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1.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669246

RESUMO

The accumulation of damaged mitochondria due to insufficient autophagy has been implicated in the pathophysiology of skeletal muscle aging. Ulk1 is an autophagy-related kinase that initiates autophagosome assembly and may also play a role in autophagosome degradation (i.e., autophagy flux), but the contribution of Ulk1 to healthy muscle aging is unclear. Therefore, the purpose of this study was to investigate the role of Ulk1-mediated autophagy in skeletal muscle aging. At age 22 months (80% survival rate), muscle contractile and metabolic function were assessed using electrophysiology in muscle-specific Ulk1 knockout mice (MKO) and their littermate controls (LM). Specific peak-isometric torque of the ankle dorsiflexors (normalized by tibialis anterior muscle cross-sectional area) and specific force of the fast-twitch extensor digitorum longus muscles was reduced in MKO mice compared to LM mice (p < 0.03). Permeabilized muscle fibers from MKO mice had greater mitochondrial content, yet lower mitochondrial oxygen consumption and greater reactive oxygen species production compared to fibers from LM mice (p ≤ 0.04). Alterations in neuromuscular junction innervation patterns as well as changes to autophagosome assembly and flux were explored as possible contributors to the pathological features in Ulk1 deficiency. Of primary interest, we found that Ulk1 phosphorylation (activation) to total Ulk1 protein content was reduced in older muscles compared to young muscles from both human and mouse, which may contribute to decreased autophagy flux and an accumulation of dysfunctional mitochondria. Results from this study support the role of Ulk1-mediated autophagy in aging skeletal muscle, reflecting Ulk1's dual role in maintaining mitochondrial integrity through autophagosome assembly and degradation.


Assuntos
Envelhecimento/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/deficiência , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/metabolismo , Contração Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Debilidade Muscular/metabolismo , Transdução de Sinais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Junção Neuromuscular/metabolismo , Fosforilação/genética , Espécies Reativas de Oxigênio/metabolismo , Adulto Jovem
2.
Arch Biochem Biophys ; 701: 108788, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33548213

RESUMO

The cholinergic neuromuscular junction is the paradigm peripheral synapse between a motor neuron nerve ending and a skeletal muscle fiber. In vertebrates, acetylcholine is released from the presynaptic site and binds to the nicotinic acetylcholine receptor at the postsynaptic membrane. A variety of pathologies among which myasthenia gravis stands out can impact on this rapid and efficient signaling mechanism, including autoimmune diseases affecting the nicotinic receptor or other synaptic proteins. Cholesterol is an essential component of biomembranes and is particularly rich at the postsynaptic membrane, where it interacts with and modulates many properties of the nicotinic receptor. The profound changes inflicted by myasthenia gravis on the postsynaptic membrane necessarily involve cholesterol. This review analyzes some aspects of myasthenia gravis pathophysiology and associated postsynaptic membrane dysfunction, including dysregulation of cholesterol metabolism in the myocyte brought about by antibody-receptor interactions. In addition, given the extensive therapeutic use of statins as the typical cholesterol-lowering drugs, we discuss their effects on skeletal muscle and the possible implications for MG patients under chronic treatment with this type of compound.


Assuntos
Colesterol/metabolismo , Miastenia Gravis/metabolismo , Junção Neuromuscular/metabolismo , Animais , Humanos , Miastenia Gravis/patologia , Junção Neuromuscular/patologia
3.
Biochem Biophys Res Commun ; 537: 125-131, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33401146

