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Lysosomal storage disorders are a group of rare neurodegenerative diseases that are collectively common, sharing many aspects with other neurodegenerative disorders, including substrate build-up and neuroinflammation. The GM2 Gangliosidoses, Tay-Sachs disease and Sandhoff disease, are pathologically overlapping lysosomal storage disorders, with high prevalence within specific ethnicities. Their effects are neurologically devastating and often fatal at young ages. Current treatments only slow or stall an inevitable decline in health. Novel treatment targets are needed for these disorders, and others with similar pathologies. In these works we demonstrate the negative effect the inflammatory cytokine tumour necrosis factor-alpha has on survival of a model of Sandhoff disease. We demonstrate its role in the upregulation of astrogliosis, and apoptosis, and we present evidence that this effect on astrogliosis occurs through an upregulation of the JAK-2/STAT3 pathway. Though fruitful, a singular focus on inflammation/gliosis in these diseases has left a vacuum in the research into neuron specific molecular processes. We observe the development of inflammation, astrogliosis and neuronal processes in our model, and demonstrate a bi-phasic disease progression, in which early onset microgliosis precedes terminal astrogliosis, apoptosis, and a decline in excitatory glutamate receptors, suggesting neuron-specific malfunction. Furthermore, we show that knockout of the synaptic protein neuronal pentraxin 1 retards neurodegeneration and extends the lifespan of Sandhoff disease mice, independent of inflammation or astrogliosis. Through electrophysiology, we provide evidence of dysregulation of glutamate receptors in Sandhoff disease, and show that knockout of neuronal pentraxin 1 provides rescue from this dysregulation. This work expands on research into gliosis in GM2 gangliosidoses, presents the finding of a novel protein isoform, and presents a new focus on non-glial disease mechanisms and treatments for these and other neurodegenerative disorders. / Thesis / Doctor of Philosophy (PhD) / Lysosomal storage disorders are a group of neurological diseases that are debilitating, and often fatal at a young age. Two diseases of this group- Tay-Sachs disease and Sandhoff disease – are similar in their causes and symptoms. Current treatments for these diseases only slow or stall an inevitable decline in health. New targets for treatment are required, and we provide data suggesting several proteins that may fit this criterion. We also provide evidence of the discovery of a new form of one of these proteins, which is found in high levels in the disease, indicating it may be important in these and other neurodegenerative diseases. Finally, we provide findings indicating that a certain cell type, which is largely ignored in current research for these diseases, may be important in the disease progress. These findings increase our knowledge of Tay-Sachs disease and Sandhoff disease, and open new avenues for medicinal intervention.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative tauopathy associated with exposure to repetitive head impacts (RHI) in contact sports. No treatments are currently available. Much of the focus in CTE has been on the microtubule-binding protein tau, which tends to accumulate within neurons and glia around blood vessels at the depths of cortical sulci. The mechanisms of tau accumulation and propagation in CTE are still unknown. The predilection for the perivascular region suggests inherent structural and/or cellular vulnerabilities in this area. Astrocytes are glial cells in this perivascular region that help form the blood brain barrier (BBB) and the neurovascular unit (NVU). Their endfeet envelop blood vessels and help transport nutrients from the blood into the brain, as well as clear harmful waste products out of the brain. Astrocytes are also vital players in many of the brain’s other normal physiological functions, including providing structural and metabolic support to neurons and maintenance of ion and water homeostasis. In response to injury or disease, astrocytes undergo a series of structural and functional changes in a process known as reactive astrogliosis. Astrogliosis is widely considered a hallmark of brain pathology, however, only recently have we begun to understand its functional implications. Astrocytes can respond heterogeneously to CNS insults, including either loss or increase of homeostatic functions, or gain of new, possibly toxic functions. These different astrocytic responses can either assist in recovery or further exacerbate injury. Our current understanding of how astrocytes are altered in RHI and CTE is limited. A degenerative phenotype has been identified in older donors with later stage CTE, but its presence in younger donors with earlier stage disease is unknown. The hypothesis of this study is that exposure to repetitive head trauma causes astrocytes to become reactive and adopt altered phenotypes, including loss of homeostatic functions, in brain areas known to be biomechanically susceptible to the shearing forces of head trauma, such as the perivascular region and interface of the grey and white matter at the depth of the cortical sulcus. These altered phenotypes are expected to be found in athletes with and without pathological tau deposition, highlighting astrocytes as potential therapeutic targets in the post-traumatic injury cascade. Specifically, I seek to characterize reactive astrocyte phenotypes and assess changes in their perivascular function in the brains of former American football players with and without a neuropathological diagnosis of CTE.

