Mutant mRNA and protein both contribute to the clinical manifestation of many repeat-associated neurodegenerative and neuromuscular disorders. The presence of nuclear RNA clusters is a feature shared amongst these diseases, such as C9ORF72/ALS and myotonic dystrophy 1/2 (DM1/2); however, this pathological hallmark has not been conclusively demonstrated in Huntington’s disease (HD) in vivo. Investigations into HD – caused by a CAG repeat expansion in exon 1 of the huntingtin (HTT) gene – have largely focused on toxic protein gain-of-function as a disease-causing feature, with fewer studies investigating the role of mutant HTT mRNA in pathology or pathogenesis. Here we report that in two HD mouse models, YAC128 and BACHD-97Q-ΔN17, mutant HTT mRNA is preferentially retained in the nucleus in vivo. Furthermore, we observed the early, widespread formation of large mutant HTT mRNA clusters (approximately 0.6 to 5 µm3 in size) present in over 50-75% of striatal and cortical neurons. Affected cells were limited to one cluster at most. Endogenous wild-type mouse Htt or human HTT mRNA containing 31 or fewer repeats did not form clusters. Additionally, the aberrantly spliced N-terminal exon 1-intron 1 RNA fragment, HTT1a, also formed clusters that fully co-localized with the mutant HTT mRNA clusters. These results suggest that multiple repeat-containing transcripts can coalesce to form a single cluster in a given cell. Treating YAC128 mice with antisense oligonucleotides efficiently silenced individual HTT mRNA foci but had limited impact on clusters. Our findings identify mutant HTT mRNA clustering as an early, robust molecular signature of HD, further supporting HD as a repeat expansion disease with suspected mRNA involvement.

La maladie de Huntington (MH) est une maladie neurodégénérative causée par une extension de répétitions du codon CAG dans le gène de la Huntingtine (Htt). Cette maladie est caractérisée par la mort des neurones striataux et la présence d’agrégats de Htt mutée (mHtt). De plus, au cours de la MH, les astrocytes, qui sont essentiels au bon fonctionnement neuronal, changent d’état et deviennent réactifs. La réactivité astrocytaire est caractérisée par des changements morphologiques et transcriptomiques mais l’impact fonctionnel de cette réactivité reste peu compris.Afin d’étudier le rôle des astrocytes réactifs dans la MH, nous avons utilisé des vecteurs viraux récemment développés par notre équipe, qui induisent ou bloquent la réactivité astrocytaire in vivo en ciblant la voie JAK2-STAT3. Nous avons montré que les astrocytes réactifs diminuent le nombre et la taille des agrégats de mHtt majoritairement présents dans les neurones. Ceci est associé à l’amélioration de plusieurs altérations neuronales observées dans ces modèles. Une analyse transcriptomique réalisée sur des astrocytes réactifs révèle des changements majeurs d’expression de gènes liés aux systèmes de protéostasie. De plus, l’activité du lysosome et du protéasome est augmentée dans les astrocytes réactifs de souris modèles de la MH. Nous montrons également que les astrocytes réactifs éliminent plus efficacement leurs propres agrégats de mHtt, suggérant qu’au cours de la MH, ces cellules pourraient dégrader plus efficacement la mHtt provenant des neurones. De plus, certaines protéines chaperonnes sont induites dans les astrocytes réactifs. En particulier, la co-chaperonne DNAJB1/Hsp40 est surexprimée dans les astrocytes réactifs et est retrouvée dans les exosomes isolés à partir de striata de souris MH. Des expériences de gain et perte de fonction suggèrent que cette chaperonne est impliquée dans les effets bénéfiques des astrocytes réactifs sur l’agrégation de la mHtt et l’état des neurones. Les astrocytes réactifs pourraient donc libérer des protéines anti-agrégantes qui favorise l’élimination de la mHtt dans les neurones.Notre étude montre que les astrocytes peuvent, en devenant réactifs au cours de la MH, acquérir des propriétés bénéfiques pour les neurones et favoriser, via un dialogue complexe avec les neurones, l’élimination des agrégats de mHtt. / Huntington’s disease (HD) is a hereditary neurodegenerative disease caused by an expansion of CAG codons in the Huntingtin gene. It is characterized by the death of striatal neurons and the presence of mutant Huntingtin (mHtt) aggregates. In pathological conditions, as in HD, astrocytes change and become reactive. Astrocyte reactivity is characterized by morphological and significant transcriptomic changes. Astrocytes are essential for the proper functioning of neurons but the functional changes associated with reactivity are still unclear.To better understand the roles played by reactive astrocytes in HD, we took advantage of our recently developed viral vectors that infect selectively astrocytes in vivo and either block or induce reactivity, through manipulation of the JAK2-STAT3 pathway. We used these vectors in two complementary mouse models of HD and found that reactive astrocytes decrease the number and the size of mHtt aggregates that mainly form in neurons. Reduced mHtt aggregation was associated with improvement of neuronal alterations observed in our mouse models of HD. A genome-wide transcriptomic analysis was performed on acutely sorted reactive astrocytes and revealed an enrichment in genes linked to proteolysis. Lysosomal and proteosomal activities were also increased in reactive astrocytes in HD mice. Moreover, we show that reactive astrocytes degrade more efficiently their own mHtt aggregates, suggesting that these cells could siphon mHtt away from neurons. Alternatively, several chaperones were induced in reactive astrocytes. In particular, the co-chaperone DNAJB1/Hsp40 was upregulated in reactive astrocytes and was present in exosomal fraction from HD mouse striatum. Loss and gain of function experiments suggest that this chaperone is involved in the beneficial effects of reactive astrocytes on mHtt aggregation and neuronal status. Therefore, reactive astrocytes could release anti-aggregation proteins that could promote mHtt clearance in neurons.Overall, our data show that astrocytes, by becoming reactive in HD, develop a protective response that involves complex bidirectional signaling with neurons to reduce mHtt aggregation.

