproteincanundergoandthechanges itcangeneratewereanalyzed.Calculationofphosphorylationsites,OglycosylationandN-glycosylationsitesweremadefromtheprimarysequencestocharacterizethetwoproteins,asshownin Table3

ForSPAG9wt,thereare257phosphorylationsitesintotal(Table4). Therepeatednumbersareduetotheirbeingphosphorylatedbyvarious kinasesatthesamepositionwhentheyexceedthenormalizedphosphorylationvalueof0.500.Themutationidentifiedatposition914,which,in thiscase,producesastopcodon,meansthattheproteincannotextend furtherandlosesstructuralinformation,whichdirectlyaffectsthethreedimensionalarrangement.Regardingphosphorylation,duetothespatial effectfromposition909,thephosphorylationsconsidereduptothetotal SPAG9proteinwith1321aminoacidswerelost(Table4).Thesekinases contemplatepositionswithatriplepossibilityofphosphorylation,such asposition1188withanonspecificenzymeandtwoessentialenzymes (proteinkinaseAandproteinkinaseC)involvedincellsignaling.Inthe sameway,position1238withthekinasesp38MAPK,cdk5andGSK3;position1249withanonspecificenzyme,proteinkinaseCandcdc2;similarly,position1262withanonspecificenzyme,DNA-PKandATM.Finally, position1264hasphosphorylationlossofanonspecificenzyme,cdk5and p38MAPK.

RegardingtheO-glycosylations,106glycosylationsiteswereconsideredforSPAG9wt,whichconsideredaprobabilitygreaterthanthethresholdat0.5.WhentheSPAG9Tyr914Termutationoccurred,therewasadecreaseinthetotalnumberofO-glycosylationsites,amongwhichchanges appearedintheN-terminalaminoacidresidueduetotheeffectofthe changeinspatialarrangementwithposition16.Thesechangesconceive effectsbyearlyproteinterminationandlossofglycosylationforpositions 905,909,911,915,937,938,949,1179,1188,1238,1241,1242,1243, 1244,1246,1249,1256,and1262.ThesealterationsinO-glycosylation affectproteinpolarityandtheadhesionofothercirculatingproteinsystems.N-glycosylationwasalsoahighlyaffectedpost-translationalmodification.Sixmodificationsweremadeatpositions309,565,694,830, 939,and1176,butduetothemutationSPAG9 Tyr914Ter, onlyonemodificationwasperformedatposition309.Molecularmodelingallowedus toobtaintheSPAG9wtprotein,asshownin Figure4A andthevariantwith thestopcodoninthree-dimensionalalignmentwiththenativestructure, in Figure4B

Figure1. FindingsreportedonbrainMRIofthreesiblings.(A)Comparisonbetweenthethreecasesaccordingto1.Measurementsofthefronto-occipitaldiameter (FOD)andparametersoftheCC(GT:Thicknessofthegenu,BT:Thicknessofthebody,IT:Thicknessoftheisthmus,ST:Thicknessofthesplenium)2.Gradient ofirondepositsintheputamennucleus,fromlowesttohighest,withcase3beinghighest.3.Cerebellumandfourthventricle.(B)1.Bilateralmalrotation hippocampal(Case1).2.Agenesisoftheseptumpellucidum(Case3).

Discussion

Thisstudyreportsanewsyndromewithcongenitalalterations,neurodevelopmentdisorder,andneurodegeneration.Theexomesanalyzedfrom 17familymembers,Sangersegregationanalysis,andstructuralandfunctionalevidencehelpdeterminepossiblepathogenicvariantswithsignificanteffects.Withtheobservationspresented,wecanconcludethat

thisrarediseasecanbeassociatedwiththehomozygousdeletionof SPAG9/JIP4gene.ThefamilywasidentifiedfromtheruralareaofnorthernAntioquia,Colombia,whereageneticisolatewaspreviouslyreported, withafoundereffectandseveralgeneticdisordersassociatedwithapossiblegeneticbottleneck(45–47).

(Continued)

Thesethreesiblingspresentsimilarclinicalcharacteristics.Theyhad closescoresontheintelligencescale(IQ)despitesuchadissimilarMiniMentalStateExamination(MMSE),probablybecauseofthedifferences inlanguage.Inotheraspects,wecanseeheterogeneityinseveritydespitehavingthesamemutation.Themalecaseistheonewhohasthe

mostseveremotordisorder,withmoreincredibledifficultyinlanguage andfunctionality,inadditiontoseverepsychiatricsymptomsandfacial dysmorphism.Theyoungersister(case3)appearstohaveotheressential alterationsonMRI.However,functionally,sheperformedbetterindaily livingactivitiesandrevealedminordifferencesinfacialfeatures.

Figure2. Family’spedigreeandSangersequencing.(A)Pedigreeshowsfourgenerationsofthecompletefamily.Squaresaremen,andcirclesarewomen.The arrowindicatestheindexcase.Romannumeralsaregenerations,andArabicnumeralsrepresentthepositionofeachindividualinthefamily.Filledsquaresand circlesrepresentaffectedfamilymembers.Slash(/)indicatesadeceasedperson.Thesamelinebetweentwoindividualsrepresentsconsanguinity.Individuals withDNAsamplesareindicatedwithaplus(+)symbol.(B)Chromatogramsofthreesubjects(parentsandtwosiblings)wereavailableandvisualizedwith novoSNP(31).Thereferencesequenceisvisualizedinthefirsttrace.Variationishighlightedinredcolor.TheunaffectedparentsIII:1andIII:2are heterozygous (G/ ),andthetwoaffectedchildrenIV:1andIV:2arehomozygous( / )forthevariant (NM_001130528.3):c.2742del

ResearchArticle Acosta-Baenaetal.

Figure1.

Figure3. Localizationof (NM_001130528.3):c.2742del(p.Tyr914Ter) inthedomainsoftheSPAG9/JIP4protein.SchemeoftheSPGA9/JIP4proteinwiththe maindomainsandthelocationoftheidentifiedvariant.

AccordingtotheMRI,microcephalyandbilateralhippocampalmalrotationincase1wereevident.Hippocampalrotationcanbeginbetween gestationweeks21–32.Asymmetricaldevelopmentisexpected,withthe rightsidefasterthantheleft(48).Amalrotationorincompleteinversion ofthehippocampuscanbeexpectedin19%.However,itcanalsobeassociatedwithstructuralvariantsassociatedwithbraindevelopmentthat predisposetoepilepsytosomeextent(49).Itisstrikingthatthiscase hasbeentheonlyoneofthethreesiblingswhohaspresentedconvulsive episodesaftertheageof34.

TheCCisthemainstructurethatconnectsthecerebralhemispheres andintegratesmotor,cognitive,andsensoryinformation.Morphological anomaliescorrelatewithalterationsincognitiveandbehavioraldevelopment.Eachsubregion(genus,body,isthmus,andsplenium)isassociatedwithdevelopmentandfunctioninthecortex.Weobservedadecreaseinthesizeofthegenuandsplenium.Genuareprojectionsfrom theprefrontalcortex.Thespleniumhasfibersfromtheoccipital-parietal andtemporalcortex(50).FullmaturityoftheCCappearstooccurinearly adulthood.IthasbeensuggestedthattheincreaseinCCsizeatthatageis relatedtotheincreaseinaxonalsize(51).Inallthreecases,thereisventriculomegaly,possiblyassociatedwithsubcorticalatrophyandironaccumulationintheputamenandthedentatenucleus.Findingsthatcould beassociatedwithneurodegenerativechanges.Ironisthemostabundant metalinneurons;transportandstoragefailuresareprocessesassociated withneurodegenerativedisorders(52).Itisinterestingtoexplorethese findingsfurthersincetheywouldprovidecluestomechanismsnotyetunderstoodindementiasyndromes.

Thesecaseswereidentifiedinthefourthdecadeoflife,sowecanobservetheevolutionofaneurodevelopmentaldisorderwhosecauseremainsandappearstobeprogressing.Insubsequentevaluations,allsiblingshadworsenedinthefunctionalscales(datanotshown)andbrain atrophy.Alsonoteworthyistheonsetofseizuresatage34andworsening motorandbehaviorimpairmentincase1.Aspectsthatsuggestpossible degenerationafterthedevelopmentaldisorder.

OurfindingsconcludethatdeletionintheSPAG9gene (NM_001130528.3):c.2742del(p.Tyr914Ter) generateschangesin theproteinregardinglengthandaminoacidcompositionduetolack ofassemblyof407aminoacids.Thisaffectsposttranslationalmodifications,affectingkeysitesforN-glycosylation,O-glycosylation,and phosphorylation.Thesethree-dimensionaleffectsandmodificationsare responsibleforchangingthechemicalenvironmentoftheprotein,which impliesanalterationincellfunction,eitherbyactivatingametabolic pathwayorcellsignaling,suchastheinteractionwithcofactorsand ligandssuchasdyneinandkinesin-1,amongothers.Theidentified homozygousdeletionproducesadoubletruncatedprotein,withtheabsenceofimportantkinasephosphorylationsites,includingtwopositions forp38MAPK,inadditiontotheabsenceof24sitesforO-glycosylation andfivesitesforN-glycosylationforitsproperfunction.

