La Sclérose Latérale Amyotrophique (SLA) est une affection neurodégénérative touchant les motoneurones, habituellement mortelle en 3 à 5 ans. La cause de la maladie n'est pas connue et le seul traitement actuellement disponible ne permet qu'un allongement modeste de la survie. Des altérations fonctionnelles de la jonction neuromusculaire (JNM) ont été rapportées dans la SLA mais leur origine physiopathologique n'est pas connue. Nous avons étudié les JNM chez 11 patients atteints de SLA, en associant étude morphologique en microscopie confocale et analyse ultrastructurale. L'analyse fonctionnelle réalisée en EMG de surface retrouvait une anomalie de transmission neuromusculaire (décrément > 10%) chez 45% des patients. Des altérations morphologiques des JNM étaient visibles chez tous les patients, y compris au stade précoce de la maladie. Associé aux anomalies en rapport avec le phénomène de dénervation, nous avons observé un aspect anormal de spiculation de la gouttière primaire dans environ un tiers des cas. Une interposition marquée de la cellule de Schwann terminale entre la terminaison nerveuse et la membrane postsynaptique, pouvant altérer la transmission synaptique, était parfois visible. Nous avons objectivé une réinnervation compensatrice significativement plus importante chez les patients présentant une SLA d'évolution lente et montré que certains facteurs moléculaires musculaires comme l'histone déacétylase 4 pourraient jouer un rôle crucial dans la capacité de réinnervation. Ce travail a mis en évidence des altérations morphologiques majeures au niveau des JNM des patients atteints de SLA et a permis d'identifier des cibles thérapeutiques potentielles. / Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder affecting motor neurons, usually leading to death in 3 to 5 years. The only treatment currently available, riluzole, has a modest effect on survival. Functional alterations of the neuromuscular junction (NMJ) have been reported in ALS, but their pathophysiological significance remains unknown. We studied the morphology of neuromuscular junctions in muscle samples collected from 11 ALS patients, using confocal and electron microscopy. Functional analysis of the NMJs was performed using surface-recording of compound motor action potentials after repetitive nerve stimulation at slow stimulus rate. A significant decrement (>10%), suggesting impairment of the neuromuscular transmission, was present in 45% of the patients. Morphological alterations of the NMJs were present in all ALS patients even at the early-stages. Beside denervation-induced morphological changes, one third of the NMJs showed abnormal spike-like areas of the outer edge of the postsynaptic primary gutter. A marked interposition of the terminal Schwann cell between the nerve terminal and the postsynaptic membrane, which was likely to alter synaptic transmission, was sometimes present. We found a significantly greater compensatory reinnervation in muscle from patients with slowly progressive ALS. Furthermore, we identified that the muscle molecular factor histone deacetylase 4 could play a key role in muscle reinnervation and disease progression in patients with ALS. This work has highlighted the presence of major morphological changes at the NMJs of ALS patients and identified potential new targets for future treatment.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative dont les premiers symptômes apparaissent généralement vers 60 ans. Elle affecte sélectivement le système moteur et provoque une paralysie progressive amenant au décès du patient par défaillance respiratoire en quelques années. À ce jour, il n’existe aucun traitement curatif, d’où la nécessité de comprendre la physiopathologie de la SLA. Bien que de nombreuses altérations dans le muscle aient été mises en évidence, sa contribution dans la SLA reste à définir. Nous avons montré que FUS est enrichi dans les noyaux sous-synaptiques de façon dépendante de l’innervation. De plus, FUS se lie au promoteur des récepteurs de l’acétylcholine et induit leur transcription de façon dépendante d’ERM. Le mutant FUS, quant à lui, est enrichi dans les noyaux extra-synaptiques et entraîne une toxicité musculaire responsable de l’altération de la jonction neuromusculaire (JNM). Au-delà de la JNM, FUS active MEF2A, de façon dépendante de PRMT1 afin de réguler les fonctions mitochondriales et la différenciation musculaire. La toxicité musculaire de FUS joue donc un rôle clé dans la physiopathologie de la SLA. / Amyotrophic lateral sclerosis is a neurodegenerative whose first symptoms generally appear around age 60. It is characterized by progressive motor neuron degeneration, paralysis and leading to death due to respiratory failure in a few years. Currently, there is no cure so the understanding of ALS physiopathology is necessary. Although many alterations in the muscle have been highlighted, its contribution in ALS remains to be defined. We showed that FUS is enriched in subsynaptic nuclei and this enrichment depended on innervation. Besides, FUS binds directly acetylcholine receptors (AchR) promoter and is required for Ermdependent induction of AChR expression. Conversely, mutant FUS is enriched on extra-synaptic nuclei and induce muscle intrinsic toxicity responsible for neuromuscular junction (NMJ) alteration. Beyond NMJ, FUS is required for muscle mitochondrial function and muscle differentiation through PRMT1-dependent MEF2A activation. Thus, FUS muscular toxicity plays a key role in the ALS physiopathology.

