La sclérose latérale amyotrophique (SLA) et la démence fronto-temporale (DFT) sont des maladies neurodégénératives fatales, actuellement sans traitement. Ces maladies entrainent la dégénérescence des neurones moteurs et corticaux, engendrant des troubles moteurs et cognitifs et ultimement menant à la mort des patients souvent par détresse respiratoire trois à cinq ans après l’apparition des premiers symptômes. À l’échelle d’une vie, le risque de développer ces pathologies est de 1 pour 300-400 pour la SLA et 1 pour 742 pour la DFT, faisant de ces pathologies un risque majeur. Avec le vieillissement de la population que nous connaissons actuellement, il est évident que l’incidence de ces maladies deviendra de plus en plus élevée. Ainsi il est essentiel de comprendre les mécanismes moléculaires sous-jacents à ces pathologies dans le but de développer des thérapies effectives et prévenir l’impact de ces pathologies dans notre société. À ce jour, l’étiologie de la SLA-DFT est encore débattue, cependant la communauté scientifique s’accorde sur le fait que l’interaction entre la génétique et l’environnement joue un rôle essentiel dans le développement de ces maladies. La caractéristique moléculaire principale de ces pathologies est la localisation cytoplasmique de la protéine, normalement, nucléaire TDP-43. TDP-43 est un régulateur clef de l’homéostasie des ARNs. Parmi ces nombreuses fonctions, TDP-43 régule la formation des granules de stress, en régulant leur protéine régulatrice G3BP1. Ces granules formés d’ARN et de protéines se forment pour protéger les cellules durant une période de stress. Récemment, ces granules ont fait l’objet de nombreuses études et leurs dysfonctions ont été associées à la SLA-DFT. Dans cette thèse, nous avons approfondi l’étude de la régulation de TDP-43 sur G3BP1. Nous avons défini que TDP-43 stabilise les transcrits de G3BP1 de par une liaison forte à une séquence conservée à travers l’évolution se situant dans le 3’UTR de G3BP1. La perte de localisation nucléaire, la présence de mutations ou de TDP-35, une isoforme pathologique de TDP-43, sont associées à une diminution des niveaux de G3BP1. Également, d’un point de vue histopathologique, dans le cortex orbitofrontal des patients atteints de SLA-DFT, les neurones présentant une localisation cytoplasmique de TDP-43 ont une perte des niveaux transcriptionnels de G3BP1, associant alors directement G3BP1 à la maladie. Par la suite, nous avons défini que la perte de fonction en tant que stabilisateur, permet la liaison de microARNs sur les transcrits de G3BP1, engendrant leur dégradation. Le blocage de la liaison de microARNs sur G3BP1 empêche la dégradation des transcrits et restaure les fonctions de la protéine. Ainsi, nous avons déterminé un moyen de contrer la perte de fonction de TDP-43 sur G3BP1. De façon intéressante, en plus de la formation des granules de stress, G3BP1 est essentielle pour l’homéostasie neuronale et la survie neuronale post-stress. Par conséquent, la restauration de la protéine est potentiellement une avenue thérapeutique multi-approche pour le traitement de ces maladies. / Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two fatal neurodegenerative diseases, currently without cure. These diseases lead to the degeneration of motor and cortical neurons, causing motor and cognitive disorders and ultimately leading to death, often from respiratory distress three to five years after the onset. Over a lifetime, the risk of developing these conditions is 1 in 300-400 for ALS and 1 in 742 for FTD, making these conditions a major risk. With the current aging of the population, it is evident that the incidence of these diseases will become increasingly high. It is therefore essential to understand the molecular mechanisms underlying these pathologies in order to develop effective therapies. To this day, the etiology of ALS-FTD is still debated. However, the scientific community agrees that the interaction between genetics and the environment play an essential role in the development of these diseases. The main molecular characteristic of these pathologies is the cytoplasmic localization of the normally nuclear protein TDP-43. TDP-43 is a key regulator of RNA homoeostasis. Among these many functions, TDP-43 regulates the formation of stress granules, by regulating their nucleator protein G3BP1. These granules of RNA and protein form to protect cells during times of stress. Recently these granules have been the subject of several studies and their dysfunction has been associated with ALS-FTD. In this thesis, we have deepened the study of the regulation of TDP-43 on G3BP1. We have defined that TDP-43 stabilizes G3BP1 transcripts by strong binding to a sequence conserved through evolution located in the 3'UTR of G3BP1. Loss of nuclear localization, the presence of mutations or of TDP-35, a pathological isoform of TDP-43, are associated with decreased levels of G3BP1. Also, histopathologically, in the orbitofrontal cortex of patients with ALS-DFT, neurons with cytoplasmic localization of TDP-43 have a loss of transcriptional levels of G3BP1, directly associating G3BP1 with the disease. Subsequently, we defined that TDP-43 loss of function as a stabilizer allows the binding of two microRNAs on the G3BP1 transcripts, causing their degradation. Blocking the binding of these microRNAs to G3BP1 prevents the degradation of the transcripts and restores the functions of the protein. Thus, we have determined a way to counter the loss of function of TDP-43 on G3BP1. Interestingly, in addition to the formation of stress granules, G3BP1 is essential for neuronal homoeostasis and post-stress neuronal survival. Therefore, the restoration of the protein is potentially a multi-approach therapeutic avenue for the treatment of these diseases.

