"Alzheimer’s disease (AD) is the most widespread neurodegenerative disorder, affecting approximately 20 million people worldwide. AD pathology is primarily characterized by the formation of extracellular amyloid plaques resulting from the aggregation of insoluble amyloid-beta 1-42 (A-beta), and neurofibrillary tangles (NFT’s) resulting from intracellular aggregation of hyperphosphorylated tau protein. The current FDA-approved AD treatments do not stop or reverse neurodegeneration, but only treat the symptoms by increasing acetylcholine neurotransmitter. Our laboratory is attempting to provide an additional therapeutic approach by using neurotrophic factors to block apoptosis or to restore neurons. We previously demonstrated that, in an in vitro model for AD, hEPN-1 neurotrophic factor mimetics can block synthetic A-beta-induced neuronal cell death when added to cultures, presumably by blocking caspase activation. In this thesis, we extended these findings to study the effect of A-beta and hEPN-1 on tau hyperphosphorylation (as measured by immunoblots with phospho-specific antibodies) and nuclear DNA fragmentation (as measured by TUNEL staining), both in vitro and in vivo in AD transgenic mice. We found that A-beta induces the hyperphosphorylation of tau in both mouse N2a and human SHSY neuronal cells, and that hEPN-1 may lower this phosphorylation in N2a cells. Furthermore, we discovered that hEPN-1 can reduce nuclear DNA fragmentation when added both simultaneously to A-beta and 3 and 6 hours post A-beta addition. Finally, in vivo hEPN-1 may lower both tau hyperphosphorylation and caspase-7 related protein (C7RP) in AD transgenic (Tg) mice. The overall results validate our in vitro AD model, show the efficacy of hEPN-1 at blocking A-beta-induced DNA fragmentation even when added post-insult, and show that hEPN-1 may work in an AD mouse model. However, more studies must be conducted to confirm these findings. "

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Le « Brain-Derived Neurotrophic Factor » (BDNF) est la neurotrophine la plus abondante et la plus répandue dans le cerveau humain. De nombreuses études se sont intéressées à son rôle dans la survie neuronale, la croissance et la plasticité synaptique. La signalisation BDNF est dépendante de deux récepteurs, le récepteur tyrosine kinase (TrkB) et le récepteur neurotrophine p75 (p75NTR). Il est bien établi que le rôle trophique du BDNF est assuré via son récepteur de haute-affinité TrkB, alors que la forme précurseur proBDNF active p75NTR vers la voie d'apoptose. Cette double signalisation est physiologiquement contrôlée par un équilibre entre les différentes voies. Les résultats obtenus à partir des études cliniques et des modèles animaux suggèrent un rôle de la signalisation BDNF dans les tauopathies, caractérisées par l'existence de dépôts intracérébraux de protéine tau, une caractéristique commune à certaines maladies neurodégénératives, notamment la maladie d'Alzheimer (MA). Cependant, aucune investigation n'a été menée jusqu'à présent sur les modifications que pouvaient induire les tauopathies dans la signalisation BDNF et si une dérégulation de l'expression du BDNF pouvait affecter ses propres récepteurs TrkB et p75NTR.Dans ce travail de thèse, nous avons utilisé une lignée de poisson-zèbre transgénique portant la mutation humaine TAUP301L retrouvée notamment dans le démence fronto-temporale. Nous avons mesuré l'expression de BDNF et de ses deux récepteurs au niveau transcriptionnel et protéique. Nous n'avons observé aucune modification des taux d'expression de BDNF et de TrkB, en revanche, nous avons noté une augmentation significative de p75NTR. A l'aide de la même lignée transgénique, nous avons induit une baisse d'expression de BDNF via la micro-injection de morpholinos. De manière remarquable, la baisse d'expression de BDNF affecte de façon différentielle TrkB et p75NTR. En effet, nous avons observé une diminution de l'expression de TrkB et parallèlement une augmentation de p75NTR. De plus, la baisse d'expression de BDNF aggrave la neurotoxicité associée au développement de la tauopathie ce qui se traduit par une augmentation de la mort neuronale et de l'hyperphosphorylation de tau, cette dernière étant concommittante à une activation de la Glycogen Synthétase Kinase 3 beta (GSK3beta).Une diminution de l'effet neuroprotecteur de BDNF à travers un déséquilibre de ces récepteurs de signalisation a été également montré en étudiant le rôle