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1.
Nat Commun ; 12(1): 1955, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782410

RESUMO

p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.


Assuntos
Autofagia/genética , Proteínas de Ciclo Celular/genética , Chaperonas Moleculares/genética , Agregados Proteicos , Proteína Sequestossoma-1/genética , Superóxido Dismutase-1/genética , Animais , Autofagia/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Leupeptinas/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Chaperonas Moleculares/metabolismo , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Superóxido Dismutase-1/metabolismo , Transgenes , Xenopus laevis
2.
Nat Commun ; 12(1): 847, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558503

RESUMO

A large G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Neuronal degeneration associated with this expansion arises from a loss of C9orf72 protein, the accumulation of RNA foci, the expression of dipeptide repeat (DPR) proteins, or all these factors. We report the discovery of a new targeting sequence that is common to all C9orf72 transcripts but enables preferential knockdown of repeat-containing transcripts in multiple cellular models and C9BAC transgenic mice. We optimize stereopure oligonucleotides that act through this site, and we demonstrate that their preferential activity depends on both backbone stereochemistry and asymmetric wing design. In mice, stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. We hypothesize that targeting C9orf72 with stereopure oligonucleotides may be a viable therapeutic approach for the treatment of C9orf72-associated neurodegenerative disorders.


Assuntos
Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Mutação/genética , Oligonucleotídeos/química , Oligonucleotídeos/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Animais , Proteína C9orf72/química , Éxons/genética , Glutamatos/toxicidade , Íntrons/genética , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estereoisomerismo
3.
J Stroke Cerebrovasc Dis ; 30(4): 105630, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33497934

RESUMO

BACKGROUND: The therapeutic effects of dimethyl fumarate (DMF) in patients with multiple sclerosis and animal models of neurologic disease were reported. The density and the distribution pattern of motor neurons are important in transmitting the signal and controlling the movement-related functions. The present study evaluated the effects of DMF treatment on the neurological functions, infarct volume, and spatial distribution of the neurons in the primary motor cortex after cerebral ischemia. METHODS: Thirty-three Sprague-Dawley rats were randomly divided into three groups: The sham group underwent surgery without middle cerebral artery occlusion (MCAO) and drug. The vehicle and treatment groups after MCAO received a vehicle or DMF for three consecutive days. Post-stroke neurological and motor functions were assessed. At the end of the third day, the brains were removed, and the cerebral infarct volume was evaluated. We used cresyl violet staining to analyze the density and the spatial arrangement of motor cortical neurons using Voronoi tessellation. RESULTS: Treatment of the brain ischemia for three days with DMF could not significantly reduce the neurological and motor function deficits and infarct volume. However, it reduced the neuronal area and death and preserved their spatial distribution in the normal regular pattern. CONCLUSION: Cerebral ischemia decreased the neuronal density of the primary motor cortex and changed their distributions to a random pattern. DMF treatment during sub-acute ischemic stroke did not significantly improve the neurological deficit scores. However, it could prevent neuronal swelling and death and preserved the spatial distribution of the cortical neurons in their normal pattern.


Assuntos
Comportamento Animal/efeitos dos fármacos , Fumarato de Dimetilo/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Atividade Motora/efeitos dos fármacos , Córtex Motor/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , /fisiopatologia , Masculino , Córtex Motor/patologia , Córtex Motor/fisiopatologia , Neurônios Motores/patologia , Ratos Sprague-Dawley , Teste de Desempenho do Rota-Rod , Fatores de Tempo
4.
Biochem Biophys Res Commun ; 533(4): 1470-1476, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33333712

