Substrate-bound agrin induces expression of acetylcholine receptor epsilon-subunit gene in cultured mammalian muscle cells.

Expression of the epsilon-subunit gene of the acetylcholine receptor (AChR) by myonuclei located at the neuromuscular junction is precisely regulated during development. A key role in this regulation is played by the synaptic portion of the basal lamina, a structure that is also known to contain agrin, a component responsible for the formation of postsynaptic specializations. We tested whether agrin has a function in synaptic AChR gene expression. Synaptic basal lamina from native adult muscle and recombinant agrin bound to various substrates induced in cultured rat myotubes AChR clusters that were colocalized with epsilon-subunit mRNA. Estimation of transcript levels by Northern hybridization analysis of total RNA showed a significant increase when myotubes were grown on substrate impregnated with agrin, but were unchanged when agrin was applied in the medium. The effect was independent of the receptor aggregating activity of the agrin isoform used, and agrin acted, at least in part, at the level of epsilon-subunit gene transcription. These findings are consistent with a role of agrin in the regulation of AChR subunit gene expression at the neuromuscular junction, which would depend on its binding to the synaptic basal lamina.

Onconeural antigens and the paraneoplastic neurologic disorders: at the intersection of cancer, immunity, and the brain.

Paraneoplastic neurologic disorders (PNDs) are believed to be autoimmune neuronal degenerations that develop in some patients with systemic cancer. A series of genes encoding previously undiscovered neuronal proteins have been cloned using antiserum from PND patients. Identification of these onconeural antigens suggests a reclassification of the disorders into four groups: those in which neuromuscular junction proteins, nerve terminal/vesicle-associated proteins, neuronal RNA binding proteins, or neuronal signal-transduction proteins serve as target antigens. This review considers insights into basic neurobiology, tumor immunology, and autoimmune neuronal degeneration offered by the characterization of the onconeural antigens.

Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles.

Immunohistochemical visualization of the rat vesicular acetylcholine transporter (VAChT) in cholinergic neurons and nerve terminals has been compared to that for choline acetyltransferase (ChAT), heretofore the most specific marker for cholinergic neurons. VAChT-positive cell bodies were visualized in cerebral cortex, basal forebrain, medial habenula, striatum, brain stem, and spinal cord by using a polyclonal anti-VAChT antiserum. VAChT-immuno-reactive fibers and terminals were also visualized in these regions and in hippocampus, at neuromuscular junctions within skeletal muscle, and in sympathetic and parasympathetic autonomic ganglia and target tissues. Cholinergic nerve terminals contain more VAChT than ChAT immunoreactivity after routine fixation, consistent with a concentration of VAChT within terminal neuronal arborizations in which secretory vesicles are clustered. These include VAChT-positive terminals of the median eminence or the hypothalamus, not observed with ChAT antiserum after routine fixation. Subcellular localization of VAChT in specific organelles in neuronal cells was examined by immunoelectron microscopy in a rat neuronal cell line (PC 12-c4) expressing VAChT as well as the endocrine and neuronal forms of the vesicular monoamine transporters (VMAT1 and VMAT2). VAChT is targeted to small synaptic vesicles, while VMAT1 is found mainly but not exclusively on large dense-core vesicles. VMAT2 is found on large dense-core vesicles but not on the small synaptic vesicles that contain VAChT in PC12-c4 cells, despite the presence of VMAT2 immunoreactivity in central and peripheral nerve terminals known to contain monoamines in small synaptic vesicles. Thus, VAChT and VMAT2 may be specific markers for "cholinergic" and "adrenergic" small synaptic vesicles, with the latter not expressed in nonstimulated neuronally differentiated PC12-c4 cells.

Mismatch repair in Escherichia coli enhances instability of (CTG)n triplet repeats from human hereditary diseases.

