As doenças neuromusculares incluem um grupo muito heterogêneo de patologias que atingem 1 em cada 1.000 indivíduos nascidos vivos. Dentre as doenças neuromusculares destacam-se as paraplegias espásticas hereditárias que acometem, aproximadamente, cerca de 1 em cada 10.000. As paraplegias espásticas hereditárias (PEH) são caracterizadas pela espasticidade e fraqueza muscular dos membros inferiores. São muito heterogêneas tanto em clínica como geneticamente. Diversas formas já foram descritas e a mais comum delas, acometendo por volta de 40% dos casos autossômicos dominantes, causada por mutações no gene SPAST (PEH do tipo 4 ou SPG4). Estudos de correlação genótipo: fenótipo têm mostrado que indivíduos da mesma família carregando a mesma mutação patogênica, podem ter quadro clínico muito distinto. A explicação para esta questão pode estar na procura por genes modificadores, no padrão de expressão, na análise proteômica (seja por ligantes a proteínas ou no dobramento das mesmas), ou em mecanismos epigenéticos. Além disso, em algumas formas observa-se uma diferença na porcentagem de pessoas afetadas de acordo com o sexo. Essa desproporção foi observada numa grande família de com PEH na qual existe um predomínio de afetados do sexo masculino. O objetivo do presente trabalho foi a análise de pacientes discordantes, ou seja, que possuam a mesma mutação, porém com quadro clínico discordante de uma grande família brasileira com SGP4. Para isso foi feito um estudo da abundância de transcritos (mRNA) e de genótipo (polimorfismos de base única) em relação a um fenótipo (sintomático ou assintomático). Os resultados sugerem que o principal sistema envolvido, que poderia explicar as diferenças entre os pacientes discordantes, é o sistema imune, com a principal atuação dos genes C2, HLA-DRB1 e LY6G6C. Esses genes podem ter papel protetor ou tóxico no desenvolvimento do quadro clínico dos pacientes analisados / The hereditary spastic paraplegia (HSP) is characterized by muscle weakness and lower limb spasticity. They are very heterogeneous both clinically and genetically. Several forms have been described and the most common one, affecting around 40% of autosomal dominant cases, is caused by mutations in the SPAST gene (HSP type 4 or SPG4). Genotype: phenotype correlation studies have shown that affected individuals from the same family, who carry the same pathogenic mutation, can have very distinct phenotypes. The underlying explanation behind this clinical heterogeneity may be found in the search for modifier genes, in expression patterns observed proteomic analyses (either by protein binding or folding), or epigenetic mechanisms. As is observed in other motor neurodisease, there is a disproportion between the number of affected males and females, with males being the predominantly affected. The objective of this study was to analyze discordant patients, i.e., those that possess the same mutation, but show discordant phenotypes, from a large Brazilian family with SGP4. For this study, the abundance of transcripts (mRNA) and genotype (single nucleotide polymorphisms) relative to a phenotype (symptomatic or asymptomatic) were analyzed. The results suggest that the main system involved, which could explain the differences between discordant patients, is the immune system, with the main activity of C2, LY6G6C and HLA-DRB1 genes. These genes may have a protective or toxic role in the development of the analyzed patients\' clinical features

Les enfants atteints de paralysie cérébrale unilatérale spastique ont un membre supérieur dont les mouvements sont dégradés. Un des symptômes en cause est la co-activation spastique, une activation excessive de certains muscles qui s’opposent au mouvement. En clinique, il n’existe pas d’outil pour distinguer quels muscles ont une activation pathologique. Les signaux électromyographiques, mesurés de façon non invasive, peuvent discriminer des activations musculaires pathologiques. Cependant, ils sont rarement mesurés au membre supérieur des enfants atteints de paralysie cérébrale, en raison d’obstacles méthodologiques et statistiques. Cette thèse aborde ces obstacles. Les signaux électromyographiques de muscles du membre supérieur ont été mesurés chez 63 enfants avec et sans paralysie cérébrale unilatérale spastique, dans le cadre de 5 études de recherche. Les 2 premières études ont consisté à détecter et caractériser les anomalies de commande musculaire chez les enfants avec une paralysie cérébrale, afin de cibler les paramètres