RESUMO

Exosc2 is one of the components of the exosome complex involved in RNA 3' end processing and degradation of various RNAs. Recently, EXOSC2 mutation has been reported in German families presenting short stature, hearing loss, retinitis pigmentosa, and premature aging. However, the in vivo function of EXOSC2 has been elusive. Herein, we generated Exosc2 knockout (exosc2-/-) zebrafish that showed larval lethality 13 days post fertilization, with microcephaly, loss of spinal motor neurons, myelin deficiency, and retinitis pigmentosa. Mechanistically, Exosc2 deficiency caused impaired mRNA turnover, resulting in a nucleotide pool imbalance. Rapamycin, which modulated mRNA turnover by inhibiting the mTOR pathway, improved nucleotide pool imbalance in exosc2-/- zebrafish, resulting in prolonged survival and partial rescue of neuronal defects. Taken together, our findings offer new insights into the disease pathogenesis caused by Exosc2 deficiency, and might help explain fundamental molecular mechanisms in neuronal diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and spinal muscular atrophy.


Assuntos
Nucleotídeos/metabolismo , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Embrião não Mamífero/anormalidades , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Larva/genética , Larva/fisiologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Proteína Básica da Mielina/genética , Nucleotídeos/genética , Sirolimo/farmacologia , Peixe-Zebra/embriologia
5.
PLoS Negl Trop Dis ; 14(9): e0008547, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898186

RESUMO

Envenomation by snakes is a major neglected human disease. Hospitalization and use of animal-derived antivenom are the primary therapeutic supports currently available. There is consensus that additional, not expensive, treatments that can be delivered even long after the snake bite are needed. We recently showed that the drug dubbed NUCC-390 shortens the time of recovery from the neuroparalysis caused by traumatic or toxic degeneration of peripheral motor neurons. These syndromes are characterized by the activation of a pro-regenerative molecular axis, consisting of the CXCR4 receptor expressed at the damaged site in neuronal axons and by the release of its ligand CXCL12α, produced by surrounding Schwann cells. This intercellular signaling axis promotes axonal growth and functional recovery from paralysis. NUCC-390 is an agonist of CXCR4 acting similarly to CXCL12α. Here, we have tested its efficacy in a murine model of neuroparalytic envenoming by a Papuan Taipan (Oxyuranus scutellatus) where a degeneration of the motor axon terminals caused by the presynaptic PLA2 toxin Taipoxin, contained in the venom, occurs. Using imaging of the neuromuscular junction and electrophysiological analysis, we found that NUCC-390 administration after injection of either the purified neuroparalytic Taipoxin or the whole Taipan venom, significantly accelerates the recovery from paralysis. These results indicate that NUCC-390, which is non-toxic in mice, should be considered for trials in humans to test its efficacy in accelerating the recovery from the peripheral neuroparalysis induced by Taipans. NUCC-390 should be tested as well in the envenomation by other snakes that cause neuroparalytic syndromes in humans. NUCC-390 could become an additional treatment, common to many snake envenomings, that can be delivered after the bite to reduce death by respiratory deficits and to shorten and improve functional recovery.


Assuntos
Venenos Elapídicos/toxicidade , Indazóis/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Paralisia/terapia , Piperidinas/farmacologia , Piridinas/farmacologia , Receptores CXCR4/agonistas , Potenciais de Ação/efeitos dos fármacos , Animais , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/efeitos dos fármacos
6.
Nat Commun ; 11(1): 3848, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737286

RESUMO

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the degeneration of upper and lower motor neurons (MNs). We find a significant reduction of the retromer complex subunit VPS35 in iPSCs-derived MNs from ALS patients, in MNs from ALS post mortem explants and in MNs from SOD1G93A mice. Being the retromer involved in trafficking of hydrolases, a pathological hallmark in ALS, we design, synthesize and characterize an array of retromer stabilizers based on bis-guanylhydrazones connected by a 1,3-phenyl ring linker. We select compound 2a as a potent and bioavailable interactor of VPS35-VPS29. Indeed, while increasing retromer stability in ALS mice, compound 2a attenuates locomotion impairment and increases MNs survival. Moreover, compound 2a increases VPS35 in iPSCs-derived MNs and shows brain bioavailability. Our results clearly suggest the retromer as a valuable druggable target in ALS.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Hidrazonas/farmacologia , Neurônios Motores/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Proteínas de Transporte Vesicular/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Hidrazonas/síntese química , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/síntese química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Relação Estrutura-Atividade , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Proteínas de Transporte Vesicular/metabolismo
7.
Am J Respir Cell Mol Biol ; 63(4): 502-509, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32603263