Long CTG triplet repeats which are associated with several human hereditary neuromuscular disease genes are stabilized in ColE1-derived plasmids in Escherichia coli containing mutations in the methyl-directed mismatch repair genes (mutS, mutL, or mutH). When plasmids containing (CTG)180 were grown for about 100 generations in mutS, mutL, or mutH strains, 60-85% of the plasmids contained a full-length repeat, whereas in the parent strain only about 20% of the plasmids contained the full-length repeat. The deletions occur only in the (CTG)180 insert, not in DNA flanking the repeat. While many products of the deletions are heterogeneous in length, preferential deletion products of about 140, 100, 60, and 20 repeats were observed. We propose that the E. coli mismatch repair proteins recognize three-base loops formed during replication and then generate long single-stranded gaps where stable hairpin structures may form which can be bypassed by DNA polymerase during the resynthesis of duplex DNA. Similar studies were conducted with plasmids containing CGG repeats; no stabilization of these triplets was found in the mismatch repair mutants. Since prokaryotic and human mismatch repair proteins are similar, and since several carcinoma cell lines which are defective in mismatch repair show instability of simple DNA microsatellites, these mechanistic investigations in a bacterial cell may provide insights into the molecular basis for some human genetic diseases.

Molecular cloning and functional characterization of the Xenopus Ca(2+)-binding protein frequenin.

Frequenin was originally identified in Drosophila melanogaster as a Ca(2+)-binding protein facilitating transmitter release at the neuromuscular junction. We have cloned the Xenopus frequenin (Xfreq) by PCR using degenerate primers combined with low-stringency hybridization. The deduced protein has 70% identity with Drosophila frequenin and about 38-58% identity with other Ca(2+)-binding proteins. The most prominent features are the four EF-hands, Ca(2+)-binding motifs. Xfreq mRNA is abundant in the brain and virtually nondetectable from adult muscle. Western blot analysis indicated that Xfreq is highly concentrated in the adult brain and is absent from nonneural tissues such as heart and kidney. During development, the expression of the protein correlated well with the maturation of neuromuscular synapses. To determine the function of Xfreq at the developing neuromuscular junction, the recombinant protein was introduced into Xenopus embryonic spinal neurons by early blastomere injection. Synapses made by spinal neurons containing exogenous Xfreq exhibited a much higher synaptic efficacy. These results provide direct evidence that frequenin enhances transmitter release at the vertebrate neuromuscular synapse and suggest its potential role in synaptic development and plasticity.

Parkinson's disease and gastrointestinal dysfunctions: morphological, neurochemical and functional modifications of the enteric nervous system associated with central dopaminergic impairment

Parkinson’s disease (PD) is frequently associated with gastrointestinal (GI) symptoms, mostly represented by abdominal distension, constipation and defecatory dysfunctions. Despite GI dysfunctions have a major impact on the clinical picture of PD, there is currently a lack of information on the neurochemical, pathological and functional correlates of GI dysmotility associated with PD. Moreover, there is a need of effective and safe pharmacological therapies for managing GI disturbances in PD patients. The present research project has been undertaken to investigate the relationships between PD and related GI dysfunctions by means of investigations in an animal model of PD induced by intranigral injection of 6-hydroxydopamine (6-OHDA). The use of the 6-OHDA experimental model of PD in the present program has allowed to pursue the following goals: 1) to examine the impact of central dopaminergic denervation on colonic excitatory cholinergic and tachykininergic neuromotility by means of molecular, histomorphologic and functional approaches; 2) to elucidate the role of gut inflammation in the onset and progression of colonic dysmotility associated with PD, characterizing the degree of inflammation and oxidative damage in colonic tissues, as well as identifying the immune cells involved in the production of pro-inflammatory cytokines in the gut; 3) to evaluate the impact of chronic treatment with L-DOPA plus benserazide on colonic neuromuscular activity both in control and PD animals. The results suggest that central nigrostriatal dopaminergic denervation is associated with an impaired excitatory cholinergic neurotransmission and an enhanced tachykininergic control, resulting in a dysregulated smooth muscle motor activity, which likely contributes to the concomitant decrease in colonic transit rate. These motor alterations might result from the occurrence of a condition of gut inflammation associated with central intranigral denervation. The treatment with L-DOPA/BE following central dopaminergic neurodegeneration can restore colonic motility, likely through a normalization of the cholinergic enteric neurotransmission, and it can also improve the colonic inflammation associated with central dopaminergic denervation.

