Transforming growth factor-βs (TGF-βs) are highly expressed in neural development but why the adult nervous system continues to express them is unclear. TGF-β2 is concentrated at mature neuromuscular junctions (NMJs) of mammalian skeletal muscle fibres, and the nerve terminal expresses TβR-II receptors. To test the role of TGF-β2 at mammalian NMJs, I performed four experiments. The first study tested whether TGF-β2 acutely modulates synaptic transmission at mature mammalian NMJs. Second, I asked if chronically reduced TGF-β2 expression disrupts synaptic transmission. Third, I asked if TGF-β2’s effects differ in terminals adapted to different activity patterns in vivo. Lastly, I asked whether TGF-β1, a related peptide to TGF-β2, is distinct in terms of its effects on transmitter release. Using single electrode potential recording, I found TGF-β2 significantly increased the amplitude of spontaneous released single neurotransmitter vesicles (miniature endplate potentials, MEPPs) and nerve stimulation evoked multi-vesicular release (endplate potentials, EPPs). These effects were blocked by L-vesamicol, a vesicular acetylcholine transporter inhibitor, and bafilomycin, a proton pump inhibitor, suggesting the increase in MEPP/EPP amplitude is due to increased vesicle filling presynaptically. These effects were also blocked by the MARK inhibitors, UO126 and PD98059, suggesting TGF-β2 acts via a MARK-dependent pathway. Postsynaptically, two electrode recording showed postsynaptic potential amplitude was enhanced by an increased fibre input resistance, suggesting TGF-β2 also acts postsynaptically. TGF-β2 reduced the number of vesicles released per stimulus (quanta content, QC) but this was blocked by atropine, showing this was indirect through autoreceptor negative feedback. Voltage clamp recording showed TGF-β2 significantly increased the miniature end plate currents (MEPCs), but not the end-plate currents (EPCs), supporting my initial hypothesis that TGF-β2 acts mainly presynaptically to increase vesicle filling. In TGF-β2+/- mice, I found greater MEPP amplitude variability. This supports my previous findings that TGF-β2 modulates vesicle filling. Unexpectedly, there was an excess in larger MEPP sizes (>0.88 mV), perhaps reflecting upregulation of either presynaptic signalling or another synaptic mediator. Two MEPP amplitude populations were induced in TGF-β2-treated TGF-β2+/- mouse NMJs, similar to the bimodal vesicle population in electroplaques. The extensor digitorum longus (EDL, ~95% fast fibres) and soleus (SOL, ~95% slow fibres) were used to investigate whether the TGF-β2-mediated effect differed between fibre types. Overall, TGF-β2 increased the quantal size (MEPP amplitude) in NMJs of both muscles, suggesting this effect is not fibre-type specific and, together with results in mice, that the TGF-β2-mediated increase in vesicle filling is common to all mammalian neuromuscular terminals. With respect to EPP amplitude and QC, the results differed between muscles. In EDL, the EPP amplitude was not significantly changed, whereas it increased in SOL. In EDL, QC was reduced but not in SOL. These difference compared to diaphragm perhaps do reflect muscle fibre-type dependent differences. TGF-β1, at 0.1 ng/ml, significantly reduced quantal size – the opposite of TGF-β2 at any concentration. One explanation would be that a receptor inhibition by TGF-β1 at low concentration interferes with endogenous TGF-β2 binding/receptor activation at the NMJ. However, when the TGF-β1 concentration was raised to 1 ng/ml, like TGF-β2, it significantly increased MEPP amplitude. This suggests that perhaps sufficient binding of TGF-β1 results in the receptor activation of TGF-β2 like signalling. Overall, I conclude that TGF-β2 enhances the size of spontaneous synaptic potentials in all types of muscle fibres, and this is much more rapid (1 hr vs 1 day) than at central neurone synapses in culture. Detailed study in the rat diaphragm shows it increased the evoked EPP amplitude, reduced QC and increased postsynaptic input resistance. Together, TGF-β2 would therefore enhance the postsynaptic depolarisation increasing synaptic strength, and by reducing QC, increase the efficiency of neurotransmission at mammalian NMJs. While unimportant for single stimuli in healthy terminals, by conserving vesicle use, it may help maintain release during stimulus trains, especially during neuromuscular disease.

