Elucidating functions of the gamma-protocadherins in CNS development

复杂的树枝状树枝的生长是神经回路形成的先决条件，降低了树枝的复杂性 在神经发育/神经精神障碍中，这是一个一致的发现。用于未来的治疗 方法是可行的，迫切需要阐明调控的分子机制 树枝状分枝。γ-Protocadherins(γ-Pcdhs)是一个由22个细胞黏附分子组成的家族，由 Pcdhg基因簇，是正常皮质树突树枝形成所必需的；它们通过哪些机制 然而，人们对不同亚型的行为知之甚少。长期目标是确定分子机制。 来控制神经回路的形成。这项更新申请的目的是阐明这两种异构体是如何 多样性和共同的信号机制参与了γ-PCDH对树突分枝的调控。这个 中心假说是γ-Pcdhs通过：1)不同的嗜同性细胞-细胞相互作用来调节树枝形成； 通过独特的可变细胞质结构域(VCD)的异构体特异性信号；以及3)通过 常见的C-末端基序。提出了三个目标，每个目标都是以当前拨款中发表的作品为前提的 每一个都利用通过CRISPR/Cas9基因组编辑产生的新的Pcdhg等位基因。目标1： 建立一个共享的γ-Pcdh C-末端基序在体内树突分枝中的重要性。PKC荧光粉-- 富含赖氨酸的C-末端基序中的丝氨酸破坏了其结合磷脂的能力，并阻止了γ- Pcdhs体外抑制FAK，促进树突状突起。为了解决这一问题的重要性， 体内，将检查2个新的小鼠品系的树枝形成、γ-Pcdh蛋白稳定性和FAK调节： PcdhgS/A，其中丝氨酸突变为丙氨酸，阻止磷酸化；以及PcdhgCTD，其中 过早的终止密码子会导致蛋白质缺乏该基序。目标2：通过以下途径确定特定异构体的机制 其中γ-Pcdh-C3VCD调控树枝分枝。γ-Pcdh-C3亚型的Vcd唯一地抑制 WNT信号通过与Axin1结合，Axin1是树突分枝的已知调节因子。使用宫内电穿孔， Axin1基因敲除，以及一种新的PcdhgC3KO单异构体基因敲除小鼠，C3在树突中的独特作用 树木化将被确定。目的3：确定γ-Pcdh异构体多样性对树枝晶的重要性 树木化和行为。目前尚不清楚是否由内源Pcdhg基因产生的异构体多样性 集群是重要的分枝方式。为了在体内测试这一点，多个新的小鼠品系中有不同数量的 Pcdhg外显子已经被打乱，降低了潜在的异构体多样性，将被分析。此外，焦虑- 并将对Pcdhg空、单一异构体过度表达和减少进行记忆相关测试 使小鼠多样化，将细胞表型与行为结果联系起来。这项拟议的研究具有创新性， 因为它利用CRISPR/Cas9基因组编辑技术来全面评估改变的影响 体内的γ-PCDH谱系和信号转导。它意义重大，因为未来的治疗方法将严重依赖于 关于确定控制树枝枝复杂性的分子和信号机制。

Growth of a complex dendrite arbor is a prerequisite for neural circuit formation, and reduced arbor complexity in the cortex is a consistent finding in neurodevelopmental/neuropsychiatric disorders. For future therapeutic approaches to be conceivable, there is a critical need to elucidate the molecular mechanisms that regulate dendrite arborization. The γ-Protocadherins (γ-Pcdhs), a family of 22 cell adhesion molecules encoded by the Pcdhg gene cluster, are required for normal cortical dendrite arborization; the mechanisms through which their diverse isoforms act are, however, poorly understood. The long-term goal is to identify molecular mechanisms that control neural circuit formation. The objective of this renewal application is to elucidate how both isoform diversity and common signaling mechanisms contribute to γ-Pcdh regulation of dendrite arborization. The central hypothesis is that γ-Pcdhs regulate arborization through: 1) diverse homophilic cell-cell interactions; 2) isoform-specific signaling via unique variable cytoplasmic domains (VCDs); and 3) shared signaling via a common C-terminal motif. Three Aims are proposed, each premised on published work from the current grant period, and each utilizing novel Pcdhg alleles generated through CRISPR/Cas9 genome editing. Aim 1: Establish the importance of a shared γ-Pcdh C-terminal motif in dendrite arborization in vivo. PKC phosphory- lation of a serine within a lysine-rich C-terminal motif abrogates its ability to bind phospholipids, and prevents γ- Pcdhs from inhibiting FAK and promoting dendrite arborization in vitro. To address the importance of this in vivo, 2 new mouse lines will be examined for arborization, γ-Pcdh protein stability, and FAK regulation: PcdhgS/A, in which the serine is mutated to an alanine, preventing phosphorylation; and PcdhgCTD, in which a premature stop codon results in proteins lacking the motif. Aim 2: Identify isoform-specific mechanisms through which the γ-Pcdh-C3 VCD regulates dendrite arborization. The VCD of the γ-Pcdh-C3 isoform uniquely inhibits Wnt signaling by binding to Axin1, a known regulator of dendrite arborization. Using in utero electroporation, Axin1 knockdown, and a novel PcdhgC3KO single-isoform knockout mouse, a distinct role for C3 in dendrite arborization will be ascertained. Aim 3: Ascertain the importance of γ-Pcdh isoform diversity for dendrite arborization and behavior. It's unknown whether isoform diversity generated by the endogenous Pcdhg gene cluster is important arborization. To test this in vivo, multiple novel mouse lines in which varying numbers of Pcdhg exons have been disrupted, reducing potential isoform diversity, will be analyzed. Additionally, anxiety- and memory-associated tests will be carried out on Pcdhg null, single-isoform overexpression, and reduced diversity mice to link cellular phenotypes to behavioral outcomes. The proposed research is innovative, because it utilizes CRISPR/Cas9 genome editing techniques to comprehensively assess the effects of altered γ-Pcdh repertoire and signaling in vivo. It is significant, because future therapeutic approaches will rely critically on identifying the molecules and signaling mechanisms that control dendrite arbor complexity.