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1.
Nat Commun ; 11(1): 1004, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081878

RESUMO

Cytoplasmic aggregation of TDP-43 characterizes degenerating neurons in most cases of amyotrophic lateral sclerosis (ALS). Here, we develop an optogenetic TDP-43 variant (opTDP-43), whose multimerization status can be modulated in vivo through external light illumination. Using the translucent zebrafish neuromuscular system, we demonstrate that short-term light stimulation reversibly induces cytoplasmic opTDP-43 mislocalization, but not aggregation, in the spinal motor neuron, leading to an axon outgrowth defect associated with myofiber denervation. In contrast, opTDP-43 forms pathological aggregates in the cytoplasm after longer-term illumination and seeds non-optogenetic TDP-43 aggregation. Furthermore, we find that an ALS-linked mutation in the intrinsically disordered region (IDR) exacerbates the light-dependent opTDP-43 toxicity on locomotor behavior. Together, our results propose that IDR-mediated TDP-43 oligomerization triggers both acute and long-term pathologies of motor neurons, which may be relevant to the pathogenesis and progression of ALS.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Esclerose Amiotrófica Lateral/genética , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Mutação , Optogenética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Multimerização Proteica , Estabilidade Proteica , Regulação para Cima , Peixe-Zebra
2.
RNA ; 26(5): 595-612, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32051223

RESUMO

Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in the maintenance of the axoplasm in a steady state. Recent studies have begun to identify the mRNAs localized in axons, which could be translated locally under different conditions. Despite that by now hundreds or thousands of mRNAs have been shown to be localized into the axonal compartment of cultured neurons in vitro, knowledge of which mRNAs are localized in mature myelinated axons is quite limited. With the purpose of characterizing the transcriptome of mature myelinated motor axons of peripheral nervous systems, we modified the axon microdissection method devised by Koenig, enabling the isolation of the axoplasm RNA to perform RNA-seq analysis. The transcriptome analysis indicates that the number of RNAs detected in mature axons is lower in comparison with in vitro data, depleted of glial markers, and enriched in neuronal markers. The mature myelinated axons are enriched for mRNAs related to cytoskeleton, translation, and oxidative phosphorylation. Moreover, it was possible to define core genes present in axons when comparing our data with transcriptomic data of axons grown in different conditions. This work provides evidence that axon microdissection is a valuable method to obtain genome-wide data from mature and myelinated axons of the peripheral nervous system, and could be especially useful for the study of axonal involvement in neurodegenerative pathologies of motor neurons such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophies (SMA).


Assuntos
Esclerose Amiotrófica Lateral/genética , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , RNA/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Humanos , Microdissecção , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Sistema Nervoso Periférico/metabolismo , Sistema Nervoso Periférico/patologia , RNA Mensageiro/genética , RNA-Seq , Transcriptoma/genética
3.
Ecotoxicol Environ Saf ; 191: 110159, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31962214

RESUMO

Mercury chloride (HgCl2) is a chemical pollutant widely found in the environment. This form of mercury is able to promote several damages to the Central Nervous System (CNS), however the effects of HgCl2 on the spinal cord, an important pathway for the communication between the CNS and the periphery, are still poorly understood. The aim of this work was to investigate the effects of HgCl2 exposure on spinal cord of adult rats. For this, animals were exposed to a dose of 0.375 mg/kg/day, for 45 days. Then, they were euthanized, the spinal cord collected and we investigated the mercury concentrations in medullary parenchyma and the effects on oxidative biochemistry, proteomic profile and tissue structures. Our results showed that exposure to this metal promoted increased levels of Hg in the spinal cord, impaired oxidative biochemistry by triggering oxidative stress, mudulated antioxidant system proteins, energy metabolism and myelin structure; as well as caused disruption in the myelin sheath and reduction in neuronal density. Despite the low dose, we conclude that prolonged exposure to HgCl2 triggers biochemical changes and modulates the expression of several proteins, resulting in damage to the myelin sheath and reduced neuronal density in the spinal cord.