de BDNF au cours du développement de la ligne latérale postérieure (PLL). Ce système est considéré comme un modèle d'étude particulièrement pertinent pour évaluer différents processus biologiques comme la migration cellulaire collective ou la régénération cellulaire. Nous avons détecté l'expression de BDNF dans plusieurs structures de la PLL. La diminution d'expression de BDNF conduit à un défaut de migration du primordium de la PLL, associé à une augmentation de la mort cellulaire. De plus, nous avons observé une réduction de la prolifération cellulaire et un défaut de repousse axonale du nerf, ce qui conduit à des anomalies de régénération à la fois du nerf de la PLL et des cellules ciliées. Nos résultats suggèrent que le BDNF joue un rôle essentiel au cours du développement de la PLL et démontrent la pertinence du système de la ligne latérale en tant que modèle d'étude des fonctions de BDNF.En conclusion, notre étude représente la première analyse du rôle in vivo de BDNF et de ses 2 récepteurs de signalisation. Nous avons ainsi montré les répercussions d'une dérégulation des voies de signalisation du BDNF. Un équilibre entre ces deux voies est essentiel pour le développement et la survie cellulaire, ce qui fait de BDNF non seulement une cible thérapeutique potentielle, mais également une neurotrophine clé pouvant activer plusieurs circuits de signalisation, potentialisant ainsi son rôle protecteur. / Brain-derived neurotrophic factor (BDNF) is the most abundant secreted and widely distributed neurotrophin in human brain. It has been extensively studied for its role in neuronal survival, growth and synaptic plasticity. BDNF signaling mediated through tryosine receptor kinase B (TrkB) and p75NTR neurotrophin receptor (p75NTR). It is well established that BDNF beneficial actions are mediated by it is high-affinity TrkB, whereas pro-BDNF activates p75NTR towards apoptosis. This diverse dual signaling is normally under a tight balance regulation. Based on clinical and animal studies, it has been suggested that BDNF signaling is involved in tauopathy, which is a pathological hallmark in several neurodegenerative diseases, including Alzheimer's disease (AD). However, what changes tauopathy may induce on BDNF signaling, and whether BDNF deregulation could affect its two signaling receptors (TrkB, p75NTR), and eventually tauopathy pathogenesis, have not been investigated. In this study we used a transgenic zebrafish line for human Tau-P301L tauopathy, and measured transcriptional and protein levels of BDNF and of its two signaling receptors. We found no modification of BDNF and TrkB expression levels, but a significant up-regulation of p75NTR. We then used the same transgenic line to generate BDNF knockdown using morpholino microinjection technique. Interestingly, BDNF knockdown differentially affects TrkB and p75NTR; we observed a reduction of TrkB expression and an increase in p75NTR expression. In addition, BDNF knockdown aggravates tauopathy-associated toxicity; we found an increase in neuronal cell death and tau hyperphosphorylation, the latter was accompanied by an activation of tau glycogen synthase kinase 3beta (GSK3beta). Attenuation of BDNF neuroprotective effects through imbalance of its signaling receptors was further highlighted through studying BDNF role in the development of zebrafish posterior lateral line system (PLL). This system has recently emerged as a powerful tool to study several dynamic biological processes, including collective cell migration and nerve/hair cells regeneration. We detected BDNF expression in different PLL components. BDNF knockdown led to an impairment of the PLL primordium migration due to concomitant increase in cell death rate. In addition, reduced cell proliferation and defect in axonal re-growth were observed , which led to major defects of PLL nerve/hair cells regeneration, respectively. These findings suggest that BDNF has an essential role in PLL development, but more important they introduce PLL as research model to study BDNF functions. This is the first study to provide a detailed in vivo analysis of BDNF and its two signaling receptors. Our findings highlight several implications of BDNF signaling deregulation. Balanced signaling clearly has essential roles in survival and development, in addition to being a therapeutic target, BDNF can itself activate diverse molecular pathways, thus setting up a potential circuitry that could enhance its protective role.

