TDP-43 est une protéine multifonctionnelle possédant des rôles dans la transcription, l'épissage des pré-ARNm, la stabilité et le transport des ARNm. TDP-43 interagit avec d'autres hnRNP, incluant hnRNP A2, via son extrémité C-terminale. Plusieurs membres de la famille des hnRNP étant impliqués dans la réponse au stress cellulaire, alors nous avons émis l’hypothèse que TDP-43 pouvait y participer aussi. Nos résultats démontrent que TDP-43 et hnRNP A2 sont localisés au niveau des granules de stress, à la suite d’un stress oxydatif, d’un choc thermique, et lors de l’exposition à la thapsigargine. TDP-43 contribue à la fois à l'assemblage et au maintien des granules de stress en réponse au stress oxydatif. TDP-43 régule aussi de façon différentielle les composants clés des granules de stress, notamment TIA-1 et G3BP. L'agrégation contrôlée de TIA-1 est perturbée en l'absence de TDP-43. En outre, TDP-43 régule le niveau d`ARNm de G3BP, un facteur de granule de stress de nucléation. La mutation associée à la sclérose latérale amyotrophique, TDP-43R361S, compromet la formation de granules de stress. Ainsi, la fonction cellulaire de TDP-43 s'étend au-delà de l’épissage; TDP-43 est aussi un composant de la réponse cellulaire au stress central et un acteur actif dans le stockage des ARNs. / TDP-43 is a multifunctional protein with roles in transcription, pre-mRNA splicing, mRNA stability and transport. TDP-43 interacts with other hnRNPs, including hnRNP A2, via its C-terminus and several hnRNP family members are involved in the cellular stress response. This relationship led us to investigate the role of TDP-43 in cellular stress. Our results demonstrate that TDP-43 and hnRNP A2 are localized to stress granules, following oxidative stress, heat shock, and exposure to thapsigargin. TDP-43 contributes to both the assembly and maintenance of stress granules in response to oxidative stress and differentially regulates key stress granules components including TIA-1 and G3BP. The controlled aggregation of TIA-1 is disrupted in the absence of TDP-43. In addition, TDP-43 regulates G3BP mRNA levels, a stress granule nucleating factor. A mutation associated with amyotrophic lateral sclerosis, TDP-43R361S, compromises stress granule formation. Thus, the cellular function of TDP-43 extends beyond splicing and places TDP-43 as a participant of the central cellular response to stress and an active player in RNA storage.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative due à une dégénérescence des motoneurones. Plus de 40 mutations du gène TARDBP ont été identifiées chez des patients SLA. Les défauts biochimiques de ces mutations étant encore inconnus, les modèles animaux sont présentement la seule mesure possible d’un phénotype. Pour étudier les conséquences physiopathologiques d’une de ces mutations, nous avons développé deux lignées transgéniques de poisson zébré, exprimant le gène humain TARDBP soit de type sauvage, soit avec la mutation G348C liée à la SLA, sous le contrôle d’un promoteur de choc thermique. Ces lignées ont été étudiées sur trois générations, après avoir établi un protocole de choc thermique induisant une expression ubiquitaire du transgène. Les embryons transgéniques de la génération F2 de la lignée exprimant la mutation développent un phénotype moteur suite à un choc thermique de 38.5°C pendant 30 minutes lorsque les embryons sont à 18 heures post-fertilisation. 60% des embryons ont une réponse anormale au toucher. De plus, une réduction de 28% de la longueur de pré-branchement des axones des motoneurones est observée. Ces résultats indiquent que notre lignée exprimant la protéine mutante TDP-43 est un modèle génétique de la SLA prometteur, qui ouvre des perspectives pour la compréhension de la physiopathologie de la maladie et la découverte de molécules thérapeutiques. / Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease due to motoneurons degeneration. More than 40 mutations of the gene TARDBP, coding for the protein TDP-43 have been found in ALS patients. As the biochemical defects of these mutations are not known, in vivo models are currently the only windows onto the pathology. To study the pathophysiological consequences of one of these mutations, we have generated two stable zebrafish transgenic lines, expressing the human gene TARDBP, either the wild-type version, or the G348C mutated version linked to ALS, under the control of a heat shock promotor. These lines were studied for three generation, after establishing a heat shock protocol sufficient to induce a ubiquitous expression of the transgene. The transgenic embryos of the F2 generation of the line expressing the mutant protein develop a motor phenotype after a 38.5°C heat shock for 30 minutes when the embryos are 18 hours post-fertilization. 60% of these embryos have an abnormal touch escaped evoked response, and a 28% reduction of the pre-branching axonal length of the motoneurons axons. These results indicate that our line expressing the mutant TDP-43 protein is a promising genetic model of ALS, opening perspectives for the pathophysiological understanding of the disease, and the discovery of new therapeutics.

