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Spinal muscular atrophy (SMA) is a devastating disorder caused by deletions and mutations in the survival of motor neuron (SMN1) gene and is marked by motor neuron loss and muscle weakness. While its genetic basis is clear, the underlying molecular mechanisms remain elusive. Decreased levels of the survival of motor neuron (SMN) protein, encoded by the SMN1 gene, are implicated in SMA pathology. Despite splicing has been under the spotlight as a major mechanism impaired in SMA, recent evidence suggests that SMN deficiency also disrupts protein translation in vivo in a mouse model of severe SMA, complicating SMA's molecular landscape. This thesis examines the impact of SMN protein loss on translation in SMA mouse models across tissues, post-natal and pre-natal disease stages, focusing on both mild (Chapter 1) and severe forms of SMA (Chapter 2) respectively. To tackle this question, in this thesis, I took advantage of multiple cutting-edge and sequencing-based techniques (ribosome profiling and RNA-seq) coupled with biochemical and molecular biology-based assay (polysome profiling, co-sedimentation profiles, qPCR, and western blotting), which applied to study in molecular detail the translational defects in the brain, spinal cord and liver at asymptomatic, pre-symptomatic and early symptomatic stages of SMA. Polysome profiling in control mice (Smn2B/+) reveals a gradual increase in SMN association with ribosomes/polysomes during postnatal development, indicating dynamic SMN function in protein translation during post-natal development. In SMA condition, where SMN protein levels drop, this binding reveals a tissue-specific decrease in the spinal cord and liver. Through ribosome profiling, numerous alterations in translation were identified at the pre-symptomatic stage of the disease, suggesting that translational defects are features of the early stages of SMA. Importantly these alterations are independent of transcriptional and splicing changes. Although no gene was found to be in common, I found that genes altered in at least 2 tissues are involved in the same processes. The dysregulated mRNAs exhibit rare codons at the beginning of coding sequences in all three tissues, as shown in the case of the severe model of SMA. 4 From these common processes I have identified specific mRNA targets that play key roles in the organization of the extracellular matrix I validated the presence of translational changes in Col1a1, Col1a2, and Spp1 highlighted effects of SMN deficiency on translational regulation, in the absence of transcriptional alterations. Validation studies in both mice and SMA patient-derived fibroblasts further underscored the potential of translational dysregulation and drop in Col1a1 protein expression during SMA progression. Finally, prenatal studies have revealed distinct translational changes in embryonic tissues from Taiwanese mice. Despite no alterations in global translation, a drop in SMN association with ribosomes/polysomes and tissue-specific differences in ribosome occupancy were observed. Also, in this case, dysregulated mRNAs exhibit rare codons at the beginning of coding sequences. These findings shed light on the unique molecular landscape of prenatal development in the context of SMN deficiency. In summary, this study provides insights into translation dysregulation in SMA pathology, emphasizing tissue-specific effects and developmental stage-dependent alterations. By elucidating the complex relationship between SMN protein function and translational dynamics, it lays the groundwork for targeted therapeutic strategies and biomarkers to improve SMA management. Ongoing investigations into prenatal development and translation dynamics are crucial for a comprehensive understanding of SMA pathogenesis and effective treatment development.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: Lorson, Christian L. "December 2008" Includes bibliographical references

