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.

Spinal Muscular Atrophy (SMA) is a neurodegenerative, inherited disease caused by an insufficient amount of functional Survival of Motor Neurone protein (SMN), though the exact mechanism underlying this is not fully understood. The primary function of SMN is assembling a ring of Sm proteins around small nuclear RNA (snRNA) in an early, cytoplasmic stage of small nuclear ribonucleoprotein (snRNP) biogenesis, a process essential in eukaryotes. SMN, together with several mRNA binding proteins, has been linked to neural transport of mRNA towards areas of growth in Motor neurons for local translation of transcripts. Previous research in our group has found that this may involve Coatomer protein-containing vesicles transported by Dynein and requiring the Sm family protein, SmB, for maintenance. Little is known, however, about what other proteins are also present and required for correct transport and localisation of these vesicles. To further investigate this, we have produced plasmids expressing each Sm protein tagged to fluorescent proteins to help track their behaviour, in some cases for the first time, and developed a detergent-free fractionation protocol to enrich for SMN containing vesicles, providing tools that can be used to further probe behaviour and interactions in the future. Using these approaches, SmN, a neural specific Sm protein, was identified to also be present in SMN-containing vesicles similarly to SmB. Analysis of the interactomes of different Sm proteins identified a novel interactor of SMN, Neurochondrin (NCDN), that appears to be required for the correct localisation of SMN in neural cells. NCDN was found to not associate with snRNPs, indicating an snRNP-independent interaction with SMN. NCDN and SMN both independently associated and co-enriched with Rab5, indicating a potential endocytic and cell polarity role for the interaction. This interaction has the potential to be key in SMA pathology and may have therapeutic potential.

L’atròfia muscular espinal (AME) és una malaltia d’origen genètic que afecta, majoritàriament a la població infantil. La malaltia cursa amb una mort de les motoneurones  i atròfia muscular. El gen implicat és el survival motor neuron (SMN) que està delecionat en un 95% dels casos. El nostre estudi està dividit en dues parts: 1- l’aprofundiment de les alteracions musculars en dos models animals murins transgènics que pateixen les formes més greus d’AME (Tipus 1-2) i 2- estudi dels possibles efectes terapèutics del liti en un d’aquests models d’AME. S’ha trobat alteracions greus en les unions neuromusculars d’animals nounats i prenatals en marcadors relacionats amb l’ancoratge de les vesícules a la membrana presinàptica, organització dels canals de calci presinàptics i altres proteïnes presinàptiques, desorganització i apoptosi de les cèl•lules musculars, apoptosi massiva del timus i alteracions generalitzades en els òrgans limfoides. L’estudi ultraestructural del múscul ens indica que hi ha una mort, per apoptosi, de les cèl•lules satèl•lit, confirmat amb la tècnica de TUNEL. L’augment de les apoptosi, però no es reflexa en un increment, per altra banda esperat, de la densitat dels macròfags. El tractament amb concentracions terapèutiques del liti no millora l’evolució de la malaltia en els ratolins que manifesten l’AME, s’observa una acumulació progressiva dels nivells de liti, provocant toxicitat en l’animal. L’efecte del liti inhibint la GSK3 no es tradueix en el increment d’expressió de SMN, tal com s’ha deduït d’alguns experiments publicats. / La atrofia muscular espinal (AME) es una enfermedad de origen genético que afecta, mayoritariamente a la población infantil. La enfermedad cursa con muerte de las motoneuronas y atrofia muscular. El gen implicado es el “survival motor neuron” (SMN) que está delecionado en un 95% de los casos. Nuestro estudio está dividido en dos partes: 1 - la caracterización de las alteraciones musculares en dos modelos animales murinos transgénicos que sufren las formas más graves de AME (Tipo 1-2) y 2 - estudio de los posibles efectos terapéuticos del litio en uno de estos modelos. Se han encontrado alteraciones pre y postnatales graves en las sinapsis neuromusculares a nivel de marcadores relacionados con el anclaje de las vesículas en la membrana presináptica, en la organización de los canales de calcio presinápticos y en otras proteínas presinápticas, Asimismo se ha hallado desorganización y apoptosis de las células musculares, apoptosis masiva del timo y alteraciones generalizadas en los órganos linfoides. El estudio ultraestructural del músculo nos revela muerte, por apoptosis, de las células satélite, confirmado con la técnica de TUNEL. El aumento de las apoptosis muscular no conlleva un incremento, por otra parte esperado, de la densidad de los macrófagos. El tratamiento con litio no mejora la evolución de la enfermedad en los ratones con AME. Se observa un incremento progresivo de los niveles de litio, provocando toxicidad en el animal. Por otra parte, el efecto del litio inhibiendo la GSK3 no se traduce en un aumento de la expresión de SMN, tal como se ha deducido de algunos experimentos publicados. / The spinal muscular atrophy (SMA) is a pediatric genetic disease. The SMA is a motor neuron disease that affects the motor neurons causing its death and muscle atrophy. The gene involved is the survival motor neuron (SMN) that is mutated in the 95% of the cases. Our study is divided into two parts: 1 – studies of the neuromuscular junction in two transgenic SMA murine models that develop the most severe forms of SMA (type 1-2) and 2 - study of the possible therapeutic effects of lithium on one of these models of SMA. We found severe alterations in the neuromuscular junctions of newborn animals and also in prenatal markers related to the vesicle docking at the presynaptic membrane, lack of organization of presynaptic calcium channels and defects in the expression of other presynaptic proteins. We found also, disruption and apoptosis of muscular cells, massive apoptosis of the thymus and widespread alterations in lymphoid organs. The ultrastructural study of muscle identifies apoptotic satellite cells that was confirmed by the TUNEL technique. The increase in apoptosis is not followed by the expected increase, in the macrophage density. Treatment with therapeutic concentrations of lithium does not improve the course of the disease in SMA mice. There was a progressive accumulation of lithium, causing toxicity in the animal. The effect of lithium inhibiting GSK3 does not determine an increased expression of SMN, as could be deduced from some published experiments.