RESUMO

Respiratory depression is the main cause of morbidity and mortality associated with opioids. Obesity increases opioid-related mortality, which is mostly related to comorbid obstructive sleep apnea. Naloxone, a µ-opioid receptor blocker, is an effective antidote, but it reverses analgesia. Like humans with obesity, mice with diet-induced obesity hypoventilate during sleep and develop obstructive sleep apnea, which can be treated with intranasal leptin. We hypothesized that intranasal leptin reverses opioid-induced sleep-disordered breathing in obese mice without decreasing analgesia. To test this hypothesis, mice with diet-induced obesity were treated with morphine at 10 mg/kg subcutaneously and with leptin or placebo intranasally. Sleep and breathing were recorded by barometric plethysmography, and pain sensitivity was measured by the tail-flick test. Excitatory postsynaptic currents were recorded in vitro from hypoglossal motor neurons after the application of the µ-opioid receptor agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin and leptin. Morphine dramatically increased the frequency of apneas and greatly increased the severity of hypoventilation and obstructive sleep apnea. Leptin decreased the frequency of apneas, improved obstructive sleep apnea, and completely reversed hypoventilation, whereas morphine analgesia was enhanced. Our in vitro studies demonstrated that [D-Ala2, N-MePhe4, Gly-ol]-enkephalin reduced the frequency of excitatory postsynaptic currents in hypoglossal motoneurons and that application of leptin restored excitatory synaptic neurotransmission. Our findings suggest that intranasal leptin may prevent opioid respiratory depression during sleep in patients with obesity receiving opioids without reducing analgesia.


Assuntos
Analgésicos Opioides/efeitos adversos , Leptina/administração & dosagem , Respiração/efeitos dos fármacos , Síndromes da Apneia do Sono/induzido quimicamente , Síndromes da Apneia do Sono/prevenção & controle , Sono/efeitos dos fármacos , Administração Intranasal/métodos , Analgesia/métodos , Animais , Modelos Animais de Doenças , Encefalinas/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Morfina/farmacologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Receptores Opioides mu/metabolismo , Síndromes da Apneia do Sono/metabolismo , Transmissão Sináptica/efeitos dos fármacos
8.
Chemosphere ; 253: 126762, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32302915

RESUMO

17ß-trenbolone (17ß-TBOH) is one of the dominant metabolites of trenbolone acetate, which is widely applied in beef cattle operations around the globe. The effects of environmental concentrations of 17ß-trenbolone on the early development of zebrafish embryos have received very little attention. Melatonin could regulate sleep-wake cycle and plays a protective role in various adverse conditions. Here, environmentally realistic concentrations of 17ß-trenbolone (1 ng/L, 10 ng/L, 50 ng/L) has been exposure to zebrafish embryos at 2 h postfertilization (hpf). The results showed that 10 ng/L and 50 ng/L 17ß-trenbolone disturbed the distribution of caudal primary motoneurons and downregulated expression of motoneuron development related genes along with locomotion decreasing. While melatonin could recover the detrimental effects caused by 17ß-trenbolone. Interestingly, 17ß-trenbolone exposure increased waking activity and decreased rest even in a low dose (1 ng/L). Moreover, it upregulated hypocretin/orexin (Hcrt) signaling which promotes wakefulness. Melatonin restored the insomnia-like alternation induced by 17ß-trenbolone exposure. Collectively, we conclude that 17ß-trenbolone disturbed motoneuron development and altered sleep/wake behavior, while melatonin could alleviate the deleterious influence on motoneuron development and recover the circadian rhythm.