Proteins are fundamental structural, enzymatic and regulatory components of cells. Analysis of proteins, such as by measuring their concentrations, characterizing their modifications, and detecting their interactions, provides insights in how biological systems work physiologically or pathologically at the molecular level. To perform such analysis, molecular tools with good sensitivity, specificity, high multiplexing and throughput capacity are needed. In this thesis, four different assays were developed and applied to detect and profile proteins and protein complexes in human body fluids, and in cells or tissues. These assays are based on targeting proteins or protein complexes by oligonucleotide-conjugated antibodies, and subsequent proximity dependent enzymatic reactions involving the attached DNA reporter sequences. In paper I, a solid-phase proximity ligation assay (SP-PLA) was applied to detect synthetic and endogenous amyloid beta protofibrils. The SP-PLA provided better sensitivity and increased dynamic range than a traditional enzyme-linked immunosorbent assay (ELISA). In paper II, in situ PLA was applied to investigate the correlation between MARK2-dependent phosphorylation of tau and Alzheimer’s disease. Greater numbers of MARK2-tau interactions and of phosphorylated tau proteins were observed in brain tissues from Alzheimer’s patients than in healthy controls. In paper III, a multiplex SP-PLA was applied to identify protein biomarker candidates in amyotrophic lateral sclerosis (ALS) disease and in the analgesic mechanism of spinal cord stimulation (SCS). Among 47 proteins in human cerebrospinal fluid (CSF) samples, four were found at significantly lower concentrations (p-values < 0.001) in the samples from ALS patients compared to those from healthy controls (follistatin, IL-1α, IL-1β, and KLK5). No significant changes of the analyzed proteins were found in the CSF samples of neuropathic pain patients in   the stimulated vs. non-stimulated condition using SCS. In paper IV, a new technology termed the proximity barcoding assay (PBA) was developed to profile individual protein complexes. The performance of PBA was demonstrated on artificially assembled streptavidin-biotin oligonucleotide complexes. PBA was also proven to be capable of profiling transcriptional pre-initiation complexes from nuclear extract of a hepatic cell line.

To identify the risk factors being associated with aetiology of amyotrophic lateral sclerosis (ALS), a series of systematic reviews based on existing observational epidemiological studies identified through searching of bibliographic databases were conducted. Associations between ALS and a number of genetic and environmental risk factors were examined using meta-analysis. Specifically we found that previous exposure to lead, pesticides, solvents, experience of trauma and electric shock were associated with relative increased risks of developing ALS of 86% [odds ratio (OR) =1.86, 95% CI: 1.39-2.48], 57% (OR=1.57,95% CI: 1.19-2.08), 47% (OR=1.47, 95%CI: 1.13-1.80), 64% (OR=1.64; 95%CI: 1.36-1.98), and 2.27% (OR=3.27, 95%CI:1.87-5.73) respectively, compared to their corresponding controls. The presence of intermediate CAG repeat expansion in the ATXN2 gene was associated with a 4.4 -fold increase in the risk of ALS (OR=4.44, 95%CI: 2.91-6.76). However, the attributable risk associated with each identified risk factor was estimated to be less than 5% of all ALS cases. These results confirm that ALS is a rare multifactorial degenerative condition of motor-neurons.

