Le vieillissement de la population est à l’origine de l’augmentation du nombre de personnes souffrant de démences, dont la plus fréquente est la maladie d’Alzheimer (MA). La MA est une maladie neurodégénérative incurable caractérisée par une atteinte progressive des fonctions cognitives, en premier lieu les fonctions mnésiques. Son diagnostic formel repose sur l’examen post-mortem du cerveau des patients. Il est basé sur la présence conjointe de deux lésions caractéristiques: des dépôts extracellulaires majoritairement composés de peptide amyloïde fibrillaire, résultat d’un clivage anormal du précurseur transmembranaire APP, et d’une dégénérescence neurofibrillaire ou pathologie tau, caractérisée par l’accumulation intra-neuronale de protéines tau hyper- et anormalement phosphorylées. Parallèlement à ces deux lésions se développe une réponse neuro-inflammatoire, notamment caractérisée par une augmentation du nombre et de l’activité des cellules microgliales et astrocytaires. Bien que les relations entre la pathologie amyloïde et la mise en place des processus neuro-inflammatoires aient fait l’objet d’intenses investigations, peu d’études se sont intéressés aux liens réciproques existants entre ces processus et la pathologie tau. A travers l’utilisation d’un modèle murin transgénique mimant le versant tau de la MA, la lignée THY-Tau22, un des objectifs de ma thèse a consisté à caractériser les différents aspects de leur réponse neuro-inflammatoire. Ces souris développent une pathologie tau hippocampique progressive associée à des altérations mnésiques. Les études transcriptomiques, biochimiques et histologiques réalisées ont mis en évidence une augmentation progressive de l’expression hippocampique de marqueurs de l’immunité innée mais également adaptative chez les souris THY-Tau22. Nous observons particulièrement l’établissement progressif de réactions microgliales et astrocytaires, une augmentation des niveaux de différentes chimiokines (CCL3, CCL4 et CCL5) conjointement à une infiltration parenchymateuse de lymphocytes T, en l’absence d’altération majeure de l’intégrité de la barrière hémato-encéphalique. Ces résultats mettent en exergue une corrélation entre le développement de troubles mnésiques et de la pathologie Tau hippocampique d’une part, et la présence d’une réponse neuro-inflammatoire d’autre part. La MA est une maladie multifactorielle dont la survenue est modulée par différents facteurs génétiques et environnementaux. Parmi les facteurs environnementaux mis en évidence par les études épidémiologiques, la consommation de caféine réduit notablement le risque de développer la MA. La caféine est une substance psychoactive dont les effets sont essentiellement médiés par le blocage des récepteurs adénosinergiques A1 et A2A, ces derniers étant particulièrement décrits pour moduler les processus neuroinflammatoires. Le rôle de ces récepteurs étant mal connus dans le contexte de la MA, et inconnu concernant ses relations à la pathologie Tau, la seconde partie de ma thèse a consisté à évaluer les effets de la caféine mais également d’un blocage spécifique des récepteurs A2A, par des approches génétiques et pharmacologiques, vis-à-vis des altérations comportementales, de la pathologie tau et de la réponse neuro-inflammatoire dans le modèle THY-Tau22. Les résultats obtenus démontrent que la caféine et le blocage spécifique des récepteurs A2A exercent des effets bénéfiques dans ce modèle de Tauopathie, avec une prévention des altérations mnésiques, une réduction de l’hyperphosphorylation de Tau et des effets anti-inflammatoires. Ces modifications sont associées à des effets bénéfiques en terme neurochimique et synaptique. L’ensemble de ces résultats démontrent pour la première fois un effet bénéfique de la caféine et du blocage des récepteurs A2A dans un modèle murin de tauopathie et suggèrent qu’un ciblage thérapeutique de ces récepteurs puisse être d’intérêt dans la MA. / Population ageing is a major risk factor for dementia, the most prevalent being Alzheimer disease (AD). AD is a neurodegenerative disorder characterized by a progressive cognitive decline, notably impacting memory functions. Its formal diagnosis is based on the post-mortem examination of AD patients’ brains and defined by the combination of two lesions: extracellular deposition of fibrillar amyloid peptide, resulting from the abnormal cleavage of transmembrane APP precursor, and neurofibrillary tangles, characterized by intraneuronal accumulation of hyper- and abnormal phosphorylated tau protein (Tau pathology). Besides these two lesions hallmarks, neuro-inflammatory processes, mainly defined by an increase of the number and the activity of microglial and astroglial cells, are considered as a third pathological component. Although the relationships between amyloid pathology and neuro-inflammatory processes had been the subject of intense investigations, few studies has been achieved with regards to tau pathology. As a first aim of this work, neuro-inflammatory processes associated with Tau pathology has been evaluated using a transgenic mouse model mimicking AD-like Tau pathology, THY-Tau22 strain.. These mice overexpress a mutated human tau protein under the control of a neuronal promoter and progressively hippocampal tau pathology associated to memory