Les protéines SREBP-1, Sterol Response Element Binding Proteins, sont des régulateurs clés du métabolisme des lipides et du cholestérol. A ce titre, ils ont été largement étudiés dans le foie et le tissu adipeux. Les facteurs SREBP-1 sont également exprimés dans le muscle squelettique au sein duquel ils sont les principaux médiateurs des effets géniques de l’insuline.Les travaux de thèse présentés dans ce manuscrit ont eu pour but de définir le rôle spécifique de SREBP-1 dans le muscle squelettique. L’étude transcriptomique de cellules musculaires humaines révèle plus de1500 gènes régulés par SREBP-1 dans le muscle squelettique humain, dont la moitié est réprimée. L’analyse fonctionnelle de ces gènes révèle l’implication de SREBP-1 dans des fonctions musculaires dépassant la cadre du métabolisme glucido-lipidique. Ainsi, SREBP-1 inhibe l’expression de plusieurs gènes impliqués dans la différenciation et le maintien du phénotype musculaire. En conséquence, la sur expression de SREBP-1 bloque la différenciation myogénique in vitro et induit une atrophie marquée in vitro, sur des myotubes différenciés et in Vivo, dans le muscle squelettique de souris.En parallèle, deux répresseurs transcriptionnels : BHLHB2 et BHLHB3 apparaissent, après étude de leur promoteur, comme deux nouvelles cibles directes de SREBP-1. Ainsi, 20% des gènes inhibés par SREBP-1sont des cibles de BHLHB2 et BHLHB3, de nombreux gènes muscle-spécifiques y compris. De plus, BHLHB2 apparaît, de la même façon que SREBP-1, comme un acteur essentiel dans l’action de l’insuline sur le muscle squelettique, et dans le développement de l’insulino-résistance musculaire chez les patients diabétiques de type2.Le blocage de la différenciation myogénique et l’atrophie induite par SREBP-1 in vitro étant reversées par l’inhibition de l’expression de BHLHB2 et BHLHB3, nous concluons que BHLHB2 et BHLHB3 sont responsables de l’effet répressif de SREBP-1 sur le phénotype musculaire.Ces résultats mettent donc en évidence un nouveau rôle pour les facteurs SREBP-1 dans la régulation de la myogenèse et le maintien de la masse musculaire. SREBP-1 intègrent ainsi la régulation métabolique au contrôle du phénotype musculaire / Transcription factors SREBP-1, Sterol Response Element Binding Proteins, are key regulators of lipid and cholesterol homeostasis. Their function has been largely studied in liver and adipose tissue, but they are also well expressed in skeletal muscle where they mediate insulin transcriptional effects.This work aims to define the muscle specific role of SREBP-1. Microarray analysis of human myotubes over-expressing SREBP-1 identifies more than 1500 SREBP-1 target genes in human skeletal muscle, including number of repressed genes. Gene ontology analysis reveals the involvement of SREBP-1 in a large variety of biological functions in muscle cells. In fact, SREBP-1 represses expression of a number of muscle-specific genes and markers of muscle differentiation. As a result, SREBP-1 over-expression leads to blockage of in vitro myogenic differentiation and marked atrophy in vitro as in Vivo.In the same time, we identified the transcriptional repressors BHLHB2 and BHLHB3 as new direct target genes of SREBP-1, by promoter analysis. 20% of SREBP-1 repressed genes are also target genes of BHLHB2 and BHLHB3. Furthermore, BHLHB2, like SREBP-1, is involved in insulin action on skeletal muscle and muscular insulin-resistance in type 2 diabetic patients.As SREBP-1 effects on atrophy and myogenic differentiation inhibition are reversed by silencing BHLHB2 and BHLHB3 expression, we can conclude that BHLHB2 and BHLHB3 mediate negative SREBP-1action on muscular phenotype.These results confer a new role for SREBP-1 in the regulation of muscle mass and muscle cell differentiation, thus linking the control of muscle mass to metabolic pathways

Bibliographic identification HAVLIŠTOVÁ, Michaela. Respiratory physiotherapy affects the quality of life of children with SMA - how, when and why? Prague: Charles University, 2nd Faculty of Medicine, Department of rehabilitation and sport medicine, 2012. 81 p. Supervisor Doc. PaedDr. Libuše Smolíková, Ph. D. Annotation This thesis deals with the influence respiratory function in children with spinal muscular atrophy (SMA). The theoretical part provides an overview of respiratory physiotherapy techniques that can be used in the care of the airways in people with SMA. The practical part deals with the question whether it is possible using the selected techniques of respiratory physiotherapy after six weeks of training to affect ventilatory parameters in children with SMA. The group of six probands with SMA I. - III. type in the range of the age from 3.5 to 12 years participated in this study. To objectively assess changes was performed spirometry efore the beginning of the therapy and after its conclusion. The main therapy was daily training with inspiratory breath simulator CliniFlo. After the finishing of therapy there was a positive change in all measured parameters except for vital capacity (VC) and maximal expiratory flow at 75% of FVC (MEF75), where the values didn't change. Statistically significant...