Assuntos
Poluentes Ambientais/toxicidade , Cloreto de Mercúrio/toxicidade , Neurônios Motores/efeitos dos fármacos , Doenças Neurodegenerativas/induzido quimicamente , Proteoma/metabolismo , Medula Espinal/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Masculino , Neurônios Motores/metabolismo , Neurônios Motores/ultraestrutura , Bainha de Mielina/ultraestrutura , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Proteômica , Ratos , Ratos Wistar , Medula Espinal/metabolismo , Medula Espinal/ultraestrutura
4.
Cell Biochem Funct ; 38(1): 21-27, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31774572

RESUMO

Desflurane is one of the commonly used general anaesthetics. Recently, it was reported that desflurane caused neurotoxicity, raising concerns in clinical use. In this study, we found desflurane could affect viability and maturation in motor neurons. Dexmedetomidine, a α2-adrenergic receptor agonist, could attenuate the effect of desflurane on motor neurons. This process was mediated by NF-KappaB signalling. Interestingly, we also found that dexmedetomidine could recover the lesion in motor function and memory impaired by desflurane. Collectively, our results showed the neurotoxic effect of desflurane in motor neurons. More importantly, this process was alleviated by dexmedetomidine, potentially showing its application in protecting motor neuron from neurotoxic agents. Significance of the study: This work provides the evidence to support the protective role of dexmedetomidine in desflurane-induced motor neuron death. Since desflurane is a widely used anaesthetic in surgery and leads to neuron death, the neuroprotective effect of dexmedetomidine holds promising clinical application.


Assuntos
Desflurano/toxicidade , Dexmedetomidina/farmacologia , Neurônios Motores/efeitos dos fármacos , NF-kappa B/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Neurônios Motores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
5.
Nat Commun ; 10(1): 5583, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31811140

RESUMO

Mutations in fused in sarcoma (FUS) lead to amyotrophic lateral sclerosis (ALS) with varying ages of onset, progression and severity. This suggests that unknown genetic factors contribute to disease pathogenesis. Here we show the identification of muscleblind as a novel modifier of FUS-mediated neurodegeneration in vivo. Muscleblind regulates cytoplasmic mislocalization of mutant FUS and subsequent accumulation in stress granules, dendritic morphology and toxicity in mammalian neuronal and human iPSC-derived neurons. Interestingly, genetic modulation of endogenous muscleblind was sufficient to restore survival motor neuron (SMN) protein localization in neurons expressing pathogenic mutations in FUS, suggesting a potential mode of suppression of FUS toxicity. Upregulation of SMN suppressed FUS toxicity in Drosophila and primary cortical neurons, indicating a link between FUS and SMN. Our data provide in vivo evidence that muscleblind is a dominant modifier of FUS-mediated neurodegeneration by regulating FUS-mediated ALS pathogenesis.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteínas do Complexo SMN/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Esclerose Amiotrófica Lateral/genética , Animais , Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Drosophila/genética , Drosophila/metabolismo , Feminino , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neurônios Motores/metabolismo , Mutação , Fenótipo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/toxicidade , Proteínas do Complexo SMN/genética , Fatores de Transcrição/metabolismo
6.
Genes (Basel) ; 10(12)2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31817338

RESUMO

Growing evidence suggests that aberrant energy metabolism could play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Despite this, studies applying advanced technologies to investigate energy metabolism in ALS remain scarce. The rapidly growing field of metabolomics offers exciting new possibilities for ALS research. Here, we review existing and emerging metabolomic tools that could be used to further investigate the role of metabolism in ALS. A better understanding of the metabolic state of motor neurons and their surrounding cells could hopefully result in novel therapeutic strategies.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Metabolismo Energético , Metabolômica , Neurônios Motores/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Humanos , Neurônios Motores/patologia
7.
EBioMedicine ; 50: 274-289, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31787569