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disorder caused by an unstable expansion of the CAG repeat sequence within the huntingtin gene. Huntingtin associates with ubiquitin-proteasome system that ensures degradation of particular proteins including β-catenin which is an important molecule whose equilibrated degradation is necessary for the proper functioning of the Wnt signaling pathway. The binding of β-catenin to the destruction complex is altered in HD, leading to the toxic stabilization of β-catenin. The main goal of my thesis was to determine whether the accumulation of β-catenin due to the presence of mutant huntingtin is also characteristic of Liběchov minipigs, a large animal model of Huntington's disease stably expressing N-truncated human mutant huntingtin. Using immunoblot and specific antibodies, we have revealed age-dependent accumulation of mutant huntingtin in transgenic minipigs. Unlike endogenous huntingtin, no decrease of the level of mutant huntingtin was observed in the striatum of transgenic animals. Surprisingly, this was followed by a decrease of phosphorylated β-catenin. Nevertheless, our results demostrate the accumulation of β-catenin in mesenchymal stem cells isolated from the oldest boars during ontogenesis. Furthermore, we have revealed a...

Huntingtons's disease (HD) is devastating neurodegenerative disorder manifesting by motor disturbances, cognitive decline and personal changes. The huge effort to find a cure for HD has brought several promising therapeutic treatments on the scene. Each of the prospective approaches needs to be investigated for safety, tolerability and efficacy. Mouse and rat models were a lot helpful in examination of pathological mechanisms of HD, but they are not sufficient for completion of pre-clinical testing. Therefore, we aimed to generate transgenic HD minipig to overcome the gap between rodents and humans. Minipig transgenic for the first 548 aminoacids of human mutant huntingtin gene (TgHD) under the control of human HD promotor was manipulated by lentiviral transduction of porcine one-cell stage embryos. Currently, six generations of minipigs expressing single copy of N-truncated human mutant huntingtin protein (mtHtt) with a repetition of 124 glutamines are at disposal. The more the model simulates the disease symptoms the better it is for translational research as the efficacy of the cure can be finer evaluated. Hence, the second aim was to demonstrate HD-like phenotype in our model. Testicular degeneration that preceded the clinical symptoms onset was observed as a consequence of expression of mtHtt....