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Trotz intensiver Forschung ist die Ätiologie der sporadischen amyotrophen Lateralsklerose (sALS) weiterhin unbekannt. Zahlreiche Anzeichen deuten allerdings auf eine Mitbeteiligung von oxidativem Streß an der Pathogenese der sALS hin. So fand sich eine verminderte Aktivität der zellulären Glutathion-Peroxidase (GPX-1), eines als Radikalenfänger fungierenden Enzyms, in den Gyrus praecentrales bei sALS-Patienten. Zusätzliche Studien fanden eine Trinukleotid-Expansion des GGG-repeats im 1. Exon des für die GPX-1 kodierenden Gens. Da Trinukleotid-Expansionen bei einer Vielzahl von neurodegenerativen Erkrankungen wie dem Kennedy-Syndrom und der spinozerebellären Ataxie nachgewiesen werden konnten, war das Ziel dieser Arbeit, eine fragliche Mitbeteiligung dieser Trinkukleotid-Expansion der GPX-1 an der Pathogenese der sALS zu klären. Nach Etablierung der Methode bestehend aus einer Kombination von Polymerase-Kettenreaktion (PCR) und Restriktions-Fragment-Längen-Polymorphismus (RFLP) zeigte sich, dass der Genotyp 4*5 bei einer Gruppe von 231 sALS-Patienten signifikant häufiger vertreten war, wohingegen der Genotyp 5*6 in der Kontrollgruppe signifikant überrepräsentiert war. Im Vergleich zu bisher veröffentlichten Ergebnissen ließ sich der Genotyp 4*4 in der Kontrollgruppe signifikant häufiger nachweisen. Ursache hierfür könnte - neben einem tatsächlich erhöhten Risiko, an sALS zu erkranken - der Zusammenhang mit einem C/T-Polymorphismus der GPX-1 sein, der zu einem Austausch von Prolin zu Leucin führt. Die für Leucin kodierende Variante tritt hierbei nur zusammen mit 5 GCG-repeats auf, während die für Prolin kodierende Variante mit dem Auftreten von 4 und 6 GCG-repeats korreliert. / In spite of intensive research efforts the ethiology of sporadic amyotrophic lateral sclerosis (sALS) remains unknown. Various indices indeed suggest an involvement of oxidative stress in the pathogenesis of sALS. Thus a decreased activity of the cellular glutathione peroxidase (GPX-1) in gyrus praecentrales of sALS patients could be detected, an enzym strongly participating in the clearence of free radicals. Additional studies uncovered a trinucleotid expansion of a GCG repeat in the 1st exon of the gene coding for GPX-1. Such trinucleotid expansions play a major role in a variety of neurodegenerative disorders like the Kennedy Syndrom and spinal-cerebellary ataxia. Goal of this work was to disclose a possible involvement of the GCG expansion in the pathogenesis of sALS. Through the successful establishment of the methodology consisting of a combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) we could demonstrate a significant decrease of the genotype 4*5 in a group of 231 sALS patients, whereas the genotype 5*6 was overrepresented in the control group. Compared to hitherto publications we detected an increased occurrence of the 4*4 genotype in the control group. Besides an effective increased risk to contract sALS, the distribution of the GCG-repeat expansion could originate from another C/T polymorphism of GPX-1-gene leading to a substitution of proline with leucine. The leucine coding mutation occurs together with 5 GCG repeats, whereas the proline coding mutant correlates with 4 and 6 GCG-repeats.