Estudo morfológico do Sistema Nervoso Central de cães com Distrofia Muscular do Golden Retriever (GRMD)

Distrofia muscular de Duchenne é uma desordem neuromuscular causada pela mutação ou deleção do gene da distrofina, a qual é ligada ao cromossomo X. Estudos recentes têm demonstrado o importante papel da distrofina no SNC, sendo sua deficiência relacionada com uma variedade de anormalidades na função do SNC, como comportamento e disfunção cognitiva. Os modelos animais mais adequados para esses estudos são os que apresentam o quadro clinico mais semelhante ao da DMD encontrada em humanos, como cães Golden Retriever com distrofia muscular (GRMD). Por não haver ainda estudos a respeito do SNC de animais GRMD, o objetivo deste trabalho foi analisar a morfologia do encéfalo dos GRMD e o de animais não distróficos, através de análise macroscópica, utilizando métodos de medição e registro fotográfico, e análise microscópica, utilizando a técnica de coloração de violeta cresil modificada. Entretanto, usando a metodologia proposta, não foi possível verificar diferenças significativas no encéfalo quando comparados os animais distróficos e os não distróficos, o que está em concordância com a literatura para a DMD usando os mesmos parâmetros. Em tempo, existe uma variação individual na morfologia do encéfalo do cão, independente de serem animais do grupo de distróficos ou controles. Outras técnicas devem ser aplicadas a fim de elucidar as consequências da ausência total ou parcial da distrofina no SNC

Responsividade da escala de avaliação funcional do sentar e levantar do solo para distrofia muscular de Duchenne (FES-DMD - D4), no período de um ano

OBJETIVO: Avaliar a responsividade da escala de avaliação funcional para pacientes com distrofia muscular de Duchenne (FES-DMD-D4), sentar e levantar do solo, no período de um ano. MÉTODO: Estudo observacional, longitudinal e retrospectivo. Foi estudada, utilizando o software FES-DMDDATA, uma amostra com 25 pacientes na atividade sentar no solo e 28 pacientes para a atividade levantar do solo. As avaliações ocorreram a cada três meses no período de um ano. Para análise estatística da capacidade de resposta foram utilizados índices de tamanho de efeito, como, effect size (ES) e Standardized Response Mean (SRM). RESULTADOS: A responsividade da atividade de sentar no solo foi considerada baixa a moderada em intervalos de três meses (ES de 0.28 a 0.54 e SRM de 0.38 a 0.71), moderada a alta em intervalos de seis meses (ES de 0.69 a 1.07 e SRM de 0.86 a 1.19), alta em intervalos de nove meses (ES de 1.3 a 1.17 e SRM de 1.26 a 1.55) e doze meses (ES de 1.9 e SRM de 1.72). Na atividade levantar do solo, a responsividade variou em baixa, moderada e alta em intervalos de três meses (ES de 0.21 a 0.33 e SRM de 0.45 a 0.83), baixa a alta em intervalos de seis meses (ES de 0.46 a 0.59 e SRM de 0.73 a 0.97), moderada a alta em intervalos de nove meses (ES de 0.76 a 0.88 e SRM de 1.03 a 1.22) e alta em doze meses (ES de 1.14 e SRM de 1.25). CONCLUSÃO: Para detectar alterações clinicamente significativas e consistentes nas atividades funcionais sentar e levantar do solo recomendamos a utilização da FES-DMD-D4 em intervalos a partir de seis meses, pois foi neste período de tempo que a capacidade de resposta variou de moderada a alta

Acute Synaptic Activity Causes Differential miRNA Expression in The Drosophila melanogaster Larval Central Nervous System