RESUMO

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons (MNs). It was shown that human astrocytes with mutations in genes associated with ALS, like C9orf72 (C9) or SOD1, reduce survival of MNs. Astrocyte toxicity may be related to their dysfunction or the release of neurotoxic factors. METHODS: We used human induced pluripotent stem cell-derived astrocytes from ALS patients carrying C9orf72 mutations and non-affected donors. We utilized these cells to investigate astrocytic induced neuronal toxicity, changes in astrocyte transcription profile as well as changes in secretome profiles. FINDINGS: We report that C9-mutated astrocytes are toxic to MNs via soluble factors. The toxic effects of astrocytes are positively correlated with the length of astrocyte propagation in culture, consistent with the age-related nature of ALS. We show that C9-mutated astrocytes downregulate the secretion of several antioxidant proteins. In line with these findings, we show increased astrocytic oxidative stress and senescence. Importantly, media conditioned by C9-astrocytes increased oxidative stress in wild type MNs. INTERPRETATION: Our results suggest that dysfunction of C9-astrocytes leads to oxidative stress of themselves and MNs, which probably contributes to neurodegeneration. Our findings suggest that therapeutic strategies in familial ALS must not only target MNs but also focus on astrocytes to abrogate nervous system injury.


Assuntos
Esclerose Amiotrófica Lateral/etiologia , Esclerose Amiotrófica Lateral/metabolismo , Astrócitos/citologia , Astrócitos/metabolismo , Proteína C9orf72/genética , Células-Tronco Pluripotentes Induzidas/citologia , Mutação , Estresse Oxidativo , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Biomarcadores , Células Cultivadas , Reprogramação Celular , Senescência Celular/genética , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Neurônios Motores/metabolismo , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo
8.
Elife ; 82019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31868588

RESUMO

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting motor neurons (MNs) during late adulthood. Here, with the aim of identifying early changes underpinning ALS neurodegeneration, we analyzed the GABAergic/glycinergic inputs to E17.5 fetal MNs from SOD1G93A (SOD) mice in parallel with chloride homeostasis. Our results show that IPSCs are less frequent in SOD animals in accordance with a reduction of synaptic VIAAT-positive terminals. SOD MNs exhibited an EGABAAR10 mV more depolarized than in WT MNs associated with a KCC2 reduction. Interestingly, SOD GABAergic/glycinergic IPSCs and evoked GABAAR-currents exhibited a slower decay correlated to elevated [Cl-]i. Computer simulations revealed that a slower relaxation of synaptic inhibitory events acts as compensatory mechanism to strengthen GABA/glycine inhibition when EGABAAR is more depolarized. How such mechanisms evolve during pathophysiological processes remain to be determined, but our data indicate that at least SOD1 familial ALS may be considered as a neurodevelopmental disease.


Assuntos
Esclerose Amiotrófica Lateral/genética , Neurônios GABAérgicos/metabolismo , Neurônios Motores/metabolismo , Superóxido Dismutase-1/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Cloretos/metabolismo , Modelos Animais de Doenças , Feto , Neurônios GABAérgicos/patologia , Glicina/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Inibição Neural/genética , Medula Espinal/metabolismo , Medula Espinal/patologia , Simportadores/genética , Transmissão Sináptica/genética , Ácido gama-Aminobutírico/genética , Ácido gama-Aminobutírico/metabolismo
9.
J Neuroinflammation ; 16(1): 218, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727149

