RESUMO
Attention deficit/Hyperactivity disorder (ADHD) is one of the most diagnosed psychiatric disorders nowadays. The core symptoms of the condition include hyperactivity, impulsiveness and inattention. The main pharmacological treatment consists of psychostimulant drugs affecting Dopamine Transporter (DAT) function. We have previously shown that genetically modified mice lacking p35 protein (p35KO), which have reduced Cdk5 activity, present key hallmarks resembling those described in animal models useful for studying ADHD. The p35KO mouse displays spontaneous hyperactivity and shows a calming effect of methylphenidate or amphetamine treatment. Interestingly, dopaminergic neurotransmission is altered in these mice as they have an increased Dopamine (DA) content together with a low DA turnover. This led us to hypothesize that the lack of Cdk5 activity affects DAT expression and/or function in this animal model. In this study, we performed biochemical assays, cell-based approaches, quantitative fluorescence analysis and functional studies that allowed us to demonstrate that p35KO mice exhibit decreased DA uptake and reduced cell surface DAT expression levels in the striatum (STR). These findings are supported by in vitro observations in which the inhibition of Cdk5 activity in N2a cells induced a significant increase in constitutive DAT endocytosis with a concomitant increase in DAT localization to recycling endosomes. Taken together, these data provide evidences regarding the role of Cdk5/p35 in DAT expression and function, thus contributing to the knowledge of DA neurotransmission physiology and also providing therapeutic options for the treatment of DA pathologies such as ADHD.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Quinase 5 Dependente de Ciclina/deficiência , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/biossíntese , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , Linhagem Celular , Quinase 5 Dependente de Ciclina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Ativação Enzimática/fisiologia , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Cerebral ischemia is a cerebrovascular episode that generates a high incidence of death and physical and mental disabilities worldwide. Excitotoxicity, release of free radicals, and exacerbated immune response cause serious complications in motor and cognitive areas during both short and long time frames post-ischemia. CDK5 is a kinase that is widely involved in the functions of neurons and astrocytes, and its over-activation is implicated in neurodegenerative processes. In this study, we evaluated the brain parenchymal response to the transplantation of CDK5-knockdown astrocytes into the somatosensory cortex after ischemia in rats. Male Wistar rats were subjected to the two-vessel occlusion (2VO) model of global cerebral ischemia and immediately transplanted with shCDK5miR- or shSCRmiR-transduced astrocytes or with untransduced astrocytes (Control). Our findings showed that animals transplanted with shCDK5miR astrocytes recovered motor and neurological performance better than with those transplanted with WT or shSCRmiR astrocytes. Cell transplantation produced an overall prevention of neuronal loss, and CDK5-knockdown astrocytes significantly increased the immunoreactivity (IR) of endogenous GFAP in branches surrounding blood vessels, accompanied by the upregulation of PECAM-1 IR in the walls of vessels in the motor and somatosensory regions and by an increase in Ki67 IR in the subventricular zone (SVZ), partially associated with the production of BDNF. Together, our data suggest that transplantation of shCDK5miR astrocytes protects the neurovascular unit in ischemic rats, allowing the motor and neurological function recovery.