ATP is a (co)transmitter and signaling molecule in the CNS. It acts at a multitude of ligand-gated cationic channels termed P2X to induce rapid depolarization of the cell membrane. Within this receptor-channel family, the P2X7 receptor (R) allows the transmembrane fluxes of Na+, Ca2+, and K+, but also allows the slow permeation of larger organic molecules. This is supposed to cause necrosis by excessive Ca2+ influx, as well as depletion of intracellular ions and metabolites. Cell death may also occur by apoptosis due to the activation of the caspase enzymatic cascade. Because P2X7Rs are localized in the CNS preferentially on microglia, but also at a lower density on neuroglia (astrocytes, oligodendrocytes) the stimulation of this receptor leads to the release of neurodegeneration-inducing bioactive molecules such as pro-inflammatory cytokines, chemokines, proteases, reactive oxygen and nitrogen molecules, and the excitotoxic glutamate/ATP. Various neurodegenerative reactions of the brain/spinal cord following acute harmful events (mechanical CNS damage, ischemia, status epilepticus) or chronic neurodegenerative diseases (neuropathic pain, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis) lead to a massive release of ATP via the leaky plasma membrane of neural tissue. This causes cellular damage superimposed on the original consequences of neurodegeneration. Hence, blood-brain-barrier permeable pharmacological antagonists of P2X7Rs with excellent bioavailability are possible therapeutic agents for these diseases. The aim of this review article is to summarize our present state of knowledge on the involvement of P2X7R-mediated events in neurodegenerative illnesses endangering especially the life quality and duration of the aged human population.

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No description available.

<p>Amyotrophic Lateral Sclerosis (ALS) is a fatal progressive neurodegenerative disease with no known cause. Despite the efforts of investigators over the past 150 years, there remains no effective cure which substantially prolongs life. Therapeutic strategies have explored all of the proposed underlying pathological pathways of the disease from increased oxidative damage to impaired axonal transport, with little to no success. In the following pages, a novel perspective will be presented outlining the preliminary investigations of a new line of research demonstrating that Sonic hedgehog (Shh) protein and its agonists have cytoprotective effects on motor neurons. To begin these investigations, initial experiments were conducted <em>in vitro</em> utilizing a mouse hippocampal cell-line (HT-22) which served as a model for transient transfection and oxidative challenge assays. The results are reported in Chapter 2. Building upon these introductory findings, further investigations were conducted exploiting the SOD1^G93A mouse model of ALS. Chapter 3 summarizes key observations pertaining to the abundance of a key cellular organelle in the sensing of Shh signalling, the primary cilium, in the spinal cord of SOD1^G93A mice. In Chapter 4, a semi-quantitative analysis of the effects of Shh and Shh agonists pre-treatment <em>in vitro </em>on primary mixed spinal cord cultures are described. Subsequent challenge with an excitotoxic NMDA treatment was also conducted, as well as an <em>in vivo</em> survival study exploring the potential therapeutic effects of chronic Shh administration on SOD1^G93A mice. The cumulative research presented here represents the very first investigation into the unique application of Shh and its agonists as potential therapeutic agents for the treatment of ALS, and our findings indicate that Shh has the potential of becoming a novel therapeutic agent for the treatment of ALS.</p> / Doctor of Philosophy (Medical Science)

T cells infiltrate the degenerating brain and influence central nervous system (CNS) inflammation and neuronal health. In mice, the choroid plexus and the meninges have been implicated in regulating T cell entry and egress from the CNS, respectively. Further, antigen presenting cells in the mouse meninges present CNS-derived antigens to T cells and may represent a method for the peripheral immune system to sense and respond to CNS immune triggers. However, whether these processes occur in the human choroid plexus and meninges has not been comprehensively studied. Further, the antigens towards which T cells in the degenerating human brain and its borders respond remain unknown. Therefore, I implemented a multi-omics approach using fresh postmortem tissue from patients diagnosed with amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), and non-neurodegenerative controls to identify not only the T cell-associated changes that occur in the degenerating human CNS and surrounding tissues but also identified a library of putative antigen targets for disease-associated T cell populations. Specifically, using single cell RNA and TCR sequencing information from paired postmortem choroid plexus, leptomeninges, and brain I lineage traced T cells using their TCR information and found that T cell access to leptomeninges and brain is likely limited and controlled by anti-inflammatory macrophage activity at the blood/CSF barrier (BCSFB). Once past the BCSFB, I present evidence that T cells access the CNS where they interact with MHC expressed by microglia. T cells also accumulate in the leptomeninges where they become tissue resident memory T cells. These tissue resident memory T cells likely serve as a reservoir for a rapid antigen-driven immune response to future CNS inflammatory insults. Finally, by performing immunopeptidomics to identify peptides presented by MHC in the same patients’ CNS and border tissues, I identified a library of putative antigenic triggers that may drive high levels of T cell clonal expansion in the brain and surrounding tissues. Altogether, this thesis serves as a resource for understanding the trajectory of T cells as they travel into the degenerating human brain and as a foundation for the development of antigen-specific precision medicines to treat neurodegeneration.

