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1.
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
2.
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
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.
Sheng Li Xue Bao ; 71(6): 809-823, 2019 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-31879736

RESUMO

Spinal α-motoneurons directly innervate skeletal muscles and function as the final common path for movement and behavior. The processes that determine the excitability of motoneurons are critical for the execution of motor behavior. In fact, it has been noted that spinal motoneurons receive various neuromodulatory inputs, especially monoaminergic one. However, the roles of histamine and hypothalamic histaminergic innervation on spinal motoneurons and the underlying ionic mechanisms are still largely unknown. In the present study, by using the method of intracellular recording on rat spinal slices, we found that activation of either H1 or H2 receptor potentiated repetitive firing behavior and increased the excitability of spinal α-motoneurons. Both of blockage of K+ channels and activation of Na+-Ca2+ exchangers were involved in the H1 receptor-mediated excitation on spinal motoneurons, whereas the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were responsible for the H2 receptor-mediated excitation. The results suggest that, through switching functional status of ion channels and exchangers coupled to histamine receptors, histamine effectively biases the excitability of the spinal α-motoneurons. In this way, the hypothalamospinal histaminergic innervation may directly modulate final motor outputs and actively regulate spinal motor reflexes and motor execution.


Assuntos
Histamina , Neurônios Motores , Animais , Histamina/farmacologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Ratos , Receptores Histamínicos H2/metabolismo , Trocador de Sódio e Cálcio/metabolismo
5.
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
6.
Brain Nerve ; 71(9): 943-952, 2019 Sep.
Artigo em Japonês | MEDLINE | ID: mdl-31506396

RESUMO

We performed drug screening using motor neurons derived from disease-specific induced pluripotent stem cells (iPSCs) for amyotrophic lateral sclerosis (ALS) and found that ropinirole hydrochloride prevented motor neuron death. We have started a randomized clinical trial testing ropinirole hydrochloride in ALS patients in December 2018. This is a phase I/IIa randomized, double-blind, placebo-controlled, single-center, open-label continuation clinical trial. The primary aim is to assess the safety and tolerability of ropinirole hydrochloride in patients with ALS. Secondary aims include the following effectiveness evaluations: ALSFRS-R, quantitative muscle strength by a hand-held dynamometer, muscle volume by CT scan, forced vital capacity, physical activity by an activity tracker, survival, ALSAQ40 scale, and a Zarit Caregiver Burden Interview. Moreover, we will perform an efficacy evaluation using subjects-derived iPSCs/motor neurons and assess plasma/CSF biomarkers (TDP-43, and ALS-related RNA/micro RNA) as exploratory research questions. Ropinirole hydrochloride potentially targets multiple mechanisms of ALS pathology (i.e., oxidative stress, mitochondrial dysfunction, and abnormal aggregation of TDP-43/FUS protein, which is representative of the ALS phenotype), with promising preclinical study results based on iPSC research. The availability of the drug suggests that rapid translation to daily clinical use might be possible. Our trial will provide reliable and important data for further potential trials. The results will appear in March 2021.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Indóis/uso terapêutico , Método Duplo-Cego , Humanos , Neurônios Motores/efeitos dos fármacos
7.
Int J Mol Sci ; 20(15)2019 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-31382568

RESUMO

(1) Background: Amyotrophic lateral sclerosis (ALS) is a multifactorial non-cell autonomous disease where activation of microglia and astrocytes largely contributes to motor neurons death. Heat shock proteins have been demonstrated to promote neuronal survival and exert a strong anti-inflammatory action in glia. Having previously shown that the pharmacological increase of the histamine content in the central nervous system (CNS) of SOD1-G93A mice decreases neuroinflammation, reduces motor neuron death, and increases mice life span, here we examined whether this effect could be mediated by an enhancement of the heat shock response. (2) Methods: Heat shock protein expression was analyzed in vitro and in vivo. Histamine was provided to primary microglia and NSC-34 motor neurons expressing the SOD1-G93A mutation. The brain permeable histamine precursor histidine was chronically administered to symptomatic SOD1-G93A mice. Spine density was measured by Golgi-staining in motor cortex of histidine-treated SOD1-G93A mice. (3) Results: We demonstrate that histamine activates the heat shock response in cultured SOD1-G93A microglia and motor neurons. In SOD1-G93A mice, histidine augments the protein content of GRP78 and Hsp70 in spinal cord and cortex, where the treatment also rescues type I motor neuron dendritic spine loss. (4) Conclusion: Besides the established histaminergic neuroprotective and anti-inflammatory effects, the induction of the heat shock response in the SOD1-G93A model by histamine confirms the importance of this pathway in the search for successful therapeutic solutions to treat ALS.