RESUMO

Four-and-a-half LIM domain protein 1 (FHL1) is a member of the FHL protein family that serves as a scaffold protein to maintain normal cellular structure and function. Its mutations have been implicated in multiple muscular diseases. These FHL1 related myopathies are characterized by symptoms such as progressive muscle loss, rigid or bent spine, even cardiac or respiratory failure in some patients, which implies pathological problems not only in muscles, but also in the nervous system. Moreover, decreased FHL1 protein level has been found in patients with FHL1 mutations, indicating the protein loss-of-function as a pathological cause of such diseases. These findings suggest the significance of understanding the systemic role of FHL1 in the homeostasis of nervous system and muscle. Here we reported that Fhl1 loss in C2C12 myotubes obscured acetylcholine receptor (AChR) clustering in addition to myotube fusion, which was associated with impaired MuSK phosphorylation. Mechanistically, myostatin-SMAD2/3 signaling was enhanced, whereas IGF-PI3K-AKT signaling was suppressed in Fhl1-/- C2C12 myotubes. Reversion of these molecular alterations rescued AChR clustering and differentiation deficits. These data outline a systemic regulation of AChR clustering and myotube fusion by FHL1, which may offer clues for mechanism study and development of therapeutic strategies to treat FHL1 related myopathies.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Miostatina/metabolismo , Junção Neuromuscular/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Folistatina/farmacologia , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/metabolismo
4.
Neurosci Lett ; 746: 135663, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33493647

RESUMO

Hevin and secreted protein acidic and rich in cysteine (SPARC) are highly homologous matricellular proteins that function in concert to guide the formation of brain synapses. Here, we investigated the role of these glycoproteins in neuromuscular junction (NMJ) maturation, stability, and repair following injury. Hevin and SPARC mRNA levels in developing (postnatal day 9), adult (postnatal days 90 and 120), and injured (fibular nerve crush) skeletal muscles were assessed with qPCR. Muscle fiber size was analyzed in developing (P9) mice lacking SPARC, Hevin, and both SPARC and Hevin. NMJ morphology was assessed in developing (P9), adult (P90) and injured (fibular nerve crush) mice lacking SPARC, Hevin, and both SPARC and Hevin skeletal muscle. Hevin and SPARC are expressed in skeletal muscles and are upregulated following nerve injury. Hevin-/- mice exhibited delayed NMJ and muscle fiber development but displayed normal NMJ morphology in adulthood and accelerated NMJ reinnervation following nerve injury. Mice lacking SPARC displayed normal NMJ and muscle fiber development but exhibited smaller NMJs with fewer acetylcholine receptor islands in adulthood. Further, SPARC deletion did not result in overt changes in NMJ reformation following nerve injury. The combined deletion of Hevin and SPARC had little effect on NMJ phenotypes observed in single knockouts, however deletion of SPARC in combination with Hevin reversed deficiencies in muscle fiber maturation observed in Hevin-/- muscle. These results identify SPARC and Hevin as extracellular matrix proteins with roles in NMJ development and repair.


Assuntos
Proteínas de Ligação ao Cálcio/biossíntese , Proteínas da Matriz Extracelular/biossíntese , Junção Neuromuscular/crescimento & desenvolvimento , Junção Neuromuscular/metabolismo , Osteonectina/biossíntese , Sinapses/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mioblastos/metabolismo , Osteonectina/genética
6.
J Vis Exp ; (159)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32478717

RESUMO

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology.  To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.


Assuntos
Dendritos/metabolismo , Drosophila melanogaster/metabolismo , Imageamento Tridimensional , Microtúbulos/metabolismo , Junção Neuromuscular/metabolismo , Imagem Individual de Molécula , Sinapses/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Larva/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Interferência de RNA , Software
7.
Nat Commun ; 11(1): 2674, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471987

RESUMO

Increasing evidence indicates that guidance molecules used during development for cellular and axonal navigation also play roles in synapse maturation and homeostasis. In C. elegans the netrin receptor UNC-40/DCC controls the growth of dendritic-like muscle cell extensions towards motoneurons and is required to recruit type A GABA receptors (GABAARs) at inhibitory neuromuscular junctions. Here we show that activation of UNC-40 assembles an intracellular synaptic scaffold by physically interacting with FRM-3, a FERM protein orthologous to FARP1/2. FRM-3 then recruits LIN-2, the ortholog of CASK, that binds the synaptic adhesion molecule NLG-1/Neuroligin and physically connects GABAARs to prepositioned NLG-1 clusters. These processes are orchestrated by the synaptic organizer CePunctin/MADD-4, which controls the localization of GABAARs by positioning NLG-1/neuroligin at synapses and regulates the synaptic content of GABAARs through the UNC-40-dependent intracellular scaffold. Since DCC is detected at GABA synapses in mammals, DCC might also tune inhibitory neurotransmission in the mammalian brain.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Moléculas de Adesão Celular/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de GABA-A/metabolismo , Transmissão Sináptica/fisiologia , Animais , Orientação de Axônios/fisiologia , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Helminto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/metabolismo , Sinapses/fisiologia
8.
Neuron ; 107(1): 95-111.e6, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32380032