Assuntos
Comportamento Animal/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Melatonina/farmacologia , Atividade Motora/efeitos dos fármacos , Distúrbios do Início e da Manutenção do Sono/prevenção & controle , Acetato de Trembolona/toxicidade , Peixe-Zebra , Animais , Bovinos , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/genética , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Orexinas/genética , Fenótipo , Distúrbios do Início e da Manutenção do Sono/induzido quimicamente
9.
Sci Rep ; 10(1): 4449, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157143

RESUMO

Pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease where no treatment exists, involves the compartmentalization of the nuclear protein TDP-43 (TAR DNA-binding protein 43) in the cytoplasm which is promoted by its aberrant phosphorylation and others posttranslational modifications. Recently, it was reported that CK-1δ (protein casein kinase-1δ) is able to phosphorylate TDP-43. Here, the preclinical efficacy of a benzothiazole-based CK-1δ inhibitor IGS-2.7, both in a TDP-43 (A315T) transgenic mouse and in a human cell-based model of ALS, is shown. Treatment with IGS-2.7 produces a significant preservation of motor neurons in the anterior horn at lumbar level, a decrease in both astroglial and microglial reactivity in this area, and in TDP-43 phosphorylation in spinal cord samples. Furthermore, the recovery of TDP-43 homeostasis (phosphorylation and localization) in a human-based cell model from ALS patients after treatment with IGS-2.7 is also reported. Moreover, we have shown a trend to increase in CK-1δ mRNA in spinal cord and significantly in frontal cortex of sALS cases. All these data show for the first time the in vivo modulation of TDP-43 toxicity by CK-1δ inhibition with IGS-2.7, which may explain the benefits in the preservation of spinal motor neurons and point to the relevance of CK-1δ inhibitors in a future disease-modifying treatment for ALS.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Caseína Quinase Idelta/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/citologia , Inibidores de Proteínas Quinases/farmacologia , Medula Espinal/citologia , Idoso , Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/metabolismo , Animais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Fosforilação , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo
10.
Muscle Nerve ; 61(6): 796-800, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32133655

RESUMO

INTRODUCTION: Cold allodynia is often seen in the acute phase of oxaliplatin treatment, but the underlying pathophysiology remains unclear. METHODS: Patients scheduled for adjuvant oxaliplatin for colorectal cancer were examined with quantitative sensory testing and nerve excitability tests at baseline and after the second or third oxaliplatin cycle at different skin temperatures. RESULTS: Seven patients were eligible for examination. All patients felt evoked pain and tingling when touching something cold after oxaliplatin infusion. Oxaliplatin decreased motor nerve superexcitability (P < .001), increased relative refractory period (P = .011), and caused neuromyotonia-like after-activity. Cooling exacerbated these changes and prolonged the accommodation half-time. DISCUSSION: The findings suggest that a combined effect of oxaliplatin and cooling facilitates nerve excitability changes and neuromyotonia-like after-activity in peripheral nerve axons. A possible mechanism is the slowing in gating of voltage-dependent fast sodium and slow potassium channels, which results in symptoms of cold allodynia.


Assuntos
Antineoplásicos/efeitos adversos , Axônios/fisiologia , Temperatura Baixa/efeitos adversos , Hiperalgesia/induzido quimicamente , Neurônios Motores/fisiologia , Oxaliplatina/efeitos adversos , Idoso , Axônios/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Feminino , Seguimentos , Humanos , Hiperalgesia/fisiopatologia , Masculino , Pessoa de Meia-Idade , Neurônios Motores/efeitos dos fármacos , Estudos Prospectivos , Resultado do Tratamento
11.
Proc Natl Acad Sci U S A ; 117(10): 5510-5515, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32094166