Assuntos
Resposta ao Choque Térmico/efeitos dos fármacos , Histamina/farmacologia , Neurônios Motores/efeitos dos fármacos , Superóxido Dismutase-1/genética , Esclerose Amiotrófica Lateral , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Morte Celular/efeitos dos fármacos , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/genética , Modelos Animais de Doenças , Resposta ao Choque Térmico/genética , Humanos , Camundongos , Microglia/metabolismo , Microglia/patologia , Neurônios Motores/patologia , Mutação , Neuroglia/efeitos dos fármacos , Neuroglia/patologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia
8.
Elife ; 82019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31318331

RESUMO

Overproduction of reactive oxygen species (ROS) is known to mediate glutamate excitotoxicity in neurological diseases. However, how ROS burdens can influence neural circuit integrity remains unclear. Here, we investigate the impact of excitotoxicity induced by depletion of Drosophila Eaat1, an astrocytic glutamate transporter, on locomotor central pattern generator (CPG) activity, neuromuscular junction architecture, and motor function. We show that glutamate excitotoxicity triggers a circuit-dependent ROS feedback loop to sculpt the motor system. Excitotoxicity initially elevates ROS, thereby inactivating cholinergic interneurons and consequently changing CPG output activity to overexcite motor neurons and muscles. Remarkably, tonic motor neuron stimulation boosts muscular ROS, gradually dampening muscle contractility to feedback-enhance ROS accumulation in the CPG circuit and subsequently exacerbate circuit dysfunction. Ultimately, excess premotor excitation of motor neurons promotes ROS-activated stress signaling that alters neuromuscular junction architecture. Collectively, our results reveal that excitotoxicity-induced ROS can perturb motor system integrity through a circuit-dependent mechanism.


Assuntos
Drosophila melanogaster/fisiologia , Retroalimentação Fisiológica , Ácido Glutâmico/toxicidade , Neurônios Motores/fisiologia , Neurotoxinas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Mutação/genética , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Estresse Oxidativo/efeitos dos fármacos
9.
J Steroid Biochem Mol Biol ; 192: 105385, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31150830

RESUMO

The Wobbler mouse has been proposed as an experimental model of the sporadic form of amyotrophic lateral sclerosis (ALS). The administration of natural progesterone (PROG) to Wobbler mice attenuates neuropathology, inhibits oxidative stress, enhances the expression of genes involved in motoneuron function, increases survival and restores axonal transport. However, current pharmacological treatments for ALS patients are still partially effective. This encouraged us to investigate if the synthetic progestin norethindrone (NOR), showing higher potency than PROG and used for birth control and hormone therapy might also afford neuroprotection. Two-month-old Wobbler mice (wr/wr) were left untreated or received either a 20 mg pellet of PROG or a 1 mg pellet of NOR for 18 days. Untreated control NFR/NFR mice (background strain for Wobbler) were also employed. Wobblers showed typical clinical and spinal cord abnormalities, while these abnormalities were normalized with PROG treatment. Surprisingly, we found that NOR did not increase immunoreactivity and gene expression for choline-acetyltransferase, drastically decreased GFAP + astrogliosis, favored proinflammatory mediators, promoted the inflammatory phenotype of IBA1+ microglia, increased the receptor for advanced glycation end products (RAGE) mRNA and protein expression and the activity of nitric oxide synthase (NOS)/NADPH diaphorase in the cervical spinal cord. Additionally, NOR treatment produced atrophy of the thymus. The combined negative effects of NOR on clinical assessments (forelimb atrophy and rotarod performance) suggest a detrimental effect on muscle trophism and motor function. These findings reinforce the evidence that the type of progestin used for contraception, endometriosis or replacement therapy, may condition the outcome of preclinical and clinical studies targeting neurodegenerative diseases.


