Μελέτη αναλόγων ενώσεων των Νευροτροφινών.

UNIVERSITY OF CRETE DEPARTMENT OF CHEMISTRY THESIS 2019-2020

STRUCTURE-FUNCTION OF NEUROTROPHIN ANALOGUES ON NEUROTROPHIN RECEPTOR ACTIVATION AND CELL SURVIVAL

Regenerative Pharmacology lab Department of medicine in collaboration with foundation for research & technology – Hellas (FORTH) lab

Stella Bitsika

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Contents Acknowledgements ...................................................................................................... 3 Περίληψη ..................................................................................................................... 4 Abstract ........................................................................................................................ 6 Abbreviations ............................................................................................................... 7 Introduction .................................................................................................................. 8 Alzheimer’s Disease ................................................................................................. 8 Amyloid beta hypothesis ....................................................................................... 8 Tau protein ...........................................................................................................10 Neurotrophin factors and tropomyosin kinase A receptor (TrkA) .............................11 Signaling Pathway ...................................................................................................12 Neurotrophins in Alzheimer’s Disease .....................................................................14 DHEA (Dehydroepiandrosterone) ............................................................................15 Microneurotrophins Analogues ................................................................................16 Aim of my Research Project ....................................................................................17 Materials and methods .................................................................................................18 Cell lines and Transfection .......................................................................................18 Immunoblotting .......................................................................................................18 Stripping Buffer .......................................................................................................19 MTT Assay ..............................................................................................................19 Results ........................................................................................................................20 Stimulation of TrkA receptor with DHEA analogs ...................................................20 Cell Survival assay...................................................................................................21 Time-course activation of TrkA receptor after ENT-A010 and NGF treatment .........22 Effect of DHEA analogues on TrkA downstream signaling pathway ........................23 Discussion ...................................................................................................................26 Bibliography ...............................................................................................................28 2

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Acknowledgements First and foremost, I want to express my deepest gratitude to my professor Ioannis Charalampopoulos, for giving me the opportunity to take place in this interesting research project. He took the risk from my first steps, because I was in third year of Chemistry department and my knowledge was limited in neurobiology. Ηe contributed to me to gain lab experience and obtain new biochemical procedures. Because of this experience, I realized that I love the research, I love the lab and of course, I would like to continue as a neuroscientist researcher, due to this opportunity. He listened and advised me all the times, you have been a fantastic person. Also my sincere thanks to Charalabos Katerinopoulos, who accepted to supervise my thesis from department of Chemistry. He advised me for my research direction, giving me motivation and encouragement to continue and dare to change and associated two research fields Chemistry and Neuroscience.

More personally, I would like to extend my appreciation to my life time supervisor Thanasis Rogdakis. Τhanasis is one of the best supervisor, that I could have had. He taught me many things, experimental assays, research way of thinking, advised me in my student route. Is a supervisor, who gives you strong motivation to reveal a new drug, as it difficult it seems to be. Thanasis I hope to succeed in your career and be proud of you. Except of supervisor, he was my good consultant friend in the lab.

Many thanks for candidate phD student Despoina Charou, who her part is stimulates my psychology, after a failure result with encouragement and her smile every day. I think that she is the most optimistic person I have ever met.

Many thanks to my friend Konstantina Mylonaki, who is my company every weekend in the lab and offers me several biscuits and homemade food. I really appreciate it Konstantina!

I am very thankful to my dear friends and phD students in our lab, Maria Kokali, Mariana Papadopoulou, Alexandros Tsibolis.

Special thanks to my friend and associate professor Georgios Kouvarakis, for support in four years graduation in the University of Crete. He advised me many times for my next steps and he stood me as a friend several moments. I am very glad that I am graduating from University of Crete, because I met you all and you contributed to evolve my knowledge, my personality and my way of thinking.