Yes / Protocols which permit the extraction of primary astrocytes from either embryonic or postnatal mice are well established however astrocytes in culture are different to those in the mature CNS. Three dimensional (3D) cultures, using a variety of scaffolds may enable better phenotypic properties to be developed in culture. We present data from embryonic (E15) and postnatal (P4) murine primary cortical astrocytes grown on coated coverslips or a 3D polystyrene scaffold, Alvetex. Growth of both embryonic and postnatal primary astrocytes in the 3D scaffold changed astrocyte morphology to a mature, protoplasmic phenotype. Embryonic-derived astrocytes in 3D expressed markers of mature astrocytes, namely the glutamate transporter GLT-1 with low levels of the chondroitin sulphate proteoglycans, NG2 and SMC3. Embroynic astrocytes derived in 3D show lower levels of markers of reactive astrocytes, namely GFAP and mRNA levels of LCN2, PTX3, Serpina3n and Cx43. Postnatal-derived astrocytes show few protein changes between 2D and 3D conditions. Our data shows that Alvetex is a suitable scaffold for growth of astrocytes, and with appropriate choice of cells allows the maintenance of astrocytes with the properties of mature cells and a non-reactive phenotype. / BBSRC

Traumatic brain injury (TBI) results from a physical insult to the head and often results in temporary or permanent brain dysfunction. However, the cellular pathology remains poorly understood and there are currently no clinically effective treatments. The overall goal of this work was to develop and characterize a novel three-dimensional (3-D) in vitro paradigm of neural trauma integrating a robust 3-D neural co-culture system and a well-defined biomechanical input representative of clinical TBI. Specifically, a novel 3-D neuronal-astrocytic co-culture system was characterized, establishing parameters resulting in the growth and vitality of mature 3-D networks, potentially providing enhanced physiological relevance and providing an experimental platform for the mechanistic study of neurobiological phenomena. Furthermore, an electromechanical device was developed that is capable of subjecting 3-D cell-containing matrices to a defined mechanical insult, with a predicted strain manifestation at the cellular level. Following independent development and validation, these novel 3-D neural cell and mechanical trauma paradigms were used in combination to develop a mechanically-induced model of neural degeneration and reactive astrogliosis. This in vitro surrogate model of neural degeneration and reactive astrogliosis was then exploited to assess factors influencing neural stem cell (NSC) survival and integration upon delivery to this environment, revealing that specific factors in an injured environment were detrimental to NSC survival. This work has developed enabling technologies for the in vitro study of neurobiological phenomena and responses to injury, and may aid in elucidating the complex biochemical cascades that occur after a traumatic insult. Furthermore, the novel paradigm developed here may provide a powerful experimental framework for improving treatment strategies following neural trauma, and therefore serve as a valid pre-animal test-bed.

