RESUMEN
BACKGROUND: Dopamine depletion of the striatum is one of the hallmarks of Parkinson's disease. The loss of dopamine upregulates GAD67 expression in the striatal projection neurons and causes other changes in the activity of the basal ganglia circuit. METHODS: To normalize the GAD67 expression in the striatum after dopamine depletion, we developed several lentiviral vectors that express RNA interference (RNAi) directed against GAD67 mitochondrial RNA. The vectors were injected into the striatum of hemiparkinsonian rats and the level of GAD67 protein as well as a marker of neuronal activity, mtCO1, was analyzed using western blots. RESULTS: Unilateral lesions of the dopamine neurons in substantia nigra resulted in an increased level of GAD67 protein in the ipsilateral striatum. Furthermore, we detected significantly higher levels of mtCO1, after dopamine depletion in the striatum. Using a lentiviral vectors with a synthetic miRNA scaffold to deliver RNAi, we were able to normalize the GAD67 protein levels in the parkinsonian rat striatum. In addition, we were able to normalize the increased neural activity, which resulted from the loss of dopamine as measured by the marker mtCO1. CONCLUSIONS: We conclude that RNAi directed against GAD67 may be a valid approach to correct the dysregulation of the basal ganglia circuit in a rat model of Parkinson's disease. The possibility to correct for a loss of dopamine using nondopamimetic tools is interesting because it may be more directed towards the casual mechanisms of the motor symptoms.
Asunto(s)
Dopamina/fisiología , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Neostriado/enzimología , Enfermedad de Parkinson/metabolismo , Animales , Western Blotting , Línea Celular , Modelos Animales de Enfermedad , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Vectores Genéticos , Proteínas Fluorescentes Verdes , Humanos , Inmunohistoquímica , Lentivirus/genética , MicroARNs/química , MicroARNs/genética , Mitocondrias/metabolismo , Neostriado/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Lentiviral vectors offer many advantages within the central nervous system. They are capable of transfecting neurons and can be readily manipulated for selective expression. This chapter describes the production and use of lentiviruses to deliver GFP to the central nervous system.
Asunto(s)
Sistema Nervioso Central/metabolismo , Expresión Génica , Vectores Genéticos/genética , Lentivirus/metabolismo , Proteínas/metabolismo , Transfección/métodos , Línea Celular , Clonación Molecular , Humanos , Lentivirus/genética , Regiones Promotoras Genéticas/genética , Proteínas/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is an incurable degenerative disorder of motoneurons. We recently reported that reduced expression of Vegfa causes ALS-like motoneuron degeneration in Vegfa(delta/delta) mice. In a meta-analysis of over 900 individuals from Sweden and over 1,000 individuals from Belgium and England, we now report that subjects homozygous with respect to the haplotypes -2,578A/-1,154A/-634G or -2,578A/-1,154G/-634G in the VEGF promoter/leader sequence had a 1.8 times greater risk of ALS (P = 0.00004). These 'at-risk' haplotypes lowered circulating VEGF levels in vivo and reduced VEGF gene transcription, IRES-mediated VEGF expression and translation of a novel large-VEGF isoform (L-VEGF) in vivo. Moreover, SOD1(G93A) mice crossbred with Vegfa(delta/delta) mice died earlier due to more severe motoneuron degeneration. Vegfa(delta/delta) mice were unusually susceptible to persistent paralysis after spinal cord ischemia, and treatment with Vegfa protected mice against ischemic motoneuron death. These findings indicate that VEGF is a modifier of motoneuron degeneration in human ALS and unveil a therapeutic potential of Vegfa for stressed motoneurons in mice.