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Περίληψη Η νόσος του Aλτσχάιμερ είναι μία νευροεκφυλιστική νόσος, η οποία επί του παρόντος πλήττει παραπάνω από πενήντα εκατομμύρια ανθρώπους παγκοσμίως. Η πρόκληση της ασθένειας οφείλεται στη συσσώρευση αβ αμυλοειδών πλακών στον εξωκυττάριο χώρο, καθώς επίσης και των νευροϊνιδίων από την tau πρωτεΐνη που συναντώνται στην ενδοκυττάρια περιοχή. Τα χαρακτηριστικά της νόσου περιλαμβάνουν σοβαρά ελλείματα μνήμης, μάθησης και εξασθένισης στην εκτέλεση των καθημερινών εργασιών. Οι νευροτροφίνες είναι μια οικογένεια πρωτεϊνών, όπου σε αυτές συμπεριλαμβάνεται ο νευρικός αυξητικός παράγοντας (NGF), που ελέγχει την ανάπτυξη και την επιβίωση των νευρώνων ρυθμίζοντας τη νευρική και συναπτική λειτουργία. Επιπλέον, σε μοντέλα και ασθενείς που εμφανίζουν τη νόσο, τα επίπεδα έκφρασης νευροτροφινών απορρυθμίζονται, γεγονός που υποστηρίχθηκε ότι συμβάλλει στην παθολογία της νόσου. Προηγούμενες έρευνες του εργαστηρίου μας έχουν δείξει ότι το νευροστεροειδές διϋδροεπιανδροστερόνη (DHEA) εμφανίζει υψηλή συγγένεια σύνδεσης με τους υποδοχείς του αυξητικού νευροτροφικού παράγοντα NGF, δηλαδή τους TrkA και p75 NTR υποδοχείς, εμφανίζοντας αντι-αποπτωτική δράση μέσω των μονοπατιών σηματοδότησης αυτών των υποδοχέων. Παρολ’αυτά η DHEA είναι μία στεροειδής ορμόνη, η οποία μεταβολίζεται in vivo σε οιστρογόνα κι ανδρογόνα, γεγονός που αναστέλλει την δυνατότητα μακροχρόνιας χορήγησής της, καθώς θα οδηγούσε σε αυξήμενο κίνδυνο για ορμονοεξαρτώμενες νεοπλασίες. Από την άλλη, ο νευρικός αυξητικός παράγοντας (NGF) είναι ένα μόριο με μεγάλο μοριακό βάρος και πολύπλοκή πεπτιδική δομή με αποτέλεσμα να μειώνεται η θεραπευτική του ικανότητα, αφού δεν έχει τη δυνατότητα όπως τα περισσότερα φάρμακα να διαπερνούν τον αιματοεγκεφαλικό φραγμό (BBB). Οι παραπάνω λόγοι οδήγησαν στη δημιουργία ενώσεων που να μιμείται τη θεραπευτική δράση των νευροτροφινών, με μοριακό βάρος και δομή παρόμοια με τη DHEA, με σαφώς λιγότερες παρενέργειες. Στο εργαστήριο μας βρέθηκε το BNN27 ανάλογο, το οποίο μπορεί επιλεκτικά να δεσμεύει και να ενεργοποιεί τους υποδοχείς νευροτροφίνης. Έτσι, στην παρούσα πτυχιακή παρουσιάζεται η μελέτη αναλόγων ενώσεων που να μιμούνται τη δράση των παραπάνω, μέσω φωσφορυλίωσης του υποδοχέα. Εν συνεχεία, ερευνήθηκαν τα ενδοκυττάρια μονοπάτια που ενεργοποιούν οι συγκεκριμένες ενώσεις με σκοπό την επιβίωση των νευρικών κυττάρων. Τα αποτελέσματα αναπαρίστανται με τεχνικές ανάλυσεις επιβίωσης κυττάρων, καθώς και ανοσοπροσδιορισμού δείχνοντας ότι αρκετές ενώσεις δρουν με παρόμοιο τρόπο με τις νευροτροφίνες, κυρίως όμως ΕΝΤ-003, ΕΝΤ-005, ΕΝΤ-010, οδηγούν

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στην επιβίωση των νευρικών κυττάρων. Οι παραπάνω πειραματικές διαδικασίες αφορούν in vitro ανάλυση, τα πειράματα συνεχίζονται in vivo για την ολοκλήρωση του αποτελέσματος.

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Abstract Alzheimer’s disease (AD) is a chronic neurodegenerative disease with well-defined pathophysiological mechanisms, mostly affecting medial temporal lobe and associative neocortical structures. Neuritic plaques and neurofibrillary tangles represent the pathological hallmarks of AD, and are respectively related to the accumulation of the amyloid-beta peptide (Aβ) in brain tissues, and to cytoskeletal changes that arise from the hyperphosphorylation of microtubule-associated Tau protein in neurons. Neurotrophins are a family of secreted proteins, for instance, Nerve Growth Factor (NGF) and Brain-Derived Neurotrophin Factor (BDNF), that control neuronal development and survival and have also been shown to modulate neuronal and synaptic function. Moreover, in AD models and patients, neurotrophins processing and expression levels are deregulated and this has been postulated to contribute to the disease pathology. Previous studies in our lab have shown that NGF neurosteroid dehydroepiandrosterone (DHEA) interacts with the nerve growth factor (NGF) receptors, namely TrkA and p75NTR, having the ability to activate the cell downstream signaling cascades. However, in vivo studies of DHEA shows that metabolized to androgens and estrogens, presenting a limiting factor for its long-term use, due to increase of the risk for hormone-dependent neoplasias. On the other hand, NGF is a big biomolecule and due to polypeptide chain has not the ability to penetrate Blood-Brain barrier (BBB). For all these limitations it is crucial to exist an analogue with mimic action, like NGF and structure like DHEA. BNN27, a synthetic analogue called microneurotrophin, can mimic the stimulation of NGF and the molecular structure penetrates the BBB. The present study focuses on the screening functional characterization of newly synthesized, neurotrophin-like, small molecules, that selectively activate TrkA receptor, causing its phosphorylation. Additionally, It is researched the cell downstream signaling mainly with Western Blotting and Survival assays. It seems that many analogues work as a microneurotrophin, via phosphorylation of TrkA receptor, but are appeared some more potent targets drugs, which could be use as therapeutic target in AD.