decline. Transcriptomic, biochemical and histological evaluations revealed a progressive increase several markers of both innate and adaptive immunity in the hippocampus of THY-Tau22 transgenic mice. We notably observed a progressive rise of microglial and astrogliale reactions, the overproduction of many chemokines (CCL3, CCL4, CCL5) in association with a parenchymatous infiltration of T cells, without major disruption of blood brain barrier (BBB). These results highlight a correlation between the establishments of memory alterations and hippocampal tau pathology on the one hand, and the occurrence of a neuro-inflammatory response on the other hand. AD is a multifactorial disorder whose occurrence depends on different genetic and environmental factors. Among the latter, epidemiological studies have shown that caffeine consumption significantly reduces the risk to develop AD. Caffeine is a psychoactive drug, whose effects are mainly ascribed to the blockade of A1 and A2A adenosinergic receptors, the latter beeing known to modulate neuro-inflammatory processes. The role of A2A receptors in AD is far from understood, and relationship with tau pathology currently unknown. The second part of my PhD aimed at evaluating effects of caffeine but also of a specific A2AR blockade, using genetic and pharmacological means, towards behavioural alterations, tau pathology and neuro-inflammatory processes in THY-Tau22 model. Results obtained demonstrate that caffeine and specific A2AR blockade lead to beneficial effects towards memory dysfunction, tau hyperphosphorylation and hippocampal neuro-inflammation. These improvements are associated with beneficial neurochemical and electrophysiological changes. Theses results demonstrate for the first time a beneficial effect of caffeine and A2A receptor blockade in a mouse model of tauopathy and support that therapeutic targeting of A2A receptors could be of interest in AD.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2014. / Alzheimer’s Disease (AD) is characterized by neurofibrillary tangles, senile plaques and neuronal loss. Although the mechanisms underlying Amyloid beta 42 (Aβ42) neurotoxicity have not been firmly established, it is proposed that the neuronal loss is elicited through associations with cell surface receptors. The cellular prion protein (PrPc) has been identified as an Aβ42 receptor and as a regulator of the amyloidogenic cleavage pathway. As Aβ42 shares common binding partners with the 37kDa/67kDa laminin receptor (LRP/LR), including PrPc, we investigated whether these proteins interact and assessed the pathological significance of this association. LRP/LR was found to co-localize with Aβ on the cell surface. The occurrence of FRET suggested that an interaction between LRP/LR and Aβ indeed exists at the cell surface. Furthermore, pull down assays and Aβ-specific ELISAs demonstrated that LRP/LR forms a physical association with endogenously shed Aβ, thereby verifying the physiological relevance of this association. Antibody blockade by IgG1-iS18 and shRNAmediated downregulation of LRP/LR significantly enhanced cell viability and proliferation and decreased apoptosis in cells co-treated with Aβ42 when compared to cells incubated with Aβ42 alone. In addition, antibody blockade and shRNA-mediated downregulation of LRP/LR significantly impeded Aβ42 internalization. These results suggest that LRP/LR acts as an internalization receptor for Aβ42 and may thereby contribute to the cytotoxicity of the neuropeptide by facilitating intracellular Aβ42 accumulation and aggregation - which has consequences for cell proliferation and may promote apoptosis. These findings recommend anti-LRP/LR specific antibodies and shRNAs as potential therapeutic tools for Alzheimer’s Disease treatment.

A!dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in the fulfillment of the requirements for the degree of Master of Science. 2013. / Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease affecting in excess of 26.6 million individuals globally. The neuropathological features of AD include extracellular deposition of amyloid beta (Aβ) plaques and intracellular neurofibrillary tangle formation. The cellular prion protein (PrPC) regulates the amyloidogenic cleavage pathway involved in Aβ shedding and interacts with the Aβ peptide. Given these interactions, the aim of this study was to investigate the influence of the 37kDa/67kDa laminin receptor (LRP/LR)- the cellular receptor for prion proteins- on Aβ shedding. Transfection of HEK293 cells with short hairpin RNAs (shRNAs) directed against LRP mRNA significantly decreased LRP levels in addition to Aβ shedding. Flow cytometric analysis revealed unchanged cell surface levels of the amyloid precursor protein (APP), β-secretase and γ-secretase after transfection of cells with shRNAs, suggesting a role of LRP/LR in Aβ shedding via a mechanism independent of gene-expression modulation of these key proteins. LRPshRNA treatment significantly reduced sAPPβ expression, implicating LRP/LR in APP processing specifically via augmenting the activity of β-secretase. Colocalisation of LRP/LR with APP, β- and γ-secretase, respectively, alludes to a possible interaction between said proteins. Therefore, LRP-shRNAs are suggested as alternative therapeutic tools for AD treatment.

