Evaluating the functional consequences of Alzheimer's disease-associated variant CICP36L

项目摘要 阿尔茨海默病（AD）是最常见的神经退行性疾病，影响超过4400万人 国际吧虽然超过95%的AD病例是迟发性AD（LOAD），但AD的确切致病机制是由阿尔茨海默病（AD）引起的。 负载仍然不清楚。由于AD是一种高度遗传性疾病，识别与LOAD相关的风险变体将有助于 对于理解AD的发病机制和确定治疗靶点至关重要。总体目标 本研究的目的是确定CAPIUA（CIC）中的部分功能缺失突变， 功能丧失导致智力残疾，可能导致AD。CIC编码一种转录抑制因子， 与共济失调蛋白1（ATXN 1）或其副产物共济失调蛋白1样（ATXN 1 L）形成共阻遏物复合物。与两者的互动 是CIC蛋白稳定性的关键。虽然ATXN 1/1 L-CIC复合物在生存中是必不可少的，但杂合性缺失 CIC由严重的截短突变引起患者智力残疾。此外，我们最近发现， ATXN 1-CIC功能受损与AD病理学有关。当我们在小鼠中敲除ATXN 1时， 由于稳定性降低，CIC蛋白减少>50%，并激活转录级联反应， 增加β-分泌酶（BACE 1），这是产生致病性淀粉样蛋白β（A β）的关键酶。这 最终导致加速AD病理学。ATXN 1-CIC复合体功能丧失与ATXN 1-CIC复合体功能增加之间的联系 在BACE 1介导的AD病理学中，导致CIC突变的总体假设是， ATXN 1-CIC复合物功能将增强AD病理。我们发现了一种罕见的杂合子错义 CICP 36 L变异，其仅出现在LOAD患者中，而未出现在对照中。CICP 36 L位于 高度保守的ATXN 1结合结构域。为了确定这种突变对ATXN 1-CIC相互作用的影响，我 在细胞中表达标记的CICP 36 L，进行免疫共沉淀，发现CICP 36 L的相互作用 与CICWT相比，ATXN 1减少了约60%。这表明CICP 36 L可能导致CIC降低 稳定性和降低的ATXN 1-CIC复合物功能。因此，我假设CICP 36 L变异导致 ATXN 1-CIC复合物功能的部分丧失，并通过上调 BACE1.为了了解生理相关系统中的功能后果，我生成了CicP 36 L 使用CRISPR/Cas9敲入小鼠，并证明CICP 36 L降低了PO脑裂解物中的CIC稳定性， 支持CICP 36 L可能导致ATXN 1-CIC复合物功能部分丧失的假设。在这 根据该提案，我将进一步确定CICP 36 L变体（Aim 1）的体内功能后果，并确定 CICP 36 L变体对AD病理学的影响（Aim 2）。在研究结束时，我们将确定CICP 36 L是否 AD风险变体。这项工作将强调需要寻找罕见的AD相关变异， 关联研究可能会错过并可能确定一种机制，即基因中不太严重的突变 导致智力残疾的基因可能会导致AD病理学。对这些变异体的鉴定可能揭示 以前未知的途径在AD发病机制中的重要性，并导致新的治疗方法的发展。

Project Summary Alzheimer's disease (AD) is the most common neurodegenerative disorder affecting more than 44 million people worldwide. While more than 95% of AD cases are late-onset AD (LOAD), the exact pathogenic mechanism of LOAD remains unclear. As AD is a highly heritable disease, identifying risk variants associated with LOAD will be critical for understanding the pathogenesis and identifying therapeutic targets for AD. The overarching goal of this study is to determine if a partial loss of function mutation in CAPICUA (CIC), a gene in which complete loss of function causes intellectual disability, can contribute to AD. CIC encodes a transcriptional repressor that forms a co-repressor complex with Ataxin1 (ATXN1), or its paralog, Ataxn1-like (ATXN1L). Interaction with both is critical for CIC protein stability. While the ATXN1/1L-CIC complex is essential in survival, heterozygous loss of CIC by severe truncating mutations in patients cause intellectual disability. Additionally, we recently found impaired ATXN1-CIC function to be implicated in AD pathology. When we knock out ATXN1 in mice, it leads to >50% reduction of CIC protein due to the decreased stability and activates a transcriptional cascade that increases −secretase (BACE1), a key enzyme in generating pathogenic amyloid beta (A) species. This ultimately leads to accelerated AD pathology. The link between loss of ATXN1-CIC complex function and increase in BACE1 mediated AD pathology led to the overall hypothesis that mutations in CIC that lead to a partial loss of the ATXN1-CIC complex function will potentiate AD pathology. We identified a rare heterozygous missense variant, CICP36L, which appeared only in the LOAD patients but not in the controls. CICP36L is located within a highly conserved ATXN1 binding domain. To determine this mutation’s effect on ATXN1-CIC interaction, I expressed tagged CICP36L in cells, performed co-immunoprecipitation, and found that the interaction of CICP36L with ATXN1 is reduced by ~60% compared to CICWT. This suggests that CICP36L could lead to decreased CIC stability and reduced ATXN1-CIC complex function. Therefore, I hypothesize that the CICP36L variant leads to a partial loss of function of the ATXN1-CIC complex and potentiates AD pathology via upregulation of BACE1. To understand the functional consequence in a physiologically relevant system, I generated CicP36L knock-in mice using CRISPR/Cas9 and demonstrated that CICP36L reduces CIC stability in P0 brain lysate, supporting the hypothesis that CICP36L could lead to a partial loss of ATXN1‑CIC complex function. In this proposal, I will further determine the in vivo functional consequences of the CICP36L variant (Aim1) and determine the impact of the CICP36L variant on AD pathology (Aim2). At the end of the study, we will determine if CICP36L is an AD risk variant. This work will highlight the need to search for rare AD-associated variants that genome-wide association studies could miss and potentially identify a mechanism for how a less severe mutation in a gene that causes intellectual disability could contribute to AD pathology. Identification of such variants may reveal previously unknown pathways important in AD pathogenesis and lead to the development of novel therapeutics.