électromyographiques pertinents. La 3ème étude a validé la mesure électromyographique au membre supérieur, en évaluant sa variabilité. Les 4ème et 5ème études ont appliqué la méthodologie développée à des problématiques cliniques : distinguer, pour chaque enfant, les muscles responsables des limitations de mouvements actifs ; évaluer les modifications d’activation musculaire à la suite de thérapies. Le développement méthodologique lié à ces études a permis d’évaluer la fiabilité de l’analyse électromyographique du membre supérieur, et a montré son fort potentiel pour le « diagnostic fonctionnel » des enfants atteints de paralysie cérébrale. / Children with unilateral spastic cerebral palsy have restricted movement of one upper limb.One of the symptoms responsible for this limitation is spastic co-activation, which is an excessive activation from specific muscles opposing the movement. In clinical routine, no tool can detect which muscles have a pathological activation.Electromyographic signals, measured non-invasively, can discriminate pathological muscle activations.However, they are rarely measured at the upper limb of children with cerebral palsy, due to methodological and statistical difficulties. This thesis addresses these difficulties. Electromyographic signals of upper limb muscles were measured in 63 children with and without unilateral spastic cerebral palsy, as part of 5 research studies.The first two studies consisted in detecting and characterizing abnormal muscle activations in children with cerebral palsy, in order to target relevant electromyographic parameters. The third study validated the measure of upper limb electromyography, and evaluated its variability.Fourth and fifth studies applied the developed methodology to clinical problems: the distinction, for each child, of the muscles responsible for active movement restrictions; the evaluation of muscle activation modifications following a therapy.Methodological development associated with these studies assessed the reliability of upper limb electromyography, and showed its strong potential for the “functional diagnosis” of children with cerebral palsy.

La parésie spastique est souvent envisagée comme une atteinte de la commande motrice, comportant une parésie de l’agoniste et une hyperactivité de l’antagoniste. Cependant, une seconde affection d’ordre musculaire - la myopathie spastique, apparait rapidement, pendant la phase aigüe après la lésion. L’hypomobilisation en position courte de certains muscles dans le contexte d’une parésie des muscles opposés s’accompagne d’une perte de leur tension longitudinale, première étape d’une cascade de transformations génétiques, structurelles, biomécaniques puis physiologiques des muscles hypomobilisés, incluant entre autres une perte de leur extensibilité et de leur longueur. Aux stades subaigu puis chronique du syndrome, les affections neurologique et musculaire coexistent, et semblent s’entretenir mutuellement.Sur le plan physiopathologique, ce travail de recherche s’est d’abord intéressé à ces intrications entre les affections neurologique et musculaire, et à la part de responsabilité potentielle de la myopathie spastique dans la fonction active. Ce premier travail a montré que le degré d’hypoextensibilité musculaire d’un antagoniste, au-delà d’un certain seuil, est corrélé au degré de perturbation de la commande motrice dirigée sur l’agoniste. La chronologie des différents événements établie dans la littérature, avec des anomalies histologiques musculaires qui apparaissent toujours avant les premières manifestions d’hyperactivité motoneuronale, amène à suggérer une responsabilité causale de la maladie du muscle dans une partie des perturbations ultérieures de la commande neuronale descendante. Ce rôle probablement essentiel de la maladie musculaire au sein de la parésie spastique doit inciter le clinicien à orienter une part significative de son traitement vers une stimulation optimale de la plasticité musculaire pour tenter de prévenir ou d’inverser le processus de myopathie spastique.Si l’étirement est depuis très longtemps une technique courante, sa capacité à augmenter durablement l’extensibilité musculaire est aujourd’hui controversée, même si ses effets à long terme, i.e. au-delà de six mois d’application quotidienne, n’ont pas été explorés.La deuxième partie, thérapeutique, de ce travail a donc été l’étude des effets à long terme d’un programme quotidien d’auto-étirements de haute intensité au sein de la méthode des Contrats d’Autorééducation