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伽马-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。中心假设是皮质神经元和星形胶质细胞上的伽马-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。这一假说是基于申请人的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检验：1)确定高嗜性伽马-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)确定星形细胞伽马-Pcdhs在皮质神经元树突树枝分枝中的作用；以及3)确定细胞内调节伽马-Pcdhs在树枝形成中的作用的信号机制。在目标1下，一个由初步体外测试产生的模型--组合不同的伽马-Pcdh顺式四聚体在反式中同亲作用--将被应用于皮质发育。神经元伽马-Pcdh四聚体的组成将通过转染法和几个新的Pcdh-Gamma敲入转基因小鼠品系来操纵，以直接解决相互作用特异性在树突树枝形成中的作用。目的2以初步数据为基础，确定星形细胞γ-Pcdhs是脊髓环路形成的关键调节因子。利用带有条件性Pcdh-Gamma突变体的星形胶质细胞限制性Cre转基因和体内敲入，将描述星形细胞Gamma-Pcdhs在皮质神经元树突树枝形成中的作用。目的3扩展的初步数据表明，PKC信号通路被伽马-Pcdhs抑制，以促进树突的分枝。所有的Gamma-Pcdhs共有的一个C-末端残基已经被确定可以被PKC磷酸化。我们假设，这破坏了Gamma-Pcdhs通过抑制FAK抑制PKC信号的能力，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和截断Pcdh-Gamma结构进行测试。这项拟议的研究意义重大，因为它将识别能够解释推动神经回路形成的关键步骤的不同细胞-细胞相互作用的分子机制， 填补了该领域现有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 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.