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Abbreviations APP Amyloid Precursor Protein, AD Alzheimer’s Disease, Aβ Amyloid-β Peptide CaMK-ll Calcium calmodulin-kinase ll, CNS Central Nervous System, GSK3β Glycogen Synthase Kinase-3β, MAPK Microtubule Associated Protein Kinases, NFT Neurofibrillary Tangle, PKA Protein Kinase A, PKC Protein Kinase C, sAPPα Soluble N-terminal Fragment, NGF nerve growth factor, BDNF brain-derived neurotrophic factor, NT-3 neurotrophin-3, DHEA dehydroepiandrosterone, BNN27, C17-spiroepoxy analog of DHEA, CHO Chinese Hamster Ovary cell line, PC12 pheochromacytoma cells, Trk tropomyosin related kinase, ERK Extracellular signal-regulated kinase, PI3K phospatidylinositol-3-OH kinase, PLCγ Phospholipase C gamma, IP3 inositol 1,4,5- trisphosphate, DAG diacylglycerol, NMDA, N-methyl-D-aspartate, Raf Rapidly Accelerated Fibrosarcoma, Ras Rat sarcoma, Frs2 factor receptor substrate 2, Src Proto-oncogene tyrosine-protein kinase, Shc Src homology domain- containing, TBS riethanolamine-buffered saline, DMEM (Dulbecco's Modified Eagle Medium), SOS son of sevenless, Grb2 Growth factor receptorbound protein 2, SHP2 SH2 containing protein tyrosine phosphatase-2, CREB cAMPresponse element binding protein, GDP Guanosine diphosphate, GTP Guanosine triphosphate, cAMP Cyclic adenosine monophosphate, PIP2 phosphatidylinositol (4,5)bisphosphate.

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Introduction Alzheimer’s Disease Amyloid beta hypothesis Alzheimer’s Disease (AD) is a chronic neurodegenerative disease with a complex pathophysiology. The accumulation of neuritic plaques extracellular, in combination with hyperphosphorylated tau protein forms neurofibrillary tangles intracellular, are the hallmarks of AD. Under physiological conditions, the transmembrane protein called Amyloid Precursor Protein (APP) undergoes proteolysis by an enzyme, α-secretase (Sakono & Zako, 2010).

Following this cleavage, are formed fragments, which are soluble and as a result easy to dispose of. The pathway is known as non amyloidogenic pathway. Furthermore, sAPPa possess neuroprotective and memory-enhancing effect, often being compared to cerebral growth stimulants. Thus, these two features of reduced Aβ generation and sAPPa-induced neuroprotection point towards APP non-amyloidogenic pathway as a suitable therapeutic target for AD. Intriguingly, although amyloidogenic pathway is fairly well explored in relation to AD therapy, the non-amyloidogenic neuroprotective pathway remained mostly ignored. The advantages of sAPPa is the ability to lead in neuronal survival and synaptic plasticity by several cell signaling cascades. The most famous pathway is the PKC signaling, which can increase the level of anti-apoptotic proteins like BCl2 and Bcl-xL Modified tyrosine kinase (TK), mitogen-activated protein kinase (MAPK)-extracellular signalregulated kinase (ERK) signaling, and Ca2+ signaling are involved in the anti-apoptotic mechanism of sAPPa Additionally, acetylcholine, serotonergic and glutamatergic receptors, hormones and cholesterol-lowering statins increased the levels of sAPPa release. The nonamyloidogenic could be used as a therapeutic target but remained mostly ignored (Bandyopadhyay et al., 2007).Amyloidogenic pathway occurs when another enzyme, β secretase, specifically, beta-site APP-cleaving enzyme 1 (BACE1) and γ secretase cleave APP and produce the amyloid-β peptide (Aβ) (Roßner et al., 2006) .

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Figure 1: Τhe amyloid precursor protein (APP) can undergoes cleavage which leads in a non amyloidogenic and amyloidogenic pathway. APP may be cleaved from a secretase and then there are two fragments which are soluble or APP can be chopped up by β secretase and γ secretase. This aforementioned cleavage leads to three fragments containing aβ peptide. The oligomer aggregate is neurotoxic. As a result the neural communication breakdown and these senile plaques can potentially get between the neurons (Report, 2008) .