Guidée, pendant un an ou plus. Il a d’abord été rétrospectivement montré que ce programme avait permis, sur trois années de suivi de sujets parétiques, une amélioration progressive importante de l’extensibilité musculaire mesurée cliniquement. Un essai randomisé contrôlé contre la kinésithérapie conventionnelle avec mesures échographiques de paramètres structuraux des muscles étirés a ensuite établi que l’application de ce programme durant un an chez des sujets atteints d’hémiparésie chronique sur AVC générait une augmentation de la longueur fasciculaire des muscles fléchisseurs plantaires, parallèlement à une amélioration de la fonction active, de façon nettement plus importante que la thérapie conventionnelle.L’ensemble de ces résultats doit participer à une meilleure prise en compte des altérations passives structurelles évolutives du muscle comme une entité nosologique, la myopathie spastique, méritant un traitement spécifique au sein de la parésie spastique, afin de limiter ses interactions avec la maladie neurologique et de réduire les déficits fonctionnels. La pratique quotidienne de postures d’auto-étirement à haute intensité, guidée par un thérapeute et auto-documentée par le patient sur un registre, peut être prescrite et réalisée sur le long terme, afin de traiter la myopathie spastique. / Spastic paresis is often understood as a neurologic disorder of the motor command that includes agonist paresis and antagonist overactivity. However, a second disorder, involving the muscle and named spastic myopathy, appears rapidly during the acute phase after the lesion. Hypo-mobilization in shortened position of some muscles of the paretic limbs, in the context of paresis of their antagonists, will reduce their longitudinal tension, which acts as the first event of a cascade of transformations involving genetic, structural, biomechanical, and then physiological mechanisms, leading to loss of muscle extensibility and length. At the subacute and chronic stages of this syndrome, the neurological and the muscular disorders coexist, seemingly feeding on each other.From a pathophysiological point of view, this research work started by exploring the entanglements between the neurological and muscular disorders, and the potential responsibility of spastic myopathy in the impairment of active function. The first study demonstrated that the loss of muscle extensibility in antagonists, when above a certain threshold, correlates with the degree of disturbance of the motor command directed to the agonist. The chronology of events established in the literature, with histological muscle abnormalities emerging always before the first expressions of motoneuronal overactivity, suggests a causal role of the muscle disorder in a part of the descending command disorder. The crucial role of the muscle disorder in the syndrome of spastic paresis should encourage clinicians to direct treatment towards techniques to stimulate muscle plasticity.Despite the classic use of muscle stretching in daily practice, its potential to increase muscle extensibility remains a subject of controversy, even though its long-term effects, i.e. over six months of daily implementation, have not been investigated. The second part of this work was thus therapeutic. We retrospectively explored the long-term effects of a daily, high load self-stretching programme within the Guided Self-rehabilitation Contract method, for at least a year. This work showed that this programme, applied over three years in paretic subjects, was increasingly associated with major gains in clinical muscle extensibility. A randomized controlled trial against conventional therapy, which used ultrasound exploration of structural parameters in the stretched muscles, demonstrated that this programme, applied over one year in subjects with stroke-induced chronic hemiparesis, enabled greater increase in plantar flexor fascicle length and active function improvement than conventional therapy.Taken together, these results will contribute to enhancing the knowledge about the evolving structural and mechanical muscle changes in spastic paresis, as a pathologic entity, spastic myopathy. Spastic myopathy needs to be specifically addressed, as it interacts with the neurological disorder and worsens functional impairment. Daily postures of high load self-stretch, guided by the therapist and self-monitored on a diary, should be prescribed and practiced over the long term, in order to treat spastic myopathy in subjects with spastic paresis.