INTRODUÇÃO: A técnica de eletromiografia de fibra única (EMGFU), mediante análise do jitter, é o método neurofisiológico mais sensível para a confirmação do distúrbio da junção neuromuscular na miastenia gravis (MG). Os registros são tradicionalmente obtidos com agulha de fibra única, de alto custo e reutilizável. Por causa da necessidade atual do uso de material descartável, a agulha concêntrica vem sendo utilizada em substituição à agulha de fibra única. A técnica utilizada é semelhante, porém os potencias de ação para a análise do jitter são obtidos com eletrodo de agulha concêntrica (Eletromiografia de fibra única - jitter com agulha concêntrica, EMGFU-JAC). Contudo, os estudos são escassos e as metodologias utilizadas são heterogêneas com a utilização dessa agulha. OBJETIVOS: Este estudo tem por objetivo mensurar os valores de jitter obtidos com agulha concêntrica, no músculo Orbicularis Oculi, em sujeitos saudáveis e em pacientes com MG autoimune adquirida e avaliar a validade do método nas formas generalizada e ocular da doença. MÉTODOS: Foram estudados 20 sujeitos saudáveis, 20 pacientes com miastenia gravis forma generalizada (grupo MGG) e 13 com a forma ocular da doença (grupo MGO). A EMGFU-JAC foi realizada em todos os participantes, idealmente com 20 medidas de jitter em cada estudo. O jitter foi expresso como a média das diferenças consecutivas (MCD). Em todos os pacientes do estudo foram realizados o teste de estimulação repetitiva e dosagem sérica de anticorpo antirreceptor de acetilcolina (ac-AChR) no momento da análise do jitter. Nos pacientes soronegativos para ac-AChR, foi pesquisado o anticorpo antimúsculo específico tirosina-quinase (ac-MuSK). Foram definidos o limite superior da normalidade (LSN) para a média do MCD de cada estudo e para valores individuais de MCD. Os critérios de anormalidade foram: (1) média do MCD acima do LSN; ou (2) mais de 10% dos valores individuais de MCD acima do LSN. A definição do LSN para valores individuais de MCD baseou-se no conceito de que dois entre 20 valores de MCD acima do LSN são aceitáveis em um músculo saudável, para a técnica de contração voluntária. Portanto, estimou-se o LSN para o 18o valor mais alto de MCD (18o par). Para análise da acurácia do método, foram construídas duas curvas ROC (Receiver Operating Characteristic) para as variáveis média do MCD e 18o par, no grupo de pacientes (MGG e MGO) versus controle. RESULTADOS: No grupo controle a média das médias do MCD foi (19,0 ± 2,4)us e a média do 18o valor mais alto de cada estudo foi (24,5 ± 3,6)us. Esses valores obtidos apresentaram distribuição Gaussiana e o LSN foi definido como a média desses valores + 2 DP. O LSN para a média do MCD foi 24us, e 32?s para valores individuais de MCD. No grupo MGG, a análise do jitter foi anormal em todos os 20 pacientes por ambos os critérios de anormalidade, exceto em um paciente que apresentou anormalidade por apenas um dos critérios. No grupo MGO, apenas um dos 13 pacientes não preencheu os critérios de anormalidade. No grupo de pacientes, a positividade da EMGFU-JAC foi maior do que o teste de estimulação repetitiva e dosagens de anticorpos. Nas curvas ROC para as variáveis médias do MCD e 18o par, o valor de melhor sensibilidade (93,9%), sem resultados falsos positivos, foi 24,7us e 33,1us, respectivamente. CONCLUSÕES: A EMGFU-JAC apresenta alta sensibilidade e especificidade na identificação de distúrbio da transmissão neuromuscular em pacientes