abstract: The Erk/MAPK pathway plays a major role in cell growth, differentiation, and survival. Genetic mutations that cause dysregulation in this pathway can result in the development of Rasopathies, a group of several different syndromes including Noonan Syndrome, Costello Syndrome, and Neurofibromatosis Type-1. Since these mutations are germline and affect all cell types it is hard to differentiate the role that Erk/MAPK plays in each cell type. Previous research has shown that individual cell types utilize the Erk/MAPK pathway in different ways. For example, the morphological development of lower motor neuron axonal projections is Erk/MAPK-independent during embryogenesis, while nociceptive neuron projections require Erk/MAPK to innervate epidermal targets. Here, we tested whether Erk/MAPK played a role in the postnatal development of lower motor neurons during crucial periods of activity-dependent circuit modifications. We have generated Cre-dependent conditional Erk/MAPK mutant mice that exhibit either loss or gain of Erk/MAPK signaling specifically in ChAT:Cre expressing lower motor neurons. Importantly, we found that Erk/MAPK is necessary for the development of neuromuscular junction morphology by the second postnatal week. In contrast, we were unable to detect a significant difference in lower motor neuron development in Erk/MAPK gain-of-function mice. The data suggests that Erk/MAPK plays an important role in postnatal lower motor neuron development by regulating the morphological maturation of the neuromuscular junction. / Dissertation/Thesis / Masters Thesis Biology 2017

No description available.

Au cours du processus neurodégénératif lié à la Sclérose Latérale Amyotrophique (SLA), les motoneurones innervant les muscles des membres sont très atteints tandis que ceux innervant les muscles axiaux sont relativement préservés. Les motoneurones diffèrent aussi dans leur réponse à plusieurs facteurs neurotrophiques (NTF). Je me suis intéressée aux mécanismes moléculaires déterminant la morphologie distincte et les réponses aux NTF de sous-populations de motoneurones pertinentes dans la SLA. J'ai démontré que les sous-populations de motoneurones murins innervant les muscles axiaux (MN-MMC) ou les muscles des membres (MN-LMC) ont de fortes différences morphologiques. Ces différences sont dues à une régulation différentielle de gènes codant pour la Péripherine et la Diacylglycérol kinase beta (DGK-β) qui sont régulés par les facteurs de transcription LIM-HD FoxP1/HB9. J'ai montré que les MN-LMC et les MN-MMC répondaient distinctement à trois facteurs neurotrophiques: le HGF (Hepatocyte Growth Factor), l'Artémine et le CNTF (Ciliary Neurotrophic Factor). J'ai étudié les motoneurones lombaires dans deux situations pathologiques: 1) les altérations de l'appareil de Golgi et du trafic axonal suite à la perte de TBCE (Tubulin Binding Cofactor E), 2) l'effet de l'augmentation de KCC2 à la membrane plasmique des motoneurones sur la transmission synaptique inhibitrice après lésion.Enfin, j'ai développé une nouvelle méthode de purification de motoneurones dérivés d'iPS (induced Pluripotent Stem Cell) humains en utilisant un vecteur rapporteur lentiviral Hb9::GFP et un anticorps dirigé contre le récepteur de faible affinité des neurotrophines, p75. / Muscles are highly vulnerable whereas motor neurons innervating axial muscles are relatively resistant. Motor neurons also seem to differ in their response to several neurotrophic factors (NTF). I investigated the molecular mechanisms determining the distinct morphology and the differential NTF response of ALS-relevant motor neuron subsets. First, I demonstrated that mouse lumbar motor neurons innervating either axial muscles (MMC-MN) or hindlimb muscles (LMC-MN) display remarkable morphological differences. These differences involve a differential regulation of genes coding for Peripherin and Diacylglycerol kinase-b (DGK-β) which are regulated by the transcription factors FoxP1/HB9. Second, I showed that LMC-MN and MMC-MN respond differentially to the three neurotrophic factors HGF (Hepatocyte Growth Factor), Artemin and CNTF (Ciliary NeuroTrophic Factor). Their differential survival is explained by the corresponding receptor gene expression in specific pools of MMC-MN and LMC-MN. Third, I studied lumbar motor neurons in two pathological conditions: 1) alteration of Golgi apparatus and axonal trafficking induced by loss of TBCE (Tubulin Binding Cofactor E) 2) the effect of KCC2 increase at motor neuron plasma membrane on inhibitory synaptic transmission after trauma.Finally, I developed a new FACS-based method for isolating human iPS (induced Pluripotent Stem Cell)-derived motor neurons with both an HB9::GFP reporter lentivirus and an antibody directed against the low-affinity neurotrophin receptor p75.