This research investigated the relationship between exercise training and cathepsin B expression in the 3xTg-AD murine model of Alzheimer’s disease. 3xTg-AD mice were assigned to control (Tg, n=10), aerobic training (Tg+AT, n=10), or resistance training (Tg+RT, n=10). RotaRod peak latency and grip strength were assessed as preand post-measurements. Skeletal muscle was collected after training and analyzed for cathepsin B protein. Tg+RT showed greater grip strength than Tg and Tg+AT at posttesting (p ≤ 0.05). Only Tg+AT showed an improvement in RotaRod peak latency (p ≤ 0.05). Gastrocnemius weight was greater in Tg+RT compared to Tg (p ≤ 0.05), and no differences were observed in cathepsin B or procathepsin B expression (p > 0.05). This data suggests that cathepsin B was not induced by either mode of exercise training, however, physical function and muscle mass were improved, therefore inclusion of both training modalities may address peripheral comorbidities in Alzheimer’s disease. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

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Amyloid β(Aβ)-induced synaptic and neuronal degeneration has been linked to the memory loss observed in Alzheimer's disease (AD). Although Aβ-induced impairment of synaptic and nonsynaptic plasticity is known to occur before any cell death, the links between these neurophysiological changes and the loss of specific types of behavioural memory are not fully understood. This thesis introduces a behaviourally and physiologically tractable animal model to the Aβ field for the first time, allowing for an in-depth approach to investigating Aβ-induced memory loss to be explored. In Aβ 1-42- and Aβ 25-35-treated Lymnaea stagnalis, retrieval of consolidated memory is disrupted after single-trial conditioning and single-injection of synthetic peptide. All succeeding work builds upon these findings using a top-down approach to investigate how Aβ disrupts retrieval of consolidated memory. Neuronal and synaptic health were monitored over a 24 hour in vivo incubation period and other memory stages were considered to determine time points of memory vulnerability. In brains that displayed healthy neurons and degenerating synapses, only animals that were exposed to Aβ during the 24-48 hour post-training time points exhibited any behavioural deficits. All other behavioural responses remained normal. Focus then shifted to investigate the peptide, as opposed to behaviour, involved in the above mentioned experiments. After systemic injection, Aβ was found to penetrate the ganglia, enter cells, and localise to specific organelles by 24 hours exposure. Aβ morphology and structure were also monitored over the 24 hour incubation period, using transmission electron microscopy (TEM), formic acid extraction, silver stain, and western blot. A large distinction between the two peptides, Aβ 1-42 and Aβ 25-35, became apparent at this point and even when peptides were prepared using the same procedure, their effects on behaviour became drastically different. However, it is interesting to note that although the two peptides used are very different, under different preparation procedures they will both produce predominantly tetramer species after 24 hour in vivo incubation. Finally, investigations into disruptions of molecular signalling cascades were considered in order to correlate these disruptions to the observed Aβ-induced behavioural deficits. Specifically, molecular, pharmacological, and biochemical techniques were used to measure protein alterations and post-translational modifications, and to inhibit key protein components, involved in cAMP response element binding protein (CREB)-signalling pathways in Lymnaea brain after 24 hour in vivo incubation of Aβ. Phosphorylated CREB was found to be decreased in both Aβ-treated groups; this decrease pattern was also found in active protein kinase A (PKA) experiments. These experiments correlate memory deficits to Aβ-induced disruptions in PKA and CREB activity; however, PKA inhibition experiments indicate that this molecular cascade disruption is not sufficient to cause the observed behavioural deficits. Taken together, this work correlates Aβ-induced changes from a wide range of components involved in learning and memory, with Aβ-disrupted memory recall. Importantly as well, this work develops Lymnaea stagnalis as a novel model for Aβ research and continues to distinguish the two commonly used peptides, Aβ 1-42 and Aβ 25-35. By linking the effects of Aβ on defined neuronal circuits to behavioural deficits in a novel model, the Aβ field has been further developed in an important and unique way.