Neuromuscular synapse formation is driven by two nerve-derived molecules, agrin and neuregulin. Agrin is believed to signal through a Muscle Specific Kinase (MuSK)/Lrp4 receptor complex to cluster existing acetylcholine receptors (AChRs) in the postsynaptic membrane via a rapsyn-mediated mechanism, while neuregulin signals via ErbB kinase receptors to induce synaptic gene transcription. Recent studies suggest that neuregulin-ErbB signalling may also cross-signal to the agrin-MuSK pathway to modulate agrin’s ability to cluster AChRs. This thesis aimed to further elucidate this idea. Results of this thesis present two novel findings. First, there is a direct interaction between two tyrosine kinase signalling pathways at the neuromuscular synapse and second, neuregulin plays an important role in modulating, modifying and refining AChRs at developing synapses. Here I show that neuregulin can modulate two distinct processes. In the presence of agrin, neuregulin was able to potentiate both agrin-induced AChR clustering and agrin-induced AChR cluster dispersal, and this modulation by neuregulin occurred independently of any transcriptional mechanism. In vitro, I observed a marked effect by neuregulin on the number and size of AChR clusters that were induced by agrin. Treatment of myotubes for 4hrs with agrin and neuregulin led to a significant potentiation in agrin-induced AChR clustering compared to agrin treatment alone. Neuregulin on its own had no measurable effect on AChR clustering. When incubation times were much longer (12hrs), neuregulin promoted a further significant decrease in AChR cluster number compared to agrin treatment alone. Thus at 12hrs, rather than inhibit AChR clustering, as has been previously suggested, neuregulin promoted the dispersal of AChRs from pre-existing agrin-induced clusters. Follow-up in vivo studies into the potentiating ability of neuregulin in agrin-induced AChR clustering showed that the injection of exogenous neuregulin into developing mouse sternomastoid musculature led to an increase in the size of AChRs. Collectively, these data suggest interactions between the signalling pathways initiated by agrin and neuregulin. Subsequent investigation into the second messengers downstream of agrin-MuSK and neuregulin-ErbB signalling revealed that cyclin-dependent kinase 5 (Cdk5) and Shp2 played a role in neuregulin’s modulation of AChR cluster formation and dispersal. It appears that neuregulin enhances the phosphorylation status of MuSK by inhibiting the Shp2-dependent negative feedback loop on MuSK phosphorylation, thereby leading to an increase in AChR cluster numbers. By contrast, the way in which neuregulin disperses agrin-induced AChR clusters seems to occur partially, via a Cdk5 signalling-dependent mechanism. While it is accepted that neuregulin acts in a transcriptional manner during neuromuscular synapse formation, real-time PCR and immunoblot results suggest that transcriptional regulation was not involved in neuregulin’s modulation of agrin-induced AChR clustering.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008 / Vita. Bibliography: p. 23-4

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p. 23-24

The neuromuscular junction (NMJ), the synapse formed between lower motor neuron and skeletal muscle fibre, is known to be a target in a number of neurodegenerative conditions, including motor neuron disease (MND). Located in an accessible part of the peripheral nervous system, the NMJ can be used as a ‘model synapse’ in the context of ‘connectomics’ – the study of synaptic connectivity throughout the nervous system as a whole. Although the NMJ has been studied in a number of species, relatively little is known about its structure in humans, complicating the translation of animal models of disease to the human condition. Described here is the first detailed cellular and molecular characterization of the human NMJ. A standardized methodology for comparative morphometric analysis of NMJs was developed and validated (‘NMJ-morph’). NMJ-morph was used to generate baseline data for 2160 NMJs from a single litter of wild type mice, representing 9 distinct muscles across 3 body regions. Principal components analysis (PCA) revealed synaptic size and fragmentation to be the key determinants of synaptic variability. Correlation data revealed the pre-synaptic cell (motor neuron) to be a stronger predictor of synaptic morphology than the post-synaptic cell (muscle fibre). Other factors influencing synaptic variability were in a clear hierarchy: muscle identity accounted for more variation in synaptic form than animal identity, with side having no effect. Human tissue was obtained from 20 patients (aged 34 to 92 years) undergoing lower limb amputation, primarily for the complications of peripheral vascular disease (PVD). Muscle samples were harvested from non-pathological regions of the surgical discard tissue. 2860 human NMJs were analyzed from 4 distinct muscles (extensor digitorum longus, soleus, peroneus longus and peroneus brevis), and compared with equivalent NMJs from wild type mice. Human NMJs displayed unique morphological characteristics, including small size, thin axons, rudimentary nerve terminals and distinctive ‘nummular’ endplates, all of which distinguished them from equivalent mouse NMJs. The previous notion of partial occupancy in human NMJs was disproved. As in mice, the pre-synaptic cell was shown to correlate more strongly with NMJ morphology; in contrast to mice, the human NMJ was found to be relatively stable throughout its 90+ year lifespan. In support of the tissue harvesting procedure, patient co-morbidities (diabetes mellitus and vascular disease) did not significantly impact NMJ morphology. Super-resolution imaging of the NMJ revealed significant differences in the functional architecture of human and mouse active zones. Despite the smaller synaptic size in humans, the total quantity of active zone material was conserved between the species, suggesting a homeostatic mechanism to preserve effective neurotransmission. Parallel proteomic profiling demonstrated further species-specific differences in the broader molecular composition of the NMJ. The cellular and molecular anatomy of the human NMJ is fundamentally different to that of other mammalian species. These differences must be taken into account when translating animal models of disease to the human condition.