RESUMO

Progressive synapse loss is an inevitable and insidious part of age-related neurodegenerative disease. Typically, synapse loss precedes symptoms of cognitive and motor decline. This suggests the existence of compensatory mechanisms that can temporarily counteract the effects of ongoing neurodegeneration. Here, we demonstrate that presynaptic homeostatic plasticity (PHP) is induced at degenerating neuromuscular junctions, mediated by an evolutionarily conserved activity of presynaptic ENaC channels in both Drosophila and mouse. To assess the consequence of eliminating PHP in a mouse model of ALS-like degeneration, we generated a motoneuron-specific deletion of Scnn1a, encoding the ENaC channel alpha subunit. We show that Scnn1a is essential for PHP without adversely affecting baseline neural function or lifespan. However, Scnn1a knockout in a degeneration-causing mutant background accelerated motoneuron loss and disease progression to twice the rate observed in littermate controls with intact PHP. We propose a model of neuroprotective homeostatic plasticity, extending organismal lifespan and health span.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Homeostase/fisiologia , Plasticidade Neuronal/fisiologia , Neuroproteção/fisiologia , Terminações Pré-Sinápticas/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Drosophila melanogaster , Camundongos , Camundongos Knockout , Junção Neuromuscular/metabolismo
9.
Am J Physiol Cell Physiol ; 319(1): C116-C128, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32374678

RESUMO

Satellite cell (SC) activation, mediated by nitric oxide (NO), is essential to myogenic repair, whereas myotube function requires innervation. Semaphorin (Sema) 3A, a neuro-chemorepellent, is thought to regulate axon guidance to neuromuscular junctions (NMJs) during myotube differentiation. We tested whether "premature" SC activation (SC activation before injury) by a NO donor (isosorbide dinitrate) would disrupt early myogenesis and/or NMJs. Adult muscle was examined during regeneration in two models of injury: myotoxic cardiotoxin (CTX) and traumatic crush (CR) (n = 4-5/group). Premature SC activation was confirmed by increased DNA synthesis by SCs immediately in pretreated mice after CTX injury. Myotubes grew faster after CTX than after CR; growth was accelerated by pretreatment. NMJ maturation, classified by silver histochemistry (neurites) and acetylcholinesterase (AchE), and α-bungarotoxin staining (Ach receptors, AchRs) were delayed by pretreatment, consistent with a day 6 rise in the denervation marker γ-AchR. With pretreatment, S100B from terminal Schwann cells (TSCs) increased 10- to 20-fold at days 0 and 10 after CTX and doubled 6 days after CR. Premature SC activation disrupted motoneuritogenesis 8-10 days post-CTX, as pretreatment reduced colocalization of pre- and postsynaptic NMJ features and increased Sema3A-65. Premature SC activation before injury both accelerated myogenic repair and disrupted NMJ remodeling and maturation, possibly by reducing Sema3A neuro-repulsion and altering S100B. This interpretation extends the model of Sema3A-mediated motoneuritogenesis during muscle regeneration. Manipulating the timing and type of Sema3A by brief NO effects on SCs suggests an important role for TSCs and Sema3A-65 processing in axon guidance and NMJ restoration during muscle repair.