A atrofia muscular espinhal (AME) é uma doença rara e neurodegenerativa, que afeta crianças e adultos. Gera atrofia muscular, prejudica os movimentos voluntários e, em muitos casos, dificulta a comunicação oral. Esta dissertação é composta por dois artigos. O Estudo 1 objetivou revisar a literatura sobre o desenvolvimento cognitivo de pessoas com AME. O Estudo 2 objetivou avaliar o desempenho visual, cognitivo e motor de crianças com AME tipo 1 (AME-I). O primeiro estudo foi uma revisão sistemática, com levantamento dos últimos 25 anos (de 1992 a 2017) nas bases de dados PUBMED/Medline, Web of Science e Scielo. Os descritores utilizados foram: spinal muscular atrophy e cognition. O estudo 2 consistiu na avaliação de 24 crianças, divididas em dois grupos: 12 crianças com AME-I (3 a 9 anos, 9 meninos e 3 meninas) e 12 crianças saudáveis, pareadas por idade e sexo. As 24 crianças responderam a quatro tarefas de associação de figuras, com dificuldade crescente. As respostas foram detectadas por meio de um rastreador de olhar. Para o primeiro estudo foram localizados 43 artigos, desses, nove foram selecionados, de acordo com os critérios de inclusão e exclusão. O estudo apontou que o desempenho cognitivo de crianças com AME é heterogêneo. Três artigos indicaram que essas crianças têm desempenho cognitivo normal, três artigos indicaram que há atraso e outros três estudos relataram desempenho acima da média. De modo geral, as crianças com maior fraqueza muscular (AME-I) apresentaram maior comprometimento e estudos mais recentes detectaram esse atraso. O segundo estudo descreveu pior desempenho do grupo AME-I, em relação ao grupo controle (maior número de erros e maior tempo para execução das tarefas). Concluímos que, até o momento, poucos estudos investigaram aspectos cognitivos em crianças e adolescentes com AME. O desenvolvimento cognitivo de crianças com AME deve ser acompanhado, principalmente em crianças com AME-I. É necessário avaliar o desempenho cognitivo e, se necessário, propor intervenções para pessoas com AME. O desenvolvimento cognitivo adequado facilita a autonomia e interação. Os rastreadores de olhar podem estimular e aprimorar esse repertório / Spinal muscular atrophy (SMA) is a rare and neurodegenerative disease, affecting children and adults. SMA causes muscular atrophy and weakness, and, in many cases, impairs the development of oral communication. This dissertation is composed of two articles. Study 1 aimed to review the literature about the cognitive development of people with SMA. Study 2 aimed to evaluate the visual, cognitive and motor performance of children with SMA type 1 (SMAI). Study 1 reviewed the findings of the last 25 years (1992 to 2017), in PUBMED/Medline, Web of Science and Sicelo databases. The descriptors were: spinal muscular atrophy and cognition. Study 2 consisted of the evaluation of 24 children divided into two groups: 12 children with SMA-I (3 to 9 years of age, 9 boys and 3 girls) and 12 healthy children, matched by age and sex. The 24 children performed four figure-association tasks, with increasing difficulty. Responses were detected by an eye tracking device. For the first study, 43 articles were identified and nine were selected because they met the inclusion and exclusion criteria. The study pointed out that the cognitive performance of children with SMA is heterogeneous. Three articles indicated that children with SMA had normal cognitive performance, three observed delayed performance and three studies described cognitive performance as above average. In general, samples with children with more severe muscular weakness (SMA-I) showed some cognitive impairment and more recent studies detected this delay. The second study described poorer performance of the SMA-I group, compared to the control group (more errors and longer times). In conclusion, few studies investigated cognitive outcomes in children and adolescents with SMA. The cognitive development of children with SMA should be followed, especially children with SMA-I. More studies should evaluate and, if necessary, intervene to prevent cognitive impairment in people with SMA. The proper cognitive development facilitates autonomy and interaction. The eye tracker can stimulate and improve their repertoire