This accumulation produce a chemically sticky derivative and as a result the neural communication is destroyed (figure 2). The first disadvantage of the αβ accumulation is the neuronal cytotoxicity in cell membrane. The mechanisms start with channels or pores by stimulation of Ca2+ channel, such as NMDA. The process like lipid peroxidation initiates from αβ oligomers and thus in intracellular space are increased the levels of ion metal (Jang et al., 2007). Under normal conditions, neurons maintain the balanace of these gradients between intracellular and extracellular space, in order to be used as vesicular transmitter store after possible mobility. Conclusively, amyloid effect is located mainly on lipid rafts or microdomains, inducing morphologic and functional alterations to synapses and synaptic plasticity (Reiss et al., 2018).

Furthermore, αβ has the ability to penetrate the mitochondrial membrane and as a result is disrupted the bioenergetic circle, via interference with the electron chain (Mark et al., 1997). Ιt has been found that the limited activation of terminal enzyme in the electron transport chain, called cytochrome c oxidase associated with αβ accumulation and consistently presents the mechanism toxicity in AD brains. There are several mechanisms, which confirm the oxidative stress (Caspersen et al., 2005). Although the pathogenesis of AD is not completely understood, the accumulation of the Aβ protein fragment is a hallmark of the disease and the association between αβ and cholesterol needs further exploration.

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Figure 2: A chemically ‘’sticky’’ beta amyloid plaque disrupt the signaling (brown colour). Monomer of amyloid beta constists of 36-43 amino acids which is insoluble. (National Institute of Aging, What Happens to the Brain in Alzheimer's Disease? viewed 26 November 2020, <https://www.nia.nih.gov/health/what-happens-brain-alzheimers-disease>)

Tau protein

In neurons, tau protein is linked with microtubules for the maintenance of neuronal structure, axonal transport, and neuronal plasticity (Binder et al., 2005).

Intracellularly, microtubules combined together in order to form a “cellular highway” which can provide the ability to transport cellular products from the soma to the axon terminal and vice versa. Tau stabilizes microtubules that make up this cellular highway. In AD amyloid beta activates an GSK3, which in turn phosphorylates tau protein. In the hyperphosphorylated state the proteins detached from highway and constitutes the initial step for an aggregation form of hyperphosphorylated proteins, called neurofibrillary tangles (figure 3)(Mandelkow & Mandelkow, 2011). Consequently, microtubules are destabilized and collapsed disabling intracellular transport, due to tau hyperphosphorylation. the interactions with microtubules is getting more limited. Thus, the balance of kinase and phosphatases acting on tau could be important for tau functions and dysfunctions (Avila et al., 2012).

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Figure 3 Presentation of healthy neuron (up) and diseased neuron (down), hyperphosphorylated Tau impairs axonal transport and synaptic metabolism, causing dysfunctions that result in loss of cell viability and ultimately lead to the collapse of

microtubular cytoskeleton and neuronal death.

Alamy Stock Photos, Alzheimer's Disease, Neurofibrillary Tangles, viewed 11 November 2020, <https://www.alamy.com/stock-photo-alzheimers-disease-neurofibrillary-tangles-135021392.html>

Neurotrophin factors and tropomyosin kinase A receptor (TrkA)

Neurotrophins are responsible for maintenance, development, and function of vertebrate nervous system. Under normal conditions neurotrophins activate two different classes of receptors the Tropomyosin receptor kinase (Trk A,B or C) family of receptor tyrosine kinases and p75NTR. They have been found four neurotrophins providing a well-characterized example of these target-derived instructive cues. The cytoplasmic domains of Trk receptors contain several sites of tyrosine phosphorylation that recruit intermediates in intracellular signaling cascades (Huang & Reichardt, 2001). There are nerve growth factor (NGF) - the first factor to be characterised, brain-derived neurotrophic factor (BDNF), and neurotrophins 3 and 4 (NT3 and NT4). It is important to mention that for many years researchers believed neurotrophins were binding to specific trk receptors, NGF to TrkA, BDNF and NT-4 to TrkB, and NT-3 to TrkC. However, it seems to be link among them, as NT-3 can also bind to TrkA and TrkB receptors that contain an additional short insert in the extracellular domain (Clary & Reichardt, 1994). Neurotrophins, bind to the Trk receptors by interacting with the most 11

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proximal immunoglobulin (Ig) domain of each receptor. The three-dimensional structures of each of these Ig domains has been solved and the structure of NGF bound to the TrkA membrane proximal Ig domain has also been determined (Ultsch et al., 1999).

As aforementioned, NGF is the first neurotrophic factor to be discovered. NGF binds to TrkA on the cell membrane causing its phosphorylation. This is the initial step in order to start a downstream signaling cascade following the phosphorylation of TrkA. Of the five domains comprising its extracellular portion, the immunoglobulin-like domain proximal to the membrane (TrkA-d5 domain) is necessary and sufficient for NGF binding with high affinity.Binding of neurotrophins leads to autophosphorylation of the receptors, which is believed to be induced by receptor homodimerization, a common trigger for tyrosine kinase activation (Urfer et al., 1995).

