RESUMO
Spasticity, one of the most frequent comorbidities of spinal cord injury (SCI), disrupts motor recovery and quality of life. Despite major progress in neurorehabilitative and pharmacological approaches, therapeutic strategies for treating spasticity are lacking. Here, we show in a mouse model of chronic SCI that treatment with nimodipine-an L-type calcium channel blocker already approved from the European Medicine Agency and from the U.S. Food and Drug Administration-starting in the acute phase of SCI completely prevents the development of spasticity measured as increased muscle tone and spontaneous spasms. The aberrant muscle activities associated with spasticity remain inhibited even after termination of the treatment. Constitutive and conditional silencing of the L-type calcium channel CaV1.3 in neuronal subtypes demonstrated that this channel mediated the preventive effect of nimodipine on spasticity after SCI. This study identifies a treatment protocol and suggests that targeting CaV1.3 could prevent spasticity after SCI.
Assuntos
Bloqueadores dos Canais de Cálcio , Espasticidade Muscular , Nimodipina , Traumatismos da Medula Espinal , Animais , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo L , Camundongos , Espasticidade Muscular/tratamento farmacológico , Espasticidade Muscular/prevenção & controle , Nimodipina/uso terapêutico , Qualidade de Vida , Medula Espinal , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/tratamento farmacológicoRESUMO
Ascorbate requiring Fe2+/2-oxoglutarate-dependent dioxygenases located in the nucleoplasm have been shown to participate in epigenetic regulation of gene expression via histone and DNA demethylation. Transport of dehydroascorbic acid is impaired in the endomembranes of fibroblasts from arterial tortuosity syndrome (ATS) patients, due to the mutation in the gene coding for glucose transporter GLUT10. We hypothesized that altered nuclear ascorbate concentration might be present in ATS fibroblasts, affecting dioxygenase activity and DNA demethylation. Therefore, our aim was to characterize the subcellular distribution of vitamin C, the global and site-specific changes in 5-methylcytosine and 5-hydroxymethylcytosine levels, and the effect of ascorbate supplementation in control and ATS fibroblast cultures. Diminished nuclear accumulation of ascorbate was found in ATS fibroblasts upon ascorbate or dehydroascorbic acid addition. Analyzing DNA samples of cultured fibroblasts from controls and ATS patients, a lower global 5-hydroxymethylcytosine level was found in ATS fibroblasts, which could not be significantly modified by ascorbate addition. Investigation of the (hydroxy)methylation status of specific regions in six candidate genes related to ascorbate metabolism and function showed that ascorbate addition could stimulate hydroxymethylation and active DNA demethylation at the PPAR-γ gene region in control fibroblasts only. The altered DNA hydroxymethylation patterns in patient cells both at the global level and at specific gene regions accompanied with decreased nuclear accumulation of ascorbate suggests the epigenetic role of vitamin C in the pathomechanism of ATS. The present findings represent the first example for the role of vitamin C transport in epigenetic regulation suggesting that ATS is a compartmentalization disease.
Assuntos
Artérias/anormalidades , Ácido Ascórbico/metabolismo , Núcleo Celular/metabolismo , Metilação de DNA/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Genoma Humano , Instabilidade Articular/genética , Dermatopatias Genéticas/genética , Malformações Vasculares/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Células Cultivadas , Epigênese Genética , Humanos , Modelos Biológicos , PPAR gama/genética , PPAR gama/metabolismoRESUMO
High fatty acid (FA) levels are deleterious to pancreatic ß-cells, largely due to the accumulation of biosynthetic lipid intermediates, such as ceramides and diglycerides, which induce ER stress and apoptosis. Toxicity of palmitate (16:0) and oleate (18:1 cis-Δ9) has been widely investigated, while very little data is available on the cell damages caused by elaidate (18:1 trans-Δ9) and vaccenate (18:1 trans-Δ11), although the potential health effects of these dietary trans fatty acids (TFAs) received great publicity. We compared the effects of these four FAs on cell viability, apoptosis, ER stress, JNK phosphorylation and autophagy as well as on ceramide and diglyceride contents in RINm5F insulinoma cells. Similarly to oleate and unlike palmitate, TFAs reduced cell viability only at higher concentration, and they had mild effects on ER stress, apoptosis and autophagy. Palmitate increased ceramide and diglyceride levels far more than any of the unsaturated fatty acids; however, incorporation of TFAs in ceramides and diglycerides was strikingly more pronounced than that of oleate. This indicates a correlation between the accumulation of lipid intermediates and the severity of cell damage. Our findings reveal important metabolic characteristics of TFAs that might underlie a long term toxicity and hence deserve further investigation.