The primary goal of this thesis was to determine if spaced synaptic stimulation induced the differential expression of microRNAs (miRNAs) in the Drosophila melanogaster central nervous system (CNS). Prior to attaining this goal, we needed to identify and validate a spaced stimulation paradigm that could induce the formation of new synaptic growth at a model synapse, the larval neuromuscular junction (NMJ). Both Channelrhodopsin- and high potassium-based stimulation paradigms adapted from (Ataman, et al. 2008) were tested. Once validation of these paradigms was complete, we sought to characterize the miRNA expression profile of the larval CNS by miRNA array. Following attainment of these data, we used quantitative real-time PCR (RT-qPCR) to determine if acute synaptic stimulation caused the differential expression of neuronal miRNAs. We found that upon high potassium spaced training in a wild type (Canton S) genotype, 5 miRNAs showed significant differential expression when normalized to a validated reference gene, the U1 snRNA. Moreover, absolute quantification of our RT-qPCR study implicated one miRNA: miR-958 as being significantly regulated by activity. Investigation into potential targets for miR-958 revealed it to be a potential regular of Dlar, a protein tyrosine phosphatase implicated in synapse development. This investigation provides the foundation to directly test our underlying hypothesis that, following spaced training, differential expression of miRNAs alters the translation of proteins required to induce and maintain these structural changes at the synapse.

Responsividade do domínio subir e descer escada da escala de avaliação funcional para pessoas com distrofia muscular de Duchenne, no período de um ano

Objetivo: Determinar a responsividade do domínio subir e descer escada da escala de avaliação funcional em distrofia muscular de Duchenne (DMD), no período de um ano. Método: Participaram do estudo 26 pacientes com DMD. A análise utilizou o Tamanho do Efeito (ES) e a Média Padronizada de Resposta (SRM). Resultados: Atividade de subir escada: o ES mostrou responsividade baixa nos intervalos de avaliação de 3 meses (0,26; 0,35; 0,13; 0,17), baixa a moderada em 6 meses (0,58, 0,48; 0,33), moderada em 9 meses (0,70; 0,68) e alta em 1 ano (0,88). A análise com SRM mostrou responsividade baixa nos intervalos de avaliação de 3 meses (0,29; 0,38; 0,18 e 0,19), baixa a moderada em intervalos de 6 meses (0,59 e 0,51, 0,36), moderada em 9 meses (0,74 e 0,70) e alta em 1 ano (0,89). Atividade de descer escada: O ES apresentou responsividade baixa nos intervalos de avaliação de 3 meses (0,16; 0,25; 0,09; 0,08) e 6 meses (0,48; 0,35; 0,18), baixa a moderada em 9 meses (0,59, 0,44) e moderada em 1 ano (0,71). Análise com SRM mostrou responsividade baixa nos intervalos de 3 meses (0,25; 0,35; 0,12 e 0,09) e 6 meses (0,47; 0,38 e 0,21), moderada a baixa em 9 meses (0,62, 0,49) e moderada em 1 ano (0,74). Conclusão: A avaliação da atividade de subir escada, por meio da FES-DMD-D3, deve ser realizada em intervalos a partir de 9 meses, pois a responsividade é de moderada a alta. A avaliação do descer escadas deve ser realizada anualmente, pois houve responsividade moderada somente a partir de 12 meses

Características electromiográficas y kinesiográficas de una población española con oclusión ideal

Tradicionalmente, la alteración de las características neuromusculares de un paciente se ha considerado como un posible factor etiológico o agravante de una maloclusión, debido a que los diferentes patrones de actividad muscular pueden modificar las relaciones estructurales y de crecimiento de la mandíbula con el maxilar. Además, numerosos estudios han confirmado que una maloclusión podría alterar la actividad funcional del paciente, de tal manera que, uno de los objetivos del tratamiento ortodóncico sería proporcionar una adecuada normalidad de las características neuromusculares del paciente a través de la corrección de su maloclusión. Actualmente, las técnicas de electromiografía (EMG) y kinesiografía permiten estudiar cuantitativamente la actividad muscular y los movimientos mandibulares de los pacientes en unas condiciones fisiológicas normales, tanto estáticas como dinámicas. Para poder identificar la anormalidad neuromuscular es necesario previamente tener sistematizados unos valores de normalidad que puedan servir como criterios de referencia para poder reconocer y cuantificar las alteraciones funcionales neuromusculares. Según nuestro conocimiento, existe una falta de estudios sobre la población española en condiciones de normalidad fisiológica que proporcionen unos parámetros de referencia sobre la actividad, simetría y patrones de actividad muscular, así como de los rangos normales de amplitud de los movimientos mandibulares. De igual manera, también existe controversia en los diferentes estudios realizados sobre el dimorfismo sexual observado en estas funciones neuromusculares...

