RESUMO
Ubiquitous classical (typical) calpains, calpain-1 and calpain-2, are Ca(+2)-dependent cysteine proteases, which have been associated with numerous physiological and pathological cellular functions. However, a clear understanding of the role of calpains in the CNS has been hampered by the lack of appropriate deletion paradigms in the brain. In this study, we describe a unique model of conditional deletion of both calpain-1 and calpain-2 activities in mouse brain, which more definitively assesses the role of these ubiquitous proteases in brain development/function and pathology. Surprisingly, we show that these calpains are not critical for gross CNS development. However, calpain-1/calpain-2 loss leads to reduced dendritic branching complexity and spine density deficits associated with major deterioration in hippocampal long-term potentiation and spatial memory. Moreover, calpain-1/calpain-2-deficient neurons were significantly resistant to injury induced by excitotoxic stress or mitochondrial toxicity. Examination of downstream target showed that the conversion of the Cdk5 activator, p35, to pathogenic p25 form, occurred only in the presence of calpain and that it played a major role in calpain-mediated neuronal death. These findings unequivocally establish two central roles of calpain-1/calpain-2 in CNS function in plasticity and neuronal death.
Assuntos
Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Encéfalo , Calpaína/deficiência , Potenciação de Longa Duração/fisiologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Biofísica , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/fisiopatologia , Bromodesoxiuridina/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Dendritos/metabolismo , Dendritos/patologia , Dendritos/ultraestrutura , Modelos Animais de Doenças , Estimulação Elétrica , Embrião de Mamíferos , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Técnicas In Vitro , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Potenciação de Longa Duração/genética , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , N-Metilaspartato/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/citologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Fosfotransferases , Desempenho Psicomotor , RNA Mensageiro/metabolismo , Coloração pela Prata , TransfecçãoRESUMO
We have earlier reported the critical nature of calpain-CDK5-MEF2 signaling in governing dopaminergic neuronal loss in vivo. CDK5 mediates phosphorylation of the neuronal survival factor myocyte enhancer factor 2 (MEF2) leading to its inactivation and loss. However, the downstream factors that mediate MEF2-regulated survival are unknown. Presently, we define Nur77 as one such critical downstream survival effector. Following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in vivo, Nur77 expression in the nigrostriatal region is dramatically reduced. This loss is attenuated by expression of MEF2. Importantly, MEF2 constitutively binds to the Nur77 promoter in neurons under basal conditions. This binding is lost following 1-methyl-4-phenylpyridinium treatment. Nur77 deficiency results in significant sensitization to dopaminergic loss following 1-methyl-4-phenylpyridinium/MPTP treatment, in vitro and in vivo. Furthermore, Nur77-deficient MPTP-treated mice displayed significantly reduced levels of dopamine and 3,4-Dihydroxyphenylacetic acid in the striatum as well as elevated post synaptic FosB activity, indicative of increased nigrostriatal damage when compared with WT MPTP-treated controls. Importantly, this sensitization in Nur77-deficient mice was rescued with ectopic Nur77 expression in the nigrostriatal system. These results indicate that the inactivation of Nur77, induced by loss of MEF2 activity, plays a critical role in nigrostriatal degeneration in vivo.