Assuntos
Modelos Animais de Doenças , Neurônios Motores/efeitos dos fármacos , Doenças Neurodegenerativas/tratamento farmacológico , Neuroproteção/efeitos dos fármacos , Noretindrona/farmacologia , Progesterona/farmacologia , Progestinas/farmacologia , Animais , Anticoncepcionais Orais Sintéticos/farmacologia , Camundongos , Neurônios Motores/patologia
10.
Aquat Toxicol ; 213: 105227, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31226596

RESUMO

The environmental impact of exposure to 3D-printed plastics as well as potential migration of toxic chemicals from 3D-printed plastics remains largely unexplored. In this work we applied leachates from plastics fabricated using a stereolithography (SLA) process to early developmental stages of zebrafish (Danio rerio) to investigate developmental toxicity and neurotoxicity. Migration of unpolymerized photoinitiator, 1-hydroxycyclohexyl phenyl ketone (1-HCHPK) from a plastic solid phase to aqueous media at up to 200 mg/L in the first 24 h was detected using gas chromatography-mass spectrometry. Both plastic extracts (LC50 22.25% v/v) and 1-HCHPK (LC50 60 mg/L) induced mortality and teratogenicity within 48 h of exposure. Developmental toxicity correlated with in situ generation of reactive oxygen species (ROS), an increase in lipid peroxidation and protein carbonylation markers and enhanced activity of superoxide dismutase (SOD) and glutathione-S-transferase (GST) in embryos exposed to concentrations as low as 20% v/v for plastic extracts and 16 mg/L for 1-HCHPK. ROS-induced cellular damage led to induction of caspase-dependent apoptosis which could be pharmacologically inhibited with both antioxidant ascorbic acid and a pan-caspase inhibitor. Neuro-behavioral analysis showed that exposure to plastic leachates reduced spontaneous embryonic movement in 24-36 hpf embryos. Plastic extracts in concentrations above 20% v/v induced rapid retardation of locomotion, changes in photomotor response and habituation to photic stimuli with progressive paralysis in 120 hpf larvae. Significantly decreased acetylcholinesterase (AChE) activity with lack of any CNS-specific apoptotic phenotypes as well as lack of changes in motor neuron density, axonal growth, muscle segment integrity or presence of myoseptal defects were detected upon exposure to plastic extracts during embryogenesis. Considering implications of the results for environmental risk assessment and the growing usage of 3D-printing technologies, we speculate that some 3D-printed plastic waste may represent a significant and yet very poorly uncharacterized environmental hazard that merits further investigation on a range of aquatic and terrestrial species.


Assuntos
Comportamento Animal , Sistema Nervoso/efeitos dos fármacos , Plásticos/toxicidade , Impressão Tridimensional , Testes de Toxicidade , Peixe-Zebra/fisiologia , Acetilcolinesterase/metabolismo , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Biomarcadores/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Glutationa Transferase/metabolismo , Larva/efeitos dos fármacos , Larva/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/embriologia
11.
Neuroscience ; 411: 37-46, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31129201