RESUMO

BACKGROUND: The development of new therapeutic strategies to treat amyotrophic lateral sclerosis (ALS) is of utmost importance. The use of cyclic nitroxides such as tempol may provide neuroprotection and improve lifespan. We investigated whether tempol (50 mg/kg) presents therapeutic potential in SOD1G93A transgenic mice. METHODS: Tempol treatment began at the asymptomatic phase of the disease (10th week) and was administered every other day until week 14, after which it was administered twice a week until the final stage of the disease. The animals were sacrificed at week 14 (initial stage of symptoms-ISS) and at the end stage (ES) of the disease. The lumbar spinal cord of the animals was dissected and processed for use in the following techniques: Nissl staining to evaluate neuronal survival; immunohistochemistry to evaluate astrogliosis and microgliosis (ISS and ES); qRT-PCR to evaluate the expression of neurotrophic factors and pro-inflammatory cytokines (ISS); and transmission electron microscopy to evaluate the alpha-motoneurons (ES). Behavioral analyses considering the survival of animals, bodyweight loss, and Rotarod motor performance test started on week 10 and were performed every 3 days until the end-stage of the disease. RESULTS: The results revealed that treatment with tempol promoted greater neuronal survival (23%) at ISS compared to untreated animals, which was maintained until ES. The intense reactivity of astrocytes and microglia observed in vehicle animals was reduced in the lumbar spinal cords of the animals treated with tempol. In addition, the groups treated with tempol showed reduced expression of proinflammatory cytokines (IL1ß and TNFα) and a three-fold decrease in the expression of TGFß1 at ISS compared with the group treated with vehicle. CONCLUSIONS: Altogether, our results indicate that treatment with tempol has beneficial effects, delaying the onset of the disease by enhancing neuronal survival and decreasing glial cell reactivity during ALS progression in SOD1G93A mice.


Assuntos
Esclerose Amiotrófica Lateral/fisiopatologia , Óxidos N-Cíclicos/uso terapêutico , Inflamação/tratamento farmacológico , Destreza Motora/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Medula Espinal/efeitos dos fármacos , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Modelos Animais de Doenças , Feminino , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/metabolismo , Masculino , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Destreza Motora/fisiologia , Fármacos Neuroprotetores/farmacologia , Teste de Desempenho do Rota-Rod , Marcadores de Spin , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
10.
Invest Ophthalmol Vis Sci ; 60(14): 4681-4690, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31725167

RESUMO

Purpose: To compare the effects of reduced inhibitory neuron function in the retina across behavioral, physiological, and anatomical levels. Methods: Inhibitory neurons were ablated in larval zebrafish retina. The Ptf1a gene, which determines inhibitory neuron fate in developing vertebrates, was used to express nitroreductase. By exposing larvae to the prodrug metronidazole, cytotoxicity was selectively induced in inhibitory neurons. Visual phenotypes were characterized at behavioral, physiological, and anatomical levels using an optomotor response (OMR) assay, electroretinography (ERG), and routine histology, respectively. Nonvisual locomotion was also assessed to reveal any general behavioral effects due to ablation of other nonvisual neurons that also express Ptf1a. Results: Injured larvae showed severely reduced OMR relative to controls. Locomotor assessment showed unaltered swimming ability, indicating that reduced OMR was due to visual deficits. For ERG, injured larvae manifested either reduced (type-I) or absent (type-II) b-wave signals originating from bipolar interneurons in the retina. Histologic analysis showed altered retinal morphology in injured larvae, with reductions in synaptic inner plexiform layer (IPL) thickness and synaptic density more pronounced in type-II than type-I larvae; type-II larvae also had smaller retinae overall. Conclusions: The consequences of inhibitory neuron ablation corresponded closely across behavioral, physiological, and anatomical levels. Inhibitory neuron loss likely increases the ratio of neural excitation to inhibition, leading to hyperexcitability. In addition to modulating visual signals, inhibitory neurons may be critical for maintaining retinal structure and organization. This study highlights the utility of a multidisciplinary approach and provides a template for characterizing other zebrafish models of neurological disease.


Assuntos
Anti-Infecciosos/toxicidade , Comportamento Animal/fisiologia , Metronidazol/toxicidade , Neurônios Motores/efeitos dos fármacos , Retina/fisiologia , Visão Ocular/fisiologia , Animais , Animais Geneticamente Modificados , Eletrorretinografia , Larva , Neurônios Motores/metabolismo , Nitrorredutases/metabolismo , Estimulação Luminosa , Transdução de Sinais , Fatores de Transcrição/metabolismo , Peixe-Zebra
11.
Mol Cell Neurosci ; 101: 103411, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31648029