Objetivo: verificar a aplicabilidade, reprodutibilidade e validade do SCIM III nos pacientes com paraparesia espástica. Método: estudo transversal incluindo 30 sujeitos (66% mulheres; 41,5±14,7 anos) com paraparesia espástica de etiologia genética, infecciosa ou a esclarecer que foram avaliados pela análise computadorizada da marcha, versão brasileira da SCIM III (0-100 pontos), MIF (18-126 pontos), por 2 examinadores (A e B) no mesmo dia e 1 semana depois (A). Resultados: o coeficiente de correlação intraclasse (CCI) para o uso da SCIM III indicou boa reprodutibilidade intra e inter-examinadores (CCI=0,9). A correlação de Spearman entre a SCIM III e a parte motora da MIF foi considerada adequada e estatisticamente significante (Spearman=0,6; p=0,001). A correlação entre as subescalas da SCIM III e os domínios da MIF foi considerada forte e significante para auto-cuidado (Spearman=0,8; p0,001) e moderada para transferências (Spearman=0,6; p=0,0005) e locomoção (Spearman=0,6; p=0,0006). A subescala mobilidade da SCIM III mostrou correlação positiva e significante para cadência (Spearman=0,8; p0,001), velocidade da marcha (Spearman=0,7; p0,001) e comprimento do passo (Spearman=0,6; p0,001). Conclusões: A SCIM III é um instrumento de avaliação funcional reprodutível intra e inter examinadores e capaz de avaliar o nível de independência dos indivíduos com paraparesia espástica. O SCIM III é mais sensível que a MIF, especialmente para pacientes com maior independência. Cadência, velocidade de marcha e comprimento do passo se correlacionaram com a subescala mobilidade / Purpose: to verify the applicability, reproducibility, and validity of the SCIM III patients with non-traumatic spastic paraplegia. Method: The cross-sectional study included 30 subjects (66% females; 41.5 ± 14.7 y) older with spastic paraparesis of any etiology were assessment by computerized gait analysis and with the Brazilian versions of SCIM III (0-100 points), FIM (18-126 points) by 2raters (A and B) at the same day and 1 week later (A). Results: The intraclass correlation coefficient (ICC) for the use of SCIM III indicated good intra and inter-evaluator reproducibility (ICC = 0.9). Correlation between the SCIM III and the motor FIM was appropriate (Spearman=0.6; p0.001). SCIM III subscales and the FIM domains correlated strongly for self-care (Spearman=0.8; p0.001), moderately for transfers (Spearman=0.6; p=0.0005) and locomotion (Spearman=0.6; p=0.0006). SCIM III mobility subscale positively correlated with the cadence (Spearman=0.8; p0.01), gait speed (Spearman=0.7; p0.01), and step length (Spearman=0.6;p0.01). Conclusions: SCIM III is a reproducible functional assessment instrument and capable of evaluating the level of independence of the individual with spastic paraplegia. The SCIM III is more sensitive than the FIM for non-traumatic spastic paraplegic patients with higher levels of independence. Linear gait parameters correlated with its mobility subscale.

Entre os nossos sentidos, a visão é um dos canais pelo qual recém-nascidos interagem com o meio ambiente, o qual exerce fundamental influência sobre o desenvolvimento de nossas funções cognitivas, habilidades motoras e comunicação social (Milner & Goodale, 2008). Assim, a visão é expressivamente requisitada na criança em fase escolar. Nas crianças com paralisia cerebral além do comprometimento motor apresentado, outras alterações sensoriais podem estar presentes, principalmente aquelas relacionadas à visão. Nosso trabalho teve como principal objetivo avaliar a discriminação cromática em crianças com paralisia cerebral espástica em idade escolar (média=10,13; DP=2,89), pois dentre tantas funções visuais importantes esta tem sido muito pouco estudada nesta população. Avaliamos a visão de cores de 43 sujeitos com paralisia cerebral espástica (5 tetraplégicos, 22 diplégicos e 16 hemiplégicos). Divididos em dois grupos: um grupo de (n=31) possui AV (média=1,13 decimal de snellen; DP=0,25) outro grupo (n=12) possui AV (média=0,39 decimal de snellen; DP=0,13) Um grupo de crianças (n=53) formou o grupo controle. As crianças dos três grupos estavam equiparadas por idade e foram submetidas aos mesmos testes. Além da comparação entre os limiares de discriminação cromática, buscamos associações e correlações entre acuidade visual, sexo, idade, prematuridade, nível de comprometimento motor dado pela escala do GMFCS e etiologia. Estendemos também a avaliação cromática para um grupo de sujeitos adultos (n=41), com idades variando de 18 a 69 anos (média=42,58; DP=14,21). A avaliação da discriminação de cores foi realizada usando o teste psicofísico computadorizado, versão adaptada por Goulart et al. (2008) do original (Cambridge Color Test). Este teste utiliza a versão curta trivector, que mede limiares de discriminação cromática em três eixos de confusão: protan, deutan e tritan. Além disso, o teste apresenta duas posições de localização do estímulo (direita ou esquerda) o que garante condições favoráveis para avaliar crianças com paralisia cerebral. Os resultados apresentaram diferenças significativas nos eixos protan e deutan (p<0,05) no grupo de sujeitos com paralisia cerebral e acuidade visual próxima em relação ao grupo controle. Porém, o mesmo não aconteceu para o eixo tritan, contrariando os achados dos poucos estudos encontrados na literatura. O grupo de sujeitos PCs com acuidade visual reduzida apresentou piores limiares cromáticos para todos os eixos e foram estatisticamente significantes (p<0,05), tanto quando comparados com os PCs com acuidade visual normal ou quando comparados com o grupo de sujeitos controle. Entretanto, subgrupos de PCs (diplégicos e hemiplégicos) apresentaram valores semelhantes de limiares cromáticos entre si, atestados pela não diferença estatística, porém os sujeitos tetraplégicos mostraram-se significativamente diferentes dos diplégicos e hemiplégicos (p<0,05). Considerando-se os níveis de GMFCS observamos uma correlação positiva e significativa para o eixo protan e deutan, mas não para o eixo tritan, bem como também para as demais variáveis testadas / Among our senses, vision is one of the channels by which infants interact with the environment, which exerts a fundamental influence on the development of our cognitive functions, motor skills and social communication (Milner & Goodale, 2008). Thus, vision is significantly required in school age child. In addition, children with cerebral palsy who present motor impairment, can also present other sensory changes, especially those related to vision. Our study aimed to evaluate chromatic discrimination in children at school age who have spastic cerebral palsy (mean= 10,13; SD=2,89), for among many important visual functions, few studies have extensively been done in this population. We evaluated the color vision of 43 subjects with spastic cerebral palsy (5 quadriplegic, 22 diplegic and 16 hemiplegic). Divided into two groups: one group (n=31) had visual acuity (mean=1,13 decimal snellen; SD=0,25) another group (n=12) had visual acuity (mean=0,39 decimal snellen; SD=0,13) A group of children (n=53) formed the control group. Children from the three groups were matched by age and underwent the same tests. Besides comparing the chromatic discrimination thresholds, we seek associations and correlations between visual acuity, sex, age, prematurity, level of motor impairment given by scale in the GMFCS and etiology. We also extended the chromatic evaluation to a group of adult subjects (n=41), with ages ranging from 18 to 69 years (mean=42,58; SD=14,21). Evaluation of color discrimination was performed using computerized psychophysical test, version adapted by Goulart et al.(2008) original (Cambridge Color Test). This test uses the short version trivector that measures chromatic discrimination thresholds in three areas of confusion: protan, deutan and tritan. In addition, the test has two positions of the stimulus location (left or right) which ensures favorable conditions for assessing children with cerebral palsy. The results showed significant differences in protan and deutan axes (p<0,05) in the group of subjects with cerebral palsy, and short visual acuity in the control group. However, it did not happen for the tritan axis, contradicting the findings of the few studies in the literature. The group of cerebral palsy subjects that had reduced visual acuity presented worse chromatic thresholds for all axes and were statistically significant (p<0,05), this is when compared to spastic cerebral palsy with normal visual acuity or when compared to the group of controls. However, subgroups of spastic cerebral palsy (diplegic and hemiplegic) had similar values of chromatic thresholds among them, certified by non-statistical difference. On the other hand, tetraplegic subjects were significantly different from the diplegic and hemiplegic (p<0,05). Considering the levels of GMFCS, we observed a positive and significant correlation for the protan and deutan axis, but not for the tritan axis and neither for the other tested variables