com MG. A utilização da agulha concêntrica é válida para a análise do jitter, como alternativa à agulha de fibra única / INTRODUCTION: Single fiber electromyography (SFEMG) technique, through jitter analysis, is the most sensitive neurophysiological method for confirmation of neuromuscular junction disorder in myasthenia gravis (MG). Records are traditionally obtained with single fiber needle, which is reusable and has a high-cost. Due to the current need of using disposable material, concentric needle has been used to replace single fiber needle. The technique is similar, but the action potential for jitter analysis is obtained with concentric needle electrode (SFEMG - concentric needle jitter, SFEMG-CNJ). However, studies are scarce and methodologies used are heterogeneous with the use of this needle. OBJECTIVES: This study aims to measure jitter values obtained with concentric needle in the Orbicularis Occuli muscle in healthy subjects and in patients with autoimmune acquired MG and to assess the validity of the method in generalized and ocular forms of the disease. METHODS: 20 healthy subjects, 20 patients with generalized myasthenia gravis (GMG group) and 13 with the ocular form of the disease (OMG group) were studied. SFEMG-CNJ was performed on all participants, ideally with 20 jitter values in each study. Jitter was expressed as the mean consecutive difference (MCD). Repetitive nerve stimulation and serum acetylcholine receptor antibody (AChR-ab) were performed in all patients in the study, by the time of jitter analysis. Tyrosine kinase specific antibody muscle antibodies (MuSK-ab) were performed in AChR-ab negative patients. The upper limit of normality (ULN) for the mean MCD and for individual jitter values were defined. The abnormality criteria were: (1) mean MCD above ULN; or (2) more than 10% of individual jitter values above ULN. The definition of ULN for individual jitter values was based on the concept that two out of 20 jitter values above ULN are acceptable in a healthy muscle for voluntary contraction technique. Therefore, the ULN for the 18th highest jitter value (18 pair) was estimated. To analyze the method\'s accuracy, two ROC curves (Receiver Operating Characteristic) for the mean MCD and 18th pair in the group of patients (MGG and MGO) versus control were constructed. RESULTS: In the control group the mean of MCD means was (19.0 ± 2.4)us and the mean of the 18 highest value of each study was (24.5 ± 3.6)us. These values showed Gaussian distribution and the ULN was set as the mean of these values + 2 SD. The ULN for the mean MCD was 24us, and 32us for individual values of MCD. In GMG group, jitter analyses were abnormal in all 20 patients based on both abnormality criteria, except in one patient, who had abnormalities in only one of the criteria. In OMG group, only one patient from 13 met neither of the abnormality criteria. In patients, the positivity of SFEMG-CNJ was higher than repetitive nerve stimulation test and antibody detection. The ROC curve threshold showing the best sensitivity (93.9%) with no false positive results was 24.7Us for the mean MCD and 33.1us for individual pairs, respectively. CONCLUSIONS: SFEMG-CNJ has high sensitivity and specificity in identifying neuromuscular transmission disorder in patients with MG. The use of concentric needle is valid for jitter analysis as an alternative to single fiber needle

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.