RESUMO

Insect nervous systems offer unique advantages for studying interactions between sensory systems and behavior, given their complexity with high tractability. By examining the neural coding of salient environmental stimuli and resulting behavioral output in the context of environmental stressors, we gain an understanding of the effects of these stressors on brain and behavior and provide insight into normal function. The implication of neonicotinoid (neonic) pesticides in contributing to declines of nontarget species, such as bees, has motivated the development of new compounds that can potentially mitigate putative resistance in target species and declines of nontarget species. We used a neuroethologic approach, including behavioral assays and multineuronal recording techniques, to investigate effects of imidacloprid (IMD) and the novel insecticide sulfoxaflor (SFX) on visual motion-detection circuits and related escape behavior in the tractable locust system. Despite similar LD50 values, IMD and SFX evoked different behavioral and physiological effects. IMD significantly attenuated collision avoidance behaviors and impaired responses of neural populations, including decreases in spontaneous firing and neural habituation. In contrast, SFX displayed no effect at a comparable sublethal dose. These results show that neonics affect population responses and habituation of a visual motion detection system. We propose that differences in the sublethal effects of SFX reflect a different mode of action than that of IMD. More broadly, we suggest that neuroethologic assays for comparative neurotoxicology are valuable tools for fully addressing current issues regarding the proximal effects of environmental toxicity in nontarget species.


Assuntos
Exposição Ambiental , Reação de Fuga/efeitos dos fármacos , Inseticidas/toxicidade , Neurônios Motores/efeitos dos fármacos , Neonicotinoides/toxicidade , Nitrocompostos/toxicidade , Piridinas/toxicidade , Compostos de Enxofre/toxicidade , Animais , Habituação Psicofisiológica/efeitos dos fármacos , Dose Letal Mediana , Locusta migratoria/efeitos dos fármacos , Movimento (Física)
12.
Oxid Med Cell Longev ; 2020: 1020673, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32089764

RESUMO

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder characterized by progressive paralysis resulting from the death of upper and lower motor neurons. There is currently no effective pharmacological treatment for ALS, and the two approved drugs riluzole and edaravone have limited effects on the symptoms and only slightly prolong the life of patients. Therefore, the development of effective therapeutic strategies is of paramount importance. In this study, we investigated whether Miyako Island Bidens pilosa (MBP) can alleviate the neurological deterioration observed in a superoxide dismutase-1 G93A mutant transgenic mouse (G93A mouse) model of ALS. We orally administered 2 g/kg/day of MBP to G93A mice at the onset of symptoms of neurodegeneration (15 weeks old) until death. Treatment with MBP markedly prolonged the life of ALS model mice by approximately 20 days compared to that of vehicle-treated ALS model mice and significantly improved motor performance. MBP treatment prevented the reduction in SMI32 expression, a neuronal marker protein, and attenuated astrocyte (detected by GFAP) and microglia (detected by Iba-1) activation in the spinal cord of G93A mice at the end stage of the disease (18 weeks old). Our results indicate that MBP administered after the onset of ALS symptoms suppressed the inflammatory activation of microglia and astrocytes in the spinal cord of the G93A ALS model mice, thus improving their quality of life. MBP may be a potential therapeutic agent for ALS.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Medicamentos de Ervas Chinesas/química , Neurônios Motores/efeitos dos fármacos , Esclerose Amiotrófica Lateral/mortalidade , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Análise de Sobrevida
13.
Biochem Biophys Res Commun ; 524(3): 589-594, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32029276

RESUMO

Previously, we found that muscarine downregulates the acetylcholine release at the frog neuromuscular junction acting via M3 muscarinic receptors. Here, the molecular mechanisms underlying the inhibitory effect of muscarine on the quantal secretion of acetylcholine were studied. Inhibition of phospholipase C (with U-73122) prevented the reduction of evoked neurotransmitter release induced by muscarine. Interruption of synthesis of phosphatidylinositol 3-phosphate by the inhibitor of phosphoinositide-3-kinase (wortmannin) did not affect the depressant action of muscarine but precluded the restoration of secretion after removal of muscarine from the bathing solution. The effect of muscarine was not significantly modified by the blockade of endocannabinoid receptors (with AM 281), but it was abolished by the inhibitor of nitric oxide synthase (L-NAME) as well as extracellular nitric oxide (NO) chelator (hemoglobin). Moreover, muscarine increased NO-sensitive dye fluorescence in junctional region, which was prevented by the M3 receptor antagonist 4-DAMP. The data obtained indicate that the attenuation of acetylcholine release mediated by muscarine is associated with a change in the activity of both lipid-metabolizing enzymes and NO synthases.


