Elucidating Functions of the Gamma-Protocadherins in CNS Development

描述（由申请人提供）：目前对神经元之间以及神经元和神经胶质之间形成神经回路所需的相互作用的理解是不完整的。基本的空白包括确定细胞粘附分子，这些分子可以产生促进发育中的哺乳动物中枢神经系统细胞之间特异性识别所需的多样性，并阐明调节几个关键步骤的相关信号通路，包括树突乔木的发育和突触发生。长期目标是确定在发育过程中控制神经回路正确形成的分子机制。这项新应用的目的是确定由22个钙粘蛋白超家族粘附分子组成的γ - pcdhs家族调节皮质树突乔木的分子机制。核心假设是皮质神经元和星形胶质细胞上的γ - pcdh四聚体之间的亲同性相互作用通过抑制PKC信号通路促进树突树突化。这一假设是基于申请人实验室在之前的资助期内产生的大量初步数据，并将通过追求3个具体目标进行测试：1)确定亲同质γ -Pcdh相互作用促进皮层神经元树突树突化的程度；2)确定星形细胞γ -Pcdhs在皮层神经元树突生长中的作用；3)确定调节γ - pcdhs在树木生长中的作用的细胞内信号传导机制。在Aim 1下，一个由初步体外实验产生的模型——组合多样的γ -Pcdh顺式四聚体在反式中同源相互作用——将被应用于皮质发育。神经元γ -Pcdh四聚体的组成将通过转染和几种新的Pcdh- γ敲入转基因小鼠系来操纵，以直接解决相互作用特异性在树突树木化中的作用。目的2建立在初步数据的基础上，建立星形细胞γ - pcdhs作为脊髓回路形成的关键调节因子。利用星形细胞限制性Cre转基因与条件Pcdh- γ突变体和体内敲入蛋白，将描绘星形细胞γ -Pcdh在皮质神经元树突树突化中的作用。Aim 3扩展了初步数据，表明PKC信号通路被γ -Pcdhs抑制，从而促进树突的形成。所有γ -Pcdhs共有的c端残基已被确定，可被PKC磷酸化。我们假设这破坏了γ -Pcdhs通过抑制FAK抑制PKC信号传导的能力，从而提供了一种信号反馈机制。这将在生化分析和神经元培养中使用一系列点突变体和截断Pcdh- γ构建体进行测试。这项提议的研究意义重大，因为它将确定能够解释不同细胞间相互作用的分子机制，这些相互作用驱动了神经回路形成的关键步骤，

DESCRIPTION (provided by applicant): Current understanding of the interactions between neurons, and between neurons and glia, required for the formation of neural circuits is incomplete. Fundamental gaps include identifying cell adhesion molecules that can generate the diversity needed to promote specific recognition between cells of the developing mammalian CNS, and elucidating associated signaling pathways that regulate several key steps, including elaboration of dendritic arbors and synaptogenesis. The long-term goal is to identify the molecular mechanisms that control the proper formation of neural circuits during development. The objective of this renewal application is to identify the molecular mechanisms by which the gamma-Pcdhs, a family of 22 cadherin superfamily adhesion molecules, regulate cortical dendrite arborization. The central hypothesis is that homophilic interactions between gamma-Pcdh tetramers on cortical neurons and astrocytes promote dendrite arborization by inhibiting a PKC signaling pathway. This hypothesis is based on extensive preliminary data generated by the applicant's laboratory during the prior funding period, and will be tested by pursuing 3 Specific Aims: 1) Determine the extent to which homophilic gamma -Pcdh interactions promote dendrite arborization in cortical neurons; 2) Identify roles for astrocytic gamma -Pcdhs in cortica neuron dendrite arborization; and 3) Identify intracellular signaling mech- anisms regulating the gamma -Pcdhs' role in arborization. Under Aim 1, a model resulting from preliminary in vitro assays--combinatorially diverse gamma -Pcdh cis-tetramers interact homophilically in trans--will be applied to cortical development. Neuronal gamma -Pcdh tetramer composition will be manipulated using transfection and several novel Pcdh- gamma knock-in transgenic mouse lines to directly address the role of interaction specificity in dendrite arborization. Aim 2 build on preliminary data establishing astrocytic gamma -Pcdhs as key regulators of circuit form- ation in the spinal cord. Using astrocyte-restricted Cre transgenics with conditional Pcdh- gamma mutants and knock- ins in vivo, the role of astrocytic gamma -Pcdhs in dendrite arborization of cortical neurons will be delineated. Aim 3 expands on preliminary data showing that a PKC signaling pathway is inhibited by the gamma -Pcdhs to promote dendrite arborization. A C-terminal residue shared by all gamma -Pcdhs has been identified that can be phosphorylated by PKC. We hypothesize that this disrupts the gamma -Pcdhs' ability to inhibit PKC signaling via inhibition of FAK, providing a signaling feedback mechanism. This will be tested using a series of point mutant and truncation Pcdh- gamma constructs in biochemical assays and neuronal cultures. The proposed research is significant, because it will identify molecular mechanisms that can account for diverse cell-cell interactions driving a key step in neural circuit formation, filling an important gap in current knowledge in the field. Such information will be critical to understanding, and eventually ameliorating, the many neurodevelopmental disorders that involve defective dendrite development and synaptogenesis.