Figure 4 Crystal structure NGF monomers presents with red and blue, TrkA-d5 is green, in different orientations (90o). In this figure presents the high affinity of trkA and NGF epitope.(Ultsch et al., 1999)

Signaling Pathway The signaling starts with NGF binding to TrkA, causing its dimerization and activation.

There are several cells downstream cascades lead to neuronal survival or differentiation. A 12

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PLC-Îł pathway activation initiates with the phosphorylation in tyrosine 785 of TrkA receptor or similar docking sites on trkB and trk C. After this recruitment, is caused hydrolysing reaction by PIP2 and are formed IP3 and DAG as a products, then intracellular Ca2+ is released (Kaplan & Stephens, 1994). The increasing levels of Ca2+ results in the activation of dependent enzymes such as CaMK. The release of Ca2+ and production of DAG leads in activation of Raf/ERK signaling pathway, by activation of PKC (Huang & Reichardt, 2003) (Clary & Reichardt, 1994).

Figure 5 Signaling pathway TrkA (Reichardt & Reichardt, 2006)

Activation of TrkA can also be caused via phosphorylation in tyrosine 490 residue.

Subsequently, the aforementioned leads to phosphorylation of fibroblast growth factor receptor substrate 2 (Frs2) and the Src homology domain- containing (Shc) adaptor proteins.

This recruitment has the ability of stimulation PI3K with Ras-dependent mechanisms, where PI3K is created for phosphorylation of Shc or Frs2 with the following recruitment Grb2, complex with Ras exchange factor son of sevenless (SOS). The activation of PI3K also plays an important role in cell survival by activation of Akt and phosphorylation and inhibition of apoptosis-promoting proteins such as Bad and GSK3b, Ras-independent activation requires phosphorylation of Grb2 with the following recruitment of Gab1. This binding leads to stimulation of PI3K (Reichardt & Reichardt, 2006). Akt is activated by PIP3, the last is a product from PIP3K stimulation via phosphorylation at the 3â&#x20AC;&#x2122; position of phosphatidylinositol (4,5)-bisphosphate (PIP2) (Skaper, 2012). Shc-Grb2 complex recruitment to Trk, which is 13

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modulated by the Frs2/SHP2 complex, also results to Erk pathway activation (Reichardt & Reichardt, 2006). As we can see in figure 5 (right side) Frs2 and Shc are crucial for the formation of complex Grb2-SOS which stimulate Ras by replacing GDP to GTP. Raf, in turn is activated by stimulation of Ras and with the phosphorylation of MEK is activated Erk via phosphorylation, too. Eventually, Erk activation leads to CREB-regulated gene expression, which involves multiple genes required for neuronal survival and differentiation (Deinhardt & Chao, 2014)(Reichardt & Reichardt, 2006).

Neurotrophins in Alzheimer’s Disease Although, it is known that neurotrophins possess a crucial neuroprotective effect in neurodegenerative diseases, like AD, has not been found investigations in pharmacokinetics and protein delivery properties. In PC12 cells and primary neurons, experimental assays present that the absence of NGF leads to αβ accumulation and apoptotic effects (Matrone et al., 2008).

It is worth mentioning CNS diseases are difficult to treat due to the presence of the blood brain barrier (BBB) that makes it almost impossible for large biomolecules to penetrate into the brain. Additionally, the cortical and subcortical circuits of the brain are interconnected resulting in crosstalk among multiple regions, so coming up with a treatment strategy that selectively targets affected neurons only, but not those unaffected ones, is a great challenge that has to be carefully considered. Further to these issues, neurotrophins are relatively large, polar molecules that cannot readily cross the BBB and therefore must be administered directly into the central nervous system (CNS). Indeed, all current delivery strategies involve invasive procedures as discussed below (Weissmiller & Wu, 2012).

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DHEA (Dehydroepiandrosterone)

Figure 6: Biosynthesis of DHEA schematic view (Pediaditakis, Efstathopoulos, et al., 2016)

Dehydroepiandrosterone (5-androsten-3β-ol-17-one) is a naturally occurring C-19 adrenal steroid derived from cholesterol by a series of cytochrome P450-dependent monooxygenase and hydroxysteroid dehydrogenase catalyzed reactions. Produced by neurons and glia its sulphate ester are the most abundant steroid hormones in humans, and DHEA was described as the first neurosteroid produced in the brain. Recent research support that DHEA presents high affinity with NGF receptors, anti- apoptotic effect and stimulates downstream signaling cascades (Charalampopoulos et al., 2008).

Elimination of DHEA(S) has been associated with neurodegenerative disease, such as AD. It has been found that interacts with neurotrophins receptors, similar to NGF activity, and stimulates downstream signaling cascades (Goodarzi et al., 2014). It has been found that the pro-survival signaling pathways are started by DHEA at the membrane level. These pathways include kinases like

MEK1/2/ERK1/2 and PI3K/Akt. Interestingly, DHEA

stimulates these signaling pathways through TrkA receptor figure 7.

