RESUMO
Nicotinic acetylcholine receptors (nAChRs) contribute significantly to hippocampal function. Alpha7-nAChRs are present in presynaptic sites in hippocampal neurons and may influence transmitter release, but the factors that determine their presynaptic localization are unknown. We report here that Wnt-7a, a ligand active in the canonical Wnt signaling pathway, induces dissociation of the adenomatous polyposis coli (APC) protein from the beta-catenin cytoplasmic complex and the interaction of APC with alpha7-nAChRs in hippocampal neurons. Interestingly, Wnt-7a induces the relocalization of APC to membranes, clustering of APC in neurites, and coclustering of APC with different, presynaptic protein markers. Wnt-7a also increases the number and size of coclusters of alpha7-nAChRs and APC in presynaptic terminals. These short-term changes in alpha7-nAChRs occur in the few minutes after ligand exposure and involve translocation to the plasma membrane without affecting total receptor levels. Longer-term exposure to Wnt-7a increases nAChR alpha7 subunit levels in an APC-independent manner and increases clusters of alpha7-nAChRs in neurites via an APC-dependent process. Together, these results demonstrate that stimulation through the canonical Wnt pathway regulates the presynaptic localization of APC and alpha7-nAChRs with APC serving as an intermediary in the alpha7-nAChR relocalization process. Modulation by Wnt signaling may be essential for alpha7-nAChR expression and function in synapses.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Receptores Nicotínicos/metabolismo , Proteínas Wnt/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Hipocampo/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/química , Terminações Pré-Sinápticas/química , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/fisiologia , Transdução de Sinais/fisiologia , Receptor Nicotínico de Acetilcolina alfa7 , beta Catenina/metabolismoRESUMO
Amyloid-Beta (Aβ) accumulation and aggregation are thought to contribute to the pathogenesis of Alzheimers disease (AD). In AD, there also is a selective decrease in numbers of radioligand binding sites corresponding to the most abundant nicotinic acetylcholine receptor (nAChR) subtype, which contains human α4 and β2 subunits (α4β2-nAChR). However, relationships between these phenomena are uncertain, and effects of Aβ on human α4β2-nAChR function have not been investigated in detail. We created SH-EP1 cells stably transfected to heterologously express human α4β2- or α7-nAChR subtypes. Whole-cell current recording confirmed heterologous expression of functional α4β2-nAChR with characteristic responses to nicotinic agonists or antagonists. Nicotine-induced whole-cell currents were suppressed by Aβ1−42 in a dose-dependent manner. Functional inhibition was selective for Aβ1−42 compared to functionally-inactive, control peptide Aβ40-1, but was mimicked by Aβ1-40. Aβ1-42-mediated inhibition of α4β2-nAChR function was non-competitive, voltage¬independent, and use-independent. Pre-loading of cells with GDP-β-S failed to prevent Aβ1-42 induced inhibition, suggesting that the down-regulation of α4β2-nAChR function by Aβ1-42 is not mediated by nAChR internalization. Sensitivity to Aβ1-42 antagonism at 1 nM was evident for α4β 2-nAChR, but not for heterologously expressed, human α7-nAChR, although both nAChR subtypes were functionally inhibited by 100 nM Aβ1-42, with the magnitude of functional block being higher for 100 nM Aβ1-42 acting at α7-nAChR. These findings suggest that α4β2-nAChR are sensitive and perhaps pathophysiologically-relevant targets for Aβ neurotoxicity in AD.