Assuntos
Acetilcolina/metabolismo , Neurônios Motores/metabolismo , Óxido Nítrico/metabolismo , Fosfolipídeos/metabolismo , Ranidae/metabolismo , Receptor Muscarínico M3/metabolismo , Sinapses/metabolismo , Animais , Canabinoides/metabolismo , Neurônios Motores/efeitos dos fármacos , Muscarina/farmacologia , Agonistas Muscarínicos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Sinapses/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo
14.
J Neuropathol Exp Neurol ; 79(4): 393-406, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32077471

RESUMO

The early neuropathological features of amyotrophic lateral sclerosis/motor neuron disease (ALS/MND) are protein aggregates in motor neurons and microglial activation. Similar pathology characterizes Guamanian ALS/Parkinsonism dementia complex, which may be triggered by the cyanotoxin ß-N-methylamino-l-alanine (BMAA). We report here the occurrence of ALS/MND-type pathological changes in vervets (Chlorocebus sabaeus; n = 8) fed oral doses of a dry powder of BMAA HCl salt (210 mg/kg/day) for 140 days. Spinal cords and brains from toxin-exposed vervets were compared to controls fed rice flour (210 mg/kg/day) and to vervets coadministered equal amounts of BMAA and l-serine (210 mg/kg/day). Immunohistochemistry and quantitative image analysis were used to examine markers of ALS/MND and glial activation. UHPLC-MS/MS was used to confirm BMAA exposures in dosed vervets. Motor neuron degeneration was demonstrated in BMAA-dosed vervets by TDP-43+ proteinopathy in anterior horn cells, by reactive astrogliosis, by activated microglia, and by damage to myelinated axons in the lateral corticospinal tracts. Vervets dosed with BMAA + l-serine displayed reduced neuropathological changes. This study demonstrates that chronic dietary exposure to BMAA causes ALS/MND-type pathological changes in the vervet and coadministration of l-serine reduces the amount of reactive gliosis and the number of protein inclusions in motor neurons.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Doença dos Neurônios Motores/patologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Serina/administração & dosagem , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Diamino Aminoácidos/toxicidade , Esclerose Amiotrófica Lateral/induzido quimicamente , Animais , Chlorocebus aethiops , Modelos Animais de Doenças , Masculino , Microglia/efeitos dos fármacos , Microglia/patologia , Doença dos Neurônios Motores/induzido quimicamente , Tratos Piramidais/efeitos dos fármacos , Tratos Piramidais/patologia
15.
Nucleic Acids Res ; 48(6): 2853-2865, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32103257

RESUMO

Spinal muscular atrophy (SMA) is a motor neuron disease. Nusinersen, a splice-switching antisense oligonucleotide (ASO), was the first approved drug to treat SMA. Based on prior preclinical studies, both 2'-O-methoxyethyl (MOE) with a phosphorothioate backbone and morpholino with a phosphorodiamidate backbone-with the same or extended target sequence as nusinersen-displayed efficient rescue of SMA mouse models. Here, we compared the therapeutic efficacy of these two modification chemistries in rescue of a severe mouse model using ASO10-29-a 2-nt longer version of nusinersen-via subcutaneous injection. Although both chemistries efficiently corrected SMN2 splicing in various tissues, restored motor function and improved the integrity of neuromuscular junctions, MOE-modified ASO10-29 (MOE10-29) was more efficacious than morpholino-modified ASO10-29 (PMO10-29) at the same molar dose, as seen by longer survival, greater body-weight gain and better preservation of motor neurons. Time-course analysis revealed that MOE10-29 had more persistent effects than PMO10-29. On the other hand, PMO10-29 appears to more readily cross an immature blood-brain barrier following systemic administration, showing more robust initial effects on SMN2 exon 7 inclusion, but less persistence in the central nervous system. We conclude that both modifications can be effective as splice-switching ASOs in the context of SMA and potentially other diseases, and discuss the advantages and disadvantages of each.