Le virus de la rougeole (MeV) est capable d'infecter et de persister dans le système nerveux central (SNC). Dans de rares cas, l'infection du SNC donne lieu à l'apparition de complications neurologiques dramatiques pour lesquelles aucun traitement n'est disponible à ce jour. Les mécanismes cellulaires et moléculaires impliqués dans l'infection et la dissémination du virus au travers du tissu cérébral sont encore mal connu. C'est pourquoi nous avons développé et caractérisé la culture organotypique d'hippocampe et cervelet de modèle animaux. Nous avons montré qu'au cours de la culture des tranches, une astrogliose se produit et que celle-ci est accompagnée d'une réaction proinflammatoire et d'un état antiviral qui les rendent réfractaires à l'infection. Par contre, les coupes infectées le jour de la préparation, sont susceptibles à l'infection, même en l'absence des récepteurs cellulaires SLAM et Nectin4 utilisés par les souches sauvages de MeV, permettant une étude comparative du tropisme cellulaire initial. De plus, le MeV est capable de se disséminer au sein des coupes au cours du temps. Cette dissémination est maintenue si le résidu Y543 de l'hémagglutine virale est muté, ce qui interdit l'utilisation de Nectine 4. Par contre la dissémination implique le résidu R533 qui est indispensable pour l'utilsation du récepteur SLAM. Celui-ci étant absent du cerveau, nos résultats suggèrent qu'un récepteur inconnu fixant l'hémagglutine permet au MeV de se disséminer dans le SNC et nous permet d'envisager son identification / Measles virus (MeV) is able to infect and persist in the central nervous system (CNS), leading to a progressive neurological disease for which no treatment are available. The cellular and molecular mechanisms involved in the infection and spreading of MeV through brain are still poorly understood. In order to elucidate these mechanisms, we have developed and characterized organotypic culture of the hippocampus and cerebellum from rodent models. Here, we showed that the culture procedure give rise to an astrogliosis, a proinflammatory response and an antiviral state which result in an infection resistant profile. When infected on the day of the preparation, slices are susceptible to MeV infection, even in the absence of expression of a known cellular receptor for this virus, namely human SLAM and nectin 4, allowing a comparative study of initial cellular tropism. Additionally, the MeV is able to further spread within the slices. MeV dissemination did not required haemagglutinin Y543 residue, a key residue required forNectin-4 usage while , the mutation of the SLAM specific residue R533 is prevent MeV spreading through brain slices . This finding indicates that the MeV haemagglutinin, would recognize, via its R533 residue, an unknown brain receptor and paves the way for its identification

Les réseaux vasculaires et nerveux présentent des similitudes frappantes (points de branchements, superposition, voies afférentes/efférentes, …) et tous deux interagissent lors du développement ou dans le cadre de pathologies.Dans un premier projet, nous avons voulu déterminer si un facteur pro-angiogénique, c'est-à-dire induisant la formation de nouveaux vaisseaux, peut avoir un effet direct sur le réseau neuronal. Des études menées in vitro ou in vivo chez l’adulte, ont montré une implication directe du Vascular Endothelial Growth Factor (VEGF) sur le système nerveux (survie, prolifération neuronale, croissance axonale, …). Nous avons cherché à savoir si ce facteur a un effet sur le développement ou l’activité des réseaux neuronaux lors de la vie embryonnaire alors que les systèmes vasculaires et nerveux se mettent progressivement en place. Avec une approche électrophysiologique, nous avons focalisé notre attention sur les motoneurones de la moelle épinière de souris entre les stades E13,5 et P0. Nos résultats montrent que le VEGF augmente de façon significative la fréquence des activités synaptiques liées à la libération de GABA et de Glycine pendant une fenêtre temporelle correspondant à la mise en place de ces mêmes activités (E13,5 et E15,5). Cet effet modulateur met en évidence un nouveau rôle du VEGF dans la maturation fonctionnelle des réseaux neuronaux et ouvre de nouvelles perspectives dans l’étude des neurodégénérescences précoces. Dans un second projet, nous nous sommes intéressés au glioblastome, cancer cérébral très invasif. Nous montrons que l’inhibition d’IRE1 (Inositol Requiring-Enzyme 1, senseur du stress du réticulum endoplasmique) dans un modèle d’implantation orthotopique chez la souris induit la formation de tumeurs plus petites, moins vascularisées et plus dispersées avec un meilleur pronostic de survie. Nous observons aussi des altérations du microenvironnement tumoral (matrice extracellulaire, réaction astrocytaire) avec des modifications de l’expression de nombreux facteurs de croissance dont le TGFß. / The nervous and the vascular systems share similarities (branching points, afferent/efferent parts …) and are closely connected during development and pathology.In the first part of this project, we questioned whether the pro-angiogenic key factor VEGF (Vascular Endothelial Growth Factor), which promotes new blood vessels formation, can directly interact with neural networks while nervous and vascular systems are developing. In the present study, using an electrophysiological approach, we focused on the effect of VEGF on embryonic spinal lumbar motoneurons (MNs). Our results demonstrate that VEGF increases the frequency of the GABA/glycinergic events at early developmental stages (E13.5 and E15.5) but not at the perinatal stage E17.5. Our data highlight a new role for VEGF which can control both the maturation of the vascular and neuronal networks and may likely be involved in early MNs degeneration.In the second part, we focused on glioblastoma, the most agressive form of brain cancer. Our results show that inhibition of IRE1 (Inositol Requiring-Enzyme 1, stress sensor of endoplasmic reticulum) leads to formation of smaller, less vascularized, more invasive tumors with a better prognosis. We also observe that tumoral microenvironnement is altered (reactive astrogliosis, extracellular matrix) and expression of several growth factors like TGFß is modified.