Synapses vary widely in the probability of transmitter release. For instance, in response to an action potential the phasic synapses of the crayfish have a 100-1000-fold higher release probability than tonic synapses. The difference in release probability is attributed to differences in the exocytotic machinery such as the degree of “zippering” of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex. I used physiological and molecular approaches to determine if the zippered state of SNAREs associated with synaptic vesicles and the interaction between the SNARE complex and Complexin influence the probability of release at the synapse. I used three Botulinum neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by SNARE interaction (zippering) or open to proteolytic attack. Under low stimulation conditions, the light-chain fragment of botulinum B (BoNT/B-LC) but not BoNT/D-LC or tetanus neurotoxin (TeNT-LC) cleaved VAMP and inhibited evoked release at both phasic and tonic synapses. In addition, a peptide based on the C-terminal half of crayfish VAMP’s SNARE motif (Vc peptide) designed to interfere with SNARE complex zippering at the C-terminal end inhibited release at both synapses. The susceptibility of VAMP to only BoNT/B-LC and interference by the Vc peptide indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end with the C-terminal end exposed under resting conditions. I used a peptide containing part of the crayfish Complexin central α-helix domain to interfere with the interaction between Complexin and the SNARE complex. The peptide enhanced phasic evoked release and inhibited tonic evoked release under low stimulation but attenuated release at both synapses under intense stimulation. Therefore, Complexin appeared to exhibit a dual function under low synaptic activity but only promoted release under high synaptic activity. The results showed that the zippered state of the SNARE complex does not determine initial release probability as a similar zippered SNARE complex structure under resting conditions is common to both phasic and tonic synapses. However, Complexin may have a role in influencing the initial release probability of a synapse. Therefore, the interaction between the SNARE complex and Complexin is important for release but other factors contribute more significantly to synaptic strength.

Synapses vary widely in the probability of transmitter release. For instance, in response to an action potential the phasic synapses of the crayfish have a 100-1000-fold higher release probability than tonic synapses. The difference in release probability is attributed to differences in the exocytotic machinery such as the degree of “zippering” of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex. I used physiological and molecular approaches to determine if the zippered state of SNAREs associated with synaptic vesicles and the interaction between the SNARE complex and Complexin influence the probability of release at the synapse. I used three Botulinum neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by SNARE interaction (zippering) or open to proteolytic attack. Under low stimulation conditions, the light-chain fragment of botulinum B (BoNT/B-LC) but not BoNT/D-LC or tetanus neurotoxin (TeNT-LC) cleaved VAMP and inhibited evoked release at both phasic and tonic synapses. In addition, a peptide based on the C-terminal half of crayfish VAMP’s SNARE motif (Vc peptide) designed to interfere with SNARE complex zippering at the C-terminal end inhibited release at both synapses. The susceptibility of VAMP to only BoNT/B-LC and interference by the Vc peptide indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end with the C-terminal end exposed under resting conditions. I used a peptide containing part of the crayfish Complexin central α-helix domain to interfere with the interaction between Complexin and the SNARE complex. The peptide enhanced phasic evoked release and inhibited tonic evoked release under low stimulation but attenuated release at both synapses under intense stimulation. Therefore, Complexin appeared to exhibit a dual function under low synaptic activity but only promoted release under high synaptic activity. The results showed that the zippered state of the SNARE complex does not determine initial release probability as a similar zippered SNARE complex structure under resting conditions is common to both phasic and tonic synapses. However, Complexin may have a role in influencing the initial release probability of a synapse. Therefore, the interaction between the SNARE complex and Complexin is important for release but other factors contribute more significantly to synaptic strength.