The current thesis investigated a potential relationship between Alzheimer disease and type-2 diabetes mellitus by analysing early gene expression related to insulin receptor signalling in the hippocampus as well as glucose metabolism in APP/PS1 mice, a model of familial Alzheimer disease. Compared to wild-type animals, a reduction in hippocampal insulin receptor and insulin-receptor substrate 2 transcripts in APP/PS1 mice three-month old as well as an increase in insulin-like growth factor 2 transcripts after six months was detected using real-time polymerase chain reaction. The alterations in hippocampal insulin signalling were accompanied by perturbation of glucose metabolism analysed by intraperitoneal glucose tolerance test. At the age of six months APP/PS1 mice developed glucose intolerance. Learning and recognition memory in APP/PS1 mice were tested using the Novel Object Recognition Test. Cognitive decline became evident in APP/PS1 mice at six months of age. Degradation of both insulin and amyloid β is mediated through insulin-degrading enzyme. However, expression of insulin-degrading enzyme in APP/PS1 mice was notdifferent from wild-type littermates. Changes in hippocampal phosphorylation of the tau phosphoepitopes serine 199, threonine 205, serine 396 and serine 404 were investigated using Western blot. Levels of three phosphoepitopes were increased significantly at either age.IV Protein expression of the phosphorylated form of glycogen synthase kinase 3β remained unchanged indicating an alternative pathway of tau phosphorylation in the APP/PS1 mouse model of familial Alzheimer disease. The current results demonstrate an increase in cyclin-dependant kinase 5 phosphoralyted at tyrosine 15 in APP/PS1 mice at three and six months of age. The correlation between elevated levels of phosphorylated tau and cyclin-dependant kinase 5 suggests that cyclin-dependant kinase 5 might contribute to tau phosphorylation in APP/PS1 mice. In general, this work corroborates common pathologic features in Alzheimer disease and diabetes mellitus. A significant cognitive decline in APP/PS1 mice was associated with changes in early gene expression of insulin-related molecules and perturbations in glucose metabolism. Cyclin-dependant kinase 5 is considered to coregulate tau phosphorylation in APP/PS1 mice, and to be part of a pathway contributing to pathology in Alzheimer disease

Neurological disorders are the biggest concern globally and ageing contributes in worsening the disease scenarios. In AD or AD like diseases, there is abnormal accumulation of extracellular amyloid beta produced due to abnormal processing of the transmembrane amyloid precursor protein, by β and γ-secretases. It spreads in the cortical and limbic regions of the brain leading to neuronal toxicity, impairment in memory and neurological functions. Aβ deposition in the CNS is common in aging HIV patients. Neurotoxic protein Tat, results in increased Aβ in combination with drugs of abuse cocaine. We examined the role of Withaferin A, against Aβ induced neurotoxicity. Our in-vitro dose optimization study demonstrates that lower concentrations (0.5–2 μM) of WA significantly reduce the Aβ40, without inducing cytotoxicity in the APP plasmid transfected SH-SY5Y cells (SHAPP). We demonstrate that Aβ secretion is increased in the presence of Tat (50 ng/ml) and coc (0.1 μM), WA reduces the Tat and coc induced increase in Aβ40. Additionally, we studied the role of WA against NF-kB mediated neuroinflammation, and observed that WA inhibits the expression of NFkB2 and RELA transcription factors, which play a major role in the expression of inflammatory chemokines. Further, to address the issue of minimal drug bioavailability in the CNS, we developed the WA loaded liposomal nanoformulation (WA-LNF) and characterized its size (499+/-50nm), toxicity and drug binding efficacy (28%). Our in-vitro 3D BBB transmigration of WA-LNF demonstrated ~40% transmigration efficiency. Furthermore, it was imperative for us to understand the mechanism of action of WA, therefore we studied the molecular mechanism of interaction of WA with Aβ protein by in-silico molecular dynamics simulations. We demonstrated that WA binds to the middle region of Aβ protein and the amino acid motif involved were FAEDVGS highlighting the mid-region Aβ capture by WA. 3 Hydrogen bonds were formed between WA and the amino acids, ASN17, GLY15 and SER16. This study reports WA as a potent neuroprotectant against amyloid induced neurotoxicity. Our study may have an immense therapeutic potential to target Aβ in the CNS, in the ageing patients and/or PLWH and/or ageing drug abusers.