While great strides have been made in treating many classes of human disease, the late-onset neurodegenerative diseases continue to elude modern medicine. These diseases, which include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), the transmissible spongiform encephalopathies (TSEs), and amyotrophic lateral sclerosis (ALS), involve accumulation of insoluble aggregates of one or more causative proteins, leading to progressive loss of central nervous system neurons, progressively worsening neurological symptoms, and eventual patient death. All of these diseases are currently incurable and fatal. In the case of ALS, progressive death of upper and lower motor neurons leads to full-body paralysis, respiratory difficulty, and patient death. Of the subset of ALS cases showing familial inheritance, approximately 20% are caused by mutations in the SOD1 gene, encoding the Cu, Zn superoxide dismutase (SOD1). These mutations do not have the common property of impairing SOD1's normal function as a free radical scavenger. Instead, they are thought to increase the protein's likelihood of misfolding and aggregating via a poorly-understood aggregation cascade. It is believed that species populated along the misfolding and aggregation pathway may prove to be good targets for therapies designed to block accumulation of downstream toxic species, or to prevent aberrant protein-protein interactions responsible for neurotoxicity. In this thesis, several new techniques are developed to enable detailed elucidation of the SOD1 unfolding and misfolding pathways. Time-resolved measurements collected during SOD1 unfolding or misfolding of release of bound Cu and Zn, of changes in intrinsic fluorescence, of exposure of hydrophobic surface area, and of alterations in the chemical environment of histidine residues, are presented. A new mathematical analysis technique named the Analytical Laplace Inversion Algorithm is developed for rapid extraction of mechanistic information from these time-resolved signals. These tools are applied to the construction of the most detailed models to date of the unfolding and misfolding mechanisms of WT and ALS-causing mutant SOD1. The models presented identify several well-populated unfolding and misfolding intermediates that could serve as good targets for therapies designed to address the fundamental molecular mechanisms underlying SOD1-associated ALS, and to treat what is currently a devastating and incurable disease.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease which currently lacks rapid and definitive diagnostic tests. Recently identified neuron specific splice variant molecules, Per28 and NFL-60, have been shown to contain unique epitopes and to have altered levels of expression in ALS patients. It is believed that these factors make Per28 and NFL-60 excellent candidate biomarkers for the ALS disease state. In this study, we attempted to develop ELISA assays directed against Per28 and NFL-60, as well as a generalized guideline for splice variant ELISA development, which could be used in a clinical setting. Limitations in currently identified antibodies to the splice variants allowed only for the completion of a Per28 ELISA, which lacked the sensitivity for clinical relevance. This assay creation process, however, did produce a guideline for similar ELISA development, which should allow for the more expeditious creation future ELISA.

Made available in DSpace on 2015-12-06T23:02:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2003 / A Doenca do Neuronio Motor (DNM) e uma doenca degenerativa, progressiva, irreversivel e incuravel, com envolvimento primario do motoneuronio. Dentre as formas de manifestacao, predominam a Esclerose Lateral Amiotrofica (ELA) e a Paralisia Bulbar Progressiva (PBP). Na evolucao clinica, a disfagia apresenta-se como um dos comprometimentos principais, levando a diminuicao da massa corporea, desidratacao e desnutricao. Associado a diminuicao da inGestão alimentar, observa-se o aumento da taxa de metabolismo basal e das necessidades nutricionais, determinando assim perda de peso. O reconhecimento da deplecao nutricional e intervencao precoce podem ser um diferencial no tratamento. Nesse trabalho foram estudados 20 pacientes com Doenca do Neuronio Motor (DNM)/Esclerose Lateral Amiotrofica (ELA), provenientes do Setor de Investigacao de Doencas Neuromusculares da Disciplina de Neurologia Clinica da Universidade Federal de São Paulo - Escola Paulista de Medicina (UNIFESP-EPM). O objetivo desse estudo foi descrever o estado nutricional dos pacientes com Doenca do Neuronio Motor (DNM) /Esclerose Lateral Amiotrofica (ELA), submetidos a suplementacao nutricional. Os pacientes receberam suplementacao nutricional durante seis meses consecutivos. Eles foram submetidos a avaliacao nutricional bimestral, com medidas