JIP4proteinhastwoknownmolecularfunctions:(1)Dynein-dynactin motoradapterforretrogradeflow.(2)Scaffoldproteinthatpotentiates thep38MAPKsignalingcascade.Murinestudieswithadoubleknockout (KO)toJIP4haveshownneurodegeneration(11),butnopreviouscases withthisphenotypeassociatedwithJIP4mutationshadbeenreported. Thealterationsinneurodevelopmentandneurodegenerationseeninour patientscouldbeexplainedbypermanentalterationretrogradeaxonal transportandsignalingdeficienciesp38MAPKcascade.

p38MAPKregulatesseveralcellularfunctionsinthecentralnervous system,suchasmetabolism,secretion,migration,differentiation,apoptosis,andsenescence(53).Thecascadeconsistsofthreephosphorylation

levelstoactivatep38,startingwithaMAPkinase(MAP3K),thenaMAP2K, andfinallyp38MAPkinase.Proteinkinasesregulateaxonaltransportby phosphorylatingmotorandadapterproteinsandcargoesdirectlyandindirectlybymodifyingthemicrotubulenetwork(54).MAPKp38cannegativelyregulateaxonaltransport.Inpatientsandmicewithamyotrophic lateralsclerosis(ALS),overactivationofthissignalingpathwayproduces alterationsinaxonaltransportinthespinalcord(55).Scaffoldingproteins,suchasJIP4,recruitupstreamMAP2KandMAP3Ktoenhancethe activationofp38kinases(10).JIP4regulatesretrogradeandconstitutivetransportoflysosomes,butunderstressconditions,itactivates thep38MAPKsignalingpathwaywithposttranscriptionalregulationof TMEM55B.TMEM55BrecruitsJIP4todeliverdynein-dynactintolysosomalmembranes(56).DepletionofTMEM55BorJIP4resultsinthedispersaloflysosomestowardthecellperiphery(57).Moreresearchisrequired tounderstandalterationsinJIP4andtheirimpactonregulatingmTORC signaling(58).

TheshorteningoftheproteinwasbeforethepositionoftheWD40 domain(12).Proteinswiththistypeofdomainareassociatedwiththe functionofinteractingwithotherproteins(59).Thisdomainwouldgive itsscaffoldingfunction,whichisfundamentaltocriticalfunctionsinsignalingpathwaysandthegatheringofmultiplepartnerstofacilitateconcertedinteractionsandmolecularfunctions.Functionalstudiesarerequiredtodeterminetheimplicationsofthesefindingsforregulation,cell typesorspecifictissues,andmachineryinvolvedineachprocesswhere retrogradetransportisinvolved.

TheJIP4homologousprotein,previouslyidentifiedasJIP3,overlaps withJIP4inregulatingaxonallysosometransportinneurons(60).Accordingtopreviousstudies,ithasbeensuggestedthatJIP3andJIP4 arefunctionallyredundantandwhosemaindifferenceistheexpressionofJIP3onlyinneuronalcells(14).Ithasbeenhypothesizedthat ifJIP3isnotexpressed,JIP4canreplaceJIP3inthekinesinactivation complex(14).DenovoheterozygousmutationsintheMAPK8IP3gene encodingtheJIP3alsoshowaphenotypeofintellectualdisabilityand brainabnormalities(61, 62).Thereportedvariantsarelocatedinthefour maindomainsoftheJIP3protein,includingthreemutationswithinthe WD40domain(62).Here,wereportthesefirstthreecaseswithpossible pathogenicmutationsinJIP4,whichdemonstratetheimportanceofJIP4 atthebrainleveldespitethepresenceofJIP3.Thiscouldagreewiththe statementthatJIP4hasdifferentfunctionsthanJIP3,anditspresence isessentialandnotreplaceableincertainbrainfunctionsorsignaling pathways(10).

Retrogradeendosomalsignalinginneuronsincludesthefollowing steps:internalizationofligand–receptorcomplexesintoaxonterminals, sortingofcomplexesintoactivesignalingvesicles,transportalongaxonal microtubulestocellbodies,signalingendosomalandthedismantlingof thecomplex(63).Thecentralmotorforretrogradetransportisacytoplasmicdyneincomplexcomposedofmultiplesubunits.Thiscomplexbinds tomicrotubulesandhydrolyzesATP.However,onitsown,itcannotcarry outtransportwithoutdissociatingfrommicrotubules,soitdependson adapterproteinsforefficientprocessivity(9).

HowDoesDefectiveRetrogradeAxonalTransportContributeto NeurodevelopmentalDisorders?

Retrogradeintracellularcommunicationisessentialforbraindevelopmentandmaintenance(2).Theneurotrophinfamilyofgrowthfactors aresynthesizedandsecretedawayfromneuronalcellbodies,propagate retrogradelyalongtheaxontothebodyoftheneuronandarerequired forproperneuronalsurvival,axonalgrowth,geneexpression,neuronal

Table3. Phosphorylationandposttranslationalmodificationssitesinwild-typeSPAG9andSPAG9Tyr914Ter

ProteinPhosphorylationsitesO-glycosylationsitesN-glycosylationsites wild-type SPAG9

257phosphorylationsites: Positions9,21,21,25,30,30,32,87, 109,109,109,119,119,119,123,123, 126,126,143,143,183,183,183,185, 185,194,194,203,203,217,217,226, 226,238,238,242,244,245,245,249, 251,268,268,268,268,268,272,272, 275,276,276,276,279,283,292,305, 305,329,330,332,332,332,339,339, 339,347,347,347,348,358,363,363, 364,365,379,381,381,381,387,388, 391,391,418,493,493,497,504,504, 504,538,538,550,550,550,551,551, 551,557,561,561,562,563,564,564, 564,566,566,567,567,578,582,582, 583,586,586,588,588,593,593,594, 594,594,595,595,595,597,611,614, 617,620,620,629,683,684,705,705, 710,713,728,730,730,730,732,732, 733,733,756,763,764,790,804,804, 806,806,813,815,822,826,829,831, 832,835,837,837,848,858,858,858, 858,858,861,865,865,865,879,879, 887,892,895,901,901,905,909,909, 909,932,935,937,944,944,966,966, 967,986,996,996,1002,1021,1036, 1049,1049,1054,1054,1069,1081, 1081,1090,1090,1105,1110,1111, 1111,1131,1138,1144,1149,1149, 1169,1173,1175,1188,1188,1188, 1198,1205,1205,1238,1238,1238, 1241,1242,1244,1249,1249,1249, 1256,1262,1262,1262,1264,1264, 1264,1273,1273,1278,1290,1290, 1302.

SPAG9

Tyr914Ter

191phosphorylationsites:

Positions9,21,21,25,30,30,32,87, 109,109,109,119,119,119,123,123, 126,126,143,143,183,183,183,185, 185,194,194,203,203,217,217,226, 226,238,238,242,244,245,245,249, 251,268,268,268,268,268,272,272, 275,276,276,276,279,283,292,305, 305,329,330,332,332,332,339,339, 339,347,347,347,348,358,363,363, 364,365,379,381,381,381,387,388, 391,391,418,493,493,497,504,504, 504,538,538,550,550,550,551,551, 551,557,561,561,562,563,564,564, 564,566,566,567,567,578,582,582, 583,586,586,588,588,593,593,594, 594,594,595,595,595,597,611,614, 617,620,620,629,683,684,705,705, 710,713,728,730,730,730,732,732, 733,733,756,763,764,790,804,804, 806,806,813,815,822,826,829,831, 832,835,837,837,848,858,858,858, 858,858,861,865,865,865,879,879, 887,892,895,901,901,905,909,909.

106O-Glicosilaciónsites: Positions16,25,128,183,185,190, 191,194,203,226,229,238,244,245, 249,251,268,272,275,276,279,280, 283,287,290,292,305,325,329,330, 332,358,363,364,365,367,387, 493,497,504,538,551,557,561, 562,563,564,566,567,582,583, 586,588,593,594,595,597,604, 617,620,658,705,710,724,728, 730,815,822,826,828,829,831, 832,835,843,848,853,857,858, 860,861,865,879,887,892,895, 901,905,909,911,915,937,938, 949,1179,1188,1238,1241,1242, 1243,1244,1246,1249,1256,1262).

6N-Glicosilaciónsites: Positions

309NKSE(0,6999)

565NTTK(0,5121)

694NLSG(0,5093)

830NSSA(0,5031)

939NDSD(0,5029)

1176NKTS(0,5162)

82O-Glicosilaciónsites: Positions25,128,183,185,190,191, 194,203,226,229,238,244,245, 249,251,268,272,275,276,279, 280,283,287,290,292,305,325, 329,330,332,358,363,364,365, 367,387,493,497,504,538,551, 557,561,562,563,564,566,567, 582,583,586,588,593,594,595, 597,604,617,620,658,710,724, 728,730,732,815,826,828,829, 835,853,857,858,860,861,865, 879,887,892,895,901.

1N-Glicosilaciónsite: Position 309NKSE(0,6928)

Comparisonbetweenprimarysequencesoftwoproteins(wild-typeandmutated)accordingtophosphorylation,O-glycosylationandN-glycosylation sites.

Table4. Positionsphosphorylatedbykinasesthatinteractwith SPAG9wt andtheeffectof SPAG9Tyr914Ter

PositionsphosphorylatedbykinasesinSPAG9(wild-type)are notpresentinSPAG9Tyr914Ter

PositionAminoacidEnzymePositionAminoacidEnzyme

909SATM1149TPKA

909SDNA-PK1149Tcdc2 909SCKI1169SPKA

932Sunsp1173TPKC 935YSRC1175TPKC

937Sunsp1188Sunsp

944Yunsp1188SPKA 944YINSR1188SPKC

966SPKA1198Sunsp

966Scdc21205TPKC 967Scdc21205TPKG

986SPKC1238Sp38MAPK

996Sunsp1238Scdk5

996SPKC1238SGSK3

1002Sunsp1241Sunsp

1021TDNA-PK1242Sunsp 1036Scdc21244Sunsp 1049Sunsp1249TPKC 1049SPKA1249Tunsp

1054Tunsp1249Tcdc2

1054TPKC1256Sunsp 1069Yunsp1262Sunsp 1081Sunsp1262SDNAPK

1081SPKC1262SATM 1090Sunsp1264Tunsp 1090SPKA1264Tcdk5

1105SPKC1264Tp38MAPK 1110SRSK1273Sunsp 1111TPKC1273SCKII 1111Tunsp1278Yunsp 1131Yunsp1290SCKII 1138TPKC1290Scdc2 1144Scdc21302Scdc2

Sixty-sixlostphosphorylationsitesofthemutatedproteinduetothe spatialeffectfromposition909(unsp = unspecificenzyme,S = serine, T = threonine,Y = tyrosine).

subtypespecification,axonextensionandbranching,dendriteformation,neurotransmitters,synaptogenesisandsynapticfunctionandaxon regeneration(64).

