Role of PAK5 in neurons and dendritic spines

描述（由申请人提供）： Rho GTP酶Cdc 42、Rac和Rho通过控制肌动蛋白细胞骨架的组织来调节细胞形态。虽然它们最初在成纤维细胞中具有特征，但它们在神经元发育中也具有关键作用。例如，它们在控制轴突导向和神经突生长中起重要作用。我们最近的工作表明Rho GTP酶的另一个功能可能是控制树突棘的密度和形态。树突棘是在大多数树突表面发现的富含肌动蛋白的突起，并且它们介导大脑中的大多数突触接触。棘密度、形状和运动性的调节被认为对新皮层的发育和功能具有重要意义。Rho GTP酶介导神经元细胞形态变化的靶点尚不完全清楚。我们已经确定了Cdc 42和Rac的一个新靶点，称为PAK 5，这是一种主要在大脑中表达的丝氨酸/苏氨酸激酶。我们已经发现PAK 5触发神经元细胞系中的丝状伪足形成和神经突生长。PAK 5的底物未知。该提案的目标是确定PAK 5靶点和调节蛋白，并确定PAK 5及其靶蛋白是否以及如何控制神经元形态。在第一个目标中，我们将确定PAK 5的分子靶点和与PAK 5相互作用的蛋白质。在第二个目标中，我们将确定这些蛋白质是否是导致N1 E-115细胞中神经突生长和丝状伪足形成的途径的一部分。在第三个目标中，我们将使用基因枪转染和双光子显微镜研究PAK 5在调节小鼠脑切片锥体神经元和浦肯野细胞中树突棘的形态、密度和运动性中的可能作用。在这个目标中，我们还将研究我们实验室中产生的PAK 5敲除小鼠的脊柱运动性和结构。总之，本研究的实验结果将有助于阐明控制神经元细胞形态和脊髓发生的分子机制，这对正常和患病大脑的发育和功能具有重要影响。

DESCRIPTION (provided by applicant): The Rho GTPases Cdc42, Rac, and Rho regulate cell morphology by controlling the organization of the actin cytoskeleton. While they were originally characterized in fibroblasts, they also have key roles in neuronal development. For example, they play important roles in controlling axon guidance and neurite outgrowth. Our recent work has indicated that another function of the Rho GTPases may be to control the densities and morphologies of dendritic spines. Dendritic spines are actin rich projections found on the surface of most dendrites, and they mediate most synaptic contacts in the brain. The regulation of spine density, shape, and motility is thought to have important implications for the development and function of the neocortex. The targets for the Rho GTPases that mediate changes in neuronal cell morphology are not entirely known. We have identified a new target for Cdc42 and Rac called PAK5, which is a serine/threonine kinase that is expressed primarily in the brain. We have found that PAK5 triggers filopodia formation and neurite outgrowth in neuronal cell lines. The substrates for PAK5 are unknown. The goal of this proposal is to identify PAK5 targets and regulatory proteins and to determine whether and how PAK5 and its target proteins control neuronal morphology. In the first aim we will identify molecular targets for PAK5 and proteins that interact with PAK5. In the second aim we will determine whether these proteins are part of the pathway that leads to neurite outgrowth and filopodia formation in N1E-115 cells. In the third aim we will use biolistic transfections and two photon microscopy to investigate the possible role for PAK5 in regulating the morphology, density, and motility of dendritic spines in pyramidal neurons and purkinje cells from mouse brain slices. In this aim we will also examine spine motility and structure in PAK5 knockout mice that are being generated in our lab. Overall, the results from the experiments in this proposal will help elucidate the molecular mechanisms that control neuronal cell morphology and spinogenesis, which have important repercussion for the development and function of the normal and diseased brain.