RESUMEN
NMDA-type glutamate receptors mediate both trophic and excitotoxic signalling in CNS neurons. We have previously shown that blocking NMDAR- post-synaptic density-95 (PSD95) interactions provides significant protection from excitotoxicity and in vivo ischaemia; however, the mechanism of neuroprotection is unclear. Here, we report that blocking PSD-95 interactions with the Tat-NR2B9c peptide enhances a Ca²âº-dependent protective pathway converging on cAMP Response Element binding protein (CREB) activation. We provide evidence that Tat-NR2B9c neuroprotection from oxygen glucose deprivation and NMDA toxicity occurs in parallel with the activation of calmodulin kinase signalling and is dependent on a sustained phosphorylation of the CREB transcription factor and its activator CaMKIV. Tat-NR2B9c-dependent neuroprotection and CREB phosphorylation are blocked by coapplication of CaM kinase (KN93 and STO-609) or CREB (KG-501) inhibitors, and by siRNA knockdown of CaMKIV. These results are mirrored in vivo in a rat model of permanent focal ischaemia. Tat-NR2B9c application significantly reduces infarct size and causes a selective and sustained elevation in CaMKIV phosphorylation; effects which are blocked by coadministration of KN93. Thus, calcium-dependent nuclear signalling via CaMKIV and CREB is critical for neuroprotection via NMDAR-PSD95 blockade, both in vitro and in vivo. This study highlights the importance of maintaining neuronal function following ischaemic injury. Future stroke research should target neurotrophic and pro-survival signal pathways in the development of novel neuroprotective strategies.
Asunto(s)
Infarto Encefálico/prevención & control , Proteína de Unión a CREB/metabolismo , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Infarto Encefálico/etiología , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Corteza Cerebral/citología , Modelos Animales de Enfermedad , Homólogo 4 de la Proteína Discs Large , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Glucosa/deficiencia , Hipoxia/patología , Técnicas In Vitro , Infarto de la Arteria Cerebral Media/complicaciones , Masculino , Neuronas/metabolismo , Nimodipina/farmacología , Fosforilación , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Cardiac arrest victims may experience transient brain hypoperfusion leading to delayed death of hippocampal CA1 neurons and cognitive impairment. We prevented this in adult rats by inhibiting the expression of transient receptor potential melastatin 7 (TRPM7), a transient receptor potential channel that is essential for embryonic development, is necessary for cell survival and trace ion homeostasis in vitro, and whose global deletion in mice is lethal. TRPM7 was suppressed in CA1 neurons by intrahippocampal injections of viral vectors bearing shRNA specific for TRPM7. This had no ill effect on animal survival, neuronal and dendritic morphology, neuronal excitability, or synaptic plasticity, as exemplified by robust long-term potentiation (LTP). However, TRPM7 suppression made neurons resistant to ischemic death after brain ischemia and preserved neuronal morphology and function. Also, it prevented ischemia-induced deficits in LTP and preserved performance in fear-associated and spatial-navigational memory tasks. Thus, regional suppression of TRPM7 is feasible, well tolerated and inhibits delayed neuronal death in vivo.