SynapticdysfunctionappearstoberelevantintheabsenceofJIP4.AlterationsintheJIP4scaffoldproteincandirectlyimpactsynapses,mainly atthepresynapticbutalsoatthepostsynapticlevels(65, 66).Thetransportofneurotrophicfactorsnecessaryfortheformationandmaintenance ofsynapsesatthepresynapticlevelandthepostsynapticlevelseemsto involvetheactivityoflysosomesresponsibleforsynapticorganization andneuronalpruning.

RecentreportdemonstratedthataxonallysosomaltransportisalteredbythelossofJIP4.JIP4/JIP3couldalsoberegulatingthestructure anddynamicsoftheneuronalcytoskeleton(58).Studiesinmousemotorneuronsconcludesthatadequatelysosomalactivityiskeytonatural synapseeliminationinmousemotorneuron(67).Authorshavesuggested possiblecommonmechanismsforthisregulationinnervoussystemboth attheperipheralandcentrallevels,duringneuronalpruningandeliminationofaxonalconnectionstocausesynapticrefinement(68).

MultipleMendelianmutations,theyhavebeenassociatedwithdefects inmotorproteins,adapters,orregulatorsofaxonaltransport(65).Some neurodevelopmentaldiseasesinvolvedspecificallywithproteinswith functionsinretrogradetransportpreviouslyidentified(9)aresummarizedbelow.Geneticalterationsinthedyneincytoplasmic1heavychain 1(DYNC1H1gene)withassociatedphenotypes:Charcot-Marie-Toothdisease,axonal,type2O,corticaldysplasia,complex,withotherbrainmalformations13andspinalmuscularatrophy,lowerextremity-predominant1. Mutationsinregulators(NDE1andBICD2)havebeenreported(69).NDE1 isassociatedtolissencephaly4(withmicrocephaly)andmicrohydranencephaly.BICD2-associatedphenotypesarespinalmuscularatrophy,lower extremity-predominant,2Aand2B.PAFAH1B1orLYS1isalsoanimportantgenerequiredfordyneinandmicrotubuledependentprocesses,and itisassociatedwithlissencephalytype1(70).Allofthesephenotypes seemtoinvolvemoresevereandearlierchangesinbraindevelopment thanthephenotypepresentedhere,withclearmalformationsofcerebral corticaldevelopment(MCD)(71).Inourcases,noMCDpatternswereevidentintheneuroimaging.

HowDoesDefectiveRetrogradeAxonalTransportContributeto NeurodegenerativeDiseases?

Lysosomesrecycleoreliminatedamagedormisfoldedproteinsasthey traveltotheneuronalsomaviaretrogradeaxonaltransport.Retrogradetransportoflysosomesisrecognizedasanimportantregulator ofautophagy.Autophagymaintainshomeostasisandpreventstheaccumulationoftoxicmaterialwithinthecell.Neuronsareparticularlysensitivetothistoxicaccumulation(72).Multipleneurodegenerativediseases

Figure4. Representationofthethree-dimensionalmodelintapes.(A)RegionoftheSPAG9wtproteinafterposition914(inyellowandorange).(B) SPAG9wt and SPAG9Tyr914Ter (violet)proteins.Therewasnoalignmentofthestructures.Theeffectofthechargesoftheaminoacidswaslost,affectingtheirfolding andinducingchangesinanglesthatalterthefunctionalinteraction.

havebeenrelatedtodefectsinaxonaltransport,includingAlzheimerdisease(AD),Parkinson’sdisease,ALS,Huntingtondisease,frontotemporaldementia,Perrysyndrome,Charcot-Marie-Toothtype2B,amongothers(73).Autophagyistheprocessbywhichagedortoxicproteinsand organellesareengulfedbythemembraneforminganautophagosome thatthenfuseswithalysosometoformanautolysosome,withtheaim ofdegradingthecontentsofthevesiclebylysosomalhydrolases.Lysosomalretrogradetransportregulatesautophagicfluxbyfacilitatingthe formationofautophagosomesandfusionbetweenautophagosomesand lysosomes(72).JIP4phosphorylationactsasaswitchthatcontrols lysosomaldistributionsignalingpathwaysdependingonthetypeof autophagy-inducingsignal(72).Axonaltransportofautophagosomesis regulatedbyJIP4(74).

ThereisevidenceofJIP3thehomologofJIP4anditsroleinneurodegeneration.TheabsenceofJIP3showedalterationinzebrafishretrograde transportandlysosomeaccumulation(75).Andindystrophicaxonsofthe Jip3KOmouse,immaturelysosomeswerefoundinthecellbody(76). IthasbeenseenthatblockingretrogradetransportleadstopoormaturationanddegradationoflysosomescontributestotheiraxonalaccumulationandalteredmaturationinaxonalinflammationsofAD.InJIP3 KOmouseneurons,AD-likeaccumulationsoflysosomeswereidentified (76)andinJIP3 +/ hadworseningamyloidplaquepathology.These resultsshowtheimportanceofJIP3-dependentaxonallysosometransportinregulatingamyloidprecursorproteinprocessing;however,inthe 19unrelatedindividualswithdenovovariantsinJIP3,withintellectual disabilityphenotypeandbrainmalformations;nosubsequentneurodegenerativechangesorsignsofdevelopmentalregressionweredescribed (61, 62).Thischaracteristicwasalsonotmentionedinarecentcase report(77).

Ourthreecasesareonaverage34yearsoldandfollow-upformore than10yearswithworseningcognitiveandbehavioralfunctionhascorroboratedprogressivecognitivedeterioration.Wedidnotfindsimilar findingsintheliteraturecausedbydysfunctionintheretrogradetransportmachineryoraxonaltransportingeneral,wherecaseswithprogressivephenotype,withneurodevelopmentaldiseaseandsubsequentcentralneurodegenerationphenotype,arereported(9).

Theseobservationsgeneratemultiplequestionswhosefutureanswerscouldexplainthedescribedfamily’scausalpathologicalmechanismsandmanyotherdiseasesinvolvedincerebralretrogradetransport, wheredevelopmentanddegenerationtautologicallyconverge.

DeclarationofPossibleConflictsofInterests: Allcontributorshave confirmedthatnoconflictofinterestexits.

AuthorContributions

N.A.-B.andC.A.V.-L.hadfullaccesstoallthedatainthestudy.

• Studyconceptanddesign:N.A.-B.,J.T.-M.,L.M.,andC.A.V.-L.

• Acquisition,analysis,orinterpretationofdata:N.A.-B.,J.T.-M.,A.S.-O., A.M.,M.P.,andJ.N.-T.

• Draftingthemanuscript:N.A.-B.andM.A.-B.

• Criticalrevisionofthemanuscriptforimportantintellectualcontent: M.A.C.,W.R.,G.P.C.,andM.A.-B.

• Bioinformaticandstructuralanalysis:N.A.-B.,J.T.-M.,andA.S.-O.

• ObtainingfundingandStudysupervision:C.A.V.-L.andM.A.-B.

DataAvailability

Thedatasetsusedandanalyzedduringthecurrentstudyareavailable fromthecorrespondingauthoruponreasonablerequest.

Acknowledgments

StudysupportedbyUniversidaddeAntioquiaandMINCIENCIASgrant number1115-807-63223,RepublicofColombia.Thisworkisdedicated tothememoryofProfessorGabrielBedoya.WeespeciallythankProfessor SergioVargasforhiscontributionsinreadingthebrainMRI,themembers oftheGENMOLandtheGNAgroupswhohelpedinsomewaytomakethis studypossible,andthefamilystudiedfortheiravailabilityandtrust.

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GenomicPsychiatry

OPEN RESEARCHREPORT

TreatmentwithshRNAtoknockdown the5-HT2Areceptorimprovesmemory invivoanddecreasesexcitabilityin primarycorticalneurons

TroyT.Rohn1 ,DeanRadin1 ,TracyBrandmeyer1 , PeterG.Seidler1 ,BarryJ.Linder1 ,TomLytle1 ,DavidPyrce1 , JohnL.Mee1 ,andFabioMacciardi1 , 2

1 Cognigenics,Eagle,Idaho,83616,USA

2 DepartmentofPsychiatryandHumanBehavior,UniversityofCalifornia, Irvine,California92697,USA

CorrespondingAuthor: TroyT.Rohn,1372S.EagleRoad,Suite197 Eagle,Idaho83616USA.E-mail: troy.rohn@cognigenics.io

GenomicPsychiatry January2025;1(1):85–93; doi: https://doi.org/10.61373/gp024r.0043

ShorthairpinRNAs(shRNA),targetingknockdownofspecificgenes, holdenormouspromiseforprecision-basedtherapeuticstotreatnumerousneurodegenerativedisorders.WedesignedanAAV9-shRNA targetingthedownregulationofthe5-HT2Areceptor,andrecently demonstratedthatintranasaldeliveryofthisshRNA(referredtoas COG-201),decreasedanxietyandenhancedmemoryinmiceandrats.In thecurrentstudy,weprovideadditionalinvivodatasupportingarole ofCOG-201inenhancingmemoryandfunctionalinvitrodata,whereby knockdownofthe5-HT2Areceptorinprimarymousecorticalneurons ledtoasignificantdecreaseinmRNAexpression(p = 0.0007),protein expression p-value = 0.0002,andinspontaneouselectricalactivityas measuredbymultielectrodearray.Inthisregard,weobservedasignificantdecreaseinthenumberofspikes(p-value = 0.002),themeanfiringrate(p-value = 0.002),thenumberofbursts(p-value = 0.015),and adecreaseinthesynchronyindex(p-value = 0.005).Thedecreasein mRNAandproteinexpression,alongwithreducedspontaneouselectricalactivityinprimarymousecorticalneurons,corroborateourinvivo findingsandunderscoretheefficacyofCOG-201indecreasing HTR2A geneexpression.ThisconvergenceofinvitroandinvivoevidencesolidifiesthepotentialofCOG-201asatargetedtherapeuticstrategy. TheabilityofCOG-201todecreaseanxietyandenhancememoryin animalmodelssuggeststhatsimilarbenefitsmightbeachievablein humans.Thiscouldleadtothedevelopmentofnewtreatmentsforconditionslikegeneralizedanxietydisorder,post-traumaticstressdisorder(PTSD),andcognitiveimpairmentsassociatedwithagingorneurodegenerativediseases.