No description available.

Le terme dégénérescence lobaire frontotemporale ou FTLD définit un groupe hétérogène de maladies neurodégénératives caractérisé par des troubles du langage, du comportement et/ou moteurs qui résultent principalement d’une dégénérescence du cortex frontal et temporal. Cette hétérogénéité tant au niveau clinique, génétique que neuropathologique rend cette pathologie très complexe et il existe aujourd’hui un véritable problème de diagnostic différentiel des FTLD. Le diagnostic final des FTLD repose ainsi sur l’examen neuropathologique, la nature des lésions observées et leurs constituants moléculaires. La caractérisation de ces lésions a permis d’établir une classification des FTLD qui ne cesse d’évoluer avec la découverte de nouveaux acteurs moléculaires. À l’instar de nombreuses maladies neurodégénératives, les FTLD sont caractérisées par la présence de protéines agrégées dans les régions cérébrales affectées. Cependant, contrairement à la maladie d’Alzheimer (MA), ces agrégats ne sont pas toujours constitués des mêmes protéines. Ainsi, approximativement 40% des cas de FTLD présentent des agrégats composés de protéines Tau hyper et anormalement phosphorylées, et forment le groupe FTLD-Tau. Lorsqu’aucune pathologie Tau n’est détectée, les patients présentent généralement des inclusions neuronales cytoplasmiques ou intranucléaires immunoréactives pour la protéine TDP-43 (transactive response DNA binding protein 43), et constituent la sous-classe FTLD-TDP. Plus rarement, la protéine FUS (Fused in Sarcoma, FTLD-FUS) ou des protéines liées au système ubiquitine protéasome peuvent également s’agréger (FTLD-UPS). La génétique représente également une composante majeure des FTLD avec 10 à 15% des cas correspondant à des formes héréditaires dominantes. Les premières mutations furent découvertes sur le gène MAPT. Le gène de la progranuline (GRN) fut ensuite identifié comme fréquemment associé aux FTLD. Plus récemment, une répétition anormale d’héxanucléotides GGGGCC au sein du gène C9ORF72 (chromosome 9 open reading frame 72) a été montrée comme étant responsable d’un grand nombre de cas familiaux de FTLD. De manière moins fréquente, d’autres gènes tels que VCP (valosin containing protein) ou CHMP2B (charged multivesicular body protein 2B) peuvent aussi être associés à des cas familiaux de FTLD. Des années avant la découverte des principaux acteurs moléculaires des FTLD, des études ont décrit une perte partielle ou totale des protéines Tau physiologiques dans le tissu cérébral. A l’origine, ce phénomène fut observé dans un groupe de démences appelées DLDH pour démences sans signe histopathologique distinctif (plus tard appelé FTLD-ni pour no inclusion). En 2006, la majorité de ces cas a été reclassée en tant que FTLD-U (présence de lésions immunoréactives pour l’ubiquitine). En revanche, aucune étude ne s’est intéressée à cette perte de Tau depuis celle de Zhukareva et collègues en 2003. Au regard des récentes avancées sur la compréhension de la base moléculaire et génétique des FTLD, la pertinence de cette perte de Tau reste ainsi encore à déterminer. Dans ce contexte, ce travail de recherche a pour principal objectif d’étudier l’expression des protéines Tau au sein du tissu cérébral d’individus sains ou atteints de différents troubles neurodégénératifs (MA, FTLD-Tau, FTLD-TDP-GRN, FTLD-TDP-C9ORF72, FTLD-TDP et FTLD-FUS sporadiques) en utilisant la technique d’immunoempreinte. De manière remarquable, nous avons mis en évidence une réduction significative de Tau, et ce, spécifiquement chez les patients FTLD-TDP-GRN. Ainsi, nos résultats démontrent que ces cas, appelés FTLD-TDP-GRNltau (pour low Tau protein level), caractérisés par une altération synaptique et une astrogliose très importante, pourraient constituer un groupe distinct dans la classification