Asunto(s)
Isquemia Encefálica/patología , Isquemia Encefálica/prevención & control , Hipocampo/metabolismo , Hipocampo/patología , Neuronas/fisiología , Canales Catiónicos TRPM/metabolismo , Análisis de Varianza , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Células Cultivadas , Modelos Animales de Enfermedad , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Miedo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Humanos , Técnicas In Vitro , Masculino , Aprendizaje por Laberinto/fisiología , Memoria/fisiología , Ratones , Microinyecciones/métodos , Neuronas/efectos de los fármacos , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/uso terapéutico , Ratas , Ratas Wistar , Transfección/métodosRESUMEN
BACKGROUND AND PURPOSE: Postsynaptic density-95 inhibitors reduce ischemic brain damage without inhibiting excitatory neurotransmission, circumventing the negative consequences of glutamatergic inhibition. However, their efficacy in permanent ischemia and in providing permanent neuroprotection and neurobehavioral improvement in a practical therapeutic window is unproven. These were tested here under conditions that included fever, which is a common occurrence in clinical stroke. METHODS: Six studies were performed in unfasted Sprague-Dawley rats. Two involved permanent pial vessel occlusion in male and female rats. Two involved permanent middle cerebral artery occlusion, which induced severe hyperthermia, and 2 involved transient middle cerebral artery occlusion. Animals were treated with a single intravenous injection of postsynaptic density-95 inhibitors (Tat-NR2B9c([SDV]) or Tat-NR2B9c([TDV])) 1 hour or 3 hours after stroke. Infarct volumes and neurobehavior were assessed in a blinded manner at 24 hours (pial vessel occlusion and permanent middle cerebral artery occlusion) or at 62 days (transient middle cerebral artery occlusion). RESULTS: Postsynaptic density-95 inhibitors dramatically reduced infarct size in male and female animals exposed to pial vessel occlusion (>50%), in hyperthermic animals with fever exceeding 39 degrees C exposed to permanent middle cerebral artery occlusion (approximately 50%), and at 62 days poststroke in animals exposed to transient middle cerebral artery occlusion (approximately 80%). Effectiveness of postsynaptic density-95 inhibitors was achieved without the drugs affecting body temperature. In transient middle cerebral artery occlusion, a single dose of postsynaptic density-95 inhibitor given 3 hours after stroke onset permanently maintained reduced infarct size and improved neurobehavior. CONCLUSIONS: Postsynaptic density-95 inhibitors administrated 3 hours after stroke onset reduced infarct volumes and improved long-term neurobehavioral functions in a wide therapeutic window. This raises the possibility that they may have future clinical usefulness.
Asunto(s)
Infarto Encefálico/tratamiento farmacológico , Isquemia Encefálica/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Ataque Isquémico Transitorio/tratamiento farmacológico , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Temperatura Corporal/efectos de los fármacos , Temperatura Corporal/fisiología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatología , Infarto Encefálico/fisiopatología , Infarto Encefálico/prevención & control , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatología , Modelos Animales de Enfermedad , Homólogo 4 de la Proteína Discs Large , Femenino , Fiebre/tratamiento farmacológico , Fiebre/etiología , Fiebre/fisiopatología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/fisiopatología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ataque Isquémico Transitorio/metabolismo , Ataque Isquémico Transitorio/fisiopatología , Masculino , Proteínas de la Membrana/metabolismo , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/fisiopatología , Degeneración Nerviosa/prevención & control , Péptidos/genética , Péptidos/farmacología , Péptidos/uso terapéutico , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Resultado del TratamientoRESUMEN
Angiogenesis within an ischemic region of the brain may increase tissue viability and act to limit the extent of an infarct. The ANG II pathway can both stimulate and inhibit angiogenesis depending on the tissue and the activated receptors. Previous work showed that 2-wk losartan administration (ANG II type 1 receptor blockade) initiates a significant cerebral angiogenic response. We hypothesized that administration of losartan in the drinking water of rats for 2 wk before initiation of focal ischemia would decrease the extent of the resulting infarct. Adult male Sprague-Dawley rats were given losartan (50 mg/day) in drinking water for 2 wk before initiation of cerebral focal ischemia produced by cauterization of cortical surface vessels. Controls received normal drinking water. In control animals, three main vessels feeding the whisker barrel cortex were cauterized, resulting in cessation of blood flow. The same protocol was followed for losartan-treated animals but did not result in cessation of blood flow in the whisker barrel cortex. Another group of losartan-treated animals received between 8 and 14 cauterizations of surface vessels feeding the whisker barrel cortex, and cessation of blood flow was verified. Rats were killed 72 h after surgery. Morphological examination revealed angiogenesis, maintained vascular delivery, and significantly decreased infarct size in losartan-treated animals compared with controls. These results demonstrate that pretreatment with losartan reduces infarct size after cerebral focal ischemia and support the hypothesis that cerebral angiogenesis may be one of the mechanisms responsible.