Keywords: RNAinterference,5-HT2Areceptor,memoryenhancement, neuronalexcitability,anxiety,cognitiveimpairment.

Introduction

Neurologicaldisorderssuchasmildcognitiveimpairment(MCI)and chronicanxietyareamajorpublicmentalhealthchallenge,affecting millionsofpeopleworldwide.MCIisoftenatransitionalstagebetween healthyaginganddementia.Dependingontheinclusioncriteria,the prevalenceofMCIhasbeenestimatedtobebetween5.0%and36.7%(1). Accordingtoasystematicreviewandmeta-analysis,theoverallpooled prevalenceofanxietyinpatientswithMCIisapproximately21%.This prevalenceratevariesbasedonthesourceofthesampleandthemethod ofdiagnosis.Forexample,theprevalenceofanxietyincommunity-based samplesofpatientswithMCIisabout14.3%,whileitisapproximately 31.2%inclinic-basedsamples(2).Basedonthesestatistics,weestimate

thatroughly1.5–2millionAmericanssufferfromMCIwithanunderlying anxietydisorder.Currently,thereisnosinglemedicationtotreatboth cognitiveimpairmentsandanxietyinthispatientpopulation.

Precision-basedtherapeuticssuchasRNAinterferenceofferapromisingnewapproachtotreatingneurologicalandneurodegenerativedisorders.ShorthairpinRNA(shRNA)representsoneclassofRNAinterference moleculesthathasamechanismbasedonthesequence-specificdegradationofhostmRNAthroughcytoplasmicdeliveryanddegradationof double-strandedRNAthroughtheRNA-inducedsilencingcomplex(RISC) pathway(3, 4).WedesignedplasmidscontainingtheRNAinstructionsto constructaspecificshRNAtosilencethe HTR2A gene(U.S.PatentApplicationNo.63/567,853).The HTR2A geneencodesforthe5-HT2Areceptor,oneofthe15knownserotoninreceptorsubtypesexpressedin thebrain,andisimplicatedinbothanxietydisorders(5, 6)andmemory (7–9).Thisplasmidcontainsaneuronalspecificpromoter,MeCP2andis packagedwithinAAV9viralparticles.IntranasaltreatmentofthisAVV9shRNA(hereintermedCOG-201)inmiceorratssignificantlydecreased anxietyandimprovedmemory(10).Inthisstudy,wepresentfurtherevidencesupportingthememory-enhancingeffectsCOG-201.Wealsoprovidefunctionaldatafromexperimentsonprimarycorticalneuronstaken frommice.OurresultsshowthattreatmentwithCOG-201leadstoreducedspontaneouselectricalactivityintheseneurons.Thiseffectoccursspecificallyafterreducingtheexpressionofthe5-HT2Areceptor. ThesefindingsbolsterthepotentialofintranasalshRNAdeliveryasanoninvasivetherapeuticmethodandestablishafoundationforcontinuedinvestigationintoitsroleintreatinganxietyandcognitivedeficitslinkedto aspectrumofneurodegenerativediseases.

Methods shRNADesignandAAV9VectorDesign

ConstructionofthemouseshRNAtotargetknockdownofthe5-HT2Areceptorwasaspreviouslydescribed(11).Themouse HTR2A geneconsists ofthreeexonsthatgiverisetotwomajorisoformsandisfoundonchromosome13.ThepredictedbindingregionoftheprimaryRNAtranscript forthissequenceisthebeginningofexon2,whichwouldleadtothepotentialknockdownofallpossibleisoforms.Thefollowingsequencewas usedforassemblyoftheshRNAbasedon invitro testingindicatinga77% knockdown:

GCTGAGCACATCCAGGTAAATCCAGGTTTTGGCCACGACTGACCTGGATTT CTGGATGTGCTCAG

Noknockdownwasobservedwiththeemptyvectorcontrolorascrambled shRNAcontrol(Figure2B).Forvalidationandscreening,knockdownwas verifiedusingHEK293cellscotransfectedwiththecDNAplasmidcontainingthe HTR2A genetarget.Forinvitrotreatmentofprimarymousecorticalneurons,shRNAdeliverysubcloningoftheshRNAwascarriedoutina modifiedpAAV cis-plasmidundertheneuronal-specificpromoter,MeCP2. TheinclusionoftheMeCP2promoterisacrucialelementdesign,asitensuresexpressionoftheshRNAplasmidonlyinneuronalpopulations.Areportergeneenhancedgreenfluorescentproteinwassubclonedupstream oftheshRNAsequence.AAV9virallarge-scaletransfectionofplasmids wascarriedoutinHEK293cellsandpurifiedthroughaseriesofCsClcentrifugations.Titerload(ingenomecopynumberpermL,orGC/mL)was determinedthroughquantitativereal-timePCR,withtypicalyieldsgiving 1–2 × 1013 GC/mL.AllAAV9vectorswerestoredinphosphatebuffered saline(PBS)with5%glycerolat 80°Cuntilused.Design,manufacturing,andpurificationofAAV9vectorsusedinthisstudywereperformed byVectorBiolabs(Malvern,PA).

NovelObjectRecognitionTest

Theobjectrecognitiontaskisusedtoassessshort-termmemory, intermediate-termmemory,andlong-termmemoryinratsandwasperformedaspreviouslydescribed(11).Thetaskisbasedonthenaturaltendencyofratstopreferentiallyexploreanovelversusafamiliarobject, whichrequiresmemoryofthefamiliarobject.Thetimedelaydesignallowsforthescreeningofcompoundswithpotentialcognitiveenhancing propertiestoovercomethenaturalforgettingprocess.Wistarmalerats

Received:24April2024.Revised:28May2024and16June2024.Accepted:19June2024. Publishedonline:5August2024.

(12animalspergroup)wererandomlyassignedtotwogroupsconsisting ofvehicle(PBS)orCOG-201treated.FollowingadministrationofthevehicleorCOG-201,ratswereassessedinthistask3weeksposttreatmentand thediscriminationindexwascalculated.Tocalculatethediscrimination index,thefollowingequationwasused:(timeexploringnovelobject-time exploringfamiliarobject)/(timeexploringnovelobject + timeexploring familiarobject),multipliedby100toconverttoapercentage.Thearena andobjectswerecleanedwith70%alcoholbetweeneachrattestsession. ThesebehavioralstudieswereperformedbyNeurofitSAS.Allanimalcare andexperimentalprocedureswereperformedinaccordancewithinstitutionalguidelinesandwereconductedincompliancewithFrenchAnimalHealthRegulation.Forallbehavioralstudies,animalswerekeyed, anddatawereblindeduntiltheendofexperiments.

PrimaryMouseCorticalNeuronCulturesandTreatmentwithCOG-201 Primarycorticalneuronsfromfreshmousebrainembryoswereisolated andplatedontocoated24-wellplatesatadensityof5 × 105 cells/ well.CorticalneuronsweremaintainedinNeurobasal-AMedium,supplementedwithB27,Glutamax,andantibiotics(100U/MIpenicillinand 100 μg/mLstreptomycin).Culturedneuronswereincubatedat37°Cand 5%CO2 andhalfthemediawereexchangedwithfresh,completemedia every3days.Onday6followingplating(DIV6),corticalneuronswere treatedexogenouslywithastockconcentrationofCOG-201at1 × 1013 (GC/mL)toafinalmultiplicityofinfection(MOI)of2 × 105 .TheMOIrefers tothenumberofviralparticlesperneuron.Alternatively,corticalneurons weretreatedatthesameMOIusingAAV9-MeCP2-GFP-scrambled-shRNA representingthe HTR2A targetsequencebutrandomlyscrambled.Culturedneuronalmediawasreplacedwithhalf,fresh,completemediaevery 3daysfor10days(DIV16)atwhichpointcellswerefixedforimmunocytochemistryoranalyzedforspontaneouselectricalactivityviaMEA.Preparationandmaintenanceofprimarymousecorticalneuroncultureswas carriedoutbyCreativeBiolabs(Shirley,NY).

EthicalAnimalTreatmentStatement

CreativeBiolabscomplieswithallprovisionsoftheAnimalWelfareAct andotherregulationsrelatedtoanimals.Everyindividualinvolvedinthe careanduseoflaboratoryanimalsfullyunderstandstheresponsibilities,suchas:avoidsorminimizesdiscomfort,distress,andpaininexperimentalanimalsconsistentwithsoundscientificpractices;usesminimum numberofanimalsnecessarytoobtainvalidresults.AllexperimentalprotocolswereapprovedbytherelevantInstitutionalAnimalCareandUse Committee.

ImmunohistochemicalFluorescenceMicroscopy

Immunofluorescencehistochemistrywasaspreviouslydescribed(10, 11).

Briefly,followingdehydration,4 μmparaffin-embedded,sagittalsections werecutjustlateraltothemidlineandusedforimmunofluorescencelabeling.Briefly,alltissuesectionswerelabeledwithanti-GFPantibody (rabbitmAB#2956)1:1,000(CellSignalingTechnology,Inc.,Danvers,MA, USA)oranti-5HT-2Areceptorantibody(rabbitpolyclonal,#24288)at 1:500dilution(Immunostar,Hudson,WI).SecondaryantibodieswereconjugatedtoFITCorCy3.DAPIwasusedasanuclearstain.WholeslidescanningwasperformedusingaPannoramicMidiIIscannerusinga40Xobjectivelenswithopticalmagnificationof98X,0.1 μm/pixel.Allsectioning, immunolabeling,andcapturingofimageswascontractedouttoiHisto (Salem,MA).

