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。根本的差距包括识别细胞粘附分子，可以产生所需的多样性，以促进发展中的哺乳动物中枢神经系统的细胞之间的特异性识别，并阐明相关的信号通路，调节几个关键步骤，包括制定树突状乔木和突触。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。本更新申请的目的是确定？- Pcdhs，一个由22个钙粘蛋白超家族粘附分子组成的家族，调节皮质树突分支。中心假设是同性恋之间的相互作用？-皮质神经元和星形胶质细胞上的Pcdh四聚体通过抑制PKC信号通路促进树突分枝。这一假设是基于研究者实验室在前一个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）确定同性恋？Pcdh相互作用促进皮层神经元树突分支; 2）确定星形胶质细胞？皮质神经元树突树枝化中的PCDHs和3）确定调节？Pcdhs及其在树枝化中的作用。根据目标1，初步体外试验产生的模型-组合多样？Pcdh顺式四聚体在反式中均亲性相互作用将应用于皮质发育。神经元？Pcdh四聚体组合物将操纵使用转染和几个新的Pcdh-？敲入转基因小鼠系，以直接解决相互作用特异性在树突树枝化中的作用。目标2建立在初步数据的基础上，Pcdhs作为脊髓回路形成的关键调节因子。使用星形胶质细胞限制性Cre转基因与条件Pcdh-？突变体和基因敲入体内，星形胶质细胞的作用？在皮层神经元树突分支中的Pcdhs将被描绘出来。目的3扩展了初步数据，表明PKC信号通路被？Pcdhs促进树突树枝状化。一个所有人都共享的膜相互作用C-末端基序？已确定Pcdhs，当被PKC磷酸化时，假设增加？胞外域脱落和胞内裂解，提供信号反馈机制。这将使用一系列的点突变和截断Pcdh-？生物化学测定中的构建体。在这一目标下，还将测试CRMP 1所发挥的作用，CRMP 1在前一个资助期内被确定为一种新的？胞内结合伴侣，树突树枝化。这项研究具有重要意义，因为它将确定可以解释不同细胞间相互作用的分子机制，从而推动神经回路形成的关键步骤，填补该领域现有知识的重要空白。这些信息将是至关重要的理解，并最终改善，许多神经发育障碍，涉及缺陷树突发育和突触。

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 ?-Pcdhs, a family of 22 cadherin superfamily adhesion molecules, regulate cortical dendrite arborization. The central hypothesis is that homophilic interactions between ?-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 investigator's laboratory during the prior funding period, and will be tested by pursuing 3 Specific Aims: 1) Determine the extent to which homophilic ?-Pcdh interactions promote dendrite arborization in cortical neurons; 2) Identify roles for astrocytic ?-Pcdhs in cortical neuron dendrite arborization and 3) Identify intracellular signaling mechanisms regulating ?-Pcdhs and their role in arborization. Under Aim 1, a model resulting from preliminary in vitro assays - combinatorially diverse ?-Pcdh cis-tetramers interact homophilically in trans - will be applied to cortical development. Neuronal ?-Pcdh tetramer composition will be manipulated using transfection and several novel Pcdh-? knock-in transgenic mouse lines to directly address the role of interaction specificity in dendrite arborization. Aim 2 builds on preliminary data establishing astrocytic ?-Pcdhs as key regulators of circuit formation in the spinal cord. Using astrocyte-restricted Cre transgenics with conditional Pcdh-? mutants and knock-ins in vivo, the role of astrocytic ?-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 ?-Pcdhs to promote dendrite arborization. A membrane-interacting C-terminal motif shared by all ?-Pcdhs has been identified that, when phosphorylated by PKC, is hypothesized to increase ?-Pcdh ectodomain shedding and intracellular cleavage, providing a signaling feedback mechanism. This will be tested using a series of point mutant and truncation Pcdh-? constructs in biochemical assays. Also tested under this aim will be the role played by CRMP1, identified during the prior funding period as a novel ?-Pcdh intracellular binding partner, in dendrite arborization. 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.