Mechanisms of DSCAM-mediated self-avoidance

项目总结/摘要 唐氏综合征细胞粘附分子（DSCAM）是粘附分子IG超家族的成员 与唐氏综合症和自闭症谱系障碍（ASD）有关。在小鼠视网膜中，DSCAM 在细胞类型水平上负责神经元的自我回避;在Dscam无效的视网膜中，神经元失去其正常的 间隔，因为它们与相同亚型的其他细胞（同型）形成簇，并且它们的树突成束状 彼此之间这是不寻常的，因为DSCAM（像大多数嗜同性粘附分子）驱动粘附， 当在异源细胞系中表达时细胞聚集。PI先前的工作发现DSCAM 钙粘蛋白超家族成员介导的“掩蔽”粘附及其在不同细胞中的掩蔽作用 亚型对DSCAM的PDZ-相互作用C-末端具有不同的依赖性。的分子机制 这种伪装仍然是未知的。在这里，我们提出实验来定义这些分子机制。我们 假设DSCAM掩盖了多种细胞粘附分子（CAM），不仅是钙粘蛋白粘附， 对C-末端相互作用的不同依赖性反映了不同细胞类型特异性粘附系统。在 具体目标1我们将使用体外粘附掩蔽试验来测试候选信号传导途径， 细胞过程该测定利用了神经元中的CAM和神经元上的CAM之间的亲同性相互作用。 珠，这导致神经元CAM在珠周围聚集。嗜同性DSCAM相互作用 防止其它CAM的群集（例如，钙粘蛋白）。我们将使用各种药理学和遗传学方法 操作，以测试候选机制，在这项试验中，我们将验证结果与体内 电穿孔在特定目标2中，我们将测试我们的假设，即C-中的差异聚集和束颤 末端突变体反映了不同的细胞类型特异性CAM，DSCAM的PDZ相互作用基序仅 需要掩蔽CAM的子集。为此，我们将利用描述特定细胞类型的新数据集 基因表达以鉴定在细胞类型中表达的候选CAM，所述细胞类型需要DSCAM C-末端 正常间距的PDZ相互作用或不需要DSCAM的C-末端。使用体内电穿孔进入 视网膜特异性条件性功能丧失突变体和C-末端截短突变体，我们将测试这些突变体是否 CAM需要DSCAM的C端进行掩蔽。在具体目标3中，我们将测试ASD相关DSCAM 通过在Dscam缺陷的神经元中挽救掩蔽，在神经元/珠测定中检测突变。细胞粘附具有 出现作为ASD病理学的一个主要主题，DSCAM被定位为该过程的关键调节器。的 这些研究的完成将为理解DSCAM如何动态地 在开发过程中调节粘合剂系统，以防止过度粘合，同时允许适当的 CAM相互作用，解决视网膜神经发育中的基本问题， 神经发育障碍比如自闭症

PROJECT SUMMARY/ABSTRACT Down syndrome cell adhesion molecule (DSCAM) is a member of the Ig superfamily of adhesion molecules associated with Down syndrome and autism spectrum disorder (ASD). In the mouse retina, DSCAM is responsible for neuronal self-avoidance at the cell type level; in Dscam-null retinas neurons lose their normal spacing as they form clusters with other cells of the same subtype (homotypic) and their dendrites fasciculate with each other. This is unusual, as DSCAM (like most homophilic adhesion molecules) drives adhesion and cell clustering when expressed in heterologous cell lines. Previous work from the PI found that DSCAM “masked” adhesion mediated by members of the cadherin superfamily and that masking in different cell subtypes had a differential dependence on DSCAM’s PDZ-interacting C-terminus. The molecular mechanisms of this masking remain unknown. Here we propose experiments to define these molecular mechanisms. We hypothesize that DSCAM masks diverse cell adhesion molecules (CAMs), not only cadherin adhesion, and that the differential dependence on C-terminal interactions reflect different cell type specific adhesion systems. In Specific Aim 1 we will use an in vitro assay of adhesive masking to test candidate signaling pathways and cellular processes. This assay takes advantage of homophilic interactions between CAMs in a neuron and on a bead, which results in clustering of the neuronal CAM around the bead. Homophilic DSCAM interactions prevent the clustering of other CAMs (e.g., cadherins). We will use a variety of pharmacological and genetic manipulations to test candidate mechanisms in this assay, and we will verify findings with in vivo electroporation. In Specific Aim 2, we will test our hypothesis that differential clustering and fasciculation in C- terminal mutants reflects different sets of cell-type-specific CAMs, and DSCAM’s PDZ-interacting motif is only required to mask a subset of CAMs. To do this, we will leverage new datasets describing cell type specific gene expression to identify candidate CAMs expressed in cell types that either require DSCAMs C-terminal PDZ interactions for normal spacing or do not require DSCAM’s C-terminus. Using in vivo electroporation into retina-specific conditional loss of function mutants and into C-terminal truncation mutants, we will test if these CAMs require DSCAM’s C-terminus for masking. In Specific Aim 3, we will test ASD-associated DSCAM mutations in the neuron/bead assay by rescuing masking in Dscam-deficient neurons. Cell adhesion has emerged as a major theme in ASD pathology and DSCAM is positioned as a key regulator of this process. The completion of these studies will provide crucial first steps towards understanding how DSCAM dynamically regulates adhesive systems during development to prevent excessive adhesion while allowing appropriate CAM interactions, addressing fundamental questions in retinal neural development with implications in neurodevelopmental disorders like ASD.