ImmunocytochemistryProtocol

Cellswereculturedunderappropriateconditionsbeforetheimmunocytochemistryprocedurewasinitiated.Forfixation,cellsweretreatedwith 4%paraformaldehydepreparedin1xPBS.Thefixationsolutionwaspreheatedto37°Cpriortouse.Cellswereincubatedwiththissolutionfor 10minutesatroomtemperaturetopreservecellulararchitectureand antigenicity.Followingfixation,cellswerewashedthricewith1xPBS,with eachwashlastingfor3minutes,toremoveexcessfixative.Topermeabilizethecellmembranes,0.1%TritonX-100(dilutedinPBS)wasaddedto thewells,andthecellswereincubatedfor15minutesatroomtemperature.Thisstepfacilitatestheentryofantibodiesintothecells.Subsequently,cellswereagainwashedthreetimeswith1xPBSfor3minutes eachtoremovethepermeabilizationagent.

Thecellswerethenblockedwith500 μLofready-to-usegoatserum for1houratroomtemperaturetopreventnon-specificbindingofthe primaryantibodies.Afterblocking,theprimaryantibodieswerediluted inthegoatserum;GFPmonoclonalantibodyfrommousewasdilutedat 1:500,and5HT2Aantibodyfromrabbitat1:100.Theprimaryantibody solutionwasaddedtothewells,andthecellswereincubatedovernight at4°C.Thenextday,thecellswerewashedthreetimeswith1xPBSfor 3minuteseachtoremoveunboundprimaryantibodies.Thesecondary antibodieswerethenprepared:AF488Goatanti-MouseIgG(H+L)and AF555Goatanti-RabbitIgG(H+L),bothdilutedat1:400ingoatserum. Thesecondaryantibodysolutionwasaddedtothewells,andcellswere incubatedfor2hoursatroomtemperatureinthedarktoprotectthe fluorophoresfromphotobleaching.Afterincubationwiththesecondary antibodies,cellswerewashedwith1xPBStoremoveanyunboundantibodies.Subsequently,nucleiwerestainedwithaHoechstsolution,which wasaddeddirectlytothewells.Thecellswereincubatedinthedarkfor 5–10minutes,followedbyafinalwashwithPBS.Finally,thestainedcells wereimagedusingappropriatefluorescencemicroscopytodetectthe signalsfromthefluorophore-conjugatedantibodies.Uponcompletionof imaging,theslidesweresealedtopreventdryingandtopreservethefluorescenceforfutureanalysis.Immunocytochemistrywasperformedby CreativeBiolabs(Shirley,NY,USA).

WesternBlotAnalysisProtocol

Followingtreatment,proteinswereextractedfromcorticalneuronsthe proteinconcentrationwasdeterminedusingastandardproteinassay. EqualamountsofproteinfromeachsamplewerethendilutedwithPBS tonormalizethevolumeacrossallsamples.Thesamplesweremixedwith loadingbufferata1:4volumeratioanddenaturedbyheatingat100°Cfor 10minutes.Forelectrophoresis,sampleswereloadedintoaprecastpolyacrylamidegelalongsideamolecularweightmarker.Theproteinswere thentransferredontoapolyvinylidenedifluoride(PVDF)membraneusingasemi-drytransfersystem.Posttransfer,thePVDFmembranewas blockedin5%non-fatmilkpreparedinTris-bufferedsalinewithTween20 (TBST)topreventnon-specificproteinbinding.Subsequently,themembranewasincubatedwithaprimaryantibodyagainstthe5-HT2Areceptor,dilutedtoaconcentrationof0.3 μg/mL,andplacedovernightat4°C onashaker.ThemembranewaswashedthreetimeswithTBSTfor10minutesandthenincubatedwithagoatanti-rabbitsecondaryantibodysolutionfor1houratroomtemperatureonashaker.Forthedetectionofthe antibody–proteincomplex,achemiluminescentsubstratewasprepared andaddedtothemembrane,whichwasthenincubatedfor5minutes.The intensityofthebandswasanalyzedbydensitometrytodeterminetherelativeamountsofthetargetproteinpresentinthesamples.Westernblot analysiswasperformedbyCreativeBiolabs(Shirley,NY,USA).

QuantitativeReal-timeqPCR

Real-timeqPCRwasperformedaspreviouslydescribed(11).Briefly, totalRNAwasextractedfromprimarycorticalmouseneuronsusing astandardextractionprotocolwithTRIzol,dissolvedindiethylpyrocarbonate(DEPC)-treateddeionizedwaterandquantified.Followingreversetranscription,qPCRwascarriedoutusingthefollowing primers:Primer-F:5’-AGAGGAGCCACACAGGTCTC-3’andPrimer-R:5’ACGACAGTTGTCAATAAAGCAG-3’.Therelativeexpressionwasdetermined bycalculatingthe2 ct value.The2ˆ( ddCt)valuewasthencalculated andnormalizedtoGAPDHforeachtreatment(AVV9-scrambledvs.COG201).TheRNAextractionandqPCRwereperformedbyCreativeBiogene (Shirley,NY,USA).

MultielectrodeArrayAnalysis

MEAanalysiswasperformedaspreviouslydescribed(11).Briefly,themicroscopeusedwasanEvosXLCore.Twenty-four-wellMEAplateswere coatedwith500 μL0.07%polyetherimide(PEI)andincubatedfor1hour. Plateswerethenwashedfourtimesinsteriledeionizedwateranddried overnightinabiosafetycabinet.Primarycorticalneuronsfromfresh mousebrainembryoswereisolatedandplatedontocoated24-wellplates atadensityof5 × 105 cells/well.Corticalneuronsweremaintainedin Neurobasal-AMedium,supplementedwithB27,Glutamax,andantibiotics(100U/MIpenicillinand100 μg/mLstreptomycin).Culturedneurons wereincubatedat37°Cand5%CO2 andhalfthemediawereexchanged

withfresh,completemediaevery3days.Treatment(MOIof2 × 105 )occurredonday6for24hoursat37°Catwhichtimethemediawasreplaced withfresh,completemedia.MEAanalysiswasthenperformedonday16, 10daysafterinfection.MEAanalysiswasperformedbyCreativeBiolabs (Shirley,NY).

StatisticalAnalysis

ForPCR,andWesternblotquantification, t testsfortwoindependent meanswerecalculatedusingexcelsoftwareusingasignificancelevelset at0.05andone-tailedhypotheses.MicroelectrodearraydatawereanalyzedviarepeatedmeasuresANOVAsusingStatistica(Version13.5,Tibco Software).Alldatausedinthesetestswerecheckedandfoundtoconform toparametricassumptions.

AcknowledgmentofGenerativeArtificialIntelligenceandArtificial Intelligence–assistedTechnologiesUsedinWriting Inthecourseofthiswork’spreparation,theauthor(s)employedChatGPT 4withtheintenttoimprovereadabilityandlanguage.Uponutilizingthis tool/service,theauthorsundertookcomprehensivereviewandmodificationasnecessaryandassumefullaccountabilityforthecontentofthe publication.

ResultsandDiscussion

Inarecentstudy,wehavedemonstratedadecreaseinanxietyandimprovementinmemoryfollowingintranasaldeliveryofCOG-201inrats ormice(10).Inthatstudy,anovelrecognitionobjecttestinnormal, 2-month-oldratswerecarriedoutfollowingtreatmentwithCOG-201. Wereportedasignificantincreaseinboththecontact-recognitionindex (92%)andtime-recognitionindex(73%).Inthecurrentstudy,wenowreportasignificantincreaseinthediscriminationindexinthenovelrecognitionobjecttest3weeksposttreatmentwithCOG-201(Figure1A),and knockdownofthe5-HT2Areceptor(Figure1C)followingintranasaldelivery.Thediscriminationindexisameasureusedinthenovelobjectrecognitiontesttoquantifythedifferenceinexplorationtimebetweenanovel andafamiliarobject.Apositivediscriminationindexsuggeststhatthe animalspentmoretimeexploringthenovelobject,whichimpliesrecognitionofthefamiliarobjectand,thus,intactmemory. Figure1A indicates thevehicle-treatedgroupexhibiteda 28.9%discriminationindex,which suggeststhat,onaverage,theratsspentmoretimewiththefamiliarobjectthanwiththenovelobjectduringtheretentiontest.Oneinterpretationoftheseresultsisthatthecontrolgroupeitherdidnotremember thefamiliarobjectortherewasanalternativefactoratplay(e.g.,anxiety,stress).Incontrasttothecontrolgroup,theratstreatedwithCOG201displayedadiscriminationindexincreaseof22.5%.Thus,onaverage,treatedratsdedicatedmoretimetointeractingwithanewobject ratherthanafamiliarone,indicativeofenhancedmemoryretention.The positiveeffectsofCOG-201onmemoryretentionarefurtherhighlighted whenconsideringthesignificantnegativediscriminationindexobserved inthegrouptreatedwiththevehiclealone.Thecontrastbetweenthe groupscouldsuggestthatCOG-201notonlyimprovesmemoryretention directlybutmayalsoimproveitindirectlybyreducinganxiety,orthrough asynergisticeffectofbothmechanisms.