We are interested in understanding the molecular mechanisms that govern synaptic growth and plasticity. Recent evidence from several laboratories suggests that small GTPases play an important role in the promotion of neurite outgrowth; however, their role in the control of synaptic growth and functional plasticity is not well understood. The goal of this thesis is to investigate the role of small GTPases (including Rac, Rho and Cdc42) in the regulation of synaptic growth in vivo, using the Drosophila larval neuromuscular junction (NMJ) synapses as a model system. Our results show that presynaptic overexpression of Rac, but not of Rho or Cdc42, positively regulates both synaptic structure and function. Genetic loss of Rac leads to embryonic lethality, making it impossible to assess the full loss-of-function phenotype using conventional mutants. To circumvent this, we use the MARCM (Mosaic Analysis with a Repressible Cell Marker) technique to generate single motor neuron clones devoid of all genetic copies of Rac. Our data suggest that Rac activity is crucial for normal synaptic development. In support of this conclusion, we demonstrate that genetic removal of trio, a guanine nucleotide exchange factor (GEF) that is known to activate Rac, leads to a drastic reduction in the number of synaptic boutons. In addition, genetic removal of one copy of trio is sufficient to suppress the gain-of-function phenotype of Rac. Moreover, we demonstrate that partial removal of the fragile X mental retardation gene (dfmr1), a known suppressor of Rac, enhances the gain-of-function phenotype of Rac. Taken together, our findings support a model in which Rac signaling positively regulates synaptic growth and function at the Drosophila larval NMJ.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 5 uppsatser.

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.

INTRODUÇÃO: A técnica de eletromiografia de fibra única (EMGFU), mediante análise do jitter, é o método neurofisiológico mais sensível para a confirmação do distúrbio da junção neuromuscular na miastenia gravis (MG). Os registros são tradicionalmente obtidos com agulha de fibra única, de alto custo e reutilizável. Por causa da necessidade atual do uso de material descartável, a agulha concêntrica vem sendo utilizada em substituição à agulha de fibra única. A técnica utilizada é semelhante, porém os potencias de ação para a análise do jitter são obtidos com eletrodo de agulha concêntrica (Eletromiografia de fibra única - jitter com agulha concêntrica, EMGFU-JAC). Contudo, os estudos são escassos e as metodologias utilizadas são heterogêneas com a utilização dessa agulha. OBJETIVOS: Este estudo tem por objetivo mensurar os valores de jitter obtidos com agulha concêntrica, no músculo Orbicularis Oculi, em sujeitos saudáveis e em pacientes com MG autoimune adquirida e avaliar a validade do método nas formas generalizada e ocular da doença. MÉTODOS: Foram estudados 20 sujeitos saudáveis, 20 pacientes com miastenia gravis forma generalizada (grupo MGG) e 13 com a forma ocular da doença (grupo MGO). A EMGFU-JAC foi realizada em todos os participantes, idealmente com 20 medidas de jitter em cada estudo. O jitter foi expresso como a média das diferenças consecutivas (MCD). Em todos os pacientes do estudo foram realizados o teste de estimulação repetitiva e dosagem sérica de anticorpo antirreceptor de acetilcolina (ac-AChR) no momento da análise do jitter. Nos pacientes soronegativos para ac-AChR, foi pesquisado o anticorpo antimúsculo específico tirosina-quinase (ac-MuSK). Foram definidos o limite superior da normalidade (LSN) para a média do MCD de cada estudo e para valores individuais de MCD. Os critérios de anormalidade foram: (1) média do MCD acima do LSN; ou (2) mais de 10% dos valores individuais de MCD acima do LSN. A definição do LSN para valores individuais de MCD baseou-se no conceito de que dois entre 20 valores de MCD acima do LSN são aceitáveis em um músculo saudável, para a técnica de contração voluntária. Portanto, estimou-se o LSN para o 18o valor mais alto de MCD (18o par). Para análise da acurácia do método, foram construídas duas curvas ROC (Receiver Operating Characteristic) para as variáveis média do MCD e 18o par, no grupo de pacientes (MGG e MGO) versus controle. RESULTADOS: No grupo controle a média das médias do MCD foi (19,0 ± 2,4)us e a