Alzheimer’s disease (AD) is the most common cause of dementia, accounting for around 60-70% of cases. AD encompasses large-scale neuronal loss, resulting in progressive memory and other cognitive decline. Presently, there is no cure for dementia and in light of the ageing population demographic, this represents a clear unmet medical and socioeconomic challenge Worldwide. Much of the current AD research focuses on studying the brain once hallmark amyloid plaque and neurofibrillary tangle pathologies have presented. However their appearance is extremely end stage and to date, any therapeutic interventions aimed at alleviating them having failed to halt symptoms progression. It may therefore be beneficial to look for earlier changes, with metabolic and oxidative stress events as well as reduced cerebral metabolism thought to occur early on in disease progression. Evidence from rare, familial AD cases suggests a causative role for A in AD pathogenesis. For this reason, the enzyme beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), the rate-limiting step in A production is currently of great therapeutic interest. With the prevailing view being that reducing BACE1 levels will be beneficial in AD, there remains a need to better understand the physiological roles of BACE1 to avoid potential side effects of BACE1 inhibition. Herein is presented data showing that, in agreement with the previous literature, BACE1 is fundamentally regulated by cell stress. Notably, both acute and prolonged bouts of oxidative and metabolic stress result in significant increases in BACE1 and APP protein expression. These changes also result in a shift in APP metabolism, with amyloidogenic processing of APP predominating during times of stress. It has also been shown that chronic elevation of BACE1 and/or manipulation of APP processing can alter cellular glucose uptake and use. These changes were determined through the use of radiolabelled substrate uptake and oxidation as well as extracellular flux assays. These data highlighted a fundamental shift in cellular metabolism, with aerobic glycolysis being utilised over oxidative metabolism of glucose. These changes were later shown to come as a result of metabolic lesions, which acted to impair substrate delivery to the electron transport chain of the mitochondria. Taken together, these data show that overexpression of the AD-associated protein BACE1 phenocopies a number of the earliest detectable changes observed in the brains of people who later develop AD. Finally, these data highlighted the potential importance of a number of novel pathways (Sirtuin, AMP-activated protein kinase, and peroxisome proliferator-activated receptor- coactivator signalling) that may underlie these changes and offer therapeutic avenues for earlier and more targeted treatment to halt AD progression.

Novel approaches are required to address Alzheimer’s disease (AD) in our ageing population, with recent interest focused on inflammation and oxidative stress (OS). Disruption of NF-E2-related factor 2 (Nrf2) signalling increases OS and promotes AD. Amyloid precursor protein (APP) is intimately linked with AD, with the Swedish mutation (<i>hAPP_swe</i>) used in numerous transgenic models. Furthermore, increasing importance has been placed on the suggested link between nutritional status and AD. To assess Nrf2 as a potential target in AD, we examined the effect of metabolic stress, by chronic high fat (HF) feeding or acute lipopolysaccharide (LPS) treatment on the brains of aged WT, <i>Nrf2^-/-</i>, hAPPswe and <i>Nrf2^-/-/hAPP_swe</i> mice. <i> </i><i>Nrf2^-/- mice displayed impaired enthorinal cortex-dependent cognition, with raised basal hippocampal inflammation. The inflammatory state was attenuated by chronic HF feeding, whilst maintaining insulin sensitivity. In contrast, <i>hAPP_swe</i> did not display an inflammatory response to HF feeding, but demonstrated impaired insulin signalling; in line with AD-associated insulin resistance. Additionally, <i>Nrf2^-/-</i> mice display increased glial cell activation and activation of mitogen activated protein kinases and mitochondrial impairment. These may be indicative of OS-induced cellular dysfunction and is supported by an aggravated response to LPS, which potentiates IL-1β production. Furthermore, despite an attenuated LPS response following the induction of tolerance, <i>Nrf2^-/-</i> mice maintain glial cell activation following treatment which may be suggestive of a primed immune environment within the brain. </i> In conclusion, these data indicate altered glucose homeostasis in both <i>Nrf2^-/-and <i>hAPP_swe</i> mice, as previously reported. Further, we advocate that Nrf2 plays a key role in mitochondrial function and health and may be important for the ameliorative effects of HF feeding. Taken together, mitochondrial dysregulation and associated OS may help explain the development of cognitive impairment in <i>Nrf2^-/-</i> mice. This may be relevant for AD given the age-dependent decline in Nrf2 expression in humans.

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