L’amyotrophie spinale infantile (SMA) est une maladie neurodégénérative rare, caractérisée par une perte progressive des motoneurones de la moelle épinière, et pour laquelle aucun traitement curatif n’est disponible. Cette maladie est causée par la mutation du gène SMN1 qui induit une diminution de l’expression de la protéine SMN. Depuis plusieurs des années, notre l’équipe examine les effets de l’exercice sur le développement ou le maintien de l’unité motrice dans des maladies neurodégénératives affectant spécifiquement le motoneurone. Ces études ont notamment permis de mettre en évidence que l’exercice physique pourrait avoir des effets bénéfiques pour l’amyotrophie spinale, dans un modèle de souris SMA de type 2 soumis à un exercice de course sur roue pendant 5 jours (Grondard et al., 2005). Dans notre étude, nous avons comparé les effets de deux programmes d’entraînement différents, d’une durée de 10 mois, basés sur un exercice de course ou sur un exercice de nage, sur des populations de souris SMA de type 3, la forme la moins sévère de la maladie. Dans nos conditions, la course est un exercice de faible intensité et de faible amplitude, mais qui induit plus de lésions musculaires, au contraire de la nage, comme le confirme les mesures de lactate et de créatine kinase circulants. Ces deux paramètres ont des valeurs anormalement hautes chez les souris SMA, suggérant des anomalies métaboliques et de fragilité musculaire, qui sont limitées par les deux programmes d’entraînement. Les analyses du comportement moteur indiquent également que les 10 mois d’entraînement améliorent significativement les capacités motrices des souris SMA, et notamment la résistance à la fatigue avec la nage. Comme attendu, la perte de 46% des motoneurones spinaux enregistrée à 12 mois chez les souris SMA sédentaires est significativement limitée par les deux types d’entrainement, mais avec des efficacités différentes sur les différentes sous‐populations de motoneurones spinaux. En effet, la course protège préférentiellement les motoneurones de faible surface et exprimant ERR‐β, assimilés à des motoneurones lents, et la nage les motoneurones de large surface et exprimant Chodl, assimilés à des motoneurones rapides. De manière surprenante, la neuroprotection induite par l’exercice est indépendante de l’expression de SMN dans la moelle épinière des souris SMA. Une étude de la forme et de la surface des jonctions neuromusculaires dans trois muscles du mollet, le soleus, le plantaris et le tibialis, et une étude du phénotype musculaire de ces mêmes trois muscles confirment le rôle bénéfique de l’entrainement mais aussi les effets différentiels des deux programmes, avec un effet plus important pour la nage. Les améliorations de l’unité motrice, induites par l’exercice, permettent un meilleur fonctionnement neuromusculaire, comme le suggère les mesures électrophysiologiques du muscle plantaire. Pris tous ensemble, ces résultats suggèrent qu’un exercice de nage, à haute intensité, dans des conditions anaérobies, et axé sur le recrutement des muscles extenseurs pourrait être bénéfique pour les patients SMA, notamment pour améliorer les capacités motrices et donc la qualité de vie des patients. / Objective: Spinal Muscular Atrophy (SMA) is a group of autosomal recessive neurodegenerative diseases differing in their clinical outcome, characterized by the specific loss of spinal motor‐neurons, caused by insufficient levels of SMN protein expression. No cure is presently available for SMA. While physical exercise might represent a promising approach for alleviating SMA symptoms, the lack of data dealing with the effects of different exercise types on diseased motor‐units still precludes the use of exercise in SMA patients. Methods: We have evaluated the efficiency of two long‐term physical exercise paradigms, either based on high intensity swimming or on low intensity running, in alleviating SMA symptoms in a mild type 3 SMA‐like mouse model. Results: We found that a 10‐month physical training induced significant benefits in terms of resistance to muscle damages, energetic metabolism, muscle fatigue and motor behavior. Both exercise types significantly enhanced motor‐neuron survival, independently of SMN expression, leading to the maintenance of neuromuscular junctions and skeletal muscle phenotypes, particularly in the soleus, plantaris and tibialis of trained mice. Most importantly, both exercises significantly improved neuromuscular excitability properties. Besides, all these training‐induced benefits are quantitatively and qualitatively related to the specific characteristics of each exercise, suggesting that the related neuroprotection is strongly dependent on the specific activation of some motor‐neuron subpopulations. Interpretation: Taken together, the present data show significant long‐term exercise benefits in a mild type 3 SMA context and provide important clues for designing rehabilitation programs in patients.

