RESUMO
The majority of excitatory synapses in the mammalian brain form on filopodia and spines, actin-rich membrane protrusions present on neuronal dendrites. The biochemical events that induce filopodia and remodel these structures into dendritic spines remain poorly understood. Here, we show that the neuronal actin- and protein phosphatase-1-binding protein, neurabin-I, promotes filopodia in neurons and nonneuronal cells. Neurabin-I actin-binding domain bundled F-actin, promoted filopodia, and delayed the maturation of dendritic spines in cultured hippocampal neurons. In contrast, dimerization of neurabin-I via C-terminal coiled-coil domains and association of protein phosphatase-1 (PP1) with neurabin-I through a canonical KIXF motif inhibited filopodia. Furthermore, the expression of a neurabin-I polypeptide unable to bind PP1 delayed the maturation of neuronal filopodia into spines, reduced the synaptic targeting of AMPA-type glutamate (GluR1) receptors, and decreased AMPA receptor-mediated synaptic transmission. Reduction of endogenous neurabin levels by interference RNA (RNAi)-mediated knockdown also inhibited the surface expression of GluR1 receptors. Together, our studies suggested that disrupting the functions of a cytoskeletal neurabin/PP1 complex enhanced filopodia and impaired surface GluR1 expression in hippocampal neurons, thereby hindering the morphological and functional maturation of dendritic spines.
Assuntos
Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Actinas/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Células Cultivadas , DNA Complementar/genética , Dimerização , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Complexos Multiproteicos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Neurônios/ultraestrutura , Fosfoproteínas Fosfatases/química , Proteína Fosfatase 1 , Estrutura Terciária de Proteína , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Interferência de RNA , Ratos , Receptores de AMPA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transmissão Sináptica , TransfecçãoRESUMO
The excitatory transmitter, glutamate has been implicated in the control of reproduction, hormone secretion and neuroendocrine regulation. The present study examined whether the hypothalamic expression of three key ionotropic glutamate receptor subunits (NMDAR1, GluR1 and GluR6) fluctuates significantly on proestrus in the rat, and whether treatment with the antiprogestin, RU486 affected glutamate receptor subunit expression. The studies revealed that NMDAR1, GluR1 and GluR6 mRNA levels in the mediobasal hypothalamus (MBH) and preoptic area (POA) fluctuate little throughout the day of proestrus. However, treatment with the antiprogestin, RU486 induced a significant elevation of GluR6 mRNA levels at 14.00 and 16.00 h on proestrus in the MBH, suggesting that endogenous progesterone (P4) may act to inhibit hypothalamic GluR6 levels. In support of this suggestion, exogenous P4 treatment to estrogen (E2)-primed ovariectomized (ovx) rats significantly suppressed GluR6 mRNA levels in the afternoon (12.00-16.00 h) in the MBH, and at 12.00 h in the POA, which preceded LH surge induction. Likewise, temporal examination of hypothalamic GluR6 protein levels in E2 + P4-treated young and middle-aged ovx rats revealed an early elevation from 12.00 to 14.00 h, which was followed by a fall from 16.00 to 20.00 h. The early elevation of GluR6 protein levels was most pronounced in the POA of the young rat, and this elevation was markedly attenuated in the middle-aged rat. As a whole, the studies suggest that glutamate receptor expression fluctuates little on proestrus in the hypothalamus, but that expression of the kainate GluR6 receptor subunit may be modulated by progesterone and aging.