RESUMO

Onecut transcription factors are required to maintain Islet1 (Isl1) expression in developing spinal motor neurons (MNs), and this process is critical for proper MN differentiation. However, the mechanisms whereby OC stimulate Isl1 expression remain unknown. CREB-binding protein (CBP) and p300 paralogs are transcriptional coactivators that interact with OC proteins in hepatic cells. In the embryonic spinal cord, CBP and p300 play key roles in neurogenesis and MN differentiation. Here, using chromatin immunoprecipitation and in ovo electroporation in chicken spinal cord, we provide evidence that CBP and p300 contribute to the regulation of Isl1 expression by the OC factors in embryonic spinal MNs. CBP and p300 are detected on the CREST2 enhancer of Isl1 where OC factors are also bound. Inhibition of CBP and p300 activity inhibits activation of the CREST2 enhancer and prevents the stimulation of Isl1 expression by the OC factors. These observations suggest that CBP and p300 coactivators cooperate with OC factors to maintain Isl1 expression in postmitotic MNs.


Assuntos
Proteína de Ligação a CREB/metabolismo , Elementos Facilitadores Genéticos , Proteínas com Homeodomínio LIM/genética , Neurônios Motores/metabolismo , Fatores de Transcrição Onecut/metabolismo , Medula Espinal/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Embrião de Galinha , Proteínas com Homeodomínio LIM/metabolismo , Medula Espinal/citologia , Fatores de Transcrição/metabolismo
12.
Cells ; 8(10)2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31597311

RESUMO

Recent findings in the understanding of amyotrophic lateral sclerosis (ALS) revealed that alteration in calcium (Ca2+) homeostasis may largely contribute to motor neuron demise. A large part of these alterations is due to dysfunctional Ca2+-storing organelles, including the endoplasmic reticulum (ER) and mitochondria. Very recently, lysosomal Ca2+ dysfunction has emerged as an important pathological change leading to neuronal loss in ALS. Remarkably, the Ca2+-storing organelles are interacting with each other at specialized domains controlling mitochondrial dynamics, ER/lysosomal function, and autophagy. This occurs as a result of interaction between specific ionic channels and Ca2+-dependent proteins located in each structure. Therefore, the dysregulation of these ionic mechanisms could be considered as a key element in the neurodegenerative process. This review will focus on the possible role of lysosomal Ca2+ dysfunction in the pathogenesis of several neurodegenerative diseases, including ALS and shed light on the possibility that specific lysosomal Ca2+ channels might represent new promising targets for preventing or at least delaying neurodegeneration in ALS.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Cálcio/metabolismo , Lisossomos/metabolismo , Neurônios Motores/metabolismo , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Retículo Endoplasmático/metabolismo , Homeostase , Humanos , Lisossomos/patologia , Lisossomos/fisiologia , Mitocôndrias/metabolismo , Neurônios Motores/patologia , Neurônios Motores/fisiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia
13.
PLoS One ; 14(10): e0223443, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31584985

RESUMO

Polyethylene glycol repair (PEG-fusion) of severed sciatic axons restores their axoplasmic and membrane continuity, prevents Wallerian degeneration, maintains muscle fiber innervation, and greatly improves recovery of voluntary behaviors. We examined alterations in spinal connectivity and motoneuron dendritic morphology as one potential mechanism for improved behavioral function after PEG-fusion. At 2-112 days after a single-cut or allograft PEG-fusion repair of transected or ablated sciatic nerves, the number, size, location, and morphology of motoneurons projecting to the tibialis anterior muscle were assessed by retrograde labeling. For both lesion types, labeled motoneurons were found in the appropriate original spinal segment, but also in inappropriate segments, indicating mis-pairings of proximal-distal segments of PEG-fused motor axons. Although the number and somal size of motoneurons was unaffected, dendritic distributions were altered, indicating that PEG-fusion preserves spinal motoneurons but reorganizes their connectivity. This spinal reorganization may contribute to the remarkable behavioral recovery seen after PEG-fusion repair.