Theseresults,takentogetherwithourpreviousfindingssupporta memoryenhancementactionofCOG-201.However,themissingcomponentisfunctionaldataconnectingtheknockdownofthe5-HT2AreceptortothebehavioralactionsofCOG-201.Anadditionalaimofthecurrentstudywastoprovidefunctionaldatatosupportthesebehavioral findings.Theserotonin5-HT2Areceptoristhemajorexcitatoryreceptorsubtypeinthecortex.Forexample,thisreceptorhasbeenlinked withstress-induceddystonia,emphasizingitsroleinmediatingneuronal excitability(12).Inaddition,the5-HT2Areceptorhasbeenassociated withexcitatoryeffectsintheneocortexandhasbeenlinkedtoworkingmemoryfunctionbyinfluencingbothexcitatoryandinhibitoryelementswithinlocalcircuitry(13).Moreover,the5-HT2Areceptorhasbeen foundtodirectlystimulatekeyexcitatoryglomerularneuronsintheolfactorybulb,furthersupportingitsroleinexcitatorysynaptictransmission (14).Overall,the5-HT2Areceptorplaysacrucialroleinmemory,anxiety,andpainmodulation,exertingexcitatoryeffectsintheseprocesses. Therefore,weexaminedwhetherexposureofCOG-201toprimaryculture

Figure1. Intranasaladeno-associatedvirusdeliveryofAAV9-MeCP2-GFPmouseHTR2A-shRNAimprovesmemoryinrats.Thetargetsequenceused tosynthesizetheshRNAis100%conservedbetweenmiceandrats.Totest whetherCOG-201knockdownoftherat5-HT2Areceptorimprovesmemory, Wistarrats(12animalspergroup)wererandomlyassignedtotwodifferent groupsconsistingofvehicle-controlsorCOG-201.Followingtreatmentonday 1,animalswereassessedbehaviorally3weekslater,andthediscrimination indexwascalculated(see Methods fordetails).(A)At3weeks,therewas asignificantdifferenceinthediscriminationindexbetweenthetwogroups (p-value = .00025),withthevehiclecontrolsat 28.8%(greenbar)versus COG-201–treated +22.5%(pinkbar).Thegreenbar(labeled“Vehicle")shows theperformancethecontrolgroup.Thisgroup’sdiscriminationindexisaround 20%,indicatingafailureofpreferenceforthenovelobjectoverthefamiliarone.Thisindicatespoorperformanceinrecognizingthenewobject.On theotherhand,thepinkbar(labeled“shRNA”)representsthegroupofmice thatreceivedtreatmentwithshRNA.Theirdiscriminationindexisaround30%, meaningtheyspentsignificantlymoretimeexploringthenovelobjectcomparedtothefamiliarone.Thisindicatesbetterperformanceinrecognizingthe newobject.(B and C)Representative,mergedimmunofluorescenceimageof vehicle-controlanimalsdepictingthepresenceof5-HT2Areceptorproteinlabeling(redfluorescence)withintheolfactorybulb.Thebluestainingreflects nuclearstainingwithDAPI.Asexpected,therewasnoexpressionofGFPin vehiclecontrols(B)whilestrongGFPlabelingwasobservedincellbodiesof neuronsofshRNA-treatedrats(C).PanelCalsodepictsagenerallackof5HT2Afluorescence,supportingaknockdownofthereceptorfollowingCOG201treatment.Imagesarerepresentativeof3separateratsforeachgroup.

Figure2. TargetingstrategytoknockdownthemouseHTR2AgeneusingshRNAandtransductionefficiencyinprimarymousecorticalneurons.(A)Schematic displayingthe HTR2A mousegeneencodesasingleprotein-codingtranscript, Htr2a-201,locatedonchromosome14(11).Thetargetsequencewasconstructed torecognizethebeginningofexon2(redarrow,A).Knockdownofexon2preventtheproductionofallknownfull-lengthisoformsofthe5-HT2Areceptorsin mice.(B)Toverifyknockdown,invitroexperimentswereundertakenusingfourdifferentshRNAs,withshmir#4givingthelargestpercentknockdownofHTR2A mRNA(77%)comparedto0%knockdownforeithertheemptyvectorcontrolorascrambledshRNAcontrol.(C–F)TransductionefficiencyofAAV9-mHTR2AshRNAinmouseprimarycorticalneurons.(C and D)depictrepresentativemicroscopicimagesinmouseneuronsfollowinga10-daytreatmentwithscrambled AAV9shRNA-AAV9viralparticles(C and D)ormHTR2AshRNA-AAV9viralparticlesataMOIof2 × 105 (D and F).PanelsCandDrepresentbright-fieldimages whilePanelsEandFarefluorescenceimagesrepresentinggreenfluorescenceproteinexpression.Forbothconstructs,strongGFPexpressionwasobserved. neuronswouldleadtoadecreaseinelectricalactivityasmeasuredby MEA.Inthiscase,wemeasuredthespontaneousactivityofnetworksfollowingtreatmentbyrecordingfieldpotentials.TheadvantageofMEAis thatitcangeneratehigh-throughputreadoutofneuronalpopulations withtheplacementofmultipleelectrodesrecordingallatoncerather thanindividually.

Asaninitialapproach,wedeterminedtherelativetransductionefficiencyinprimaryculturedmouseneuronsfollowing invitro treatment witheitherCOG-201,orascrambledAAV9-shRNAversion. Figure2A outlinesthe HTR2A genetargetingstrategy,whereshRNAisdesignedtobind

atthestartofexon2,effectivelyhaltingthesynthesisofallknownfulllength5-HT2Areceptorisoforms. Figure2B demonstratestheefficacy ofourtargetedsilencingapproach,whereshmir#4induceda77%decreaseinHTR2AmRNAlevels.Thisreductionisinstarkcontrasttothe negligibleimpactobservedwiththescrambledshRNAcontrol.ThecomparisonwasmadeinHEK293cellsthatwerecotransfectedwithacDNA plasmidspecificallyengineeredtocontainthe HTR2A genesequencetargetedbytheshRNAs.Asexpected,followingtreatmentofmouseprimary corticalneurons,hightransductionefficiencyofAAV9-mediatedshRNA deliveryforboththeAVV9-scrambledshRNA(Figure2E)andCOG-201

Figure3. TreatmentofmouseprimarycorticalneuronswithshRNAleadsto knockdownofthe5-HT2Areceptor.Primarycorticalneuronsweretreated onday6ofplatingwithscrambledAAV9shRNA-AAV9viralparticles(A)or COG-201(B)atMOIof3 × 105 for10days(day16,10dpi)atwhichtime, mRNAwasisolatedforreal-timeqPCRexperiments.ResultsdisplaytherelativechangeinexpressionusingGAPDH(Continuedonthenextcolumn)

(Continued)asaninternalcontrol.Real-timeqPCRresultsrepresentatotalof threeseparatetreatmentsforeachconditioninwhichcellswerepooledand frozenat 80°C.PCRexperimentswereperformedintriplicate.Theresultsindicatedasignificant38%decreaseinHTR2AmRNAexpressionascompared tovehiclecontrols, p = .0007.(B–D)Corticalneuronsweretreatedatvarious concentrationsandcellhomogenateswerepreparedforWesternblotanalysis.Transferredmembraneswereincubatedwith0.3 μg/mLofanti-5HT2A receptorantibodyovernightat4°Cfollowedbygoatanti-rabbitsecondary antibodyfor1houratroomtemperature.PanelBdisplaystheresultsindicating5-HT2ARproteinbandinscrambled-treatedneurons(lanes1–3)orin AAV9-shRNA–treatedneurons(lanes4–6).Densitometryanalysisindicateda decreaseinbandintensityforCOG-201treatedneurons(C).InpanelD,data fromlanes1–3and4–6werecombinedandtheresulteddataindicatedanoverall34%decreasein5-HT2Areceptorproteinintreatedneuronsversusscrambledcontrols(p-value = .0002).

(Figure2F)wasobservedbyfluorescencemicroscopy,asindicatedbyrobustGFPexpression.

Wenextdeterminedtheextentof5-HT2Areceptorknockdownby real-timeqPCRorWesternblotanalysis(Figure3).Inthisinvestigation,primarycorticalneuronsunderwenttreatmentwithshRNAtoassesstheknockdownofthe5-HT2Areceptorexpression,asillustratedin Figure3.NeuronstreatedwithCOG-201exhibitedasignificant38%reductioninHTR2AmRNAexpressionascomparedtothescrambledAAV9 shRNA-AAV9controls,afindingconfirmedbyreal-timeqPCRwithGAPDH asareference(p = 0.0007).ThisknockdownofHTR2AmRNAwasfurther substantiatedattheproteinlevelthroughWesternblotanalysis.After incubationwithanti-5HT2Areceptorantibody,theresultingcombined densitometryresultsrevealedacorresponding34%decreasein5HT2AreceptorproteinlevelsinneuronstreatedwithCOG-201,ascomparedtoscrambledcontrols(Figure3C and D),(p-value = 0.0002), thusconfirmingtheknockdownatbothtranscriptionalandtranslational levels.

Furtherconfirmationof5-HT2AreceptorknockdownbyCOG-201 wasobtainedbyimmunocytochemistry.Neuronstreatedwithscrambled AAV9-shRNAviralparticlesshowedstrongexpressionofthe5-HT2Areceptorprotein,asevidencedbytherobustredfluorescence(Figure4A and D).Incontrast,neuronstreatedwithCOG-201exhibitamarkeddecrease in5-HT2Areceptorexpression(Figure4E and H),indicatingsuccessfulreceptorknockdown.Collectively,theresultspresentedin Figures3 and 4 confirmthesuccessfultargetingandsubsequentknockdownof the5-HT2AreceptorbyCOG-201,establishingtherationaleforthenext phaseofthestudy,whereweaimedtoelucidatetheimplicationsof 5-HT2AreceptorknockdownonneuronalexcitabilityemployingMEA analysis.