Assuntos
Desenvolvimento Muscular/fisiologia , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Junção Neuromuscular/metabolismo , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/metabolismo , Animais , Diferenciação Celular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Junção Neuromuscular/patologia , Células Satélites de Músculo Esquelético/patologia
10.
J Neurosci ; 40(18): 3504-3516, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32265260

RESUMO

The action potential (AP) waveform controls the opening of voltage-gated calcium channels and contributes to the driving force for calcium ion flux that triggers neurotransmission at presynaptic nerve terminals. Although the frog neuromuscular junction (NMJ) has long been a model synapse for the study of neurotransmission, its presynaptic AP waveform has never been directly studied, and thus the AP waveform shape and propagation through this long presynaptic nerve terminal are unknown. Using a fast voltage-sensitive dye, we have imaged the AP waveform from the presynaptic terminal of male and female frog NMJs and shown that the AP is very brief in duration and actively propagated along the entire length of the terminal. Furthermore, based on measured AP waveforms at different regions along the length of the nerve terminal, we show that the terminal is divided into three distinct electrical regions: A beginning region immediately after the last node of Ranvier where the AP is broadest, a middle region with a relatively consistent AP duration, and an end region near the tip of nerve terminal branches where the AP is briefer. We hypothesize that these measured changes in the AP waveform along the length of the motor nerve terminal may explain the proximal-distal gradient in transmitter release previously reported at the frog NMJ.SIGNIFICANCE STATEMENT The AP waveform plays an essential role in determining the behavior of neurotransmission at the presynaptic terminal. Although the frog NMJ is a model synapse for the study of synaptic transmission, there are many unknowns centered around the shape and propagation of its presynaptic AP waveform. Here, we demonstrate that the presynaptic terminal of the frog NMJ has a very brief AP waveform and that the motor nerve terminal contains three distinct electrical regions. We propose that the changes in the AP waveform as it propagates along the terminal can explain the proximal-distal gradient in transmitter release seen in electrophysiological studies.


Assuntos
Potenciais de Ação/fisiologia , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Feminino , Previsões , Masculino , Junção Neuromuscular/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Rana pipiens , Bloqueadores dos Canais de Sódio/farmacologia , Fatores de Tempo
11.
Sci Rep ; 10(1): 5689, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32231214

RESUMO

Drosophila melanogaster is a useful and highly tractable model organism for understanding the molecular mechanisms of human diseases. We previously characterized a new dUbqn knockdown model that induces learning-memory and locomotive deficits mediated by impaired proteostasis. Although proteinopathies are the main causes of neurodegenerative diseases, limited information is currently available on the relationship between proteostasis and neurodegenerative-related behavioral perturbations, such as locomotion, wakefulness, and sexual activities. Thus, the present study aimed to elucidate the mechanisms by which dUbqn depletion which is known to cause proteinopathies, affects neurodegenerative-related behavioral perturbations. Pan-neuronal dUbqn-depleted flies showed significantly reduced evening activity along with altered pre- and postsynaptic structural NMJ's proteins by attenuating signals of Bruchpilot puncta and GluRIIA clustering. In addition, the neurochemical profiles of GABA, glutamate, dopamine, and serotonin were disturbed and these changes also affected courtship behaviors in dUbqn-depleted flies. Collectively, these results extend our understanding on how dUbqn depletion affects neurochemical regulation to drive behavioral disturbances that are generally found in the early stage of neurodegenerative diseases. Moreover, the present study may contribute a novel finding to the design of new agents that prevent disease progression or even treat diseases related to neurodegeneration.


Assuntos
Drosophila melanogaster/metabolismo , Ubiquitina/deficiência , Animais , Corte , Dopamina/metabolismo , Dopamina/fisiologia , Drosophila melanogaster/fisiologia , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Ácido Glutâmico/metabolismo , Ácido Glutâmico/fisiologia , Larva , Masculino , Espectrometria de Massas , Atividade Motora/fisiologia , Sistema Nervoso/metabolismo , Sistema Nervoso/fisiopatologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiologia , Serotonina/metabolismo , Serotonina/fisiologia , Espectrometria de Massas em Tandem , Ubiquitina/metabolismo , Ubiquitina/fisiologia , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/fisiologia
12.
Neurobiol Aging ; 90: 93-98, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32169355