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Figure 7: Downstream signaling cascades of DHEA and the anti-apoptotic effect via receptorâ&#x20AC;&#x2122;s stimulation. (Lazaridis et al., 2011)

However, DHEA is metabolized to estrogens, androgens and related metabolites, affecting the endocrine system. Recent reports claim that long-term use of neurosteroid can cause serious side effects, especially in patients with genetic predisposition to hormonedependent tumors (Lazaridis et al., 2011).

Microneurotrophins Analogues It is well documented that large biomolecules like NGF has neuroprotective effects and the deprivation of them could lead to apoptotic effects. Unfortunately, due to the large size neurotrophins are not permeable from Blood-Brain Barrier (BBB). Surprisingly, DHEA was shown to bind and activate all Trk and p75NTR neurotrophin receptors in various neuronal cell

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types. However, there are several side effect of DHEA and it works as a precursor steroid in the biosynthesis of androgens and estrogens.

To overcome these obstacles a large chemical library of 17-carbon derivatives of DHEA was synthesized, in order to mimic the neurotrophin action. BNN-27 analogue presents similar affinity with NGF working via TrkA and p75 receptor. As a result, BNN-27 causes phosphorylation for TrkA receptor, a crucial signal to initiate the downstream signaling cascade. This microneurotrophin, a lipophilic small molecule, presents insignificant toxicity in in vivo and in vitro studies. For that reason, could be suggested as therapeutic target drug in neurodegenerative disease. Conclusively, all these compounds which were tested in our experiments, are based in BNN-27, with different functional groups, maintaining and exploiting the aforementioned benefits (Gravanis et al., 2017).

Figure 8: Synthetic microneurotrophins offer a potential pharmacological tool with non-toxic, blood-brain-barrier permeable neurotrophin receptor agonists and antagonists, effective in the treatment of neurodegenerative diseases like AD (Gravanis et al., 2017).

Aim of my Research Project In order to overcome the side effects of DHEA, we used several synthetic analogues with high permeability of BBB (brain-blood barrier) due to molecular structure. We used various techniques to screen these compounds and assess their ability to phosphorylate TrkA and its downstream targets Akt and Erk1/2. Additionally, we evaluated the efficacy of these compounds in protecting cells against Serum deprivation induced cell death.

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Materials and methods Cell lines and Transfection PC12 and CHO cell lines were cultured under specific conditions for each cell line. CHO cells were cultured in DMEM medium (Cat. No. 11965092, Thermo Fisher) containing 10% fetal bovine serum (Cat. No.10270-106, Thermo Fisher Scientific), 100 units/ml penicillin, and 0.1 mg/ml streptomycin (Cat. No. 15140-122, Gibco), at 5% CO2 and 37oC. Cells were transfected with TurboFect (Cat. No. R0532, Thermo Fischer) according to manufacturer’s instructions in 12-well tissue culture plates (Cat. No. 150628, Thermo Fischer) and were typically used on the 2nd day after transfection..

PC12 were grown in DMEM medium (Cat. No. 11965092, Thermo Fisher), 100 units/ml penicillin, 0.1 mg/ml streptomycin (Cat. No. 15140-122, Gibco), and 10% horse serum (Cat. No.16050130, Thermo Fisher Scientific), 5% Fetal Bovine Serum FBS (Cat. No.10270-106, Thermo Fisher Scientific) at 5% CO2 and 37°C.

Immunoblotting After overnight transfection, CHO cells were starved from serum and stimulated with compounds (500 nM) or with NGF (100ng/ml) (Cat. No. N-100, Alomone Labs) for the indicated times. Cells were suspended in Pierce™ IP Lysis Buffer (Cat. No. 87787, Thermo Fischer), supplemented with protease and phosphatase inhibitors (Cat. No. 539131, CalBiochem, 524629, Millipore). Then, heating our samples in 95 degrees and loading with 5X Laemli buffer containing 10% SDS, 2.5% b-mercaptoethanol, 50% glycerol and 0.25M Tris-HCL. SDS PAGE ran in running buffer 25mM Trizma Base (Cat. No. T6066, Sigma),192mM Glycine; 0.1% SDS, initiated in 80mV and when the marker are separated changed to 120mV. Proteins from the gel were then transfered in nitrocellulose membrane with transfer buffer 25mM Tris-HCl (Cat. No. T5941, Sigma), 192mM Glycine; 20% Methanol (Cat. No. 32213-251, Honeywell) , 350mA for 2h. . Afterwards, nitrocellulose membranes (Cat. No. 10600002, GE Healthcare Life Sciences) were blocked with 5% BSA (Cat. No. A2153, Sigma) for 1h. Membranes were then incubated with primary antibodies overnight at 4oC. For image analysis the membrane is washed with TBS-T for 10 minutes, 3 times and then incubated with secondary anti-rabbit, Goat anti-Rabbit HRP (Cat. No. 656120, Invitrogen) 1:5000 at room temperature. Adding ECL Western Blotting Substrate, which is a highly 18

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sensitive non-radioactive, enhanced luminol-based chemiluminescent substrate for the detection of horseradish peroxidase (HRP) (ECL Super Signal TM West Pico PLUS Chemiluminescent Substrate, Cat. No. 34580, Thermo Fisher Scientific) .