RESUMO

As an adult-onset neurodegenerative disease, amyotrophic lateral sclerosis (ALS) results in progressive muscular atrophy and paralysis. However, the mechanism of ALS has not yet been elucidated, and no cure has been found. Research has revealed that a mutation of the Cu/Zn superoxide dismutase (SOD1) gene is linked to familial ALS and that potential sex discrepancies exist in ALS incidence. Here, NSC-34 cells stably expressing hSOD1-G93A (hSOD1-G93A cells) were transiently transfected with Cyp19a1 mouse open reading frame (ORF) cDNA or a short hairpin RNA (ShRNA) plasmid. Overexpression of aromatase resulting from Cyp19a1 mouse ORF cDNA plasmid transfection enhanced cell proliferation and reduced cell damage in hSOD1-G93A cells. This protective effect occurred through anti-apoptotic pathways related to estrogen receptor-alpha (ER-α) activation. Meanwhile, knockdown of aromatase with Cyp19a1 ShRNA plasmid transfection reduced cell proliferation, increased cell damage, promoted apoptosis, and decreased ER-α expression in hSOD1-G93A cells, and the induced apoptotic effects could be reversed by estradiol (E2). In brief, the results of our study suggest that aromatase plays a neuroprotective role against apoptosis in hSOD1-G93A cells by activating ER-α and may become a new intervention target for ALS treatment.


Assuntos
Apoptose/fisiologia , Aromatase/metabolismo , Neurônios Motores/metabolismo , Superóxido Dismutase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Aromatase/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Estradiol/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Técnicas de Silenciamento de Genes , Camundongos , Neurônios Motores/efeitos dos fármacos , Superóxido Dismutase/genética
12.
Neuroscience ; 409: 130-141, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31051215

RESUMO

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective loss of motor neurons. Statins are widely used as cholesterol-lowering drugs and significantly reduce the risk of cardiovascular and cerebrovascular diseases. Increasing evidence indicates the protective effects of statins against certain neurodegenerative diseases. However, in ALS, many studies have found that statins might accelerate disease progression and shorten survival, although the exact mechanism is unclear. In the present study, we investigated the effect of simvastatin on NSC34cells stably transfected with the G93A mutation in human SOD1 (NSC34-hSOD1G93A cells), a recognized in vitro model of ALS. Our results showed that simvastatin caused a decrease in cell viability and the accumulation of autophagic vacuoles with elevated levels of LC3 II/I and P62 in NSC34-hSOD1G93A cells. Conversely, these outcomes were completely reversed by co-incubation with mevalonate, farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) but not cholesterol. In addition, inhibition of geranylgeranyl transferase I by GGTI-286 led to similar alterations in cell viability and autophagic marker levels. These results indicated that the cytotoxic effect of simvastatin on NSC34-hSOD1G93A cells might be due to the aggravation of autophagic flux impairment through the inhibition of GGPP synthesis.


Assuntos
Autofagia/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neurônios Motores/efeitos dos fármacos , Fosfatos de Poli-Isoprenil/metabolismo , Sinvastatina/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Camundongos , Neurônios Motores/metabolismo
13.
Genesis ; 57(7-8): e23301, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31038837

RESUMO

Retinoic acid (RA), the active metabolite of vitamin A, functions through nuclear receptors, one of which is the retinoic acid receptor (RAR). Though the RAR is essential for various aspects of vertebrate development, little is known about the role of RAR in nonchordate invertebrates. Here, we examined the potential role of an invertebrate RAR in mediating chemotropic effects of retinoic acid. The RAR of the protostome Lymnaea stagnalis is present in the growth cones of regenerating cultured motorneurons, and a synthetic RAR agonist (EC23), was able to mimic the effects of retinoic acid in inducing growth cone turning. We also examined the ability of the natural retinoids, all-trans RA and 9-cis RA, as well as the synthetic RAR agonists, to disrupt embryonic development in Lymnaea. Developmental defects included delays in embryo hatching, arrested eye, and shell development, as well as more severe abnormalities such as halted development. Developmental defects induced by some (but not all) synthetic RAR agonists were found to mimic those induced by addition of high concentrations of the natural retinoid isomers. These pharmacological data support a possible physiological role for the RAR in axon guidance and embryonic development of an invertebrate protostome species.


