Mechanisms of DSCAM-mediated self-avoidance

项目摘要/摘要 唐氏综合征细胞黏附分子(DSCAM)是Ig超家族黏附分子中的一员 与唐氏综合症和自闭症谱系障碍(ASD)相关。在小鼠视网膜中，DSCAM是 负责神经元类型水平的自我回避；在DSCAM缺失的视网膜中，神经元失去正常 当它们与相同亚型(同型)的其他细胞形成簇状并且它们的树突成束时，它们之间的间隔 和彼此在一起。这是不寻常的，因为DSCAM(像大多数亲水性黏附分子一样)驱动黏附和 在异源细胞系中表达时的细胞聚集。PI之前的工作发现DSCAM 钙粘蛋白超家族成员介导的“掩蔽”黏附及其在不同细胞中的掩蔽 亚型对DSCAM与PDZ相互作用的C末端有不同程度的依赖性。分子机制 这种伪装的部分仍不为人所知。在这里，我们建议用实验来定义这些分子机制。我们 假设DSCAM掩盖了不同的细胞黏附分子(CAM)，不仅是钙粘附素黏附，而且 对C末端相互作用的不同依赖反映了不同类型的细胞特异性黏附系统。在……里面 具体目标1我们将使用粘附性掩蔽的体外测试来测试候选信号通路和 细胞过程。这种分析方法利用了神经元中的CaM和一种 珠子，这导致神经元CAM聚集在珠子周围。同嗜性DSCAM相互作用 防止其他凸轮聚集(例如钙粘附素)。我们将使用多种药理和遗传方法 在这项测试中测试候选机制的操作，我们将在体内验证发现 电穿孔。在特定的目标2中，我们将检验我们的假设，即C- 末端突变体反映了不同的细胞类型特异性凸轮，而DSCAM的PDZ相互作用基序仅 需要掩蔽凸轮的子集。为此，我们将利用描述特定单元格类型的新数据集 基因表达以确定在需要DSCAM C-末端的细胞类型中表达的候选CAM 正常间距的PDZ相互作用或不需要DSCAM的C端子。利用体内电穿孔技术 视网膜特异的条件性功能丧失突变体和C末端截断突变体，我们将测试这些 凸轮需要DSCAM的C端子来遮盖。在特定目标3中，我们将测试与ASD相关的DSCAM 通过挽救DSCAM缺陷神经元的掩蔽，神经元/珠粒分析中的突变。细胞黏附有 作为ASD病理学的一个主要主题，DSCAM被定位为这一过程的关键调节因子。这个 这些研究的完成将为理解DSCAM如何动态地 在显影过程中调整粘合系统，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 像自闭症这样的神经发育障碍。

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.