Los cuerpos nucleares de Cajal (CBs) son estructuras nucleares implicadas en la biogénesis de ribonucleoproteínas nucleares y nucleolares de pequeño tamaño (snRNPs y snoRNPs) requeridas para el procesamiento nuclear de pre-mRNAs y pre-rRNAs, respectivamente. El CB concentra la proteína coilina, un marcador molecular de esta estructura, snRNPs, el factor de supervivencia de las neuronas motoras (SNM) y las proteínas que comparte con el nucleolo Nopp140 y fibrilarina. Los CB son estructuras dependientes de transcripción, pero los mecanismos de ensamblaje molecular de estos cuerpos nucleares son poco conocidos.En este estudio se utilizan métodos de inmunofluorescencia, expresión ectópica de proteínas del CB y métodos bioquímicos para analizar la importancia de dos modificaciones postraduccionales, la metilación de la coilina y la conjugación con SUMO1 del factor SMN para el ensamblaje molecular de los CBs. Se ha utilizado la línea celular MCF7 como un modelo de hipometilación endógena debido al déficit del gen MTAP. La hipometilación de la coilina conduce al desensamblaje de los CBs y a la relocalización nucleolar de la coilina no metilada. Este efecto revierte en células transfectadas que expresan el gen MTAPwt, indicando que el grado de metilación de la coilina marca su destino nuclear.Respecto a la importancia de la sumoilación en el ensamblaje de los CBs, hemos demostrado la existencia de un subtipo de CBs que concentran SUMO1 y la conjugasa de SUMO Ubc9. En neuronas, hemos detectado la presencia de SUMO durante la fase de reformación de CBs, en la respuesta al estrés. Los experimentos de inmunoprecipitación confirman la interacción de SUMO-1 con el factor SMN y demuestran que la lisina K119, portadora de una secuencia consenso de sumoilación, es esencial para la regulación del número de CBs. / Cajal bodies (CBs) are nuclear structures involved in the biogenesis of small nuclear and nucleolar ribonucleoproteins (snRNPs and snoRNPs) required for nuclear processing of pre-mRNAs and pre-rRNAs, respectively. CBs concentrate the protein coilin, a molecular marker of this structure, snRNPs, the survival of motor neurons factor (SMN) and proteins shared with the nucleolus Nopp140 and fibrillarin. CBs are transcription-dependent structures, but the mechanisms of molecular assembly of these structures are poorly understood.In this study we used inmunofluorescence, ectopic expresion of CB proteins and biochemical methods to analyze the importance of two posttranslational modifications, methylation of coilin and conjugation of SMN with SUMO1, for the molecular assembly of CBs. The cell line MCF7 has been used as a model of endogenous hypomethylation due to the lack of MTAP gene. Coilin hypomethylation leads to the disassembly of CBs and nucleolar relocation of unmethylated coilin. This effect reverses in transfected cells expressing the gene MTAPwt, indicating that the degree of methylation of coilin directs its nuclear destination.On the importance of sumoylation in the assembly of CBs, we have demonstrated the existence of a subset of CBs which concentrate SUMO1 and the SUMO1 conjugase Ubc9. In neurons, we detected the presence of SUMO1 during the reformation of CBs in response to stress. Immunoprecipitation experiments confirm the molecular interaction of SUMO1 with SMN and demonstrate that lysine 119, carrying the SMN sumoylation consensus sequence, is essential for regulating the number of CBs.

No description available.

Spinal muscular atrophy (SMA) and Charcot-Marie-Tooth disease type 2D (CMT2D)/distal SMA type V (dSMAV) are two incurable neuromuscular disorders that predominantly manifest during childhood and adolescence. Both conditions are caused by mutations in widely and constitutively expressed genes that encode proteins with essential housekeeping functions, yet display specific lower motor neuron pathology. SMA results from recessive inactivating mutations in the survival motor neuron 1 (SMN1) gene, while CMT2D/dSMAV manifests due to dominant point mutations in the glycyl-tRNA synthetase (GlyRS) gene, GARS. Using a number of different model systems, ranging from Caenorhabditis elegans to the mouse, this thesis aimed to identify potential novel therapeutic compounds for SMA, and to increase our understanding of the mechanisms underlying both diseases. I characterised a novel C. elegans allele, which possesses a point mutation in the worm SMN1 orthologue, smn-1, and showed its potential for large-scale screening by highlighting 4-aminopyridine in a screen for compounds able to improve the mutant motility defect. Previously, the gene encoding three isoforms of chondrolectin (Chodl) was shown to be alternatively spliced in the spinal cord of SMA mice before disease onset. I performed functional analyses of the three isoforms in neuronal cells with experimentally reduced Smn levels, and determined that the dysregulation of Chodl likely reflects a combination of compensatory mechanism and contributor to pathology, rather than mis-splicing. Finally, working with two Gars mutant mice and a new Drosophila model, I have implicated semaphorin-plexin pathways and axonal guidance in the GlyRS toxic gain-of-function disease mechanism of CMT2D/dSMAV.