Assuntos
Amidas/química , Morfolinos/uso terapêutico , Atrofia Muscular Espinal/tratamento farmacológico , Oligonucleotídeos Antissenso/uso terapêutico , Ácidos Fosfóricos/química , Animais , Modelos Animais de Doenças , Éxons/genética , Humanos , Camundongos Transgênicos , Morfolinos/farmacologia , Atividade Motora/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Músculos/patologia , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/fisiopatologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Oligonucleotídeos Antissenso/farmacologia , Fenótipo , Processamento de RNA/efeitos dos fármacos , Processamento de RNA/genética , Medula Espinal/patologia , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Resultado do Tratamento
16.
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
17.
Int J Biol Sci ; 16(2): 284-297, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31929756

RESUMO

Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, the brain stem, and the motor cortex. So far, there is still a lack of effective drugs. Nicotinamide adenine dinucleotide (NAD+) takes part in redox reactions and the NAD-dependent signaling pathway. The NAD+ decline is related with many neurological diseases, leading to the accumulation of neurotoxic protein in the central nervous system. Moreover, the NAD+ supplementation is shown to promote neural stem cells/neuronal precursor cells (NSCs/NPCs) pool maintenance. Regulatory mechanisms and functions of NAD+ metabolism in ALS are still unknown. Thus, we hypothesized the aggregation of human SOD1 toxic protein and the fate of NSCs/NPCs in the ALS disease could be improved by the administration of nicotinamide riboside (NR), an NAD+ precursor. In this study, we treated SOD1G93A transgenic and wild-type mice by the oral administration of 20 mg/ml NR starting at 50 days of age. Effects of NR on the body weight, the motor function, the onset and the survival were assessed during the experiment. The expression of mutant hSOD1 protein, mitochondrial unfolded protein response (UPRmt) related protein, mitophagy markers and NAD+ metabolism related protein were detected by immunoblotting. Effects of NR on the NSCs/NPCs in neurogenic niches of brain were identified by the immunofluorescence staining. Our investigation elucidated that the NR treatment exhibited better hanging wire endurance but did not postpone the onset or extend the life span of SOD1G93A mice. Besides, we observed that the NR repletion promoted the clearance of mitochondrial hSOD1 neurotoxic protein. Meanwhile, the mitochondrial function pathway was disrupted in the brain of SOD1G93A mice. What's more, we demonstrated that the inadequate function of NAD+ salvage synthesis pathway was the primary explanation behind the decline of NAD+, and the NR treatment enhanced the proliferation and migration of NSCs/NPCs in the brain of SOD1G93A mice. At last, we found that levels of UPRmt related protein were significantly increased in the brain of SOD1G93A mice after the NR treatment. In summary, these findings reveal that the administration of NR activates UPRmt signaling, modulates mitochondrial proteostasis and improves the adult neurogenesis in the brain of SOD1G93A mice.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Niacinamida/análogos & derivados , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Niacinamida/farmacologia , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase-1/metabolismo
18.
Nat Biomed Eng ; 4(1): 52-68, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31937944

RESUMO

A small percentage of the short interfering RNA (siRNA) delivered via passive lipid nanoparticles and other delivery vehicles reaches the cytoplasm of cells. The high doses of siRNA and delivery vehicle that are thus required to achieve therapeutic outcomes can lead to toxicity. Here, we show that the integration of siRNA sequences into a Dicer-independent RNA stem-loop based on pre-miR-451 microRNA-which is highly enriched in small extracellular vesicles secreted by many cell types-reduces the expression of the genes targeted by the siRNA in the liver, intestine and kidney glomeruli of mice at siRNA doses that are at least tenfold lower than the siRNA doses typically delivered via lipid nanoparticles. Small extracellular vesicles that efficiently package siRNA can significantly reduce its therapeutic dose.