Spinal muscular atrophy (SMA) is a genetic disorder characterized by muscle weakness and atrophy and death of motor neurons in humans. Although almost all cases of SMA occur due to mutations in a gene called survival motor neuron 1 (SMN1), SMA patients present with a wide range of severities of the symptoms. The most severe cases never achieve any developmental motor milestone and die within a few years after birth. On the other hand, mild cases of SMA have a normal life span and show trivial motor deficits. This suggests the role of other factors (rather than the function of SMN1) in the outcome of the disease. Indeed, the copy number of an almost identical gene, called SMN2, is the main determining factor for the severity of SMA. In addition, a few other genes (e.g. Plastin 3) are proposed as disease modifiers in SMA. SMN1 is a housekeeping gene, but due to unknown reasons, the most prominent pathologies in SMA are atrophy of myofibers and death of motor neurons. However, recent studies showed that some other cell types are also affected in the course of SMA disease. We investigated the alterations of central synapses in Smn2B/- mice, a model of SMA. We did not observe any degeneration of central synapses in these mice until a post symptomatic stage. However, mass spectrometry (MS) analysis on isolated synaptosomes from spinal cords of these animals revealed widespread alterations in the proteome of their central synapses at a presymptomatic stage. Functional cluster analysis on MS results suggested that several molecular pathways are affected within synapses of spinal cords of Smn2B/- mice prior to the onset of any obvious pathology in their motor units. The affected molecular pathways are involved in basic cell biological functions including energy production, protein synthesis, cytoskeleton regulation and intracellular trafficking. We showed that the levels of several proteins involved in actin cytoskeleton regulation are altered in synaptosomes isolated from spinal cords of Smn2B/- mice. More investigations are required to determine the exact functional abnormalities of affected pathways in central synapses of these mice. We also generated congenic Smn2B/- mice in two different mouse genetic backgrounds; FVB and BL6. Using a systematic approach, we showed that congenic Smn2B/- mice in the FVB background show a more severe SMA phenotype than Smn2B/- mice in a BL6 background. Smn2B/- mice in the FVB background had a shorter survival, higher rate of weight loss, earlier and more severe pathologic changes compared to Smn2B/- mice in the BL6 background. We investigated the levels of several actin binding proteins in spinal cords of these animals and found higher induction of plastin 3 in Smn2B/- mice in the BL6 background. More investigations are underway to determine the role of plastin 3 in the severity of the phenotype of Smn2B/- mice, and to find other possible SMA modifier genes in these animals.

This study demonstrates the direct delivery of plasmid gene constructs into spinal motor neurons utilizing retrograde axoplasmic transport. The plasmid vectors contained the Lac Z gene under the control of both the Rous sarcoma virus (RSV) and Simian virus (SV)40 promoters. β-Galactosidase expression was observed in α and γ motor neurons by histochemical staining following direct injection into the sciatic nerve or gastrocnemius muscle. The presence of LacZ gene constructs was confirmed by the polymerase chain reaction (PCR). The ability to introduce gene constructs into motor neurons allows for the study of gene regulation and permits the development of gene therapy strategies for motor neuron diseases including the spinal muscular atrophies (SMA) and amyotrophic lateral sclerosis (ALS).