Huntington's chorea is a dominantly inherited disease caused by trinucleotide (Cytosine-Adenine -Guanine) expansion in a gene coding huntingtin protein. Carriers of these mutation show symptoms associated with motor impairment, a cognitive and psychiatric disturbance, which is called Huntington's disease (HD). The major sign of HD is striatal atrophy in the middle age of life. Since it is known that huntingtin protein participates in a lot of cellular processes, such as transcriptional regulation and metabolism, these processes change by its mutation. One of the features observed in HD pathogenesis is the presence of oxidative stress. The aim of the work was to monitor the molecular changes preceding the HD manifestation in the knock-in minipig model. As a material for monitoring molecular changes leading to this condition, primary fibroblasts were used. Whereas, the oxidative stress arises from an imbalance between oxidants and antioxidants, level of reactive species and lipid peroxidation together with expression of antioxidant response associated genes was measured. At the same time, expression of metabolic and DNA repair related genes was monitored. Although the differences in oxidative stress level or the expression of antioxidative response genes were not detected, the changes in the...

Huntington's disease (HD) is an autosomal dominant inherited neuro-psychiatric disease with usual onset in the middle age. The mutation, located on the short shoulder of chromosome 4, is an expansion of a nucleotide triplet, containing cytosine, adenine, guanine (CAG), with critical limit of 40+ repetitions. The principal symptoms include motor symptoms (chorea, dystonia, disorders of voluntary movements), progressive cognitive deterioration and neuropsychiatric symptoms (behaviour disorders, affective symptoms and so on). The clinical diagnosis is confirmed by a genetic test, which may also be carried out presymptomatically in offsprings of the diseased person. The objective of the 1st study consisted in the characterization of differences in psychiatric examination and neuropsychological testing among the people at risk (PAR), in whom it was recommended to delay the test, and people at risk, who were recommended to continue in the so-called predictive protocol. The total of 52 people have been examined (32 females, 20 males). In addition to the common psychiatric examination we have also administered the Eysenck Personality Questionnaire (EPQ-A), self-rating scale of general psychopathology (SCL- 90), three short cognitive tests - Trail making test, test of Verbal fluency and...

No description available.