média do 18o valor mais alto de cada estudo foi (24,5 ± 3,6)us. Esses valores obtidos apresentaram distribuição Gaussiana e o LSN foi definido como a média desses valores + 2 DP. O LSN para a média do MCD foi 24us, e 32?s para valores individuais de MCD. No grupo MGG, a análise do jitter foi anormal em todos os 20 pacientes por ambos os critérios de anormalidade, exceto em um paciente que apresentou anormalidade por apenas um dos critérios. No grupo MGO, apenas um dos 13 pacientes não preencheu os critérios de anormalidade. No grupo de pacientes, a positividade da EMGFU-JAC foi maior do que o teste de estimulação repetitiva e dosagens de anticorpos. Nas curvas ROC para as variáveis médias do MCD e 18o par, o valor de melhor sensibilidade (93,9%), sem resultados falsos positivos, foi 24,7us e 33,1us, respectivamente. CONCLUSÕES: A EMGFU-JAC apresenta alta sensibilidade e especificidade na identificação de distúrbio da transmissão neuromuscular em pacientes com MG. A utilização da agulha concêntrica é válida para a análise do jitter, como alternativa à agulha de fibra única / INTRODUCTION: Single fiber electromyography (SFEMG) technique, through jitter analysis, is the most sensitive neurophysiological method for confirmation of neuromuscular junction disorder in myasthenia gravis (MG). Records are traditionally obtained with single fiber needle, which is reusable and has a high-cost. Due to the current need of using disposable material, concentric needle has been used to replace single fiber needle. The technique is similar, but the action potential for jitter analysis is obtained with concentric needle electrode (SFEMG - concentric needle jitter, SFEMG-CNJ). However, studies are scarce and methodologies used are heterogeneous with the use of this needle. OBJECTIVES: This study aims to measure jitter values obtained with concentric needle in the Orbicularis Occuli muscle in healthy subjects and in patients with autoimmune acquired MG and to assess the validity of the method in generalized and ocular forms of the disease. METHODS: 20 healthy subjects, 20 patients with generalized myasthenia gravis (GMG group) and 13 with the ocular form of the disease (OMG group) were studied. SFEMG-CNJ was performed on all participants, ideally with 20 jitter values in each study. Jitter was expressed as the mean consecutive difference (MCD). Repetitive nerve stimulation and serum acetylcholine receptor antibody (AChR-ab) were performed in all patients in the study, by the time of jitter analysis. Tyrosine kinase specific antibody muscle antibodies (MuSK-ab) were performed in AChR-ab negative patients. The upper limit of normality (ULN) for the mean MCD and for individual jitter values were defined. The abnormality criteria were: (1) mean MCD above ULN; or (2) more than 10% of individual jitter values above ULN. The definition of ULN for individual jitter values was based on the concept that two out of 20 jitter values above ULN are acceptable in a healthy muscle for voluntary contraction technique. Therefore, the ULN for the 18th highest jitter value (18 pair) was estimated. To analyze the method\'s accuracy, two ROC curves (Receiver Operating Characteristic) for the mean MCD and 18th pair in the group of patients (MGG and MGO) versus control were constructed. RESULTS: In the control group the mean of MCD means was (19.0 ± 2.4)us and the mean of the 18 highest value of each study was (24.5 ± 3.6)us. These values showed Gaussian distribution and the ULN was set as the mean of these values + 2 SD. The ULN for the mean MCD was 24us, and 32us for individual values of MCD. In GMG group, jitter analyses were abnormal in all 20 patients based on both abnormality criteria, except in one patient, who had abnormalities in only one of the criteria. In OMG group, only one patient from 13 met neither of the abnormality criteria. In patients, the positivity of SFEMG-CNJ was higher than repetitive nerve stimulation test and antibody detection. The ROC curve threshold showing the best sensitivity (93.9%) with no false positive results was 24.7Us for the mean MCD and 33.1us for individual pairs, respectively. CONCLUSIONS: SFEMG-CNJ has high sensitivity and specificity in identifying neuromuscular transmission disorder in patients with MG. The use of concentric needle is valid for jitter analysis as an alternative to single fiber needle