Role of ASTN2 in cerebellar circuit function and ASD-related behaviors

项目总结 这项拟议的研究解决了自闭症谱系障碍(Asd)中一个至关重要的问题。 研究：小脑回路缺陷如何导致自闭症。特别是，它考察了 主要是小脑基因ASTN2在小脑回路功能和ASD相关行为中的作用。复制 ASTN2中的数目变异(CNV)已被确定为ASD(Lionel ET)的重要危险因素 Al，2014)，这表明ASTN2突变，如在ASTN2CNV患者中发现的突变，导致 改变小脑的突触功能。另外，我们最近报告了一家人的父亲 遗传性ASTN2基因内重复，导致ASTN2功能杂合性丧失。这个 家庭表现出一系列的神经发育障碍，包括自闭症，学习困难和 言语和语言延迟(Behesti等人，2018年)。我们对小鼠的细胞和分子研究 小脑显示ASTN2结合并调节多个突触蛋白的运输， 包括神经连接蛋白，它在基因上与自闭症有关，并调节小脑浦肯野 细胞(PC)突触活动(Behesti等人，2018年)。为研究小脑环路提供遗传学模型 函数，我们为条件生成了全局函数损失ASTN2行和浮点ASTN2行 淘汰赛实验。新的初步证据表明，缺乏ASTN2的小鼠中的PC具有 PC中诱发的兴奋相对于抑制的减少和PC树突棘密度的降低， 说明有特定的小脑回路缺陷。此外，初步证据表明，轻度运动 USV的缺陷和缺陷以及开放领域的测试，与ASD相关的行为。作为其他初步报告 研究结果并不表明小脑发育存在重大缺陷，我们假设 我们观察到的缺陷与小脑回路的缺陷以及受体的潜在变化有关 贩卖人口。 在拟议的研究中，我们将1)测试ASTN2的缺失如何改变PC和PC的内在兴奋性 来自GC和分子层中间神经元的突触前传入的突触效能，2)使用 蛋白质组学来识别突触蛋白水平的变化，并通过实时成像来评估 这些变化与内吞速率的变化有关，3)比较PC树突状细胞的变化 野生型和突变型动物中PC脊椎的分支以及区域分布为 洞察在建立过程中个人计算机投入的组织是否有变化 4)分析ASTN野生鼠社会行为和超音波发声的变化 类型、杂合子和突变的动物。综上所述，拟议的研究将提供一种新的 小鼠模型，允许我们将ASD相关基因联系起来，该基因主要在 小脑具有特定的小脑回路功能和分子通路。

PROJECT SUMMARY The proposed research addresses a critically important question in autism spectrum disorder (ASD) research: how defects in cerebellar circuits contribute to ASD. In particular, it examines the role of the predominantly cerebellar gene ASTN2 in cerebellar circuit function and ASD-related behaviors. Copy number variations (CNVs) in ASTN2 have been identified as a significant risk factor for ASD (Lionel et al, 2014), suggesting that ASTN2 mutations such as those found in patients with ASTN2 CNVs, lead to altered cerebellar synaptic function. In addition, we recently reported a family with a paternally inherited intragenic ASTN2 duplication, which caused a heterozygous loss of function of ASTN2. The family manifested a range of neurodevelopmental disorders, including ASD, learning difficulties and speech and language delay (Behesti et al, 2018). Our cellular and molecular studies on mouse cerebellum show that ASTN2 binds to and regulates the trafficking of multiple synaptic proteins, including Neuroligins, which have been genetically linked to ASDs, and modulates cerebellar Purkinje cell (PC) synaptic activity (Behesti et al, 2018). To provide a genetic model to study cerebellar circuit function, we generated both a global loss of function Astn2 line and a floxed Astn2 line for conditional knockout experiments. New, preliminary evidence indicates that PCs in mice lacking Astn2 have a decrease in evoked excitation relative to inhibition in PCs and reduced PC dendritic spine density, suggesting specific cerebellar circuit defects. In addition, preliminary evidence shows mild motor deficits and defects in USVs and an open field assay, ASD-related behaviors. As other preliminary findings do not indicate major defects in cerebellar development, we hypothesize that the behavioral defects we observed relate to defects in the cerebellar circuitry with underlying changes in receptor trafficking. In the proposed research, we will 1) test how loss of Astn2 alters intrinsic excitability in PCs and the synaptic efficacy of their presynaptic inputs from GCs and molecular layer interneurons, 2) use proteomics to identify changes in the levels of synaptic proteins and live imaging to assess whether such changes relate to changes in the rate of endocytosis, 3) compare changes in PC dendritic branching as well as the regional distribution of PC spines in wild type and mutant animals to provide insight on whether there are changes in the organization of PC inputs during the establishment of the cerebellar circuitry, and 4) analyze changes in social behavior and ultrasonic vocalization in Astn2 wild type, heterozygous and mutant animals. Taken together, the proposed research will provide a new mouse model that allows us to link an ASD-related gene that is predominantly expressed in the cerebellum with specific cerebellar circuit function and molecular pathways.