Assuntos
Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/metabolismo , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , MicroRNAs/química , Neurônios Motores/efeitos dos fármacos , Nanopartículas/administração & dosagem , Interferência de RNA , RNA Interferente Pequeno/química
19.
Neurotox Res ; 37(2): 298-313, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31721049

RESUMO

The neurotoxin 1-methyl,4-phenyl-1,2,3,6-tetrahydropiridine (MPTP) is widely used to produce experimental parkinsonism in rodents and primates. Among different administration protocols, continuous or chronic exposure to small amounts of MPTP is reported to better mimic cell pathology reminiscent of Parkinson's disease (PD). Catecholamine neurons are the most sensitive to MPTP neurotoxicity; however, recent studies have found that MPTP alters the fine anatomy of the spinal cord including motor neurons, thus overlapping again with the spinal cord involvement documented in PD. In the present study, we demonstrate that chronic exposure to low amounts of MPTP (10 mg/kg daily, × 21 days) significantly reduces motor neurons in the ventral lumbar spinal cord while increasing α-synuclein immune-staining within the ventral horn. Spinal cord involvement in MPTP-treated mice extends to Calbindin D28 KDa immune-reactive neurons other than motor neurons within lamina VII. These results were obtained in the absence of significant reduction of dopaminergic cell bodies in the Substantia Nigra pars compacta, while a slight decrease was documented in striatal tyrosine hydroxylase immune-staining. Thus, the present study highlights neuropathological similarities between dopaminergic neurons and spinal motor neurons and supports the pathological involvement of spinal cord in PD and experimental MPTP-induced parkinsonism. Remarkably, the toxic threshold for motor neurons appears to be lower compared with nigral dopaminergic neurons following a chronic pattern of MPTP intoxication. This sharply contrasts with previous studies showing that MPTP intoxication produces comparable neuronal loss within spinal cord and Substantia Nigra.


Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/efeitos adversos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/patologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL
20.
Exp Neurol ; 323: 113067, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31629857

RESUMO

Respiratory motor neuron survival is critical for maintenance of adequate ventilation and airway clearance, preventing dependence to mechanical ventilation and respiratory tract infections. Phrenic motor neurons are highly vulnerable in rodent models of motor neuron disease versus accessory inspiratory motor pools (e.g. intercostals, scalenus). Thus, strategies that promote phrenic motor neuron survival when faced with disease and/or toxic insults are needed to help preserve breathing ability, airway defense and ventilator independence. Adenosine 2A receptors (A2A) are emerging as a potential target to promote neuroprotection, although their activation can have both beneficial and pathogenic effects. Since the role of A2A receptors in the phrenic motor neuron survival/death is not known, we tested the hypothesis that A2A receptor antagonism promotes phrenic motor neuron survival and preserves diaphragm function when faced with toxic, neurodegenerative insults that lead to phrenic motor neuron death. We utilized a novel neurotoxic model of respiratory motor neuron death recently developed in our laboratory: intrapleural injections of cholera toxin B subunit (CtB) conjugated to the ribosomal toxin, saporin (CtB-Saporin). We demonstrate that intrapleural CtB-Saporin causes: 1) profound phrenic motor neuron death (~5% survival); 2) ~7-fold increase in phrenic motor neuron A2A receptor expression prior to cell death; and 3) diaphragm muscle paralysis (inactive in most rats; ~7% residual diaphragm EMG amplitude during room air breathing). The A2A receptor antagonist istradefylline given after CtB-Saporin: 1) reduced phrenic motor neuron death (~20% survival) and 2) preserved diaphragm EMG activity (~46%). Thus, A2A receptors contribute to neurotoxic phrenic motor neuron death, an effect mitigated by A2A receptor antagonism.


Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Toxina da Cólera/toxicidade , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Nervo Frênico/efeitos dos fármacos , Nervo Frênico/metabolismo , Saporinas/toxicidade , Animais , Apoptose/efeitos dos fármacos , Diafragma/inervação , Masculino , Purinas/farmacologia , Ratos , Ratos Sprague-Dawley
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