antropometricas - peso, estatura, circunferencia do braco e dobra cutanea do triceps - e analise da composicao corporal atraves da bioimpedancia eletrica. O diagnostico de ELA foi estabelecido de acordo com -os criterios propostos pelo El Escorial, modificado era 1998, atraves do quadro clinico e do auxilio de exames como eletroneuromiografia, ressonancia magnetica e tomografia computadorizada. As idade variou entre 36 e 70 anos, com media de 55,0, mediana de 59,5 e desvio padrao de 12,4 anos. O tempo referido de doenca pelo paciente variou de 8 a 218 meses, com media de 31,5 meses. Quinze pacientes (75 por cento) apresentavam ELA como forma de manifestacao, sendo onze (73,3 por cento) do sexo masculino e 4 (26,6 por cento) do sexo feminino...(au) / The Motor Neurone Disease (MND) is a degenerative disease with involvement of the motor neurone. It is a evolutionary, irreversible and incurable disease. Among the manifestation forms, prevail Amyotrophic Lateral Sclerosis (ALS) and Progressive Bulbar Paralysis (PBP). In the clinical evolution, dysphagia stands out as one of the main compromisings, inducing decrease of body mass, dehydration and malnutrition. Associated to the decrease of the food ingestion, is observed increase of the rate of basal metabolism and nutritional needs, and these determine weight loss. The early recognition of the nutritional depletion and early intervention can be a differential one in the treatment. We presented 20 patient with Motor Neuron Disease / Amyotrophic Lateral Sclerosis (ALS), coming from the Section of Neuromuscular Diseases Investigation of the Clinical Neurology Departament, Sao Paulo Federal University (UNIFESP). The objective of this study was to describe the nutritional condition of the patients with Motor Neurone Disease (MND)/ Amyotrophic Lateral Sclerosis (ALS). The patients received nutritional supplements by six consecutive months. They were submitted to bimonthly nutritional evaluation, with anthropometric measurements - weight, stature, upper arm circumference and triceps skinfold - and bioelectrical impedance analysis to evaluate corporal composition. The diagnosis of Amyotrophic Lateral Sclerosis (ALS) was established in agreement with the criteria proposed by the El Escorial, in 1998, including clinical criteria and the aid of electromyographic studies, MR and CT scan. (au) / BV UNIFESP: Teses e dissertações

Neurons connect to their remote targets via axons, which usually survive for the lifetime of an organism. Spatiotemporal regulation of the axonal proteome by local protein synthesis (LPS) plays a critical role in neuronal wiring and axon survival, raising the intriguing possibility that some neurological disorders involve LPS dysfunction. To visualise LPS in situ, I optimised multiple imaging techniques to investigate Netrin-1-induced translation in cultured retinal axons. Total axonal protein synthesis measured by metabolic and puromycin labelling indicates axons experience stage-dependent alterations in translation rate upon Netrin-1 stimulation. Remarkably, Netrin-1 triggers a burst of β-actin synthesis starting within 20 seconds of cue application at multiple non-repetitive sites visualised by single molecule translation imaging, an approach that allows direct visualisation of translation dynamics in response to external stimuli. Further studies have shown that local translation can occur on Rab7a-associated late endosomes, where mRNA recruitment and translation are coordinately regulated. Notably, mRNAs encoding mitochondria-related proteins are found translating on late endosomes docking in the vicinity of mitochondria, suggesting late endosomes act as ‘platforms’ for the localised synthesis of mitochondrial proteins necessary for maintaining mitochondrial integrity. Moreover, this process is affected in axons expressing the Charcot-Marie-Tooth disease type 2B (CMT2B)-related Rab7a mutants, leading to abnormal mitochondrial biogenesis and activity and compromised axon survival. Finally, attenuated de novo protein synthesis is observed in axons expressing amyotrophic lateral sclerosis (ALS)-associated fused in sarcoma (FUS) mutants and hypomethylated wild-type FUS. Live imaging reveals mislocalised mutant or hypomethylated FUS granules are transported along axons and accumulate at growth cones, possibly irreversibly trapping RNA molecules, resulting in reduced distance travelled by RNA granules in axons. Furthermore, mutant FUS expression results in defective retinal projections in vivo, highlighting the importance of RNA metabolism and local translation in axonal homeostatic mechanisms. In conclusion, aberrant translational activity in axons leads to prominent axonopathy, which recapitulates features of early stages of neurological diseases, providing the basis for novel therapeutic strategies.