Assuntos
Orientação de Axônios , Embrião não Mamífero/metabolismo , Receptores do Ácido Retinoico/genética , Animais , Células Cultivadas , Embrião não Mamífero/efeitos dos fármacos , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/metabolismo , Lymnaea , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/metabolismo , Tretinoína/metabolismo , Tretinoína/farmacologia
14.
Neurochem Res ; 44(7): 1773-1779, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31102025

RESUMO

Spinal muscular atrophy (SMA) is an inherited disease characterized by progressive motor neuron death and subsequent muscle weakness and is caused by deletion or mutation of survival motor neuron (SMN) 1 gene. Protecting spinal motor neuron is an effective clinical strategy for SMA. The purpose of this study was to investigate the potential effect of an anti-epileptic drug levetiracetam on SMA. In the present study, we used differentiated spinal motor neurons (MNs) from SMA patient-derived induced pluripotent stem cells (SMA-iPSCs) to investigate the effect of levetiracetam. Levetiracetam promoted neurite elongation in SMA-iPSCs-MNs. TUNEL-positive spinal motor neurons were significantly reduced by levetiracetam in SMA-iPSCs-MNs. In addition, the expression level of cleaved-caspase 3 was decreased by levetiracetam in SMA-iPSCs-MNs. Furthermore, levetiracetam improved impaired mitochondrial function in SMA-iPSCs-MNs. On the other hand, levetiracetam did not affect the expression level of SMN protein in SMA-iPSCs-MNs. These findings indicate that levetiracetam has a neuroprotective effect for SMA.


Assuntos
Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Levetiracetam/uso terapêutico , Neurônios Motores/efeitos dos fármacos , Atrofia Muscular Espinal/prevenção & controle , Neuritos/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Apoptose/efeitos dos fármacos , Chaperonina 60/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Neuritos/patologia
15.
Nutrients ; 11(5)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096690

RESUMO

Vitamin E family is composed of different tocopherols and tocotrienols that are well-known as antioxidants but that exert also non-antioxidant effects. Oxidative stress may be involved in the progression of neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), characterized by motor neuron death. The aim of the study was the evaluation of the changes induced in the transcriptional profile of NSC-34 motor neurons treated with α-tocopherol. In particular, cells were treated for 24 h with 10 µM α-tocopherol, RNA was extracted and transcriptomic analysis was performed using Next Generation Sequencing. Vitamin E treatment modulated MAPK signaling pathway. The evaluation revealed that 34 and 12 genes, respectively belonging to "Classical MAP kinase pathway" and "JNK and p38 MAP kinase pathway", were involved. In particular, a downregulation of the genes encoding for p38 (Log2 fold change -0.87 and -0.67) and JNK (Log2 fold change -0.16) was found. On the contrary, the gene encoding for ERK showed a higher expression in cells treated with vitamin E (Log2 fold change 0.30). Since p38 and JNK seem more involved in cell death, while ERK in cell survival, the data suggested that vitamin E treatment may exert a protective role in NSC-34 motor neurons. Moreover, Vitamin E treatment reduced the expression of the genes which encode proteins involved in mitophagy. These results indicate that vitamin E may be an efficacious therapy in preventing motor neuron death, opening new strategies for those diseases that involve motor neurons, including ALS.


Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Transcriptoma/efeitos dos fármacos , Vitamina E/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Regulação para Cima
16.
World Neurosurg ; 129: e6-e15, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30981793