La maladie d'Alzheimer (MA) est une maladie neurodégénérative dévastatrice qui touche un grand nombre de personnes. Elle entraîne des troubles de la mémoire et, éventuellement, une perte complète des fonctions cognitives. Le peptide amyloïde-β (Aβ) et la protéine associée aux microtubules tau sont généralement associés à la perte progressive de la mémoire. Dans les stades précoces, la MA se caractérise par une perturbation générale de l'efficacité synaptique. Les effets perturbateurs d'Aβ sur la plasticité à long terme sont bien documentés, par contre nos connaissances des effets immédiats du peptide sur la transmission synaptique sont limitées. Mon hypothèse est qu’Aβ ne nécessite pas une période prolongée pour promouvoir des changements de la transmission synaptique et qu’il peut modifier la fonction synaptique même après une exposition aiguë. Dans l’étude I, je test cette hypothèse à l’aide d’une exposition aiguë d'oligomères Aβ sur des tranches organotypiques d'hippocampe. Mes résultats indiquent qu’Aβ favorise une dépression synaptique sur les neurones pyramidaux hippocampiques avec une cinétique relativement rapide. Je démontre également que la dépression synaptique dépend de l'activation des récepteurs NMDA (NMDAR), mais ne dépend pas du flux d'ions à travers son canal ionique. Puisqu’il a été démontré que l'activation des NMDAR entraîne la phosphorylation de tau, il est plausible qu’Aβ modifie l'excitabilité des neurones en modulant la phosphorylation de cette protéine. Étant donné que les NMDAR jouent un rôle important dans la plasticité synaptique à long terme, cette chaîne d’événements moléculaires pourrait contribuer aux déficits de plasticité observés dans les phases initiales de la MA. Mon hypothèse est qu’Aβ modifie l’activité synaptique en modulant la phosphorylation de tau. Pour tester cette hypothèse, j’induis, dans des neurones de tranches de l’hippocampe, l’expression de formes de tau contenant des mutations qui bloquent la phosphorylation de la protéine aux sites ciblés. Dans l’étude II, j’observe que la phosphorylation de tau aux sites AT8 et AT180 régule l’expression de la plasticité synaptique ainsi que le dysfonctionnement de la transmission synaptique induits par les oligomères d’Aβ. Je démontre aussi que la phosphorylation du site PHF-1 ne contribue pas à la régulation de la plasticité et de la transmission synaptique. Puisque les sites AT8 et AT180 régulent l’interaction de la protéine tau avec la tyrosine kinase Fyn, mes résultats suggèrent que l’interaction entre tau et Fyn est importante pour l’expression de la plasticité synaptique et de la dépression favorisées par les oligomères d’Aβ. En effet, je démontre que l’inhibition de l’activité de la kinase Fyn résulte en un blocage de la dépression synaptique à long terme et un sauvetage de la fonction synaptique en présence d’Aβ. Je conclus que la phosphorylation de la protéine tau à des sites spécifiques est indispensable à l’expression de la plasticité synaptique et j’émets l’hypothèse que les oligomères d’Aβ modifient l'activité synaptique en influençant la stabilité du complexe Fyn-tau. Je propose donc que la perturbation de la stabilité de ce complexe peut être utilisée en thérapie pour inverser les déficits synaptiques dans les stades précoces de la MA. / Alzheimer disease is the most common form of dementia; it is characterized by problems in memory formation, which with time leads to a complete loss of cognitive functions. The peptide amyloid-β (Aβ) and the microtubule-associated protein tau are commonly believed to be responsible for the decline in memory formation. In the early stages of AD, this is thought to happen through a general disruption in synaptic efficiency. The disruptive effects of Aβ on long-term plasticity are well documented; however, little is known about the immediate effects of the peptide on synaptic transmission. My hypothesis is that Aβ does not need a prolonged period to promote changes in synaptic transmission, and that the peptide is able to affect synaptic function even after an acute exposure. In study I, I investigate this hypothesis using an acute exposure of Aβ oligomers to organotypic hippocampal slices. I report that Aβ promotes synaptic depression on hippocampal pyramidal neurons with a fairly rapid kinetic. I also show that the synaptic depression is dependent on the activation of the NMDAR, but independent on the ion flux through the channel. Because it was shown that the activation of the NMDAR leads to phosphorylation of tau, it appears feasible that Aβ modifies neuronal excitability by modulating tau phosphorylation. Since the NMDAR plays a critical role in the induction of long-term plasticity, this cascade of events could contribute to the deficits in plasticity observed during the initial stages of AD. My hypothesis is that Aβ modifies synaptic activity by modulating phosphorylation on tau. To test this hypothesis, I express in hippocampal neurons tau mutants in which phosphorylation on specific sites is blocked. In study II, I report that phosphorylation on tau at the AT8 and AT180 sites regulates the expression of synaptic plasticity as well as the dysfunction in synaptic transmission induced by Aβ oligomers. I also show that phosphorylation at the PHF-1 site is not involved in mediating either effects. Since the AT8 and AT180 sites regulate the interaction of tau protein with the tyrosine kinase Fyn, my results suggested that the interaction between tau and Fyn is important for the expression of synaptic plasticity and the depression mediated by Aβ oligomers. Indeed, I show that inhibiting the activity of Fyn kinase results in a block of LTD and a rescue of synaptic function in presence of Aβ. I conclude that phosphorylation of tau at specific sites is mandatory for the expression of synaptic plasticity, and suggest that Aβ oligomers promote changes of synaptic activity by influencing the stability of the tau-Fyn complex. I therefore propose that disrupting the stability of this complex can be exploited therapeutically to rescue synaptic deficits in the initial stages of AD.