Les facteurs de transcription de la famille p53 dans l’atrophie musculaire - Implications dans la Sclérose Latérale Amyotrophique et la cachéxie. L’atrophie musculaire est un symptôme dangereux retrouvé dans plusieurs maladies. Dans la sclérose latérale amyotrophique (SLA), une maladie neuromusculaire rare, ainsi que dans le cancer (phénomène de cachexie), l’atrophie musculaire cause le décès des patients. Les facteurs de transcription de la famille p53 sont impliqués dans de nombreux processus cellulaires, faisant face à des situations de « stress » pour les cellules. Notamment, ils peuvent induire la mort cellulaire ou promouvoir la différentiation.Nous avons constaté, à partir de modèles cellulaire et animaux de SLA et cachéxie cancéreuse, que des membres de la famille p53 sont activés dans les muscles atrophiques. Cette activation entraine l’expression de gènes cibles impliqués dans la mort cellulaire. De manière intéressante, TAp73, mais surtout TAp63, sont capables d’activer la transcription d’un effecteur de l’atrophie musculaire appelé MuRF1, démontrant que la famille p53 peut participer activement à l’atrophie en induisant la dégradation des fibres musculaires.De plus, nous avons utilisé nos modèles animaux pour identifier une nouvelle approche contre l’atrophie musculaire. Ainsi, nous avons identifié un dérivé de tocophérol avec des propriétés thérapeutiques intéressantes. / The p53 family of transcription factors in muscular atrophy - Involvements in Amyotrophic Lateral Sclerosis and cachexia Muscular atrophy is a dangerous condition found in several diseases. In amyotrophic lateral sclerosis (ALS), a rare neuromuscular disease, as well as in cancer (phenomenon of cachexia), muscular atrophy can be fatal to patients.The transcription factors from the p53 family are involved in several cellular processes, facing cellular “stress” situations. Most notably, they can induce dell death or promote differentiation.We found, using cellular and mouse models of ALS and cachexia, that members of the p53 family are induced during muscular atrophy. This induction leads to the expression of canonnic target genes involved in cell death. Interestingly, TAp73, but especially TAp63, are able to activate the transcription of an effector or muscular atrophy called MuRF1. This proves that the p53 family cand participate in muscular atrophy by promoting the breakdown of muscle fibres.In addition, we used our mouse models to identifiy a new approach agains muscular atrophy.Indeed, we identified a derivative of tocopherol with interesting therapeutical proprieties.

Application of Diffusion Tensor Imaging to Brain Gray and White Ma er A In the present work we explore the gray and white ma er applicability of diffusion tensor imaging (DTI). To evaluate effect of ferritin-bound iron on gray ma er contrast in DTI, we created an in vitro model consisting of agarose gel phantoms doped with ferritin, and validated our results in vivo on healthy volunteer subjects - years of age in the basal ganglia. We further explored the application of DTI to amyotrophic lateral sclerosis (ALS) and multiple system atrophy (MSA); neurodegenerative diseases with gray and white ma er pathophysiological components. In the ALS study, patients and age- and sex-matched controls were recruited, while the MSA study included probable MSA subjects ( MSA-P, MSA-C) and age- and sex-matched controls. We found that ferritin-bound iron may make a signi cant contribution to DTI scalars in gray ma er regions of the brain, mediated by eigenvalue repulsion. is has important implications for DTI studies targeting gray ma er regions, especially in adolescence and in diseases associated with altered brain-iron load. In ALS, we found altered diffusion in the corona radiata and callosal body, and changes in R in the caudate nucleus and frontal white ma er. In MSA, we observed widespread white ma er changes associated...