A síndrome SPOAN (acrônimo do inglês spastic paraplegia, optic atrophy and neuropathy) é uma doença neurodegenerativa de herança autossômica recessiva que tem como achados clínicos a atrofia ótica congênita não progressiva, paraplegia espástica e neuropatia ambas progressivas. Ela havia sido mapeada na região cromossômica 11q13, porém a variante patogênica e o gene associados à síndrome não haviam sido identificados. Após execução do sequenciamento do genoma completo de um paciente foi detectada a deleção de 216-pb (chr11.hg19:g.66,024,557_66,024,773del) em homozigose localizada em região regulatória upstream do gene KLC2. Surpreendentemente, essa deleção causa superexpressão do KLC2, detectada em estudos de Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) utilizando fibroblastos e neurônios motores de pacientes comparados com controles. Ensaios utilizando o Danio rerio como modelo in vivo mostraram que tanto o knockdown quanto a superexpressão do klc2 em embriões de zebrafish causa o fenótipo de cauda curvada (leve ou grave); fenótipo esse associado às doenças neurodegenerativas e HSPs. Superexpressão de um gene causada por uma pequena deleção em região regulatória é um novo mecanismo que até então não havia sido descrito na condição autossômica recessiva. Estudos funcionais por meio de gene reporter de LacZ avaliando o padrão de expressão espaço-temporal da região regulatória wild-type e com a deleção de 216-pb foram realizados nesse trabalho em modelo de camundongo, porém, não foi possível identificar um padrão de expressão reprodutível do gene reporter nesse modelo. Por fim, camundongos transgênicos para a superexpresão do KLC2 humano foram gerados, no entanto não foram realizados testes físicos e comportamentais para validar o transgênico como modelo para síndrome SPOAN / SPOAN (the acronym of its clinical symptoms) syndrome is a neurodegenerative disorder mainly characterized by a progressive spastic paraplegia, congenital non-progressive optic atrophy and progressive neuropathy. A potential causative gene was mapped at 11q13, but so far no gene and mutation were identified. Whole-genome sequencing allowed to detect a homozygous 216-bp deletion (chr11.hg19:g.66,024,557_66,024,773del) located at the regulatory upstream region of the KLC2 gene. Surprisingly, this deletion causes KLC2 overexpression detected by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) using fibroblasts and motor-neurons from patients compared with controls. Assays using Danio rerio as in vivo model showed that the klc2 knockdown either its overexpression in zebrafish embryos causes mild to severe curly-tail phenotype; phenotype that is already well defined as suggestive of a neurodegenerative disorder and HSP. Overexpression of a gene caused by a small deletion in the regulatory region is a novel mechanism, which to the best of our knowledge, was never reported before in a recessive condition. Functional studies using LacZ reporter assay evaluating the spatiotemporal expression pattern of wild-type regulatory region and with the deletion of 216-bp were performed in this work using mouse, but was not possible to identify an especific gene reporter expression pattern in this animal model. As a last experiment, transgenic mice for human KLC2 overexpression were generated, though behavioral tests were not performed to validate this transgenic animal as a model for SPOAN syndrome

Autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS) is an early onset neurodegenerative disorder resulting from mutations in the SACS gene that encodes the protein sacsin. Sacsin is a 520kDa multi-domain protein localised at the cytosolic face of the outer mitochondrial membrane with suggested roles in proteostasis and most recently in the regulation of mitochondrial morphology. An excessively interconnected mitochondrial network was observed as a consequence of reduced levels of sacsin protein following SACS knockdown in neuroblastoma cells as well as in an ARSACS patient carrying the common Quebec homozygous SACS mutation 8844delT. Moreover, it was suggested that sacsin has a role in mitochondrial fission as it was found to interact with mitochondrial fission protein Dynamin related protein 1 (Drp1). The aim of this thesis was to explore sacsin’s role in the regulation of mitochondrial morphology and dynamics in non-Quebec ARSACS patients and sacsin knockdown fibroblasts. This study shows that loss of sacsin function promotes a more interconnected mitochondrial network in non-Quebec ARSACS patients and in sacsin knockdown fibroblasts. Moreover, recruitment of the essential mitochondrial fission protein Drp1 to the mitochondria was significantly reduced in ARSACS patient cells and in sacsin knockdown fibroblasts. This reduced recruitment of Drp1 to mitochondria also occurred when cells were treated to induce mitochondrial fission. Furthermore, both the size and intensity of Drp1 foci localised to the mitochondria were significantly reduced in both sacsin knockdown and patient fibroblasts. Finally, reduced ATP production, decreased respiratory capacity of mitochondria and an increase in mitochondrial reactive oxygen species demonstrated impaired mitochondrial function in ARSACS patient and sacsin knockdown fibroblasts. These results suggest a role for sacsin in the stabilisation or recruitment of cytoplasmic Drp1 to prospective sites of mitochondrial fission similar to that observed by other mitochondrial fission accessory proteins.