<p>Tauopathies are neurodegenerative diseases defined by the accumulation of pathological tau protein in neurons and glia. Alzheimer’s disease (AD), the most common tauopathy, is characterized by the presence of neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein aggregates in neurons. Emerging evidence suggests that the NFT burden correlates with neuron death and cognitive decline, contributing to disease progression. Besides AD, a similar deposition of tau inclusions is found to be associated with neurodegeneration in the brains of patients with other tauopathies including progressive supranuclear palsy, corticobasal degeneration, and Pick’s disease. These diseases display clinical, biochemical, and neuropathological heterogeneity. Little is known about how tau aggregation can lead to varied phenotypes in tauopathies, and there is no disease-modifying treatment. Thus, it is necessary to understand the role of diverse tau proteoforms in tauopathies for the development of new therapeutics to treat tauopathies, including AD.</p> <p>In the studies summarized in Chapter 2, we investigated how the molecular diversity of tau proteoforms could impact antibody-based assays of a phospho-tau variant serving as an AD biomarker. A tau variant phosphorylated on threonine 181 (pT181-tau) has been widely investigated as a potential AD biomarker in cerebrospinal fluid (CSF) and blood. pT181-tau is present in NFTs of AD brains, and CSF levels of pT181-tau correlate with overall NFT burden. Various immuno-based analytical methods, including Western blotting and ELISA, have been used to quantify pT181-tau in human biofluids. The reliability of these methods depends on the affinity and binding specificity of the antibodies used to measure pT181-tau levels. Although both of these properties could in principle be affected by phosphorylation within or near the antibody’s cognate antigen, such effects have not been extensively studied. Here, we developed a bio-layer interferometry (BLI)-based analytical assay to assess the degree to which the affinity of pT181-tau antibodies is altered by the phosphorylation of serine or threonine residues near the target epitope. Our results revealed that phosphorylation near T181 negatively affected the binding of pT181-tau antibodies to their cognate antigen to varying degrees. In particular, two of three antibodies tested showed a complete loss of affinity for the pT181 target when S184 or S185 was phosphorylated.</p> <p>In the studies outlined in Chapter 3, we examined the relative abilities of different tau proteoforms to induce seeded tau aggregation and to themselves undergo seeded aggregation in cultured cells. Accumulating evidence suggests that tau aggregates, including NFTs, spread in a stereotypical pattern across neuroanatomically connected brain regions. This spreading phenomenon is thought to occur via a prion-like mechanism involving the release of tau aggregates from a diseased neuron into the extracellular space, aggregate uptake by neighboring healthy neurons, and the formation of new aggregates in the cytosol of the recipient cells via a seeding process. Although research over the past decade has revealed key molecular events involved in the cell-to-cell transmission of tau aggregates, the impact of the protein’s domain structure and phosphorylation profile on the efficiency of prion-like propagation remains poorly defined. Here, we compared three tau variants – K18, 0N4R, and 2N4R – in terms of their aggregation and seeding efficiencies in recombinant protein solutions and in cell culture models. Our results revealed that K18 had the highest fibrillization rate and yield among the three tau variants. Recombinant preformed fibrils (PFFs) derived from all three variants had similar seeding efficiencies. Additionally, we investigated the relationship between tau phosphorylation and aggregation. We found that hyperphosphorylated tau did not undergo self-assembly in the absence of heparin, whereas it formed fibrils at low yield in the presence of the cofactor. Moreover, hyperphosphorylated tau PFFs produced under these conditions induced seeded tau aggregation in cell culture.</p> <p>Taken together, these results point to critical roles of tau proteoforms as both AD biomarkers and drivers of disease progression. Our results indicate that the presence of different combinations of phosphorylated residues near a target phospho-tau antigen can affect the accuracy of antibody-based biomarker assays. In addition, the domain structure and phosphorylation profiles of tau proteoforms associated with AD and other tauopathies likely have a profound influence on the evolution of tau pathology in these disorders. Our findings highlight the importance of accounting for the molecular diversity of tau proteoforms in tauopathies and provide valuable insights into molecular determinants influencing tau aggregation and propagation in the brains of patients.</p>