RESUMO

BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) has been observed to have direct protective effects on neurons after stroke in experimental models and in humans. In the present study, the antiapoptotic effects of G-CSF on spinal α-motoneurons after inducement of peripheral sciatic nerve lesions were evaluated in a rat model. METHODS: Of 48 rats, 24 were treated with G-CSF and 24 were treated with glucose 5% solution (control group). The spinal cord of 6 rats in each group were removed at days 1, 4, 7, and 14. The α-motoneurons of spinal cord section L4-L6 were counted and investigated for the expression of choline acetyltransferase (ChAT), G-CSF receptor (G-CSFR), and Bcl-2 and Bax proteins. Additionally, α-motoneuron fluorescence double staining was performed for ChAT/Bcl-2, ChAT/Bax, and ChAT/G-CSFR. RESULTS: Without G-CSF treatment, the number of ChAT-positive α-motoneurons on the lesion side was significantly decreased (P < 0.001). The number of α-motoneurons with Bcl-2 and G-CSFR positivity on the lesion side was significantly decreased (P < 0.05). In contrast, the number of α-motoneurons with Bax positivity was significantly greater (P < 0.05). After G-CSF treatment, the differences in the number of α-motoneurons on the 2 sides were not statistically significant. Fluorescence double staining of α-motoneurons was positive for ChAT/Bcl-2, ChAT/Bax, and ChAT/G-CSFR. CONCLUSION: The results indicated that G-CSF has neuroprotective properties in spinal α-motoneurons and contributes to antiapoptotic effects after peripheral nerve lesions. The relevance of G-CSF, its precise mode of action, and the effect of these findings in clinical situations remains to be elucidated and require examination in further studies.


Assuntos
Apoptose/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Neurônios Motores/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Traumatismos dos Nervos Periféricos/patologia , Animais , Masculino , Neurônios Motores/patologia , Ratos , Ratos Sprague-Dawley
17.
Mol Neurobiol ; 56(10): 7144-7158, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30989631

RESUMO

Current strategies to enhance regeneration of peripheral neurons involve broad activation of sensory, autonomic, and motor axons. Peripheral neuron regeneration is limited in persons with damage or disease of peripheral axons. Here, we provide evidence that subtoxic activation of TRPV1 channels in sensory neurons is associated with activation of growth and subtle changes in skin reinnervation. We identify a bidirectional, dose-related impact of capsaicin, a TRPV1 agonist, on sensory neurons and their axons with rises in their outgrowth plasticity at low doses and toxic neurodegeneration at high doses. Moreover, its impact on growth added to that of preconditioning. Neither outcome was observed in TRPV1 null neurons. We confirmed that low dose activation was associated with rises in neuronal calcium, as well as rises in TRPV1 mRNA transcripts. In mice with a sciatic nerve crush followed by a single application of capsaicin directly to the injury site, there was no impact on motor or myelinated axon recovery but there was evidence of better recovery of thermal sensation toward baseline with hyperalgesia. Moreover, skin reinnervation by epidermal axons approached contralateral levels. TRPV1 null mice displayed loss of thermal sensation during later recovery. In sensory axons innervating the pinna of the ear, local capsaicin rendered early axon loss followed by later hyperinnervation. Taken together, TRPV1 activation alters the regenerative behavior of adult neurons and their axons both in vitro and during epidermal reinnervation in vivo. The findings identify a selective manipulation that augments cutaneous innervation by thermosensitive axons.


Assuntos
Axônios/metabolismo , Ativação do Canal Iônico , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Axônios/efeitos dos fármacos , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Capsaicina/farmacologia , Citosol/metabolismo , Epiderme/efeitos dos fármacos , Epiderme/inervação , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Regeneração Nervosa/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Remielinização/efeitos dos fármacos , Células Receptoras Sensoriais/efeitos dos fármacos , Temperatura
18.
Mater Sci Eng C Mater Biol Appl ; 101: 243-253, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31029317

