Use of Innovative Multi-Omics Platforms to Study Neurodegenerative Disease Kerry Shea1,3, Lucy, Frost1, TJ Allen1, Eleanor Platt1, Irma O’Meara1, Ana-Maria Nastase1, Tara Bowen1, Maike Langini1, Régine Anderson1, Caitlin Shaw1, Paul Sharp1, Jim McCarthy2, Donna Finch2, Gayle Marshall1 1 Medicines Discovery Catapult, Alderley Park, Cheshire, SK10 4ZF, UK; 2 Alchemab Therapeutics, Whittlesford, Cambridgeshire, CB22 4WL, UK. 3 Corresponding Author: info@md.catapult.org.uk
Abstract This study aimed to explore biomarkers in Huntington's disease (HD) using various multi-omics platforms to profile a selection of both clinical and pre-clinical samples. By employing both targeted and untargeted approaches in proteomics, lipidomics, and metabolomics, we sought to identify molecular signatures that could enhance understanding of disease progression and inform the development of targeted treatments.
Clinical Samples •
Predict-HD is a clinical research project collating a biobank of Huntington’s Disease (HD) patient and healthy age-matched control biological samples alongside clinical data (dbGaP Study No. phs000222.v6.p2).
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Plasma and CSF samples were obtained from PREDICT-HD for biomarker assay development and screening.
Pre-Clinical Samples Plasma
CSF
Age Matched Control
26
17
Pre-symptomatic
38
45
Symptomatic
35
15
Total
99
77
• R6/1 is a Huntington’s disease transgenic mouse model that overexpresses exon 1 of the human HD gene with a polyglutamine repeat expansion in the region of 115-150 length.
R6/1 HD Model C57 WT Model
Strains B6.Cg-Tg(HDexon1)61Gpb/J C57BL/6J
• The model displays a progressive neurological phenotype involving molecular, behavioural and motor disturbances that mimic many of the features of Huntington Disease in humans. • Plasma and brain tissue samples were taken at three timepoints: pre-symptomatic (6 weeks), early symptomatic (12 weeks) and late symptomatic (16 weeks) from the R6/1 model in addition to the non-transgenic control background C57BL6J model.
Spatial Analysis
Bulk Analysis Profiling of CSF, plasma and brain homogenates was carried out with a combination of targeted proteomic approaches using Luminex and Olink multiplex assays and an untargeted approach examining proteomic, lipidomic and metabolomic profiles.
Consecutive sections from 2 areas of flash frozen brain tissue, encompassing the hippocampus or striatum, from the R6/1 transgenic and C57 wildtype models used for novel biomarker discovery.
• •
Targeted proteomic analysis of 388 proteins was examined in the plasma and CSF samples using Luminex and Olink multiplex assays. Luminex platform (26 proteins), including novel assay builds
Luminex
•
Olink
Targeted Proteomic Multiplex Immunohistochemistry
Explore 384 Neurology 1 Panel
9 Protein Multiplex
Olink platform (367 proteins)
Amyloid beta 1-42 BDNF YKL-40 (CH13L1) CNTF FGF-21 GDNF GFAP Kallikrein-6 (KLK6) MIF
NCAM-1 Neurogranin (NRGN) NF-H NGF beta S100B Tau (Total) Tau (pT181) TDP-43 UCHL1
4 Protein Multiplex
Apolipoprotein E4 Clusterin (Apo J) Complement Factor H Fetuin-A
2 Protein Multiplex
mHtt exon 1 Neurofilament (L/M/H)
Singleplex
mHtt 1-1220
Singleplex
Aggregated Htt
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A dual immunofluorescence assay was designed and optimised to detect huntingtin protein, both total and protein in an aggregated conformation.
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An algorithm was developed alongside to quantify the staining patterns observed using Visiopharm AI-driven precision pathology software (Data not shown).
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This assay was used to profile huntingtin protein in brain tissue from the pre-clinical models with both found to increase in the R6/1 model at the late symptomatic 16 week period.
Novel Luminex Assay Build •
4 Novel assays were designed and optimised for 3 species of huntingtin protein and combined neurofilament light, medium and heavy.
1) mHTT 1-1220 Fragment or 2) mHTT exon 1 Fragment
2) mHTT exon 1 Fragment
1) mHTT 1-1220 Fragment mHTT 1220 Std Curve No Lines
mHTT exon1 Std Curve No Lines
100000
10000
10000
Lipidomics and Metabolomics
3) Aggregated HTT protein
1000
MFI
MFI
1000
100 100
10
10 10 -13
10 -12
10 -11
10 -10
10 -9
10 -13
10 -12
[HTT-Q48] (pM)
10 -11
10 -10
10 -9
[HTT-Q48] (pM)
4) Neurofilament-H/M/L
3) Aggregated HTT Protein Aggregated HTT Quantification 1.2
Neurofilament (L/M/H) Std Curve
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Mass spectrometry imaging was used to profile the metabolomic and lipidomic profile of the striatum region of the brain in the R6/1 model of HD and compare it with the wildtype C57 model.
•
A selection of lipid species, all directly or indirectly involved in neuroinflammation were found to be upregulated in the R6/1 model.
1.0
4) Neurofilament-H/M/L protein
1.0 0.8
Normalised net MFI
Normalised MFI
0.8
0.6
0.4
0.6
Transcriptomics
0.4
0.2
0.2
0.0
0.0 R6/1
10
C57
100
1000
10000
•
The GeoMx Digital Spatial Profiler was used to perform spatially resolved transcriptomic profiling.
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Several regions of the brain tissue from the pre-clinical models were selected for profiling: hippocampus (dentate gyrus, CA1, CA3), striatum and cortex.
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Further segmentation to enrich or deplete the profile for microglial cells was additionally performed in the cortex area.
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The numbers of Differentially Expressed Genes (DEGs) between the 2 models varied by the anatomical brain region, timepoint or enrichment/ depletion processes for microglia.
100000
[NF-L/M/H] (pg/mL)
Untargeted Lipidomics/ Metabolomics/ Proteomics •
Mass Spectrometry was used to profile proteins, lipids and metabolites in the same sample sets in an untargeted manner.
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This data was used to identify significant differences in between the patient cohorts and in the R6/1 model.
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Untargeted analysis allows the unbiased detection of molecules in a sample, enabling the discovery of novel and unexpected biomarkers.
A panel of proteins, lipids and metabolites were identified as potential biomarkers in Huntington’s disease