Mechanisms of DSCAM-mediated self-avoidance

DSCAM介导的自我回避机制

基本信息

批准号：
10614602
负责人：
Andrew Garrett
金额：
$ 19.25万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10614602
关键词：
Address Adhesions Adhesives Binding Binding Proteins Biological Assay Biology C-terminal Cadherins Cell Adhesion Cell Adhesion Molecules Cell Line Cell Surface Proteins Cell physiology Cells Complement Data Data Set Dendrites Dependence Development Developmental Process Disease Down Syndrome Down Syndrome Cell Adhesion Molecule Electroporation Endocytosis Event Fascicle Future Gene Expression Genes Goals Health Human In Vitro Individual Learning Masks Mediating Membrane Modeling Molecular Mus Muscle fasciculation Mutation NRCAM gene Nervous System Neural Retina Neurites Neurodevelopmental Disorder Neurons P-Cadherin Pathology Positioning Attribute Process Proteins Public Health RAPGEF2 gene Research Retina Role Sampling Signal Pathway Signal Transduction Specificity Surface System Testing Transfection Variant Vision Work autism spectrum disorder candidate identification cell type developmental disease experimental study genetic manipulation in vitro Assay in vivo insight loss of function member mosaic mutant neural neural circuit neurodevelopment pharmacologic prevent receptive field retinal neuron

项目摘要

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.

项目概要/摘要唐氏综合症细胞粘附分子 (DSCAM) 是粘附分子 Ig 超家族的成员 DSCAM 与唐氏综合症和自闭症谱系障碍 (ASD) 相关。负责细胞类型水平的神经自我回避；在 Dscam 缺失的视网膜神经元中失去正常功能当它们与相同亚型（同型）的其他细胞形成簇并且它们的树突束状时，间距这是不寻常的，因为 DSCAM（像大多数亲同性粘附分子）驱动粘附和 PI 之前的工作发现 DSCAM 在异源细胞系中表达时会发生细胞聚集。由钙粘蛋白超家族成员介导的“掩蔽”粘附以及不同细胞中的掩蔽亚型对 DSCAM 的 PDZ 相互作用 C 末端有不同的依赖性。这种掩蔽的作用仍然未知。在这里，我们提出实验来定义这些分子机制。 DSCAM 掩盖了多种细胞粘附分子 (CAM)，不仅是钙粘蛋白粘附，而且对 C 端相互作用的不同依赖性反映了不同细胞类型的特异性粘附系统。具体目标 1 我们将使用粘合剂掩蔽的体外测定来测试候选信号通路和该测定利用了神经元和细胞上 CAM 之间的同质相互作用。珠子，这导致神经 CAM 聚集在珠子周围的同质 DSCAM 相互作用。我们将使用多种药理学和遗传学方法来防止其他 CAM（例如钙粘蛋白）的聚集。操作来测试该测定中的候选机制，我们将在体内验证结果在具体目标 2 中，我们将检验我们的假设，即 C- 中的差异聚类和成束作用。末端突变体反映了不同的细胞类型特异性 CAM 集合，而 DSCAM 的 PDZ 相互作用基序仅是为此，我们将利用描述特定细胞类型的新数据集。基因表达来识别在需要 DSCAM C 末端的细胞类型中表达的候选 CAM PDZ 相互作用为正常间距或不需要 DSCAM 的 C 末端使用体内电穿孔。视网膜特异性条件性功能丧失突变体和 C 端截断突变体，我们将测试这些是否 CAM 需要 DSCAM 的 C 末端进行掩蔽。在特定目标 3 中，我们将测试 ASD 相关的 DSCAM。通过拯救 Dscam 缺陷神经元中的细胞粘附来挽救神经元/珠测定中的突变。成为 ASD 病理学的一个主要主题，而 DSCAM 被定位为该过程的关键调节者。完成这些研究将为理解 DSCAM 如何动态地迈出关键的第一步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的基本问题及其影响神经发育障碍，如 ASD。