RESUMO

Developing a simple produces for efficient derivation of motor neurons (MNs) is essential for neural tissue engineering studies. Stem cells with high capacity for neural differentiation and scaffolds with the potential to promote motor neurons differentiation are promising candidates for neural tissue engineering. Recently, human olfactory ecto-mesenchymal stem cells (OE-MSCs), which are isolated easily from the olfactory mucosa, are considered a new hope for neuronal replacement due to their neural crest origin. Herein, we synthesized conducting hydrogels using different concentration of chitosan-g-aniline pentamer, gelatin, and agarose. The chemical structures, swelling and deswelling ratio, ionic conductivity and thermal properties of the hydrogel were characterized. Scaffolds with 10% chitosan-g-aniline pentamer/gelatin (S10) were chosen for further investigation and the potential of OE-MSCs as a new source for programming to motor neuron-like cells investigated on tissue culture plate (TCP) and conductive hydrogels. Cell differentiation was evaluated at the level of mRNA and protein synthesis and indicated that conductive hydrogels significantly increased the markers related to motor neurons including Hb-9, Islet-1 and ChAT compared to TCP. Taken together, the results suggest that OE-MSCs would be successfully differentiated into motor neuron-like cells on conductive hydrogels and would have a promising potential for treating motor neuron-related diseases.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Quitosana/farmacologia , Condutividade Elétrica , Gelatina/farmacologia , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/citologia , Neurônios Motores/citologia , Sefarose/farmacologia , Compostos de Anilina/síntese química , Compostos de Anilina/farmacologia , Fosfatos de Cálcio/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quitosana/síntese química , Quitosana/química , Força Compressiva , Gelatina/química , Humanos , Hidrogéis/química , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/ultraestrutura , Neurônios Motores/efeitos dos fármacos , Bulbo Olfatório/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Termogravimetria , Tecidos Suporte/química
19.
J Colloid Interface Sci ; 549: 50-62, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31015056

RESUMO

Spinal cord injury (SCI) can cause locomotor dysfunctions and sensory deficits. Evidence shows that functional nanodrugs can regulate macrophage polarization and promote anti-inflammatory cytokine expression, which is feasible in SCI immunotherapeutic treatments. Molybdenum disulfide (MoS2) nanomaterials have garnered great attention as potential carriers for therapeutic payload. Herein, we synthesize MoS2@PEG (MoS2 = molybdenum disulfide, PEG = poly (ethylene glycol)) nanoflowers as an effective carrier for loading etanercept (ET) to treat SCI. We characterize drug loading and release properties of MoS2@PEG in vitro and demonstrate that ET-loading MoS2@PEG obviously inhibits the expression of M1-related pro-inflammatory markers (TNF-α, CD86 and iNOS), while promoting M2-related anti-inflammatory markers (Agr1, CD206 and IL-10) levels. In vivo, the mouse model of SCI shows that long-circulating ET-MoS2@PEG nanodrugs can effectively extravasate into the injured spinal cord up to 96 h after SCI, and promote macrophages towards M2 type polarization. As a result, the ET-loading MoS2@PEG administration in mice can protect survival motor neurons, thus, reducing injured areas at central lesion sites, and significantly improving locomotor recovery. This study demonstrates the anti-inflammatory and neuroprotective activities of ET-MoS2@PEG and promising utility of MoS2 nanomaterial-mediated drug delivery.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Dissulfetos/química , Etanercepte/farmacologia , Macrófagos/efeitos dos fármacos , Nanopartículas Metálicas/química , Molibdênio/química , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Portadores de Fármacos , Liberação Controlada de Fármacos , Etanercepte/uso terapêutico , Feminino , Humanos , Interleucina-10/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Tamanho da Partícula , Permeabilidade , Polietilenoglicóis , Células RAW 264.7 , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Propriedades de Superfície
20.
Dokl Biol Sci ; 484(1): 5-9, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31016495

RESUMO

When studying a preparation of the isolated spinal cord segment of an adult frog, damaged and intact lumbar motoneurons were found to differ significantly in the membrane potential, input resistance and the action potential properties (amplitude, duration, fast and medium phases of the afterhyperpolarization, and the frequency of spikes). Serotonin (5-HT) reduced the amplitude of afterpolarization and increased the frequency of the spikes of the intact neurons, while in the damaged motoneurons, 5-HT increased the amplitude of afterpolarization and had no effect on the frequency of discharges.


Assuntos
Neurônios Motores/efeitos dos fármacos , Agonistas do Receptor de Serotonina/farmacologia , Serotonina/farmacologia , Traumatismos da Medula Espinal/fisiopatologia , Medula Espinal/efeitos dos fármacos , Potenciais de Ação , Animais , Neurônios Motores/fisiologia , Ranidae , Medula Espinal/fisiopatologia
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