RESUMO
Nedd4-2 (NEDD4L in humans) is a ubiquitin protein ligase best known for its role in regulating ion channel internalization and turnover. Nedd4-2 deletion in mice causes perinatal lethality associated with increased epithelial sodium channel (ENaC) expression in lung and kidney. Abundant data suggest that Nedd4-2 plays a role in neuronal functions and may be linked to epilepsy and dyslexia in humans. We used a mouse model of Nedd4-2 haploinsufficiency to investigate whether an alteration in Nedd4-2 levels of expression affects general nervous system functions. We found that Nedd4-2 heterozygous mice are hyperactive, have increased basal synaptic transmission and have enhanced sensitivity to inflammatory pain. Thus, Nedd4-2 heterozygous mice provide a new genetic model to study inflammatory pain. These data also suggest that in human, SNPs affecting NEDD4L levels may be involved in the development of neuropsychological deficits and peripheral neuropathies and may help unveil the genetic basis of comorbidities.
Assuntos
Hipercinese/enzimologia , Hipercinese/genética , Ubiquitina-Proteína Ligases Nedd4/deficiência , Ubiquitina-Proteína Ligases Nedd4/genética , Animais , Região CA1 Hipocampal/enzimologia , Região CA1 Hipocampal/fisiopatologia , Modelos Animais de Doenças , Epilepsia/enzimologia , Epilepsia/genética , Epilepsia/fisiopatologia , Potenciais Pós-Sinápticos Excitadores , Haploinsuficiência , Heterozigoto , Humanos , Hipercinese/fisiopatologia , Potenciação de Longa Duração , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dor/enzimologia , Dor/genética , Dor/fisiopatologia , Transmissão SinápticaRESUMO
Nerve growth factor (NGF) is synthesized as a precursor, proNGF that undergoes post-translational processing to generate the biologically active mature NGF. While the neurotrophic function of NGF is well established, the activity of the proNGF precursor is still unclear. In this study, we have cloned the pro-domain of the precursor NGF molecule and have elucidated its function. We have used both mature and the furin resistant pro((R/G))NGF as controls in our experiments. Both pro((R/G))NGF and mature NGF (NGF) exhibited neurotrophic activity on PC12 cells while the pro-domain itself promoted cell death. The pro-domain, has been found to mediate apoptosis possibly by promoting the formation of a signaling complex comprising of endogenous p75(NTR) receptor, Bim/Bcl2 group of proteins and JNK and MEK1/2 signaling pathways.
Assuntos
Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Fatores de Crescimento Neural/química , Células PC12 , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Phospholipase A2 liberates free fatty acids and lysophospholipids upon hydrolysis of phospholipids and these products are often associated with detrimental effects such as inflammation and cerebral ischemia. The neuroprotective effect of neutral phospholipase from snake venom has been investigated. RESULTS: A neutral anticoagulant secretory phospholipase A2 (nPLA) from the venom of Naja sputatrix (Malayan spitting cobra) has been found to reduce infarct volume in rats subjected to focal transient cerebral ischemia and to alleviate the neuronal damage in organotypic hippocampal slices subjected to oxygen-glucose deprivation (OGD). Real-time PCR based gene expression analysis showed that anti-apoptotic and pro-survival genes have been up-regulated in both in vivo and in vitro models. Staurosporine or OGD mediated apoptotic cell death in astrocytoma cells has also been found to be reduced by nPLA with a corresponding reduction in caspase 3 activity. CONCLUSION: We have found that a secretory phospholipase (nPLA) purified from snake venom could reduce infarct volume in rodent stroke model. nPLA, has also been found to reduce neuronal cell death, apoptosis and promote cell survival in vitro ischemic conditions. In all conditions, the protective effects could be seen at sub-lethal concentrations of the protein.
Assuntos
Apoptose/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Encéfalo/patologia , Hipocampo/efeitos dos fármacos , Fosfolipases A2/uso terapêutico , Análise de Variância , Animais , Encéfalo/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Venenos Elapídicos/química , Glucose/deficiência , Hipocampo/metabolismo , Hipocampo/patologia , Hipóxia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Análise em Microsséries , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Técnicas de Cultura de Órgãos , Fosfolipases A2/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativador de Plasminogênio Tecidual/metabolismo , Células Tumorais CultivadasRESUMO
The NGF (nerve growth factor) from Naja sputatrix has been purified by gel filtration followed by reversed-phase HPLC. The protein showed a very high ability to induce neurite formation in PC12 cells relative to the mouse NGF. Two cDNAs encoding isoforms of NGF have been cloned and an active recombinant NGF, sputa NGF, has been produced in Escherichia coli as a His-tagged fusion protein. Sputa NGF has been found to be non-toxic under both in vivo and in vitro conditions. The induction of neurite outgrowth by this NGF has been found to involve the high-affinity trkA-p75NTR complex of receptors. The pro-survival mechanism of p75NTR has been mediated by the activation of nuclear factor kappaB gene by a corresponding down-regulation of inhibitory kappaB gene. Real-time PCR and protein profiling (by surface-enhanced laser-desorption-ionization time-of-flight) have confirmed that sputa NGF up-regulates the expression of the endogenous NGF in PC12 cells. Preliminary microarray analysis has also shown that sputa NGF is capable of promoting additional beneficial effects such as the up-regulation of arginine vasopressin receptor 1A, voltage-dependent T-type calcium channel. Hence, sputa NGF forms a new and useful NGF.