RESUMO

Partitioning-defective 1 (PAR-1), a conserved cell polarity regulator, plays an important role in synaptic development, and its mutation affects the formation of synaptic boutons and localization of postsynaptic density protein Discs large (Dlg) at the neuromuscular junction (NMJ) in Drosophila. Drosophila PAR-1 and its human homolog, Microtubule affinity-regulating kinases (MARK), are also known to be implicated in Alzheimer's disease (AD) by controlling tau-mediated Aß toxicity. However, the molecular mechanisms of PAR-1 function remain incompletely understood. Here we identified Pod-1, an actin-microtubule crosslinker, which functionally and physically interacts with PAR-1 in Drosophila. Pod-1 prominently co-localizes with PAR-1 in the postsynaptic region and regulates PAR-1 activity at the NMJ. Synaptic defects, including the reduction of boutons and delocalization of Dlg caused by PAR-1 overexpression, were rescued by Pod-1 knockdown. Conversely, the reduction of synaptic boutons in PAR-1 overexpressed NMJ was synergistically enhanced by the overexpression of Pod-1. Furthermore, Pod-1 increases the PAR-1 dependent S262 phosphorylation of tau, which is known to contribute to tau-mediated Aß toxicity. In line with the change of tau phosphorylation, Pod-1 knockdown rescued tau-mediated synaptic toxicity at the NMJ. Our results suggest that Pod-1 may act as a modulator of PAR-1 in synaptic development and tau-mediated toxicity.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Sinapses/metabolismo , Sinapses/fisiologia , Proteínas tau/metabolismo , Animais , Drosophila , Proteínas de Drosophila/genética , Epistasia Genética , Quinase 3 da Glicogênio Sintase/genética , Junção Neuromuscular/metabolismo , Fosforilação/genética
13.
PLoS One ; 15(3): e0221006, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32187190

RESUMO

Homeodomain-interacting protein kinases (Hipks) have been previously associated with cell proliferation and cancer, however, their effects in the nervous system are less well understood. We have used Drosophila melanogaster to evaluate the effects of altered Hipk expression on the nervous system and muscle. Using genetic manipulation of Hipk expression we demonstrate that knockdown and over-expression of Hipk produces early adult lethality, possibly due to the effects on the nervous system and muscle involvement. We find that optimal levels of Hipk are critical for the function of dopaminergic neurons and glial cells in the nervous system, as well as muscle. Furthermore, manipulation of Hipk affects the structure of the larval neuromuscular junction (NMJ) by promoting its growth. Hipk regulates the phosphorylation of the synapse-associated cytoskeletal protein Hu-li tai shao (Hts; adducin in mammals) and modulates the expression of two important protein kinases, Calcium-calmodulin protein kinase II (CaMKII) and Partitioning-defective 1 (PAR-1), all of which may alter neuromuscular structure/function and influence lethality. Hipk also modifies the levels of an important nuclear protein, TBPH, the fly orthologue of TAR DNA-binding protein 43 (TDP-43), which may have relevance for understanding motor neuron diseases.


Assuntos
Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/enzimologia , Drosophila melanogaster/fisiologia , Músculos/anatomia & histologia , Músculos/metabolismo , Sistema Nervoso/anatomia & histologia , Sistema Nervoso/metabolismo , Proteínas Quinases/isolamento & purificação , Animais , Padronização Corporal , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Olho/embriologia , Larva/metabolismo , Masculino , Músculos/citologia , Sistema Nervoso/citologia , Junção Neuromuscular/metabolismo , Tamanho do Órgão , Fosforilação , Sinapses/metabolismo
14.
PLoS One ; 15(2): e0228348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32032373

