Spinophilin Signaling in the Striatum

描述(申请人提供)：帕金森病(PD)是由含有多巴胺的神经元变性引起的，这些神经元从实质黑质投射到纹状体。这些神经元与树突棘结构形成突触连接，树突棘位于中棘神经元(MSN)的两个亚群上，它们分别表达D1R和D2R多巴胺受体。树突棘也接受来自皮质的谷氨酸能突触输入，激活脊椎中的钙依赖信号。因此，多巴胺的释放不同地调节了含有D1R和D2R的MSN树突棘内谷氨酸的活动。在帕金森病患者和帕金森病动物模型中，多巴胺缺乏会导致纹状体MSN树突棘的丢失；然而，在短期啮齿动物研究中，脊椎丢失仅限于含有D2R的MSN。此外，来自几个实验室的新数据表明，含有D1R和D2R的MSN上的皮质纹状体突触受到多巴胺和其他神经递质的不同调节。然而，对纹状体MSN亚群之间的生化差异知之甚少，这可能是这些差异的原因。本项目研究亲刺素在含有D1R和D2R的MSN中的作用。刺亲素是一种支架蛋白，它结合蛋白磷酸1(PP1)、F-肌动蛋白和其他几种参与调节细胞信号和形态的蛋白质。在含有D1R和D2R的MSN中，SPIN基因的全局敲除扰乱了皮质纹状体突触功能，并以一种年龄依赖的方式影响纹状体MSN的形态。我最近的研究表明，多巴胺的耗竭增强了亲刺蛋白与PP1？1的相互作用，可能调节了其他相关树突状蛋白的去磷酸化。为了确定Spin-PP1复合体的潜在底物，我进行了蛋白质组学筛选，在正常成熟的纹状体中鉴定了多个Spaps，已知这些蛋白调节细胞形态，包括CaMKII。本实验室以前的研究表明，多巴胺耗竭会导致CaMKII的过度磷酸化。在这个职业发展奖中，我将开发创新的转基因分子工具来解决我的总体假设：多巴胺耗竭和衰老在纹状体MSN亚型中以不同的方式调节亲刺蛋白依赖的信号。两个AIMS将开始检验这一假说：Aim 1将检验这一假说，即多巴胺缺乏会以一种与年龄相关的方式改变亲刺蛋白相互作用网络。目的2将利用新型转基因动物在含有D1R或D2R的MSN中表达不同标记形式的刺亲素，以验证多巴胺耗竭以细胞特异性方式改变亲刺素相互作用网络的假说。这些研究将极大地提高我们对亲刺素介导的帕金森病动物模型中纹状体信号的理解，并将为治疗这种疾病的潜在药物靶点提供信息。

DESCRIPTION (provided by applicant): Parkinson disease (PD) is proximally caused by degeneration of dopamine containing neurons that project from the Substantial Nigra to the striatum. These neurons form synaptic connections with structures termed dendritic spines that reside on two subpopulations of the medium spiny neuron (MSN) that express either D1 dopamine receptors or D2 dopamine receptors (D1R and D2R, respectively). The dendritic spines also receive glutamatergic synaptic inputs from the cortex that activates calcium-dependent signaling in spines. Thus, the release of dopamine differentially modulates the actions of glutamate within the dendritic spines of D1R- and D2R-containing MSNs. Dopamine-depletion in PD patients and in parkinsonian animal models results in loss of dendritic spines from striatal MSNs; however, spine loss is restricted to D2R-containing MSNs in short-term rodent studies. Moreover, emerging data from several labs indicate that corticostriatal synapses onto D1R- and D2R-containing MSNs are differentially regulated by dopamine and other neurotransmitters. However, very little is known about the biochemical differences between striatal MSN subpopulations that presumably account for these differences. This project investigates the roles of spinophilin in D1R- and D2R-containing MSNs. Spinophilin is a scaffolding protein that binds protein phosphates 1 (PP1), F-actin, and several other proteins involved in regulating cell signaling and morphology. The global knockout of spinophilin disrupts corticostriatal synaptic function in both D1R- and D2R-containing MSNs, and also affects the morphology of striatal MSNs in an age- dependent manner. My recent studies showed that dopamine depletion enhances the interaction of spinophilin with PP1¿1; presumably modulating the dephosphorylation of other associated dendritic proteins. In order to identify potential substrates of the spinophilin-PP1 complex, I performed a proteomics screen, identifying multiple spinophilin-associated proteins (SpAPs) in normal, mature striatum that are known to regulate cell morphology, including CaMKII. Previous studies in this lab showed that dopamine depletion leads to hyper- phosphorylation of CaMKII. In this career development award I will develop innovative transgenic molecular tools to address my over-arching hypothesis that: dopamine depletion and aging differentially regulate spinophilin-dependent signaling in striatal MSN subtypes. Two aims will begin to test this hypothesis: Aim 1 will test the hypothesis that dopamine depletion alters the spinophilin interaction network in an age-dependent manner. Aim 2 will use novel transgenic animals expressing differentially tagged forms of spinophilin in D1R- or D2R- containing MSNs to test the hypothesis that dopamine depletion alters the spinophilin interaction network in a cell-specific manner. These studies will greatly enhance our understanding of spinophilin-mediated striatal signaling in animal models of PD and will inform potential drug targets for this disorder.