des FTLD [...] / FTLD is a clinical syndrome mainly characterized by progressive deterioration in behavior, personality and/or language resulting from progressive frontal and temporal degeneration. In addition, movement disorder can also be frequently observed. Given this phenotype variability, FTLD clinical diagnosis remains difficult and uneasy to establish with certainty.The final diagnosis relies on neuropathological examination of the brain, the characteristics of these brain lesions and their molecular basis. Indeed, as many neurodegenerative diseases, FTLD are characterized by the presence of protein aggregates in the affected brain regions. However, in contrast to the well-characterized nature of protein inclusions in Alzheimer’s disease (AD), proteinaceous aggregates in FTLD can be composed of different proteins. Thus, approximatively 40% of FTLD cases display aggregates made of abnormally and hyperphosphorylated Tau proteins and constitute the FTLD-Tau subclass. However, most of FTLD brains are negative for Tau inclusions and exhibit neuronal cytoplasmic and/or nuclear inclusions immunoreactive for transactive response DNA binding protein 43 (TDP-43) and constitute the FTLD-TDP subclass). To a lesser extent, another protein called FUS (Fused in Sarcoma protein) is found in aggregates that are Tau and TDP-43 negative. This subclass is thus named FTLD-FUS. Finally, inclusions negative for Tau, TDP-43 or FUS are observed in rare cases of FTLD and associated with ubiquitin-proteasome system related proteins (FTLD-UPS).Gene mutations also play an important role in FTLD with 30 to 50% of patients reporting a positive family history of FTD and 10 to 15% of patients corresponding to dominantly inherited form. Firstly described are the MAPT mutations. Mutations in the progranulin gene GRN were then found to be the most frequent mutations associated with FTLD. More recently, two studies demonstrated that expanded hexanucleotide GGGGCC repeats in a noncoding region of the chromosome 9 open reading frame 72 (C9ORF72) gene was responsible for a large proportion of FTLD. Less frequently mutations in the valosin containing protein (VCP) gene or charged multivesicular body protein 2B (CHMP2B) gene are also found associated with FTLD.Prior to the discovery of the main molecular actors of FTLD, studies described a partial or total loss of soluble or physiological Tau protein expression in both grey and white matter. This loss of Tau was originally found in a subset of dementia called DLDH for Dementia Lacking Distinctive Histopathology (renamed later FTLD-ni for FTLD with no inclusion). In 2006, most of these cases were reclassified as FTLD-U (presenting with ubiquitin positive inclusions). However, additional investigation with specific regards to this loss of Tau expression has not been reported since Zhukareva et al. in 2003. With the progress in genetics and neuropathology of FTLD, the question of whether this reduction of Tau expression is seldom remains ill-defined.This work takes place in this context whose primary goal was to investigate human brain Tau protein expression in Control, AD, FTLD-Tau, FTLD-TDP-GRN, FTLD-TDP-C9ORF72, sporadic FTLD-TDP and sporadic FTLD-FUS brains using western blot analysis. Remarkably, we demonstrated a huge reduction of all six human brain Tau isoforms only in a subset of FTLD-TDP brains with mutation on the GRN gene. Thus, our data clearly suggest that these specific cases, referred to as FTLD-TDP-GRNltau (for low levels of Tau protein), could be part of the current classification as a distinct entity with more severe synaptic dysfunction and astrogliosis. Beside this, we also performed a comparative proteomic study between the different FTLD sub-classes in order to find common physiopathological mechanisms.

No description available.