No description available.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by reduced levels of the survival motor neuron (SMN) protein. SMA results in degeneration of motor neurons, progressive muscle atrophy, and death in severe forms of the disease. Currently, there is a lack of inexpensive, readily accessible, accurate biomarkers to study the disease. Furthermore, the current FDA approved therapeutic is neither 100 % effective nor accessible for all patients, thus more research is required. Tiny cell derived vesicles known as exosomes have been evaluated in an attempt to identify novel biomarkers for many disease states and have also shown therapeutic promise through their ability to deliver protein and nucleic acid to recipient cells. The research presented herein investigates whether (1) the level of SMN protein in exosomes isolated from the medium of cells, and serum from animal models and patients of SMA is indicative of disease, to serve as a biomarker for monitoring disease progression and therapeutic efficacy; (2) SMN-protein loaded exosomes can be utilized to deliver SMN protein to SMN-deficient cells; (3) adenoviral vectors are effective at creating SMN protein-loaded exosomes in situ for body wide distribution of SMN protein. This research has shown SMN protein is naturally released in extracellular vesicles, and the level of exosomal SMN protein is reflective of the disease state. Exosomes can also be modified to hold enhanced levels of SMN protein and deliver them to both the cytoplasm and nucleus of SMN-deficient cells. Furthermore, adenoviral vectors expressing luciferase-tagged SMN1 cDNA, targeted to the liver, results in SMN protein-loaded exosomes and detectable luciferase activity, body-wide. Thus, exosomes present as an effective biomarker and potentially a novel approach to treat SMA.

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder, which causes dysfunction/loss of lower motor neurons and muscle weakness as well as atrophy. While SMA is primarily considered as a motor neuron disease, recent data suggests that survival motor neuron (SMN) deficiency in muscle causes intrinsic defects. We systematically profiled secreted proteins from control and SMN deficient muscle cells with two combined metabolic labeling methods and mass spectrometry. From the screening, we found lower levels of C1q/TNF-related protein 3 (CTRP3) in the SMA muscle secretome and confirmed that CTRP3 levels are indeed reduced in muscle tissues and serum of an SMA mouse model. We identified that CTRP3 regulates neuronal protein synthesis including SMN via mTOR pathway. Furthermore, CTRP3 enhances axonal outgrowth and protein synthesis rate, which are well-known impaired processes in SMA motor neurons. Our data revealed a new molecular mechanism by which muscles regulate the physiology of motor neurons via secreted molecules. Dysregulation of this mechanism contributes to the pathophysiology of SMA.

Esclerose lateral amiotrófica (ELA) é uma doença neurológica progressiva e fatal, caracterizada por perda dos neurônios motores, levando à fraqueza muscular global. As funções sensitivas e mentais são preservadas durante todo o curso da doença. A esclerose lateral amiotrófica tem uma prevalência de 6 por 100.000 pessoas e o início da doença é geralmente entre os 40 a 60 anos de idade. O prognóstico é reservado e em média os pacientes vivem de 3 a 5 anos após o diagnóstico médico. Com a progressão da doença outros sintomas surgem como fraqueza dos membros, comprometimento da fala, aumento da salivação, dificuldades de deglutição, dificuldades para deambular e fadiga muscular. As alterações dos músculos respiratórios levam à falência respiratória, que é a maior causa de óbito nos pacientes com esclerose lateral amiotrófica. Fadiga é definida como a queda da máxima contração isométrica voluntária e falta de tolerância do músculo sob esforço. A máxima contração isométrica voluntária depende de uma cadeia de eventos que se inicia no córtex motor - condutor excitatório dos neurônios motores superior e inferior, e se continua na transmissão pela junção neuromuscular, no acoplamento excitação-contração e na contração da fibra muscular que depende de um suprimento energético metabólico. A fadiga muscular ocorre em pacientes com esclerose lateral amiotrófica prejudicando a função e a qualidade de vida dos pacientes. O objetivo deste trabalho foi: 1) Quantificar a freqüência da fadiga na esclerose lateral amiotrófica; 2) Analisar a evolução da fadiga nos pacientes; 3) Correlacionar a presença da fadiga com fatores como a funcionalidade, a qualidade de vida, a depressão, a dispnéia, e a sonolência, idade e duração da doença em meses. O grupo controle compôs-se de 60 indivíduos (familiares de funcionários do hospital e da equipe multidisciplinar) que não apresentavam história de doenças pregressas. O grupo teste constitui-se de 60 pacientes com diagnóstico de esclerose lateral amiotrófica. O diagnóstico foi realizado por dois neurologistas independentes e baseou-se na presença de história clínica, exame neurológico e estudos neurofisiológicos