Similar process followed for PC12 cells with the difference in starvation was 4-6h, without transfection because PC12 cell culture has endogenously TrkA.

Stripping Buffer Membranes were stripped using stripping buffer (15 g glycine, 1 g SDS (Cat. No.13771, Sigma Aldrich), 10 mL Tween 20 (Cat. No. P7949, Sigma), Dissolve in 800 mL distilled water, Adjust pH to 2.2).

Bring volume up to 1 L with distilled water . Membranes were incubated for 5-10 minutes twice at room temperature with stripping buffer followed by two 10 minute washes with PBS and two 5 minute washes with TBS-Tween. Subsequently, they were ready for blocking and incubation with primary antibody.

MTT Assay For MTT assay, PC12 cells were plated in 96-well plates at density of 20,000 cells/well.

The following day following 3-4hrs serum starvation cells were treated with NGF or compounds for 48hrs. Cells were then washed twice with warm PBS and MTT 0.5mg/ml added in the wells. After 4hrs incubation at 37oC medium was removed and DMSO (Cat. No. D5879, Sigma-Aldrich) /isopropanol 1:1 added to each well. After 30’ incubation (15’ at room temperature and 15’ at 4oC) absorbance was measured using a plate reader at 545 nm with reference wavelength at 630nm. Each condition performed in triplicates.

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Results Stimulation of TrkA receptor with DHEA analogs To begin with, we tested some newly synthesized DHEA analogs for their ability to induce phopsphorylation of the TrkA receptor. In order to achieve that, we treated cells with the respective compounds and NGF for 20â&#x20AC;&#x2122; and assessed the phosphorylation of the receptor

by Western Blot. Figure 8 shows that compounds ENTA005 and ENTA010 can strongly phosphorylate TrkA at Y490.

Figure 8: Upper: Immunoblot of CHO cells transfected with TrkA, blotted against phosphorylated TrkA(up) total TrkA (down). Bottom: Quantification of n=5-7 independent experiments. Error bars represent SEM. *p<0.05, **p<0.001 *

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Cell Survival assay In addition to the phosphorylation assay we also investigated whether the compounds have any protective effect on cell survival. PC12 cells under Serum free conditions were treated for 48 hours with the compounds and their viability was quantified using MTT assay.

The MTT assay is a quantitative and sensitive detection of cell proliferation as it measures the growth rate of cells by virtue of a linear relationship between cell activity and absorbance.

Figure 2 shows that there is no significant difference between DHEA analogs.

Figure 9: MTT assay. PC12 cells treated for 48hrs

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Time-course activation of TrkA receptor after ENT-A010 and NGF treatment We next sought to investigate whether phosphorylation of TrkA receptor by ENTA010 is sustained over long time or it is a fast transient effect, like NGF. Preliminary results in figure 3 shows that ENTA010, like NGF, cause TrkA phosphorylation after 10, 20 and 30 minutes treatment and not after 1 or 2 hour treatment.

Figure 10: Upper: Immunoblot of CHO cells transfected with TrkA and treated with ENTA010 or NGF for different timepoints, blotted against phosphorylated TrkA (up) and total TrkA (down). Bottom: Quantification of one experiment.

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Effect of DHEA analogues on TrkA downstream signaling pathway Furthermore, the compounds were tested for the ability to initiate TrkA downstream signaling. Upon binding of NGF to TrkA a cascade of downstream signaling occurs which promote cell survival and differentiation. The most potent of these downstream targets are the Akt and ERK1/2 pathways. Here, we used PC12 cells treated with NGF or compounds for 30â&#x20AC;&#x2122; and tested for increased phosphorylation of Akt and Erk1/2. Thus, figure 11-14 shows that ENT-A003, ENT-A015, ENTA021, ENTA022 and ENTA023 can activate Akt and Erk1/2.

Figure 11: Upper: Immunoblot of PC12 cells treated with NGF or compounds for 30â&#x20AC;&#x2122;, blotted against phosphorylated Akt (up) total Akt (down). Bottom: Quantification of one experiment.

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Figure 12: Upper: Immunoblot of PC12 cells treated with NGF or compounds for 30â&#x20AC;&#x2122;, blotted against phosphorylated Erk1/2 (up) total Erk1/2 (down). Bottom: Quantification of one experiment.