Vesicle trafficking provides the solution to the 'sorting problem' - how the eukaryotic cell maintains the distinct identities, and thus functional properties, of its membrane-bound organelles. During vesicle trafficking, proteins are selectively sorted into membrane bound transport intermediates by vesicle adaptors, which include those of the highly conserved adaptor protein (AP) complex family. Each AP complex has a distinct subcellular localisation and functions in the sorting of a specific subset of transmembrane cargo proteins. Adaptor protein complex 4 (AP-4) is one of the more recently identified AP complexes, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder, suggesting an important role in neuronal development and homeostasis. However, the pathomechanisms that underly the neuronal pathology in AP-4 deficiency are currently unknown. AP-4 is proposed to function in protein sorting at the trans-Golgi network (TGN), so AP-4 deficiency can be thought of as a disease of missorting. The aim of this study was to apply unbiased global proteomic approaches to define the composition of AP-4 vesicles and to identify physiological cargo proteins of the AP-4 pathway. Using 'Dynamic Organellar Maps' and comparative analysis of vesicle-enriched fractions from wild-type and AP-4-depleted cells, three ubiquitously expressed transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, were found to be mislocalised in AP-4-deficient cells. Two novel cytosolic AP-4 accessory proteins, RUSC1 and RUSC2, were also identified. Further proteomic analyses confirmed the interactions between these proteins. AP-4 deficiency was found to cause missorting of ATG9A in diverse cell types, including patient derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A and SERINC-positive vesicles from the TGN to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the 'ATG9 reservoir' required for autophagosome biogenesis. This study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

Sacsin, which is mutated in the neurodegenerative disease Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS), is a 520 kDa modular protein with regions of homology to molecular chaperones and domains linking to the ubiquitin proteasome system. This suggests a role in proteostasis. Previously, sacsin has been shown to partially localise with mitochondria, and loss of sacsin results in elongated and dysfunctional mitochondria. Moreover, alterations in neurofilaments have recently been reported in a mouse model of ARSACS. Despite these findings, pathophysiological mechanisms of ARSACS are poorly understood. The aim of this thesis was to elucidate the cellular role of sacsin by determining how loss of its function leads to the observed mitochondrial and intermediate filament defects. This hoped to shed light on the mechanism of disease in ARSACS. The results indicate that the mitochondrial elongation seen in ARSACS is likely due to reduced mitochondrial localisation of the essential fission factor DRP1. This may be mediated by loss of function of a complex involving sacsin and dynactin-6, a subunit of the dynein-dynactin motor complex, which has previously been shown to be required for DRP1 mitochondrial recruitment. DRP1-mediated mitochondrial fission is necessary for mitochondrial quality control; hence a disruption to mitochondrial quality control is likely to occur in sacsin deficient cells, which may explain the mitochondrial dysfunction in ARSACS. Furthermore, sacsin null cells display a dramatic collapse and perinuclear bundling of the vimentin intermediate filament network. This is coupled with the displacement of cellular organelles, particularly mitochondria, early endosomes and the Golgi, which accumulate at the periphery of the vimentin bundle. These are characteristic features of aggresome formation, indicating an aggregation of misfolded protein, which occurs due to disrupted proteostasis. Further supporting this, the proteostasis components ubiquitin, HSP70, LAMP2 and p62 are recruited to the perinuclear vimentin bundles. In summary, the findings of this thesis indicate a role for sacsin in mitochondrial and protein quality control, the dysfunction of which is likely to be particularly detrimental in neurons. Mitochondrial dysfunction along with protein misfolding and aggregation are implicated in many neurodegenerative diseases, and ARSACS is no exception.