RESUMO

The synaptic vesicle protein, synaptotagmin, is the principle Ca2+ sensor for synaptic transmission. Ca2+ influx into active nerve terminals is translated into neurotransmitter release by Ca2+ binding to synaptotagmin's tandem C2 domains, triggering the fast, synchronous fusion of multiple synaptic vesicles. Two hydrophobic residues, shown to mediate Ca2+-dependent membrane insertion of these C2 domains, are required for this process. Previous research suggested that one of its tandem C2 domains (C2B) is critical for fusion, while the other domain (C2A) plays only a facilitatory role. However, the function of the two hydrophobic residues in C2A have not been adequately tested in vivo. Here we show that these two hydrophobic residues are absolutely required for synaptotagmin to trigger vesicle fusion. Using in vivo electrophysiological recording at the Drosophila larval neuromuscular junction, we found that mutation of these two key C2A hydrophobic residues almost completely abolished neurotransmitter release. Significantly, mutation of both hydrophobic residues resulted in more severe deficits than those seen in synaptotagmin null mutants. Thus, we report the most severe phenotype of a C2A mutation to date, demonstrating that the C2A domain is absolutely essential for synaptotagmin's function as the electrostatic switch.


Assuntos
Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Transmissão Sináptica , Sinaptotagminas/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Potenciais Pós-Sinápticos Excitadores , Humanos , Larva/metabolismo , Larva/fisiologia , Mutagênese Sítio-Dirigida , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Alinhamento de Sequência , Sinaptotagminas/química , Sinaptotagminas/genética
15.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32040548

RESUMO

Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. Here, we show that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator of a plasticity gene that is strongly associated with human ataxias.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Junção Neuromuscular/metabolismo , Plasticidade Neuronal , Proteínas de Ligação a RNA/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Músculo Esquelético/embriologia , Junção Neuromuscular/embriologia , Junção Neuromuscular/genética , Proteínas de Ligação a RNA/genética , Fatores de Tempo
16.
Med Sci Sports Exerc ; 52(7): 1477-1484, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31985575

RESUMO

PURPOSE: Doxorubicin (DOX) is a highly effective antitumor agent widely used in cancer treatment. However, it is well established that DOX induces muscular atrophy and impairs force production. Although no therapeutic interventions exist to combat DOX-induced muscle weakness, endurance exercise training has been shown to reduce skeletal muscle damage caused by DOX administration. Numerous studies have attempted to identify molecular mechanisms responsible for exercise-induced protection against DOX myotoxicity. Nevertheless, the mechanisms by which endurance exercise protects against DOX-induced muscle weakness remain elusive. In this regard, impairments to the neuromuscular junction (NMJ) are associated with muscle wasting, and studies indicate that physical exercise can rescue NMJ fragmentation. Therefore, we tested the hypothesis that exercise protects against DOX-induced myopathy by preventing detrimental changes to key proteins responsible for maintenance of the NMJ. METHODS: Female Sprague-Dawley rats were assigned to sedentary or exercise-trained groups. Exercise training consisted of a 5-d treadmill habituation period followed by 10 d of running (60 min·d, 30 m·min, 0% grade). After the last training bout, exercise-trained and sedentary animals were paired with either placebo (saline) or DOX (20 mg·kg i.p.) treatment. Two days after drug treatment, the soleus muscle was excised for subsequent analyses. RESULTS: Our results indicate that endurance exercise training prevents soleus muscle atrophy and contractile dysfunction in DOX-treated animals. These adaptations were associated with the increased expression of the following neurotrophic factors: brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, nerve growth factor, and neurotrophin-3. In addition, exercise enhanced the expression of receptor-associated protein of the synapse and the acetylcholine receptor (AChR) subunits AChRß, AChRδ, and AChRγ in DOX-treated animals. CONCLUSION: Therefore, upregulating neurotrophic factor and NMJ protein expression may be an effective strategy to prevent DOX-induced skeletal muscle dysfunction.


Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas Musculares/genética , Junção Neuromuscular/genética , Condicionamento Físico Animal/fisiologia , Animais , Antineoplásicos/efeitos adversos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Doxorrubicina/efeitos adversos , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Debilidade Muscular/induzido quimicamente , Debilidade Muscular/prevenção & controle , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/prevenção & controle , Junção Neuromuscular/metabolismo , Neurotrofina 3/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos Sprague-Dawley , Receptores Colinérgicos/metabolismo , Regulação para Cima
17.
J Neurosci ; 40(8): 1611-1624, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31964719

RESUMO

The dogma that the synaptic cleft acidifies during neurotransmission is based on the corelease of neurotransmitters and protons from synaptic vesicles, and is supported by direct data from sensory ribbon-type synapses. However, it is unclear whether acidification occurs at non-ribbon-type synapses. Here we used genetically encoded fluorescent pH indicators to examine cleft pH at conventional neuronal synapses. At the neuromuscular junction of female Drosophila larvae, we observed alkaline spikes of over 1 log unit during fictive locomotion in vivo. Ex vivo, single action potentials evoked alkalinizing pH transients of only ∼0.01 log unit, but these transients summated rapidly during burst firing. A chemical pH indicator targeted to the cleft corroborated these findings. Cleft pH transients were dependent on Ca2+ movement across the postsynaptic membrane, rather than neurotransmitter release per se, a result consistent with cleft alkalinization being driven by the Ca2+/H+ antiporting activity of the plasma membrane Ca2+-ATPase at the postsynaptic membrane. Targeting the pH indicators to the microenvironment of the presynaptic voltage gated Ca2+ channels revealed that alkalinization also occurred within the cleft proper at the active zone and not just within extrasynaptic regions. Application of the pH indicators at the mouse calyx of Held, a mammalian central synapse, similarly revealed cleft alkalinization during burst firing in both males and females. These findings, made at two quite different non-ribbon type synapses, suggest that cleft alkalinization during neurotransmission, rather than acidification, is a generalizable phenomenon across conventional neuronal synapses.SIGNIFICANCE STATEMENT Neurotransmission is highly sensitive to the pH of the extracellular milieu. This is readily evident in the neurological symptoms that accompany systemic acid/base imbalances. Imaging data from sensory ribbon-type synapses show that neurotransmission itself can acidify the synaptic cleft, likely due to the corelease of protons and glutamate. It is not clear whether the same phenomenon occurs at conventional neuronal synapses due to the difficulties in collecting such data. If it does occur, it would provide for an additional layer of activity-dependent modulation of neurotransmission. Our findings of alkalinization, rather than acidification, within the cleft of two different neuronal synapses encourages a reassessment of the scope of activity-dependent pH influences on neurotransmission and short-term synaptic plasticity.


Assuntos
Ácido Glutâmico/metabolismo , Junção Neuromuscular/metabolismo , Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Animais , Drosophila , Feminino , Concentração de Íons de Hidrogênio , Plasticidade Neuronal/fisiologia , Vesículas Sinápticas/metabolismo
18.
Sci Rep ; 10(1): 517, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949274

RESUMO

Wnt is a conserved family of secreted proteins that play diverse roles in tissue growth and differentiation. Identification of transcription factors that regulate wnt expression is pivotal for understanding tissue-specific signaling pathways regulated by Wnt. We identified pdm3m7, a new allele of the pdm3 gene encoding a POU family transcription factor, in a lethality-based genetic screen for modifiers of Wingless (Wg) signaling in Drosophila. Interestingly, pdm3m7 larvae showed slow locomotion, implying neuromuscular defects. Analysis of larval neuromuscular junctions (NMJs) revealed decreased bouton number with enlarged bouton in pdm3 mutants. pdm3 NMJs also had fewer branches at axon terminals than wild-type NMJs. Consistent with pdm3m7 being a candidate wg modifier, NMJ phenotypes in pdm3 mutants were similar to those of wg mutants, implying a functional link between these two genes. Indeed, lethality caused by Pdm3 overexpression in motor neurons was completely rescued by knockdown of wg, indicating that Pdm3 acts upstream to Wg. Furthermore, transient expression of Pdm3 induced ectopic expression of wg-LacZ reporter and Wg effector proteins in wing discs. We propose that Pdm3 expressed in presynaptic NMJ neurons regulates wg transcription for growth and development of both presynaptic neurons and postsynaptic muscles.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Junção Neuromuscular/metabolismo , Fatores do Domínio POU/metabolismo , Proteína Wnt1/genética , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica , Masculino , Neurônios Motores/metabolismo , Mutação , Especificidade de Órgãos , Fatores do Domínio POU/genética , Fenótipo , Transcrição Genética
19.
Sci Rep ; 10(1): 99, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919382

