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。该更新申请的目的是确定γ-Pcdhs（22种钙粘蛋白超家族粘附分子的家族）调节皮质树突树枝化的分子机制。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枝化。该假设基于申请人的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个特定目的进行测试：1）确定嗜同性γ-Pcdh相互作用促进皮质神经元树突树枝化的程度; 2）鉴定星形胶质细胞γ-Pcdh在皮质神经元树突树枝化中的作用; 3）确定调控γ-Pcdhs在树枝化中作用的细胞内信号转导机制。根据目标1，从初步的体外试验-组合不同的γ-Pcdh顺式-四聚体相互作用的反式-将被应用于皮质发育的模型。神经元γ-Pcdh四聚体组成将使用转染和几种新的Pcdh-γ敲入转基因小鼠系来操作，以直接解决相互作用特异性在树突树枝化中的作用。目的2建立在星形胶质细胞γ-Pcdhs作为脊髓回路形成关键调节因子的初步数据基础上.使用星形胶质细胞限制性Cre转基因与条件性Pcdh-γ突变体和体内敲入，将描绘星形胶质细胞γ-Pcdh在皮质神经元树突树枝化中的作用。目的3扩展了初步数据，表明γ-Pcdhs抑制PKC信号通路以促进树突树枝化。所有γ-Pcdhs共有的C-末端残基已被鉴定为可被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，提供了信号反馈机制。这将在生物化学测定和神经元培养中使用一系列点突变体和截短Pcdh-γ构建体进行测试。这项拟议中的研究意义重大，因为它将确定可以解释多种细胞间相互作用的分子机制，这些细胞间相互作用是神经回路形成的关键一步， 填补了该领域现有知识的一个重要空白。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 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.