Toaccomplishthis,primarycorticalneuronsweretreatedonday6 withCOG-201orthescrambledAAV9-shRNAversionandspontaneous electricalactivity(MEAmeasurements)wererecorded10dayslater. Severalparametersweremeasuredincluding(a)thenumberofspikes (Figure5A),whichisdefinedasthetotalcountofactionpotentials (spikes)recordedbytheMEAovera5-minuteperiod,whereeachspike isabriefelectricalimpulsethatrepresentsasingleneuronalfiringevent; (b)themeanfiringrate(Figure5B)definedastheaveragerateatwhich aneuronfiresactionpotentials(spikes)measuredinhertz(Hz);(c)the numberofbursts(Figure5C),definedasaclusterofactionpotentials (spikes)thatoccursinquicksuccession,followedbyaperiodofsilence; (d)thesynchronyindex(Figure5D),definedashowinsyncthefiringof differentneuronsorgroupsofneuronsiswithvaluescloserto1indicatingstrongsynchrony;(e)numberofnetworkbursts(Figure5E)representingcoordinatedactivityacrosstheneuralnetwork,thoughttobe crucialforvariousneuralprocesses,includinglearningandmemoryand areindicativeofthenetwork’sabilitytoengageincoordinatedprocessingandcommunication;andfinally,(f)thenumberofactiveelectrodes (Figure5F).Ahighernumberofactiveelectrodestypicallysuggestsa morewidespreadorsynchronizedactivityacrossthenetwork,indicating robustinterneuronalcommunicationandnetworkintegration.Tosummarizetheresultsin Figure5,weobservedasignificantdecreaseinthe

Figure4. TreatmentofmouseprimarycorticalneuronswithCOG-201leadstoknockdownofthe5-HT2Areceptor.Representativeimmunofluorescenceimagesinmouseneuronsfollowinga10-daytreatmentwitheitherscrambledAAV9shRNA-AAV9viralparticles(A–D)orCOG-201atMOIof3 × 105 (E–H).Green fluorescencerepresentsgreenfluorescenceproteinexpressiondetectedusingaGFPmonoclonalantibody(mouse,1:500)(B and F),whileredfluorescence isindicativeof5-HT2Areceptorproteinfollowingimmunocytochemistryusingananti-rabbit5-HT2Areceptorantibody(Immunostar,1:100).Panel Aand Ddisplayrobustexpressionofthe5-HT2Areceptorproteininneuronalcellsfollowingtreatmentwiththescrambledcontrol.Incontrast,asignificantreduction in5-HT2AfluorescenceintensitywasevidentfollowingtreatmentwithCOG-201(E and H).PanelsCandGrepresentHoechstnuclearlabelingwhilepanels DandHrepresentmergedimages.Allscalebarsrepresent50 μm.

numberspikes,meanfiringrate,numberofbursts,andsynchronyindex butanincreaseinthenumberofnetworkburstsfollowingtreatmentwith COG-201innon-stimulatedneurons.Thesedatacouldbeinterpretedto suggestthatareductioninoverallexcitabilitysupportedtheactionsof COG-201onknockdownoftheexcitatory5-HT2Areceptor.Inaddition,the significantdecreaseinthenumberofburstsofisolatedneuronsandin thesynchronyindexsuggeststhatneuronswithareducedexpressionof 5-HT2Apresentwithalowerfrequencyofspontaneouselectricalactivity (from12to ∼6Hz).Ontheotherhand,asignificantincreaseinthenumber ofnetworkbursts,thatis,acoordinatedelectricalspikingwithingroups ofneurons,isindicativeofcollectivenetworkbehavior.Anincreaseinnetworkburstsamidstdecreasesinindividualspikes,meanfiringrate,and synchronysuggeststhatwhileoverallactivityandglobalbaselinecoordinationarereduced,theseeffectsmaybecompensatedbyincreasingthe instancesofglobalsynchronizationacrossneuronsforminganewnetwork(15).Thepresenceofdesynchronizednon-burstfiringandpartially synchronizedburstsindevelopingnetworksofcorticalneuronssupports thenotionofnetworkcompensationandadaptation(16),suggestinga Hebbianfield.Synchronizationofburstingneuronsisacriticalfactorin understandingnetworkbehavior,andithasbeenshownthatburstfiringcanpromotesynchronizationbetweeninterconnectedlociincentral nervoussystemnetworks(17).Insummary,theobservedchangesinmultielectrodearray(MEA)recordingsfollowingthetreatmentofprimary corticalmouseneuronswithCOG-201suggestacompensatorymechanism.Specifically,anincreaseinnetworkbursts,despitedecreasesinindividualspikes,meanfiringrate,andsynchrony,mayindicateenhanced globalsynchronizationwithinnewlyformingneuronalnetworks.Thiscontrastswiththespontaneousglobalsynchronizationobservedinallneuronstreatedwithscrambled-AAV9-shRNA,whichsuggeststheabsence ofdistinctneuronalnetworks.Thisadaptiveresponseatthenetwork levelmayhaveimplicationsforconditionssuchasanxietyandmemory impairments.

AnimportantcaveatofthecurrentstudyisconnectingtheMEAdata withtheunderlyingbehavioralobservationsofadecreaseinanxietyand improvementinmemory.Inthecurrentstudy,wefocusedoncortical neurons;however,importantneuralnetworksimplicatedinmemoryand anxietyarefoundinthehippocampusandothersubcorticalareasincludingtheinterpeduncularnucleus(IPN).Previously,weidentifiedageneralpatternofguideRNAexpressionintheCA2/CA3regionsofthehippocampusinmicetreatedwithCRISPR/Cas9(10).Additionally,therewas

anoticeablereductionin5-HT2Areceptorexpression,particularlyinthe apicaldendritesofglutamatergicneurons.Previousresearchhasdocumentedthepresenceof5-HT2AreceptormRNAintheCA3regionofthe hippocampus(18, 19).Asthe5HT-2Areceptorisexcitatory,itsdownregulationintheapicaldendritesmayenhancememorybyinfluencinghippocampalneuronaloscillatoryrhythms(20, 21).Inthecontextofthecurrentstudy,projectionstotheapicaldendritesofCA3pyramidalneurons couldoriginatefromthecortex,particularlytheentorhinalcortex,which isessentialforsensoryintegrationandmemoryformation.Intermsof howourmolecularfindingscouldconnectbehaviorallytoenhancedmemory,itisessentialtoconsiderthebroaderneuralcircuitsinvolved.Thehippocampusplaysacriticalroleinmemoryformationandanxietyregulation.Previousstudies,includingourown,haveshownthattheCA2/CA3 regionsofthehippocampusarevitalfortheseprocesses.Ourfindings havedemonstratedareductionin5-HT2Areceptorexpressionintheapicaldendritesofglutamatergicneurons.The5-HT2Areceptorisknown tobeexcitatory,anditsdownregulationcanmodulatehippocampalneuronaloscillatoryrhythms,whicharecrucialformemoryconsolidation.The CA3region,inparticular,receivesprojectionsfromtheentorhinalcortex,whichisessentialforsensoryintegrationandmemoryformation. Thefindingsofreduced5-HT2Areceptorexpressionsuggestapotentialmechanismwherealteredserotonergicsignalinginthehippocampuscaninfluencecorticalinputs,therebyenhancingmemoryfunctions. Thisalignswiththeobservedbehavioralimprovementsinthecurrent study.

Wehavealsopreviouslydemonstratedadecreasein5-HT2AreceptordensityintheIPN(10),anareaimplicatedasamajorconnectomefor stress-mediatedpathways(22).Serotonergiccorticalneuronsareknown toconnecttotheIPNviathehabenulapathway(23, 24).Therefore,the downregulationofthe5-HT2AreceptorinIPNneurons,alongwithacorrespondingdecreaseinelectricalexcitability,couldleadtoareduction inanxiety-relatedbehaviors.Togetherwiththeeffectsofthesecorticalprojections,however,wecannotexcludeapossiblemodulatoryrole of5-HT2Areceptorsexpressedonlocalintrahippocampalinterneurons thatregulatethefiringofpyramidalhippocampalsubfieldneurons,a rolenotspecificallyaddressedinthecurrentstudy.Previousstudieshave demonstratedthat5-HT2AreceptorsonGABAergicinterneuronsstimulateGABArelease,andtherebyhaveanimportantroleinregulatingnetworkactivityandneuraloscillationsintheamygdalaandhippocampal region(25–27).

Figure5. Invitro exposureofprimarymousecorticalneuronswithAAV9-mHTR2A-shRNAleadstoadecreaseinspontaneouselectricalactivity.MEAanalysiswasperformedinmousecorticalneuronsfollowingtreatmentwitheitherCOG-201(redbars,labeled“shRNA”)orascrambledshRNAversion(black bars,labeled“Scram”)ataMOIof2 × 105 .Neuronsweretreatedatday6andMEAanalyseswereperformedonday16.(A–F)QuantificationofMEAanalysisshowingthenumberofspikesover5minutes(A),meanfiringrate(B),thenumberofbursts(C),thesynchronyindex(D),whichindicatesaunitless measureofsynchronybetween0and1.Valuescloserto1indicatehighersynchrony,thenumberofnetworkburstsdefinedasaclusterofspikesacross allelectrodes(E),thesynchronyindex,whichindicatesaunitlessmeasureofsynchronybetween0and1(valuescloserto1indicatehighersynchrony), andthenumberofactiveelectrodes(F).Exposureofneuronsto2 × 105 MOIledtoasignificantdecreaseinthenumberofspikes(50%decreasecomparedtoscrambledcontrols, p-value = .002)(A),themeanfiringrate(50%decrease, p-value = .002)(B),inthenumberofbursts(27%decrease, p-value= .015),(C),andadecreaseinthesynchronyindex(38%decreasecomparedtovehiclecontrols, p-value = .005)(D).Anincreaseinthenumberofnetwork burstswasobserved(20%increase, p-value = .04)(E).Forthenumberofactiveelectrodes,therewasnosignificantdifferencebetweenthetwogroups (F), p-value = .09.Datarepresent N of6forallparameters, ±S.E.M.