RESUMO

Nicotinamide adenine dinucleotide (NAD+) plays a critical role in energy metabolism and bioenergetic homeostasis. Most NAD+ in mammalian cells is synthesized via the NAD+ salvage pathway, where nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme, converting nicotinamide into nicotinamide mononucleotide (NMN). Using a Thy1-Nampt-/- projection neuron conditional knockout (cKO) mouse, we studied the impact of NAMPT on synaptic vesicle cycling in the neuromuscular junction (NMJ), end-plate structure of NMJs and muscle contractility of semitendinosus muscles. Loss of NAMPT impaired synaptic vesicle endocytosis/exocytosis in the NMJs. The cKO mice also had motor endplates with significantly reduced area and thickness. When the cKO mice were treated with NMN, vesicle endocytosis/exocytosis was improved and endplate morphology was restored. Electrical stimulation induced muscle contraction was significantly impacted in the cKO mice in a frequency dependent manner. The cKO mice were unresponsive to high frequency stimulation (100 Hz), while the NMN-treated cKO mice responded similarly to the control mice. Transmission electron microscopy (TEM) revealed sarcomere misalignment and changes to mitochondrial morphology in the cKO mice, with NMN treatment restoring sarcomere alignment but not mitochondrial morphology. This study demonstrates that neuronal NAMPT is important for pre-/post-synaptic NMJ function, and maintaining skeletal muscular function and structure.


Assuntos
Citocinas/fisiologia , Mitocôndrias/patologia , Músculo Esquelético/patologia , Junção Neuromuscular/patologia , Neurônios/patologia , Nicotinamida Fosforribosiltransferase/fisiologia , Transmissão Sináptica , Animais , Feminino , Homeostase , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Junção Neuromuscular/metabolismo , Neurônios/metabolismo
20.
J Clin Invest ; 130(3): 1461-1465, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31794435

RESUMO

Cancer cachexia is a major cause of patient morbidity and mortality, with no efficacious treatment or management strategy. Despite cachexia sharing pathophysiological features with a number of neuromuscular wasting conditions, including age-related sarcopenia, the mechanisms underlying cachexia remain poorly understood. Studies of related conditions suggest that pathological targeting of the neuromuscular junction (NMJ) may play a key role in cachexia, but this has yet to be investigated in human patients. Here, high-resolution morphological analyses were undertaken on NMJs of rectus abdominis obtained from patients undergoing upper GI cancer surgery compared with controls (N = 30; n = 1,165 NMJs). Cancer patients included those with cachexia and weight-stable disease. Despite the low skeletal muscle index and significant muscle fiber atrophy (P < 0.0001) in patients with cachexia, NMJ morphology was fully conserved. No significant differences were observed in any of the pre- and postsynaptic variables measured. We conclude that NMJs remain structurally intact in rectus abdominis in both cancer and cachexia, suggesting that denervation of skeletal muscle is not a major driver of pathogenesis. The absence of NMJ pathology is in stark contrast to what is found in related conditions, such as age-related sarcopenia, and supports the hypothesis that intrinsic changes within skeletal muscle, independent of any changes in motor neurons, represent the primary locus of neuromuscular pathology in cancer cachexia.


Assuntos
Caquexia , Neoplasias Gastrointestinais , Junção Neuromuscular , Reto do Abdome , Caquexia/metabolismo , Caquexia/patologia , Feminino , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Humanos , Masculino , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Reto do Abdome/metabolismo , Reto do Abdome/patologia
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