Les mutations du gène SPG4 codant la spastin sont responsables de la forme la plus fréquente de Paraplégies Spastiques Héréditaires (PSH), des maladies neurologiques caractérisées par une dégénérescence des faisceaux cortico-spinaux. La spastin, ainsi que son homologue p60-katanin sont des enzymes de cassure des microtubules (MSE) essentielles à la croissance des neurones moteurs spinaux (NMS) chez l'embryon de poisson-zèbre mais dont le rôle dans les processus de guidage axonal également dépendant des microtubules (MTs) demeurent énigmatiques. Les principaux objectifs de ma thèse ont consisté à préciser le rôle et le degré de redondance fonctionnelle existant entre ces deux MSE lors de l'établissement des circuits moteurs chez ce téléoste et de clarifier les mécanismes pathogéniques à l¿origine des PSH liées au gène SPG4.J'ai tout d'abord contribué à montrer que la p60-Katanin contrôle la trajectoire des axones des NMS et la mobilité des larves de façon dose-dépendante et non redondante avec la spastin. De plus, notre étude identifie la polyglutamylation des MTs par TTLL6 comme un élément clé de l'activité de la p60-Katanin lors de ce processus. Sur le même modèle, j'ai révélé un rôle différentiel des isoformes majoritaires de la spastin (résultant d¿une traduction alternative, M1 et M61) au cours du développement des NMS en démontrant un rôle coopératif de M1 et d'autres protéines de PSH dans l'inhibition de la voie des BMPs et révélant un rôle pour M61 en aval de la signalisation Neuropilin-1. Ces données suggèrent que l'altération de ces deux grandes voies de signalisation essentielles au développement des NMS pourrait contribuer à la pathogénèse des formes SPG4. / Mutations in SPG4, encoding spastin cause the major form of Hereditary Spastic Paraplegias (HSP), a paralytic disorder characterised by the degeneration of the corticospinal tracts. Spastin and its close homologue p60-katanin are microtubule-severing enzymes (MSE) required for spinal motor neuron (SMN) axon extension during zebrafish development. However, their roles in SMN axon navigation which also rely on microtubules (MTs) remain elusive. My PhD work aimed at refining the functional specificity and redundancy of these MSE during motor circuit wiring and clarifying the physiopathology of SPG4-linked HSP. I have first contributed to show that p60-Katanin controls SMN axon targeting and larval locomotion in a dose-dependent manner. We also demonstrated that Spastin and p60-Katanin play differential roles in SMN navigation and identified TTLL6-mediated MT polyglutamylation as a key event in regulating p60-Katanin activity in this process. Concomitantly, I have conducted a functional analysis of spastin main isoforms (resulting from alternative translation, M1 and M61) during zebrafish development, which reveals their critical and specific involvement in two distinct signalling pathways that are both essential for motor circuit wiring and locomotor behaviours. This study has provided compelling evidences for a concerted role for M1 and other HSP proteins in the down-regulation of the BMP pathway and reveals a specific role for M87 as a downstream effector of Neuropilin-1 signalling. Altogether, our study emphasizes defective BMP signalling as a key pathogenic mechanism in HSP, and shows that dysregulation of the Neuropilin-1 pathway may equally contribute to SPG4-linked HSP.