Assuntos
Neurônios Motores/metabolismo , Condução Nervosa , Neuropatia Ciática/etiologia , Neuropatia Ciática/metabolismo , Potenciais de Ação , Aloenxertos , Animais , Contagem de Células , Dendritos/metabolismo , Modelos Animais de Doenças , Fenômenos Eletrofisiológicos , Feminino , Imuno-Histoquímica , Neurônios Motores/citologia , Regeneração Nervosa , Polietilenoglicóis , Ratos , Recuperação de Função Fisiológica , Neuropatia Ciática/reabilitação
14.
Genetics ; 213(4): 1447-1464, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31585954

RESUMO

Correct scaling of body and organ size is crucial for proper development, and the survival of all organisms. Perturbations in circulating hormones, including insulins and steroids, are largely responsible for changing body size in response to both genetic and environmental factors. Such perturbations typically produce adults whose organs and appendages scale proportionately with final size. The identity of additional factors that might contribute to scaling of organs and appendages with body size is unknown. Here, we report that loss-of-function mutations in Drosophila Activinß (Actß), a member of the TGF-ß superfamily, lead to the production of small larvae/pupae and undersized rare adult escapers. Morphometric measurements of escaper adult appendage size (wings and legs), as well as heads, thoraxes, and abdomens, reveal a disproportional reduction in abdominal size compared to other tissues. Similar size measurements of selected Actß mutant larval tissues demonstrate that somatic muscle size is disproportionately smaller when compared to the fat body, salivary glands, prothoracic glands, imaginal discs, and brain. We also show that Actß control of body size is dependent on canonical signaling through the transcription-factor dSmad2 and that it modulates the growth rate, but not feeding behavior, during the third-instar period. Tissue- and cell-specific knockdown, and overexpression studies, reveal that motoneuron-derived Actß is essential for regulating proper body size and tissue scaling. These studies suggest that, unlike in vertebrates, where Myostatin and certain other Activin-like factors act as systemic negative regulators of muscle mass, in Drosophila, Actß is a positive regulator of muscle mass that is directly delivered to muscles by motoneurons. We discuss the importance of these findings in coordinating proportional scaling of insect muscle mass to appendage size.


Assuntos
Tamanho Corporal , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/metabolismo , Neurônios Motores/metabolismo , Envelhecimento , Animais , Núcleo Celular/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Larva/anatomia & histologia , Larva/crescimento & desenvolvimento , Masculino , Músculos/anatomia & histologia , Mutação/genética , Tamanho do Órgão , Pupa/anatomia & histologia , Transdução de Sinais
15.
Nat Commun ; 10(1): 4147, 2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31515480

RESUMO

Energy metabolism has been repeatedly linked to amyotrophic lateral sclerosis (ALS). Yet, motor neuron (MN) metabolism remains poorly studied and it is unknown if ALS MNs differ metabolically from healthy MNs. To address this question, we first performed a metabolic characterization of induced pluripotent stem cells (iPSCs) versus iPSC-derived MNs and subsequently compared MNs from ALS patients carrying FUS mutations to their CRISPR/Cas9-corrected counterparts. We discovered that human iPSCs undergo a lactate oxidation-fuelled prooxidative metabolic switch when they differentiate into functional MNs. Simultaneously, they rewire metabolic routes to import pyruvate into the TCA cycle in an energy substrate specific way. By comparing patient-derived MNs and their isogenic controls, we show that ALS-causing mutations in FUS did not affect glycolytic or mitochondrial energy metabolism of human MNs in vitro. These data show that metabolic dysfunction is not the underlying cause of the ALS-related phenotypes previously observed in these MNs.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Diferenciação Celular , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação/genética , Proteína FUS de Ligação a RNA/genética , Estudos de Casos e Controles , Respiração Celular , Glucose/metabolismo , Glicólise , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Ácido Láctico/metabolismo , Análise do Fluxo Metabólico , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neurônios Motores/ultraestrutura , Proteína FUS de Ligação a RNA/metabolismo
16.
eNeuro ; 6(5)2019.
Artigo em Inglês | MEDLINE | ID: mdl-31541001

