RESUMEN
BACKGROUND AND PURPOSE: Hypoxia inducible factor-1 (HIF-1) promotes transitory neuronal survival suggesting that additional mechanisms such as the endoplasmic reticulum (ER) stress might be involved in determining neuronal survival or death. Here, we examined the involvement of ER stress in hypoxia-induced neuronal death and analysed the relationship between ER stress and the HIF-1 pathways. EXPERIMENTAL APPROACH: Cultures of rat cortical neurons were exposed to chemical hypoxia induced by 200 µM CoCl2 , and its effect on neuronal viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and counting apoptotic nuclei. Protein levels were determined by Western blot analysis. RT-PCR was performed to analyse the content and the t1/2 of HIF-1α mRNA. KEY RESULTS: Chemical hypoxia induced neuronal apoptosis in a time-dependent manner and activated the ER stress PRK-like endoplasmic reticulum kinase (PERK)-dependent pathway. At later stages, chemical hypoxia increased the expression of the C/EBP homologous protein (CHOP) and caspase 12 activity. CoCl2 reduced HIF-1α mRNAâ t1/2 leading to a decrease in HIF-1α mRNA and protein content, simultaneously activating the ER stress PERK-dependent pathway. Salubrinal, a selective inhibitor of phospho-eIF2α phosphatase, protected neurons from chemical hypoxia by reducing CHOP levels and caspase 12 activity, and increasing the t1/2 of HIF-1α mRNA and the levels of HIF-1α protein. Knocking down HIF-1α blocked the neuroprotective effects of salubrinal. CONCLUSIONS AND IMPLICATIONS: Neuronal apoptosis induced by chemical hypoxia is a process regulated by HIF-1α stabilization early on and by ER stress activation at later stages. Our data also suggested that HIF-1α levels were regulated by ER stress.
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Apoptosis/genética , Estrés del Retículo Endoplásmico/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Hipoxia/genética , Neuronas/metabolismo , ARN Mensajero/metabolismo , Animales , Caspasa 12/efectos de los fármacos , Caspasa 12/metabolismo , Supervivencia Celular/genética , Células Cultivadas , Cinamatos/farmacología , Cobalto/toxicidad , Factor 2 Eucariótico de Iniciación/metabolismo , Técnicas de Silenciamiento del Gen , Hipoxia/inducido químicamente , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Tiourea/análogos & derivados , Tiourea/farmacología , Factor de Transcripción CHOP/efectos de los fármacos , Factor de Transcripción CHOP/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
Many studies have focused on expanding our knowledge of the structure and diversity of peripheral and central nicotinic receptors. Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of pentameric ligand-gated ion channels, which include GABA (A and C), serotonin, and glycine receptors. Currently, 9 alpha (α2-α10) and 3 beta (ß2-ß4) subunits have been identified in the central nervous system (CNS), and these subunits assemble to form a variety of functional nAChRs. The pentameric combination of several alpha and beta subunits leads to a great number of nicotinic receptors that vary in their properties, including their sensitivity to nicotine, permeability to calcium and propensity to desensitize. In the CNS, nAChRs play crucial roles in modulating presynaptic, postsynaptic, and extrasynaptic signaling, and have been found to be involved in a complex range of CNS disorders including Alzheimer's disease (AD), Parkinson's disease (PD), schizophrenia, Tourette´s syndrome, anxiety, depression and epilepsy. Therefore, there is growing interest in the development of drugs that modulate nAChR functions with optimal benefits and minimal adverse effects. The present review describes the main characteristics of nAChRs in the CNS and focuses on the various compounds that have been tested and are currently in phase I and phase II trials for the treatment of neurodegenerative diseases including PD, AD and age-associated memory and mild cognitive impairment.
RESUMEN
Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that plays a key role in regulating the adaptive response to hypoxia. HIF-1α is stabilised during hypoxia and, after dimerisation with hypoxia-inducible factor 1ß (HIF-1ß), triggers the expression of various genes involved in cell cycle control and energy metabolism associated with cell survival. However, HIF-1α also regulates the expression of proapoptotic genes. The aim of this study was to ascertain the influence of HIF-1α on neurotoxicity evoked by hypoxia in rat cortical neurons. We found that mild hypoxia induces time-dependent neuronal death involving free radical production, mitochondrial depolarisation, cytochrome c release and caspase-3 activation. Lentivirus-mediated HIF-1α knockdown markedly strengthened all of these effects during the initial 24h of hypoxia, which suggests that HIF-1α plays a neuroprotective role in hypoxia-mediated neuronal death. After this initial period, the protective actions of HIF-1α disappeared over the course of the hypoxia-mediated HIF-1α stabilisation. Moreover, lentiviral-mediated overexpression of HIF-1α increased lactate dehydrogenase (LDH) A, one of the target genes for HIF-1α, but did not show protective actions on hypoxia-mediated neuronal death, indicating that the level of endogenous HIF-1α stabilisation achieved during hypoxia was already the maximum required for HIF-1α transcription activities. These results indicate that HIF-1α is neuroprotective in the early phases of hypoxia.
Asunto(s)
Corteza Cerebral/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/patología , Neuronas/efectos de los fármacos , Animales , Animales Recién Nacidos , Muerte Celular , Células Cultivadas , Ciclooxigenasa 1/metabolismo , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Depuradores de Radicales Libres/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Glutatión/metabolismo , Proteínas Fluorescentes Verdes/genética , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , L-Lactato Deshidrogenasa/metabolismo , Potencial de la Membrana Mitocondrial/fisiología , Proteínas de la Membrana/metabolismo , Metaloporfirinas/farmacología , Neuronas/ultraestructura , Oxígeno/metabolismo , Fenantridinas , Interferencia de ARN/fisiología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno , Sales de Tetrazolio , Tiazoles , Factores de TiempoRESUMEN
PURPOSE: To study the effect of a non-viral vector (carbosilane dendrimer) to efficiently deliver small interfering RNA to postmitotic neurons to study the function of hypoxia-inducible factor-1alpha (HIF1-alpha) during chemical hypoxia-mediated neurotoxicity. METHODS: Chemical hypoxia was induced in primary rat cortical neurons by exposure to CoCl(2). HIF1-alpha levels were determined by Western Blot and toxicity was evaluated by both MTT and LDH assays. Neurons were incubated with dendriplexes containing anti-HIF1-alpha siRNA and both uptake and HIF1-alpha knockdown efficiency were evaluated. RESULTS: We report that a non-viral vector (carbosilane dendrimer) can deliver specific siRNA to neurons and selectively block HIF1-alpha synthesis with similar efficiency to that achieved by viral vectors. Using this method, we have found that this transcription factor plays a neuroprotective role during the early phase of chemical hypoxia-mediated neurotoxicity. CONCLUSION: This work represents a proof-of-concept for the use of carbosilane dendrimers to deliver specific siRNA to postmitotic neurons to block selected protein synthesis. This indicates that this type of vector is a good alternative to viral vectors to achieve very high transfection levels in neurons. This also suggests that carbosilane dendrimers might be very useful for gene therapy.