Made available in DSpace on 2014-06-11T19:25:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-15Bitstream added on 2014-06-13T18:26:28Z : No. of bitstreams: 1 cardoso_ff_me_botib.pdf: 693511 bytes, checksum: ce598a499b044fd23ed66dd6f42f2f0a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os acidentes envolvendo as serpentes do gênero Bothrops se destacam no Brasil e em outros países da América Latina, representando 90% das notificações. O envenenamento botrópico é caracterizado por intensa mionecrose local que não é eficientemente neutralizada pelo único tratamento disponível, isto é, a soroterapia. Como conseqüência, em casos graves, este acidente pode levar a amputação de membros, desabilitando a vítima. Os principais responsáveis pelo desenvolvimento da mionecrose são proteínas com estruturas homólogas às enzimas fosfolipases A2 (PLA2s). Entre essas se destacam as variantes cataliticamente inativas que apresentam como característica um resíduo de lisina na posição 49 (Lys49-PLA2s). Tradicionalmente, as Lys49-PLA2s são consideradas miotoxinas não-neurotóxicas, uma vez que não induzem paralisia in vivo. No entanto, em preparações isoladas, tal efeito é observado. Recentemente, sugeriu-se que a paralisia muscular in vitro, da mesma forma que a lesão muscular, resultaria da atividade desestabilizadora de membrana da fibra muscular induzida por estas toxinas. O presente estudo teve como objetivo investigar a relação entre os efeitos miotóxico e paralisante das Lys49-PLA2s e contribuir para o esclarecimento do mecanismo de ação e da relação estrutura/atividade dessas toxinas. Sendo assim, realizaram-se estudos miográficos e morfológicos em preparações neuromusculares de camundongos utilizando a PrTX-I (Lys49-PLA2 isolada do veneno de Bothrops pirajai) e potenciais inibidores vegetais (ácido rosmarínico, ácido caféico e ácido aristolóquico). Os resultados obtidos mostraram as diferentes capacidades dos compostos vegetais em neutralizar os efeitos miotóxico e paralisante da PrTX-I. Assim, o ácido rosmarínico neutralizou ambos os efeitos eficientemente... / Accidents caused by Bothrops snake genus stand out in Brazil and other Latin American countries, representing 90% of notifications. Bothropic envenoming is characterized by intense local myonecrosis, not effectively neutralized by serum therapy, the only available treatment. As a result, in severe cases, these accidents can lead to amputation of limbs, disabling the victim. The main responsibles for myonecrosis development are proteins with homologous structures from enzymes phospholipase A2 (PLA2s). Among these stand out catalytically inactive variants that have a characteristic lysine residue at position 49 (Lys49-PLA2s). Traditionally, Lys49-PLA2s myotoxins are considered non-neurotoxic, since they do not induce paralysis in vivo. However, this effect is observed in isolated preparations. It was recently suggested that muscle paralysis in vitro, as well as muscle injury, would result from the destabilizing activity of the muscle fiber membrane induced by these toxins. This study aimed to investigate the relationship between paralyzing and myotoxic effects of Lys49-PLA2s and contribute to the elucidation of the mechanism of action and structure/activity relationship of these toxins. Therefore, myographical and morphological studies were performed in neuromuscular preparations of mice using PrTX-I (Lys49-PLA2 isolated from Bothrops pirajai venom) and potential inhibitors from plants (rosmarinic acid, caffeic acid and aristolochic acid) as experimental tools. The results showed the different skills of plant compounds to neutralize myotoxic and paralyzing effects of PrTX-I. Thus, the rosmarinic acid efficiently neutralized both effects, whereas caffeic acid only partially inhibited the... (Complete abstract click electronic access below)