Sinterização flash do condutor catiônico beta-alumina sintetizada pelo método dos precursores poliméricos.

A beta-alumina de sódio é uma cerâmica condutora de íons Na+ utilizada como eletrólito sólido em baterias de sódio para armazenamento de energias intermitentes como energia solar e eólica. Devido ao alto teor de sódio, esse material é instável a altas temperaturas, podendo sofrer variações de composição durante a etapa de sinterização convencional que utiliza altas temperaturas por longos períodos de tempo. A sinterização flash é uma técnica de sinterização ativada por corrente elétrica que proporciona a densificação de compactos cerâmicos em poucos segundos, a temperaturas notavelmente mais baixas que as convencionais. Uma vez obrigatória a passagem de corrente elétrica através da amostra, a sinterização flash de qualquer material condutor parece bastante razoável. Não obstante, até o presente momento a maioria dos trabalhos publicados sobre o assunto aborda apenas condutores de vacância de oxigênio ou semicondutores, materiais compatíveis com eletrodos de platina (Pt). Nesse trabalho a sinterização flash de um condutor catiônico foi estudada utilizando-se a beta-alumina como material modelo. A beta-alumina foi sintetizada pelo método dos precursores poliméricos, caracterizada e então submetida à sinterização flash. O material de eletrodo padrão (platina) provou ser um eletrodo bloqueador em contato com a beta-alumina. O sucesso da sinterização flash foi determinado pela troca do material de eletrodo por prata (Ag), o que possibilitou uma reação eletroquímica reversível nas interfaces eletrodo-cerâmica e possibilitou a obtenção de um material densificado com morfologia e composição química homogêneas. Devido à metaestabilidade da beta-alumina, a atmosfera dos experimentos precisou ser alterada para manter a integridade desse material rico em um metal alcalino (Na+). A sinterização flash de um condutor catiônico é apresentada pela primeira vez na literatura e ressalta a importância da reação de eletrodo, que é um fator limitante para o sucesso da sinterização flash e precisa ser estudada e adaptada para cada tipo de material.

RNA-Seq expression profile of genes related to neurodegenerative disorders affecting the human retina

Human neurodegenerative diseases, such as Parkinson’s disease (PD) and the neuromuscular disorders called dystroglycanopathies (DGPs), cause retinal impairments. We have used RNA-Seq technology to catalog all known genes linked to PD and DGPs expressed in the human retina and quantitate their mRNA levels in terms of FPKM. We have also characterized their expression profiles in the retina by determining their exonic, intronic and exon-intron junction expression levels, as well as the alternative splicing pattern of particular genes. We believe these data could pave the way toward understanding the molecular bases of sight deficiencies associated with neurodegenerative disorders.

Agings and the parameters in static postural way

There is strong scientific evidence from research trials that aging is associated with loss of muscle mass and decline of neuromuscular abilities. Postural stability is an important neuromuscular ability for the maintenance of upright posture as well as maintaining equilibrium or balance while performing movements and everyday activities. Postural stability is also an important factor in elderly people where postural instability is a major contributor to falls. In our study young and elderly subjects stood quietly in upright posture with parallel positions of their feet and opened eyes on a force platform and performed 3 trials with each trial lasting 30 s. The effects of healthy aging on postural sway parameters were studied. We found that age-related changes in postural sway mostly affect the velocity of the center of pressure movement and the mean amplitude center of pressure movement during static postural sway test.

Escoliose no contexto de distrofia muscular

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014