Estudos anteriores de nosso laboratório demonstraram que a estimulação aguda dos receptores 2-adrenérgicos (2-AR) atenua a perda de massa muscular induzida pela desnervação motora (DEN) por meio de uma via dependente de AMPc/PKA. No entanto os mecanismos moleculares envolvidos na ativação crônica destes receptores ainda são pouco conhecidos. Por outro lado, a ativação desta via de sinalização também está envolvida no controle da estabilidade dos receptores nicotínicos (AChR) na junção neuromuscular (JNM), sugerindo que a densidade dos AChR possa estar sob controle neuro-humoral. Desta forma, aventou-se a possibilidade de que além dos efeitos protetores na massa muscular, a ativação dos receptores 2-AR pudesse mediar a estabilização dos AChR na placa motora. Para testar essa hipótese, camundongos foram submetidos à DEN através da secção do nervo ciático, um protocolo clássico de indução de atrofia muscular e desestabilização dos AChR, e tratados com salina ou clembuterol (CB), um 2-agonista seletivo, por até 14 dias. Após 3 dias de DEN, observou-se redução da massa muscular e aumento do conteúdo proteico e expressão do RNAm de genes relacionados à ativação do sistema Ubiquitina-Proteassoma (atrogina-1 e MuRF1) e do sistema autofágico/lisossomal (catepsina L e LC3). A DEN também promoveu aumento no turnover dos AChR, no número de vesículas endocíticas e na expressão do RNAm para a subunidade 1 dos AChR. Após 7 dias, a DEN reduziu a expressão dos genes relacionados à atrofia e aumentou a atividade da via do AMPc/PKA independentemente do tratamento com CB. Na tentativa de elucidar os sinais extracelulares que produziam esta resposta adaptativa, foi demonstrado que neurônios catecolaminérgicos trafegam ao longo do nervo ciático e sua ablação pela DEN reduziu o conteúdo de noradrenalina muscular. Baseados nestes resultados, foi postulado a existência de uma hipersenbilidade às catecolaminas em músculos desnervados cronicamente. O tratamento com CB por 3 dias aboliu o aumento da expressão dos atrogenes induzido pela DEN e este efeito foi associado ao maior conteúdo de AMPc e de substratos fosforilados pela PKA. Além disso, o CB diminuiu a hiperexpressão do RNAm para catepsina L e LC3 induzida pela DEN de 7 dias. Embora o CB não tenha alterado a meia-vida dos AChR em músculos inervados e desnervados, houve um total bloqueio do aumento do número de vesículas endocíticas contendo o AChR em músculos desnervados e tratados com CB. Corroborando estes dados, o CB aumentou a incorporação de AChR novos nas JNM e este efeito foi também associado à maior expressão do RNAm para a subunidade 1-AChR em músculos desnervados. Esta ação do CB no turnover dos AChR parece ser direta uma vez que neuroniôs catecolaminérgicos presentes no nervo ciático ativam receptores 2-ARe a produção de AMPc especificamente na JNM. Em estudos in vitro, foi demonstrado que a estimulação colinérgica produzida pelo carbacol (10-4M) diminuiu a velocidade de síntese de proteínas, aumentou a proteólise total e a atividade do sistema proteolítico Ca2+-dependente em músculos soleus de ratos por meio da ativação dos receptores nicotínicos. Este efeito catabólico do carbacol foi completamente bloqueado pela adição de CB (10-4M) ao meio de incubação. Os dados obtidos no presente estudo permitem sugerir que a estimulação crônica dos 2-AR no músculo esquelético induz um efeito anti-catabólico pela supressão dos sistemas proteolíticos proteassomal e lisossomal, provavelmente através da via de sinalização do AMPc/PKA. A inibição destes sistemas pode estar relacionada ao aumento do turnover dos AChR, uma vez que a velocidade de incorporação destes receptores na JNM foi aumentada pelo CB. Além disso, os achados que mostram a associação entre neurônios noradrenérgicos e