Assuntos
Ceramidas/metabolismo , Gorduras Insaturadas na Dieta/toxicidade , Diglicerídeos/metabolismo , Ácido Oleico/toxicidade , Ácidos Oleicos/toxicidade , Ácidos Graxos trans/toxicidade , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Gorduras Insaturadas na Dieta/análise , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , MAP Quinase Quinase 4/metabolismo , Necrose/induzido quimicamente , Ácido Oleico/análise , Ácidos Oleicos/análise , Ácidos Palmíticos/análise , Ácidos Palmíticos/toxicidade , Fosforilação , Ratos , Ácidos Graxos trans/análiseRESUMO
CSF-contacting (CSF-c) cells are present in the walls of the brain ventricles and the central canal of the spinal cord and found throughout the vertebrate phylum. We recently identified ciliated somatostatin-/GABA-expressing CSF-c neurons in the lamprey spinal cord that act as pH sensors as well as mechanoreceptors. In the same neuron, acidic and alkaline responses are mediated through ASIC3-like and PKD2L1 channels, respectively. Here, we investigate the functional properties of the ciliated somatostatin-/GABA-positive CSF-c neurons in the hypothalamus by performing whole-cell recordings in hypothalamic slices. Depolarizing current pulses readily evoked action potentials, but hypothalamic CSF-c neurons had no or a very low level of spontaneous activity at pH 7.4. They responded, however, with membrane potential depolarization and trains of action potentials to small deviations in pH in both the acidic and alkaline direction. Like in spinal CSF-c neurons, the acidic response in hypothalamic cells is mediated via ASIC3-like channels. In contrast, the alkaline response appears to depend on connexin hemichannels, not on PKD2L1 channels. We also show that hypothalamic CSF-c neurons respond to mechanical stimulation induced by fluid movements along the wall of the third ventricle, a response mediated via ASIC3-like channels. The hypothalamic CSF-c neurons extend their processes dorsally, ventrally, and laterally, but as yet, the effects exerted on hypothalamic circuits are unknown. With similar neurons being present in rodents, the pH- and mechanosensing ability of hypothalamic CSF-c neurons is most likely conserved throughout vertebrate phylogeny.SIGNIFICANCE STATEMENT CSF-contacting neurons are present in all vertebrates and are located mainly in the hypothalamic area and the spinal cord. Here, we report that the somatostatin-/GABA-expressing CSF-c neurons in the lamprey hypothalamus sense bidirectional deviations in the extracellular pH and do so via different molecular mechanisms. They also serve as mechanoreceptors. The hypothalamic CSF-c neurons have extensive axonal ramifications and may decrease the level of motor activity via release of somatostatin. In conclusion, hypothalamic somatostatin-/GABA-expressing CSF-c neurons, as well as their spinal counterpart, represent a novel homeostatic mechanism designed to sense any deviation from physiological pH and thus constitute a feedback regulatory system intrinsic to the CNS, possibly serving a protective role from damage caused by changes in pH.
Assuntos
Canais Iônicos Sensíveis a Ácido/fisiologia , Líquido Cefalorraquidiano/fisiologia , Concentração de Íons de Hidrogênio , Hipotálamo/citologia , Mecanorreceptores/fisiologia , Neurônios/fisiologia , Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Potenciais de Ação , Animais , Conexinas/antagonistas & inibidores , Conexinas/fisiologia , Feminino , Junções Comunicantes/fisiologia , Lampreias , Masculino , Movimento (Física) , Técnicas de Patch-Clamp , Estimulação Física , Somatostatina/análise , Estresse Mecânico , Terceiro Ventrículo , Ácido gama-Aminobutírico/análiseRESUMO
In scrapie-infected cells, the abnormal isoform of the prion protein, PrP(Sc), accumulates in endosomes/lysosomes. In this study, the involvement of two lysosomal proteases, cathepsin B and L, in cellular processing of PrP(Sc) was analyzed in immortalized neuronal gonadotropin-releasing hormone cells (GT1-1) infected with scrapie. Treatment with inhibitors of either cathepsin B or L resulted in accumulation of PrP(Sc). Such an increased accumulation also occurred when the activities of both cathepsins were inhibited using RNA interference. We conclude that cathepsin B and L are involved in the degradation of PrP(Sc) in scrapie-infected GT1-1 cells and that they can compensate for each other's functions. This study shows that specific proteases, abundantly present in neurons, have the capacity to degrade PrP(Sc).