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Figure 13: Upper: Immunoblot of PC12 cells treated with NGF or compounds for 30â&#x20AC;&#x2122;, blotted against phosphorylated Akt(up) total Akt (down). Bottom: Quantification of one experiment.

Figure 14: Upper: Immunoblot of PC12 cells treated with NGF or compounds for 30â&#x20AC;&#x2122;, blotted against phosphorylated Erk1/2 (up) total Erk1/2 (down). Bottom: Quantification of one experiment.

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Discussion It is known that neurotrophin factors regulate the neuronal cell survival and promote cell survival after injury and thus they could be used as a therapeutic agents against neurodegenerative diseases. Experimental assays present in PC12 cell lines, that elimination of NGF leads in ιβ accumulation plaques in case of AD (Matrone et al., 2008). However, biomolecules like NGF has high molecular weight and there is no ability to penetrate the BBB. Previous studies in our lab, have shown a smaller molecule, called DHEA, which can be used as an activator of TrkA receptor (Pediaditakis, Efstathopoulos, et al., 2016). Although DHEA has small size and simple structure, it belongs in category of neurosteroids. The disanvatage of neurosteroids, is that they metabolized into estrogens, androgens and related metabolites and thus affecting the endocrine system. So, a large chemical library of 17-carbon derivatives of DHEA was synthesized, in order to mimic the neurotrophin action while omitting the endocrine effects (Pediaditakis, Kourgiantaki, et al., 2016).

Our experiments, Figure 8 and 11, suggest that microneurotrophin ENT-A010 shows a similar pattern of TrkA activation with NGF. First experiments were carried out using CHO cell lines, which are transfected with TrkA receptor. Intriguingly, in CHO cells there were many results, with obvious phopshotylation of TrkA receptor. In PC12 cells, with TrkA receptor, there were no obvious results. PC12 is derived from a pheochromocytoma of the rat adrenal medulla, that have an embryonic origin from the neural crest that has a mixture of neuroblastic cells and eosinophilic cells, so could simulate better the signaling cascades after phosphorylation, because respond to NGF by extending neurites and acquiring neuronal phenotype (Drubin et al., 1985) .

We tested if the compounds can activate TrkA downstream signaling pathway via phosphorylation of Akt and MAPK. Figure 11 shows that compounds ENTA015 and ENTA003 strongly activate Akt. In MAPK pathway (figure 12) however, ENTA003 shows strong activation while ENTA015 does not. Similarly, in figures 13 and 14 compounds ENTA021, ENTA022 and ENTA023 show activation of both Akt and Erk1/2 The problem with this way of screening, using Western Blot, is that the result is often variable.

There are many factors, which can be affect the final result. Treatment of cells, problems in transfer procedure, incubation of antibody etc. So, more experiments could increase the certainty.

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Results shown in figure 8 suggest that ENT-A010 activates TrkA receptor. For this reason, we examined the activation of TrkA after time course exposure of ENT-A010 and NGF in CHO cell lines. Preliminary results in figure 10 shows that ENTA010, like NGF, cause TrkA phosphorylation after 10, 20 and 30 minutes treatment and not after 1 or 2 hour treatment.

Simultaneously, with immunoblotting assay, were carried out survival assays, in order to investigate if the compounds can rescue cells from Serum deprivation induced cell death.

Figure 9 shows that the tested compounds do not have any protective effect on cell survival.

Perhaps, MTT assay did not work efficiently, which owing to in the intermediate experimental steps. Because MTT formazan is insoluble in water, and it forms purple needle-shaped crystals in the cells. Therefore, prior to measuring the absorbance, an organic solvent such as dimethyl sulfoxide (DMSO) or isopropanol is required to solubilize the crystals. Additionally, the cytotoxicity of MTT formazan makes it difficult to remove cell culture media from the plate wells due to floating cells with MTT formazan needles, giving significant well-to-well error.

To conclude, ENT-003, ENT-015, ENT-021, ENT-022 and ENT-023 could be used as a potent therapeutic drug targets, in order to mimic neurotrophic factors, for instance, NGF and BDNF. We tested the cell signaling pathways of these compounds and it seems activation of TrkA receptor and downstream signals. The research project of neurotrophins should be continued with variable experiments , of survival assays, immunoprecipitation studies and of course after effective results in vivo models of promising compounds. In my opinion, it could be useful to change the way of screening, for instance using AlamarBlue, which is a survival assay similar to MTT, but the difference is there is no crystal, so better precision and it is nontoxic for cells as MTT. Additionally, I suggest to test the promising compounds with ELISA sandwich protocol, a more specific antibody procedure. Furthermore, trkA inhibitors, would be a solution for identifying which of these compounds have better affinity with the receptor, or using PC12 nnr5 cell line, can no longer respond to NGF because it lacks TrkA expression.

It would be a good idea, for the most potent activators, to have alongside in vivo studies with in vitro studies, for better comparison. I strongly believe that aforementioned promising compounds, could be used as therapeutic targets with no serious side effects.

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