Elucidating Functions of the Gamma-Protocadherins in CNS Development

描述(申请人提供)：目前对神经回路形成所需的神经元之间以及神经元和神经胶质细胞之间的相互作用的理解是不完整的。基本的差距包括识别细胞黏附分子，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，以及阐明调节几个关键步骤的相关信号通路，包括树突状乔木和突触发生的阐述。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。这一更新应用的目的是确定β-Pcdhs-22个钙粘附素超家族黏附分子家族调节皮质树突树枝形成的分子机制。中心假设是皮层神经元和星形胶质细胞上的β-PcdH四聚体之间的亲和性相互作用通过抑制PKC信号通路促进树突树枝形成。这一假说是基于研究人员实验室在之前的资助期间产生的大量初步数据，并将通过追求三个具体目标进行验证：1)确定同嗜性？-Pcdh相互作用在多大程度上促进皮质神经元树突的树枝分枝；2)确定星形细胞？-Pcdhs在皮质神经元树突树枝分枝中的作用；3)确定细胞内调节？-Pcdhs的信号机制及其在树枝分枝中的作用。在目标1下，一个由初步体外分析-组合多样性？-Pcdh顺式四聚体在反式中同亲作用而产生的模型将被应用于皮质发育。神经细胞？-Pcdh四聚体的组成将通过转染法和几种新的Pcdh-？敲入转基因小鼠品系直接解决相互作用专一性在树突树枝形成中的作用。目的2建立在初步数据的基础上，确定星形细胞β-Pcdhs是脊髓环路形成的关键调节因子。将星形胶质细胞限制性Cre转基因与条件性Pcdh-？在活体中，星形细胞？-Pcdhs在皮质神经元树突树枝形成中的作用将被描述。目的3在初步数据的基础上展开，表明？-Pcdhs抑制PKC信号通路以促进树突分枝。一个膜相互作用的C末端基序被发现，当被PKC磷酸化时，假设增加了β-Pcdh胞外结构域的脱落和细胞内的切割，提供了一种信号反馈机制。这将使用一系列点突变和截断Pcdh-？生化分析中的结构。在这一目标下，还将测试CRMP1在树突树枝形成中所扮演的角色。CRMP1在之前的资助期间被确定为一种新的？-Pcdh细胞内结合伙伴。这项拟议的研究意义重大，因为它将确定能够解释不同细胞-细胞相互作用的分子机制，这些机制推动了神经回路形成的关键步骤，填补了该领域当前知识的一个重要空白。这些信息将对理解并最终改善许多神经发育障碍至关重要，这些障碍涉及树突发育和突触发生缺陷。

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.