Orientador: Lea Rodrigues Simioni, Yoko Oshima Franco / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T10:25:13Z (GMT). No. of bitstreams: 1 RochaJunior_DimasdosSantos_M.pdf: 1074208 bytes, checksum: 78f54f332c4a0788a9a1522ffdfffefa (MD5) Previous issue date: 2008 / Resumo: O dimetilaminoetanol (DMAE) é comercializado em vários países como suplemento da dieta, amplificador da cognição e também como matéria-prima de produtos cosméticos. No Brasil, a Agência Nacional de Vigilância Sanitária aprovou o uso cosmético em 2002, visando a uma ação antienvelhecimento, após a observação do efeito colateral decorrente da administração oral do medicamento Deanol-®: contração involuntária da musculatura do rosto e do pescoço. Pesquisou-se, neste trabalho, a ação do DMAE em preparação isolada do nervo frênico-diafragma de camundongos, utilizando-se técnicas miográfica e eletrofisiológica. DMAE, sob o parâmetro miográfico, mostrou dois efeitos neuromusculares principais, dependentes da concentração: o facilitador da neurotransmissão (1 mg/mL, n = 6 e 4 mg/mL, n = 11) e o bloqueador neuromuscular completo aos 40 minutos (20 mg/mL, n = 4). Em preparações previamente curarizadas (d-Tubocurarina, 10 µg/mL) e sob estímulo direto, o DMAE (8 mg/mL, n = 3) aumentou a amplitude da contração muscular (30%), mostrando uma ação direta sobre a fibra muscular; enquanto sob estímulo indireto, demonstrou não possuir ação anticolinesterásica. O DMAE, sob o parâmetro eletrofisiológico, agiu pré-sinapticamente sobre a terminação nervosa, constatado pelo aumento gradativo dos valores de conteúdo quântico (1 mg/mL, n = 4). As implicações desses resultados são discutidas neste trabalho. / Abstract: The dimethylaminoethanol (DMAE) is commercialized in several countries as diet supplement, amplifier of the cognition and also as substance in the constitution of cosmetic products. In Brazil, the cosmetic use was approved by the Agência Nacional de Vigilância Sanitária, in 2002, aiming at an antiaging action, after the collateral effect from oral Deanol® administration: the involuntary contraction of the face and the neck muscles. The objective of this work was to study the DMAE action in isolated mouse phrenic nerve-diaphragm preparation, using myographical and electrophysiological techniques. DMAE, under myographical parameter, showed two main neuromuscular and concentration-dependents effects: the facilitatory (1 mg/mL, n = 6 and 4 mg/mL, n = 11) and the neuromuscular blocker at 40 min (20 mg/mL, n = 4). In pre-curarized preparations (d-Tubocurarine, 10 µg/mL) and under direct stimuli, DMAE (8 mg/mL, n = 3) increased the amplitude of muscular contraction (30%), showing a direct action on the muscular fiber; whereas under indirect stimuli, showed to have no anticholinesterasic action. DMAE, under electrophysiological parameter did act presynaptically on the nervous terminal by a gradual increase of quantum content values (1 mg/mL, n = 4). The implications of these results are argued in this work. / Mestrado / Mestre em Farmacologia

No description available.

At motor nerve terminals, Hg2+ causes (a) irreversible depolarization, (b) increase in transmitter release, and (c) subsequent irreversible block of transmitter release. All effects are antagonized when a Na channel blocker (tetrodotoxin, TTX) and a Ca channel blocker (Co2+) are present, but not when either blocker is used alone. The effects are not antagonized by TTX plus Co2+ when the mercurial is lipid-soluble (methylmercury). This indicates that the neurotoxic action of Hg2+ is at an intracellular site and that entry is gained through both Na and Ca channels. The results suggest that metals may inhibit transmitter release at either the Ca channel or at the release site, but that irreversible toxicity is due to an intracellular action, possibly involving SH groups.

No description available.