Le réseau neuronal se base sur l’axone et les dendrites pour former des milliards de connexions, ce qui fait du cerveau l'une des structures les plus complexes existantes. Pour que ce réseau fonctionne bien, il doit être régulé et maintenu. Cela pose de grands défis au cerveau lors du vieillissement, particulièrement dans le cadre d’une maladie neurodégénérative. Les premiers symptômes de plusieurs maladies neurodégénératives corrèlent d’ailleurs plus fréquemment avec le début de la dégénérescence axonale qu’avec la mort cellulaire des neurones. Une meilleure compréhension des mécanismes qui régulent cette dégénérescence axonale pourrait permettre de trouver de nouveaux traitements potentiels agissant dans la phase précoce de la manifestation de ces maladies. Dans cette thèse, nous étudions les mécanismes de dégénérescence et régénérescence axonale impliqués dans la sclérose latérale amyotrophique (SLA) et la maladie de Huntington (MH) en utilisant le nématode Caenorhabditis elegans (C. elegans). Nous nous basons sur les connaissances acquises sur les régulateurs de la régénérescence axonale pour investiguer leur implication et leur potentiel thérapeutique dans la SLA et la MH. Le C. elegans permet d’étudier la dégénérescence axonale dans le cadre des maladies neurodégénératives, puisque la visualisation des axones par fluorescence en facilite l’étude in vivo. Les modèles transgéniques C. elegans de la SLA et de la MH démontrent des bris axonaux pathologiques spontanés lors du vieillissement, permettant ainsi d’évaluer les mécanismes qui influencent cette dégénérescence axonale. Le modèle C. elegans permet aussi de mener des études à plus grande échelle. Nous avons donc pu effectuer un criblage génétique d’environ 40 gènes connus pour être des inhibiteurs de la régénérescence axonale après un dommage axonal mécanique. Selon notre hypothèse, l’inhibition de gènes inhibiteurs de la régénérescence axonale devrait augmenter le potentiel régénérateur des axones et, ainsi, réduire la dégénérescence axonale caractéristique de notre modèle SLA. Effectivement, nous avons pu identifier plusieurs voies de signalisation capables de réduire la dégénérescence axonale pathologique, notamment Dual zipper kinase DLK, la régulation des phosphoinositides et la signalisation de stress des ARNt par le stalled ribosome sensor GCN1. La voie de signalisation de DLK est indispensable dans la régénérescence axonale. Il a été démontré que sa suractivation permet de stimuler la régénérescence. Nous avons prouvé que la suractivation de DLK-1 par l’inhibition de ses inhibiteurs RPM-1 et FSN-1 réduit la dégénérescence axonale ainsi que la paralysie dans le contexte de la SLA. Pour évaluer la meilleure approche thérapeutique, nous avons investigué plus en détail les différents membres de cette voie de signalisation. Ainsi, nous avons trouvé que l’inhibition génétique et, surtout pharmacologique, de PARP1 et PARP2 peut réduire la dégénérescence axonale dans nos modèles de la SLA et de la MH. Les inositol polyphosphate phosphatases (INPP) sont des régulateurs des messagers secondaires d’inositol phosphates et de phosphatidylinositols, qui agissent dans la même voie de signalisation que PTEN, un inhibiteur de régénérescence axonale bien documenté. Dans nos études, nous avons identifié des nouvelles approches et cibles génétiques afin de réduire la dégénérescence axonale reliée à l’ALS et la MH. En résumé, nous avons identifié de multiples gènes qui agissent dans des voies de signalisation qui régulent la dégénérescence axonale, spécifiquement lors du vieillissement, dans le cadre de l’ALS et la MH. Il est primordial de mieux comprendre la signalisation intrinsèque qui régule l’axonopathie dans les maladies neurodégénératives pour établir de nouvelles approches thérapeutiques. Dans cette thèse, nous avons donc identifié plusieurs cibles thérapeutiques potentielles dans la voie de signalisation de DLK, et dans la voie de signalisation des phosphatidylinositols. / The neural network relies on axons and dendrites to form billions of connections, making the brain one of the most complex structures. These connections are both stable and highly dynamic, keeping the network in place while allowing connections to be modulated as needed. This network must be perfectly regulated and maintained for proper functioning, which can be a great challenge for the brain during aging and in the context of neurodegenerative diseases. Indeed, most neurodegenerative diseases show some form of degeneration of their axonal projections in the early stages. Increasingly, it is observed that the first symptoms of many neurodegenerative disorders correlate with the onset of axonal degeneration rather than cell death of neurons. A better understanding of the mechanisms regulating this axonal degeneration could lead to new treatments that could act in this particularly interesting therapeutic window. In this thesis, we aimed to study the genetic mechanisms of axonal degeneration involved in amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). More specifically, we investigated if axonal regeneration-associated genes can be targeted to reduce or even repair the age-dependent axonal damage observed in ALS and HD. To do this, we used Caenorhabditis elegans (C. elegans) models of ALS and HD, as they reproduce age-dependent axonal degeneration well. In addition, the use of fluorescent markers allows the visualization of axons in living animals, which makes it a unique model to study in vivo the dynamics of axonal damage and degeneration. We hypothesized that the stimulation of axonal regeneration pathways should increase the regenerative potential of axons and thus reduce the axonal degeneration characteristic of our ALS and HD models. Another advantage of using C. elegans is the possibility of large-scale genetic screens, allowing us to perform an RNAi-based genetic screen of 40 genes known as inhibitors of axonal regeneration. We identified multiple genes that act as drivers of axonal degeneration. Further analysis allowed us to identify an age-specific signaling network that regulates axonal degeneration through several main pathways, such as the Dual zipper kinase DLK pathway, the regulation of phosphatidylinositol phosphate, and the tRNA-related GCN1 stress response. The DLK signaling pathway is essential for axonal regeneration, and its overactivation has been shown to stimulate regeneration. We demonstrated that overactivation of DLK-1 by inhibiting its inhibitors RPM-1 and FSN-1 reduces axonal degeneration and paralysis in ALS. Furthermore, amongst the DLK pathway, we identified that genetic and pharmacological inhibition of PARP1/2 can consistently reduce axonal degeneration in our models of ALS and HD. Furthermore, we identified that the dysregulation of membrane-bound phosphoinositides is another major regulator of age-dependent axonal degeneration. We identified therapeutic targets similar to PTEN, a well-documented inhibitor of axonal regeneration and modulator of neurodegeneration. In our studies, we identified an alternative therapeutic target to PTEN and a new approach to trat ALS and HD-related axonal degeneration. In summary, we have identified multiple genes acting in an age-dependent network that drives axonal degeneration in ALS and HD. A better understanding of the intrinsic signaling that regulates axonopathy in neurodegenerative diseases is essential to establish new therapeutic approaches. In this thesis, we identified several potential therapeutic targets in the DLK signaling pathway and in the phosphoinositide signaling pathway.