RESUMO

The potassium-chloride cotransporter (KCC2) maintains the low intracellular chloride found in mature central neurons and controls the strength and direction of GABA/glycine synapses. We found that following axotomy as a consequence of peripheral nerve injuries (PNIs), KCC2 protein is lost throughout the somatodendritic membrane of axotomized spinal cord motoneurons after downregulation of kcc2 mRNA expression. This large loss likely depolarizes the reversal potential of GABA/glycine synapses, resulting in GABAergic-driven spontaneous activity in spinal motoneurons similar to previous reports in brainstem motoneurons. We hypothesized that the mechanism inducing KCC2 downregulation in spinal motoneurons following peripheral axotomy might be mediated by microglia or motoneuron release of BDNF and TrkB activation as has been reported on spinal cord dorsal horn neurons after nerve injury, motoneurons after spinal cord injury (SCI), and in many other central neurons throughout development or a variety of pathologies. To test this hypothesis, we used genetic approaches to interfere with microglia activation or delete bdnf from specifically microglia or motoneurons, as well as pharmacology (ANA-12) and pharmacogenetics (F616A mice) to block TrkB activation. We show that KCC2 dysregulation in axotomized motoneurons is independent of microglia, BDNF, and TrkB. KCC2 is instead dependent on neuromuscular innervation; KCC2 levels are restored only when motoneurons reinnervate muscle. Thus, downregulation of KCC2 occurs specifically while injured motoneurons are regenerating and might be controlled by target-derived signals. GABAergic and glycinergic synapses might therefore depolarize motoneurons disconnected from their targets and contribute to augment motoneuron activity known to promote motor axon regeneration.


Assuntos
Neurônios Motores/metabolismo , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/metabolismo , Simportadores/metabolismo , Animais , Axotomia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Membrana Celular/metabolismo , Feminino , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Junção Neuromuscular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Nervo Isquiático/lesões , Transdução de Sinais/fisiologia
17.
Int J Mol Sci ; 20(18)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500113

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease (MND) with no cure. Recent advances in gene therapy open a new perspective to treat this disorder-particularly for the characterized genetic forms. Gene therapy approaches, involving the delivery of antisense oligonucleotides into the central nervous system (CNS) are being tested in clinical trials for patients with mutations in SOD1 or C9orf72 genes. Viral vectors can be used to deliver therapeutic sequences to stably transduce motor neurons in the CNS. Vectors derived from adeno-associated virus (AAV), can efficiently target genes and have been tested in several pre-clinical settings with promising outcomes. Recently, the Food and Drug Administration (FDA) approved Zolgensma, an AAV-mediated treatment for another MND-the infant form of spinal muscular atrophy. Given the accelerated progress in gene therapy, it is potentially a promising avenue to develop an efficient and safe cure for ALS.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/terapia , Terapia Genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Proteína C9orf72/genética , Modelos Animais de Doenças , Edição de Genes , Expressão Gênica , Técnicas de Transferência de Genes , Predisposição Genética para Doença , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Terapia de Alvo Molecular , Neurônios Motores/metabolismo , Mutação , Superóxido Dismutase-1/genética , Transgenes , Resultado do Tratamento
18.
Nat Commun ; 10(1): 4197, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519892

RESUMO

In all vertebrates, excitatory spinal interneurons execute dynamic adjustments in the timing and amplitude of locomotor movements. Currently, it is unclear whether interneurons responsible for timing control are distinct from those involved in amplitude control. Here, we show that in larval zebrafish, molecularly, morphologically and electrophysiologically distinct types of V2a neurons exhibit complementary patterns of connectivity. Stronger higher-order connections from type I neurons to other excitatory V2a and inhibitory V0d interneurons provide timing control, while stronger last-order connections from type II neurons to motor neurons provide amplitude control. Thus, timing and amplitude are coordinated by distinct interneurons distinguished not by their occupation of hierarchically-arranged anatomical layers, but rather by differences in the reliability and probability of higher-order and last-order connections that ultimately form a single anatomical layer. These findings contribute to our understanding of the origins of timing and amplitude control in the spinal cord.