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Les modèles murins de la Sclérose Latérale Amyotrophique (SLA) ont permis des avancées dans la compréhension des mécanismes pouvant conduire à la mort sélective et progressive des motoneurones (Mns) mais ils présentent des disparités dans la sévérité et le décours temporel de la maladie. Parmi les hypothèses avancées figurent des modifications des propriétés intrinsèques des motoneurones conduisant à des modifications de l’excitabilité et de l’homéostasie du calcium intracellulaire et à la mort du motoneurone.Nous avons donc étudié les propriétés électrophysiologiques des Mns lombaires de souris SOD1G85R et SOD1G93A, deux modèles à faible nombre de copies du gène humain muté, durant les deux premières semaines postnatales afin d’identifier d’éventuelles anomalies pré-symptomatiques précoces. Nos travaux ont été réalisés sur deux préparations in vitro de moelle entière isolée et de tranches de moelle épinière. Les Mns mutants présentent, sur les deux types de préparations, une altération des propriétés du potentiel d’action se traduisant par un allongement de la durée associée à une diminution des vitesses maximales de dépolarisation et repolarisation et une réduction d’amplitude. Ces altérations apparaissent entre P2-P5 dans les Mns SOD1G85R et entre P6-P10 dans les Mns SOD1G93A et suggèrent une diminution de la densité des canaux sodiques et potassiques associés au potentiel d’action. Nous avons aussi observé sur des tranches de moelle épinière entre P6-P10 que le gain de fréquence des Mns SOD1G85R diminue et celui des SOD1G93A augmente sans aucune modification des densités des courants entrants persistants sodiques et calciques. On note également que, sur tranches de moelle épinière, les Mns SOD1G93A présentent un potentiel de repos diminué. En présence d’une surcharge calcique extracellulaire, les propriétés membranaires des Mns SOD1G85R entre P6-P10 sont moins affectées que celles des Mns témoins. Les effets différentiels de cette surcharge peuvent être dus à des modifications différentes de la dépendance au voltage des canaux voltage-dépendants et/ou à la modulation de certains types de canaux activés par le calcium extracellulaire. Une arborisation dendritique plus ramifiée que celle de Mns témoins, comparable à celle précédemment décrite dans les Mns SOD1G85R, a été observée dans les Mns SOD1G93A à P8-P9 avec des altérations du potentiel d’action citées plus haut et une réduction de la rhéobase. Ces altérations morphologiques et électriques pourraient indiquer des modifications de cinétiques et/ou de densités de canaux sur des sites différents dans ces Mns. Nos travaux montrent donc, d’une part que les mutations SOD1G85R et SOD1G93A induisent dans ces deux modèles murins des altérations des propriétés des Mns lombaires comparables mais décalées dans le temps et d’autre part que certaines altérations semblent être spécifiques à une mutation SOD1 donnée. / The SOD1 murine models of Amyotrophic Lateral Sclerosis (ALS) allowed major progress in the understanding of mechanisms which could lead to a selective loss of motoneurons (Mns), but these models display differences in the severity and time course of the disease. Changes in intrinsic properties of motoneurons may induce changes in excitability and intracellular calcium homeostasis leading to motoneuron death.Therefore, we studied electrophysiological properties of lumbar Mns from SOD1G85R and SOD1G93A mice, low expressor lines, during the first two postnatal weeks in order to identify possible early presymptomatic abnormalities. Our studies were carried out on two in vitro preparations: the whole isolated spinal cord and acute spinal cord slices. Mutant Mns display, in the two preparations, a modified action potential characterized by an increased duration due to a decrease of the maximal speeds of depolarisation and repolarisation and a reduction of the spike amplitude. These alterations appeared between P2-P5 in SOD1G85R Mns and between P6-P10 in SOD1G93A Mns and suggest a decrease of the density of sodium and potassium channels related to action potential. We also showed on spinal cord slices between P6-P10 that the gain of frequency decreases for SOD1G85R Mns and increases for SOD1G93A Mns without any change in the density of persistent inward sodium or calcium currents in these different mutant Mns. We observed also that the resting membrane potential of SOD1G93A Mns on spinal cord slices is decreased. The membrane properties of SOD1G85R Mns between P6-P10 were less susceptible to changes in presence of an extracellular calcium overload. Differential effects of this extracellular calcium overload on membrane properties of WT and SOD1G85R Mns could be due to different alterations of the potential dependence of voltage-gated channels and/or to the modulation of some types of channels sensitive to extracellular calcium. An over-branching of dendritic arborization, similar to that previously described in SOD1G85R Mns, was observed in SOD1G93A at P8-P9 with the above-mentioned action potential alterations and a weak rheobasic current. These morphogical and electrical changes could indicate together alterations of kinetics and/or density of channels on different sites on these Mns. In conclusion, our work shows on one hand that SOD1G85R and SOD1G93A mutations induce similar alterations of lumbar Mns properties but time-shifted in these two murine models and on the other hand that some alterations seem to be specific to a given SOD1 mutation.