colinérgicos no nervo ciático, que conjuntamente inervam as JNM, e a co-localização de receptores 2-AR e AChR na sinapse permitem sugerir a existência de uma interação funcional entre o sistema colinérgico e adrenérgico na manutenção da massa muscular e da placa motora. / Previous studies from our laboratory have shown that the acute stimulation of 2-adrenergic receptor (2-AR) attenuates the muscle loss induced by motor denervation (DEN) through a cAMP/PKA dependent pathway. However, the molecular mechanisms involved in the chronic activation of these receptors are poorly understood. Furthermore, the activation of this signaling pathway is also involved in controlling the stability of nicotinic receptors (AChR) at the neuromuscular junction (NMJ), suggesting that the density of AChR may be under neurohumoral control. Thus, we postulated that besides the protective effects on muscle mass the activation of 2-AR receptors could mediate the stabilization of AChR in the motor plate. To test this hypothesis, mice were submitted to DEN through of the sciatic nerve section, a classical protocol of induction muscle atrophy and destabilization of AChR, and were treated with saline or clenbuterol (CB), a selective 2-agonist for 14 days. DEN decreased the muscle mass and increased the protein content and mRNA expression of genes related to the activation of the ubiquitin-proteasome system (atrogin-1 and MuRF1) and autophagic/lysosomal system (cathepsin L and LC3). DEN also promoted an increase in the turnover of AChR, number of endocytic vesicles and the expression of mRNA for the 1 subunit of AChR. Interestingly, chronic DEN induced down-regulation of atrophy related-genes, and increased the activity of cAMP/PKA pathway independently of CB treatment. In an attempt to elucidate the extracellular signals that produced this adaptive response, it was demonstrated that catecholaminergic neurons travels along the sciatic nerve and its ablation by DEN reduces muscle norepinephrine content. Based on these results, it was postulated the existence of a muscle adrenergic hypersensitivity to circulating catecholamines induced by chronic DEN. CB treatment for 3 days completely abolished the higher expression of atrogenes and this effect was associated with increased Camp content and PKA phosphorylated substrates. Furthermore, CB decreased the DEN-induced hyperexpression of cathepsin L and LC3 mRNA at 7 days. Although CB has not altered the half-life of AChR in innervated and denervated muscles, it produced a total blockage of the increased number of endocytic vesicles containing the AChR in denervated muscles. Consistently, CB increased the incorporation of new AChR and this effect was associated with an increased expression of the 1-subunit AChR mRNA in denervated muscles. This action of CB on AChR turnover appears to be direct, since catecholaminergic neurons are present in the sciatic nerve stimulating 2-AR and cAMP production specifically in the NMJ. Furthermore, in vitro studies demonstrated that cholinergic stimulation produced by carbachol (10-4M) decreased the rate of protein synthesis and increased the proteolytic activity of Ca2+-dependent system in rat soleus muscle through activation of nicotinic receptors. This catabolic effect of carbachol was completely blocked by the addition of CB (10-4M) to the incubation medium. These data suggest that chronic stimulation of 2-AR in skeletal muscle induces an anti-catabolic effect by suppressing proteasomal and lysosomal proteolytic systems, probably through the cAMP/PKA signaling. The inhibition of these systems seems to be related to the increased AChR incorporation into NMJ induced by CB treatment. Moreover, the association between noradrenergic and cholinergic neurons in the sciatic nerve, both of which innervate the motor endplates, and the co-localization of AChR and 2-ARat the synapse suggest the existence of a functional interaction between cholinergic and adrenergic systems in the maintenance of muscle mass and motor endplate.