Progressive degeneration in the central nervous system (CNS) of Huntington’s disease (HD) patients is a relentless debilitating process, resulting from the inheritance of a single gene mutation. With limited knowledge of the underlying pathological molecular mechanisms, pharmaceutical intervention has to-date not provided any effective clinical treatment strategies to attenuate or compensate the neuronal cell death. Attention has therefore turned to biotherapeutic molecules and novel treatment approaches to promote restoration and protection of selectively vulnerable populations of neurons in the HD brain. Rapid advances in vectorology and gene-based medicine over the past decade have opened the way for safe and efficient delivery of biotherapeutics to the CNS. With numerous factors known to regulate the development, plasticity and maintenance of the mammalian nervous system many proteins have emerged as potential therapeutic agents to alleviate HD progression. This investigative study utilised gene delivery vectors derived from the non-pathogenic adeno-associated virus (AAV) to direct high-level expression of brain-derived neurotrophic factor (BDNF), glial cell-line derived neurotrophic factor (GDNF), Bcl-xL or X-linked inhibitor of apoptosis protein (XIAP) within the rodent striatum. Maintenance of the basal ganglia and functional behaviour deficits were assessed following excitotoxic insult of the striatum by quinolinic acid (QA), a neurotoxic model of HD pathology. Enhanced striatal expression of BDNF prior to QA-induced lesioning provided maintenance of the striosome-matrix organisation of the striatum, attenuating impairments of sensorimotor behaviour with a 36-38% increase in the maintenance of DARPP-32 / krox-24 expressing striatal neurons, reduced striatal atrophy and increased maintenance of striatonigral projections. Higher levels of BDNF however induced seizures and weight-loss highlighting the need to provide regulatable control over biotherapeutic protein expression. Continuous high-expression of BDNF or GDNF resulted in a downregulation of intracellular signal mediating proteins including DARPP-32, with AAV-GDNF not found to enhance the overall maintenance of striatal neurons. Neither of the anti-apoptotic factors provided significant protection of transduced striatal neurons but tended towards ameliorating QA-induced behavioural deficits, displaying behaviour – pathology correlations with the survival of parvalbumin-expressing neurons in the globus pallidus. The results of this thesis suggest BDNF as a promising putative biotherapeutic for HD, but emphasises the requirement to control expression following gene delivery, and for further elucidation of the physiological impact that enhanced expression of endogenous factors has on the host cells. Additionally the maintenance of neural networks beyond the caudate-putamen will be vital to ensuring efficient clinical outcomes for HD. / Auckland Medical Research Foundation. Foundation for Research, Science and Technology. The University of Auckland.

Paradoxically enhanced cognitive processes in neurological disorders provide vital clues to understanding neural function. However, what determines whether the neurological damage is impairing or enhancing is unclear. Here we use the performance of patients with two disorders of the striatum to dissociate mechanisms underlying cognitive enhancement and impairment resulting from damage to the same system. In a two-choice decision task, Huntington\'s disease patients were faster and less error prone than controls, yet a patient with the rare condition of benign hereditary chorea (BHC) was both slower and more error prone. EEG recordings confirmed significant differences in neural processing between the groups. Analysis of a computational model revealed that the common loss of connectivity between striatal neurons in BHC and Huntington\'s disease impairs response selection, but the increased sensitivity of NMDA receptors in Huntington\'s disease potentially enhances response selection. Crucially the model shows that there is a critical threshold for increased sensitivity: below that threshold, impaired response selection results. Our data and model thus predict that specific striatal malfunctions can contribute to either impaired or enhanced selection, and provide clues to solving the paradox of how Huntington\'s disease can lead to both impaired and enhanced cognitive processes.