Conclusions

OurstudyprovidescompellingevidencethatCOG-201invivoimproves memorycomparedtovehiclecontrolthroughapotentialcombinedactionofimprovingretentionandloweringanxiety.Invitro,COG-201led toasignificantknockdownofthe5-HT2AreceptoratbothmRNAand proteinlevelsinprimarymousecorticalneurons,asconfirmedbyrealtimeqPCR,Westernblotanalysis,andimmunocytochemistry.Thereductioninreceptorexpressioncorrelatedwithadecreaseinneuronalexcitability,asindicatedbyMEAassessmentsofelectricalactivity.Specifically,asignificantreductioninspikes,meanfiringrate,andsynchrony index,coupledwithanincreaseinnetworkbursts,impliesthatCOG-201 inducesareductioninoverallexcitability.However,theincreasednum-

berofnetworkburstsalsosuggestsacompensatorymechanismwithin theneuralnetwork,thatpotentiallyenhancesglobalsynchronization. Ourinterpretationisthattheincreasednetworkburstsinneuronswith reduced5-HT2Aexpressionatbaseline,isindicativeofanewlyformed neuronalnetworkthathasthepotentialofalsoincreasinglong-termpotentiation.Whilethishypothesiscanonlybeconfirmedbyfurtherexperiments,forexample,applyingelectricalstimulitoinvitroneurons,thus mimickingtheinvivoeffectsoftheintegrationofsensoryinformation. Nonetheless,thesefindingsarealignedwithpreviousbehavioralobservationsofreducedanxietyandimprovedmemoryfollowingCOG-201administrationandunderlinethepotentialofCOG-201asaneffectivetherapeuticagent(10).Byelucidatingsomeofthefunctionalconsequences

of5-HT2Areceptorknockdown,thisstudyprovidesacriticallinkbetweenmolecularchangesandtheresultantalterationsinneuralcircuitry thatunderpintheobservedbehavioraloutcomes.FutureresearchiswarrantedtoexploretheprecisemechanismsbywhichCOG-201modulates networkbehaviorandtoassesstheimpactofthesefindingsontherapeuticstrategiesfordisordersincludingMCIthatischaracterizedbyanxietyandmemoryimpairments.However,thecurrentstudyutilizingmice toassesstheefficacyofCOG-201isnotwithoutits’limitations.While theseanimalmodelsareinformative,therearesignificantphysiological andgeneticdifferencesbetweenrodentsandhumansthatmayaffectthe translatabilityofthesefindingstohumantherapeutics.Inthiscontext, theintranasaldeliveryofshRNA(COG-201)inanimalmodelsmaynotdirectlytranslatetohumansduetodifferencesinnasalanatomyandabsorptionefficiency.Moreresearchisneededtodeterminewhetherthis deliverymethodisviableforhumanpatients.Therefore,conductingstudiesinlargeranimalmodelsthataremorephysiologicallysimilartohumans(e.g.,non-humanprimates)couldprovidebetterinsightsintothe potentialtranslationalimpactofCOG-201.

Inconclusion,ifCOG-201proveseffectiveinhumans,itcouldoffera newtreatmentoptionforpatientswithconditionslikeMCI,whichoften involveanxietyandmemoryproblems.Thiswouldbeparticularlybeneficialgiventhelimitedtreatmentoptionscurrentlyavailable.

DeclarationofInterests

J.L.M.,B.J.L.andD.R.areco-foundersofCognigenics,membersofitsscientificadvisoryboard,andholdequityinthecompany.T.T.R.isapart-time consultantservingasDirectorofPreclinicalResearchatCognigenicsand inadditiontoreceivingasalary,ownssharesofthecompany’scommon stockandoptionsforcommonshares.F.M.isapart-timeconsultantservingasChiefScienceOfficeratCognigenics,Inc.,andisamemberofits scientificadvisoryboard.

AuthorContributions

T.T.R.,D.R.,andF.M.designedresearch,analyzedandinterpreteddata andwrotethemanuscript.Allotherauthorsreviewedtheresultsandapprovedthefinalversionofthemanuscript.Allexperimentswerecarried outindependentlybycontractresearchorganizations.

FundingStatement

Thisresearchreceivednospecificgrantfromanyfundingagencyinthe public,commercial,ornot-for-profitsectors.

GenerativeArtificialIntelligenceStatement

Generativeartificialintelligencewasnotusedtowritethemanuscript, butinsteadwasusedasafinaltooltoimproveahuman-generatedtext.

Acknowledgments

WethankourresearchpartnersatNeurofitandCreativeBiolabsfortheir insightfulsuggestionsandexperimentalexpertise.

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BREVIA

Rethinkingtheconnectionbetweenbipolardisorderandepilepsyfrom geneticperspectives

GenomicPsychiatry January2025;1(1):94–95;doi: https://doi.org/10.61373/gp024l.0061

Epilepsyandbipolardisorder(BD)exhibit considerablebiochemicalandgenetic overlap.Ourstudyunveiledasignificant geneticcorrelation(rg = 0.154, P = 9.24 × 10–6 )betweenBD-Iandepilepsy,indicatinga meaningfulcausaleffectofepilepsyonBD-I (P = 0.0079,bxy = 0.1721,SE = 0.0648). Additionally,weidentified1.3kshared geneticvariantsand6significantloci, demonstratingsubstantialpolygenicoverlap. Notably,thers9639379variantwithinthe SP4geneexhibitedstrongassociationswith bothBD-Iandepilepsy,implicatingSP4inthe etiologyofbothdisorders.

Epilepsyandbipolardisorder(BD)ormania arepostulatedtoshareacommonbiologicalunderpinning.Alteredintracellularcalcium ionconcentration([Ca2+ ])isaconsistentbiochemicalfindinginBDandepilepsy(1, 2). Certainantiepilepticdrugsactasmoodstabilizersbyinhibitingcalciumcurrentsandare

effectiveintreatingpatientswithepilepsyas wellaspatientswithBD.Thesefindingsimply apotentiallinkbetweenmoodpolarity(particularlymania)andseizures.Asbothepilepsyand BDhavewell-describedgeneticsubstrates,in thisanalysisweascertainedsharedgeneticunderpinningsandcausaleffectsandunveiledsix independentgenomiclocisignificantlylinkedto BDandepilepsy.

Utilizinggenome-wideassociationstudy (GWAS)datafromEuropeanpopulations,comprising26,352epilepsycasesand774,517controls(3),aswellas25,060BDtypeI(BD-I)cases and307,499controls(4),weobservedasignificantpositivegeneticcorrelation(rg = 0.154, P = 9.24 × 10–6 )betweenBD-Iandepilepsy. Furthermore,weindicatedameaningfulcausal effectofepilepsyonBD-I(P = 0.0079, bxy = 0.1721,SE = 0.0648).

OurMiXeRanalysisidentifiedapproximately 7.8KvariantsinfluencingBD-Iand3.0Kimpact-

ingepilepsy,with1.3Kvariantsimplicatedin bothconditions(Figure1A).Weunveiledsix independentgenomicloci(r2 < 0.2)significantlylinkedtoBD-IandepilepsyusingConjunctionalFalseDiscoveryRate(conjFDR)analysis(conjFDR < 0.05, Figure1B),amongwhich fourlociexhibitedconsistentalleliceffectdirectionbetweenBD-Iandepilepsy,whilethe remainingtwolocishowedoppositedirection. Moreover,wefoundthatfiveofthesixriskloci showedexpressionquantitativetraitlociassociationsincortextissuesorspecificcelltypes (P < 1.00 × 10–5 ,SupplementalTableS1). Wefocusedonrs9639379intheSP4gene, findingstrongassociationswithbothriskofBDI(oddsratio(OR) = 1.0638, P = 1.41 × 10–6 ) andepilepsy(OR = 1.0437, P = 2.31 × 10–5 ) (conjFDR = 1.24 × 10–2 , Figure1C).ThestabilityofSP4proteinwasmodulatedbyneuronal activity,withlithiumdemonstratingtheabilitytostabilizeSP4levels,therebysuggesting

Figure1. (A)VennplotshowsthenumberofspecificandsharedcausalvariantsbetweenBD-Iandepilepsy.Thegeneticcorrelationof rg wasestimatedbyLinkageDisequilibriumScore Regression(LDSC).(B)ManhattanplotofconjFDRresult.LeadSingleNucleotidePolymorphisms(SNPs)ineachindependentrisklociwiththesamedirectionofalleliceffectsbetween BD-Iandepilepsyaremarkedinred,andleadSNPsineachindependentrisklociwithoppositedirectionofalleliceffectsbetweenBD-Iandepilepsyare markedinblack.(C)LocusZoom plotsshowthegeneticassociationswithBD-IandepilepsyintheSP4locus.Physicalmapsblowtheplotsdepictknowngeneswithintheregion,andtheLinkageDisequilibrium(LD)is definedbasedontheSNPrs9639379.

Received:9June2024.Revised:14August2024and2September2024and17September2024and18September2024.Accepted:18September2024. Publishedonline:30September2024.

therapeuticbenefitsinmooddisordermanagement(5).Whilethedirectassociationbetween SP4andepilepsyremainsunclear,theinvolvementofSP4inthetranscriptionalregulationof neuronalenergymetabolismsuggestedaplausiblelinktoepilepticseizures(6).

Thisstudyprovidesanovelrethinkingofthe connectionbetweenepilepsyandBD,whichis inlinewiththefactthatmoodstabilizersare effectiveinthetreatmentofbothillnesses.Althoughtherelationshipbetweensharedrisk genesandmoodstabilizersisstillunclear,their potentialinvolvementindrug-mediatedneurobiologicalmechanismsisworthfurtherinvestigation.Limitationsincludethefocuson Europeanpopulations,whichmayconstrainthe generalizabilityofthefindings,andthereliance onpublicGWASdatawithoutsex-specificinformationrestrictingusfromconductingagenderbasedanalysis.

AuthorContributions

MLoversawtheproject,conceivedanddesigned thestudyandJHHperformedtheprimary analysisanddraftedthefirstversionofthe manuscript.Allauthorsrevisedthemanuscript criticallyandapprovedthefinalversion.

ConflictsofInterest

Nonedeclared.

Funding

ThisworkwassupportedbygrantsfromSpring CityPlan:theHigh-levelTalentPromotionand TrainingProjectofKunming(2022SCP001).ML

wassupportedbytheYunnanRevitalizationTalentSupportProgramYunlingScholarProject.

Jin-HuaHuo1 , 2 ,andMingLi1 , 2 , 3

1 KeyLaboratoryofGeneticEvolutionandAnimal Models,KunmingInstituteofZoology,Chinese AcademyofSciences,Kunming,Yunnan650201,China

2 YunnanKeyLaboratoryofAnimalModelsandHuman DiseaseMechanisms,KunmingInstituteofZoology, ChineseAcademyofSciences,Kunming,Yunnan 650201,China

3 KIZ-CUHKJointLaboratoryofBioresourcesand MolecularResearchinCommonDiseases,Kunming InstituteofZoology,ChineseAcademyofSciences, Kunming,Yunnan650201,China

e-mail: limingkiz@mail.kiz.ac.cn

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