compatíveis com esclerose lateral amiotrófica, segundo os critérios de El Escorial da Federação Mundial de Neurologia; além disso, houve a investigação complementar por meio de testes hematológicos, bioquímicos, sorológicos, genéticos e radiológicos para excluir outras patologias. Os pacientes dos grupos controle e teste foram entrevistados pela pesquisadora para aplicação de questionários com escalas para verificação da presença de funcionalidade, de qualidade de vida, de depressão, de dispnéia, de sonolência e de fadiga; e os pacientes do grupo teste foram submetidos à avaliação fisioterapêutica no início do estudo e a cada 3 meses, totalizando 12 meses de coletas. O grupo teste apresentou fadiga significantemente maior em relação ao grupo controle, bem como alterações nos questionários de funcionalidade, de qualidade de vida, de depressão, de dispnéia e de sonolência. Percebeu-se que a fadiga foi evolutiva durante os meses de acompanhamento da pesquisa. A fadiga correlacionou-se com a idade, mostrando que os pacientes mais jovens apresentaram maior grau de fadiga que os pacientes mais idosos. Os resultados desta pesquisa sugerem que a fadiga é um dos problemas que afetam os pacientes com ELA; o fato de não ter correlação com outros problemas estudados sugere que a fadiga deve merecer pesquisa e tratamento individualizados no paciente com ELA, principalmente pelo fato de que os resultados sugeriram piora da fadiga no decorrer da evolução da ELA / Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurological disease, characterized by loss of the motor neurons, taking to the global muscular weakness. The sensitive and mental functions are preserved during whole the course of the disease. The ALS has a prevalence of 6 for 100.000 people and the beginning of the disease is usually among the 40 to 60 years of age. The prognostic is reserved and on average the patients live from 3 to 5 years after the medical diagnosis. With the progression of the disease other symptoms they appear as weakness of the members, compromising of the speech, increase of the salivation, deglutition difficulties, difficulties to stroll and it fatigues muscular. The alterations of the breathing muscles take to the breathing bankruptcy, that is the largest death cause in the patients with ALS. Fatigue is defined as the fall of the maxim voluntary isometric contraction and lack of tolerance of the muscle under effort. The maxim voluntary isometric contraction depends on a chain of events that begins in the motor cortex - driver excitatory of the neurons motor superior and inferior, and it is continued in the transmission by the junction neuromuscular, in the joining excitement-contraction and in the contraction of the muscular fiber that depends on a metabolic energy supply. The muscular fatigue happens in patients with ALS harming the function and the quality of the patients\' life. The objective of this work was: 1) to quantify the frequency of the fatigue in the ALS; 2) to analyze the evolution of the fatigue in the patients; 3) to correlate the presence of the fatigue with factors as the functionality, the life quality, the depression, the dispnéa, and the sleepiness, age and duration of the disease in months. The group control was composed of 60 individuals (family of employees of the hospital and of the team multidisciplinar) that didn\'t present history of past diseases. The group test is constituted of 60 patients with diagnosis of ALS. The diagnosis was accomplished by two independent neurologists and he/she based on the presence of clinical history, neurological exam and studies compatible neurophisiologycs with ALS, according to the criteria of El Escorial of the World Federation of Neurology; besides, there was the complement investigation through tests and exams to exclude other pathologies. The patients of the groups control and test were interviewed by the researcher for application of questionnaires with scales for verification of the functionality presence, of life quality, of depression, of dispnéa, of sleepiness and of fatigue; and the patients of the group test they were submitted to the evaluation physiotherapy in the beginning of the study and every 3 months, totaling 12 months of collections. The group test presented fatigue larger significantly in relation to the group it controls, as well as alterations in the functionality questionnaires, of life quality, of depression, of dispnéa and of sleepiness. It was noticed that the fatigue was evolutionary during the months of accompaniment of the research. The fatigue was correlated with the age, showing that the youngest patients presented larger degree of fatigue than the most senior patients. The results of this research suggest that the fatigue is one of the problems that affect the patients with ALS; the fact of not having correlation with other studied problems suggests that the fatigue should deserve research and treatment individualized in the patient with ALS, mainly for the fact that the results suggested worsening of the fatigue in elapsing of the evolution of the ALS