Assuntos
Interneurônios/metabolismo , Locomoção/fisiologia , Animais , Interneurônios/citologia , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Peixe-Zebra
19.
Am J Physiol Gastrointest Liver Physiol ; 317(5): G569-G579, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31411893

RESUMO

Propulsion of luminal content along the gut requires coordinated contractions and relaxations of gastrointestinal smooth muscles controlled by the enteric nervous system. Activation of excitatory motor neurons (EMNs) causes muscle contractions, whereas inhibitory motor neuron (IMN) activation causes muscle relaxation. EMNs release acetylcholine (ACh), which acts at muscarinic receptors on smooth muscle cells and adjacent interstitial cells of Cajal, causing excitatory junction potentials (EJPs). IMNs release ATP (or another purine) and nitric oxide to cause inhibitory junction potentials (IJPs) and muscle relaxation. We used commercially available choline acetyltransferase (ChAT)-channelrhodopsin-2 (ChR2)-yellow fluorescent protein (YFP) bacterial artificial chromosome (BAC) transgenic mice, which express ChR2 in cholinergic neurons, to study cholinergic neuromuscular transmission in the colon. Intracellular microelectrodes were used to record IJPs and EJPs from circular muscle cells. We used blue light stimulation (BLS, 470 nm, 20 mW/mm2) and electrical field stimulation (EFS) to activate myenteric neurons. EFS evoked IJPs only, whereas BLS evoked EJPs and IJPs. Mecamylamine (10 µM, nicotinic cholinergic receptor antagonist) reduced BLS-evoked IJPs by 50% but had no effect on electrically evoked IJPs. MRS 2179 (10 µM, a P2Y1 receptor antagonist) blocked BLS-evoked IJPs. MRS 2179 and Nω-nitro-l-arginine (100 µM, nitric oxide synthase inhibitor) isolated the EJP, which was blocked by scopolamine (1 µM, muscarinic ACh receptor antagonist). Immunohistochemistry revealed ChAT expression in ~88% of enhanced YFP (eYFP)-expressing neurons, whereas 12% of eYFP neurons expressed nitric oxide synthase. These data show that cholinergic interneurons synapse with EMNs and IMNs to cause contraction and relaxation of colonic smooth muscle.NEW & NOTEWORTHY Electrical stimulation of interganglionic connectives has been used widely to study synaptic transmission in the enteric nervous system. However, electrical stimulation will activate many types of neurons and nerve fibers, which complicates data interpretation. Optogenetic activation of enteric neurons using genetically modified mice expressing channelrhodopsin-2 in cholinergic neurons offers a new approach that provides more specificity for nerve stimulation when studying myenteric plexus nerve circuitry.


Assuntos
Colina O-Acetiltransferase/metabolismo , Colo/fisiologia , Junção Neuromuscular/metabolismo , Potenciais Sinápticos , Animais , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Colina O-Acetiltransferase/genética , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/fisiologia , Colo/inervação , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Contração Muscular , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Junção Neuromuscular/fisiologia , Optogenética
20.
Methods Mol Biol ; 2036: 221-236, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31410800

RESUMO

Antisense oligonucleotides (ASOs) are a widely used form of gene therapy, which is translatable to multiple disorders. A major obstacle for ASO efficacy is its bioavailability for in vivo and in vitro studies. To overcome this challenge we use cell-penetrating peptides (CPPs) for systemic delivery of ASOs. One of the most advanced clinical uses of ASOs is for the treatment of spinal muscular atrophy (SMA). In this chapter, we describe the techniques used for in vitro screening and analysing in vivo biodistribution of CPP-conjugated ASOs targeting the survival motor neuron 2, SMN2, the dose-dependent modifying gene for SMA.


Assuntos
Peptídeos Penetradores de Células/química , Técnicas de Transferência de Genes , Atrofia Muscular Espinal/genética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Administração Intravenosa , Linhagem Celular , Sistemas de Liberação de Medicamentos , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/terapia , Oligonucleotídeos Antissenso/química , Processamento de RNA
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