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，影响着4400万人 全世界。虽然95%以上的AD病例是晚发性AD(LOAD)，但其确切的发病机制 载客量仍不清楚。由于阿尔茨海默病是一种高度可遗传的疾病，识别与负载相关的风险变量将 对于了解AD的发病机制和确定治疗靶点至关重要。首要目标是 这项研究的目的是确定Capicua(CIC)基因的部分功能丧失突变是否完全 功能丧失会导致智力残疾，可能会导致AD。CIC编码一种转录抑制因子 与ATAXIN1(ATXN1)或其类似物Aaxn1(ATXN1L)形成共抑制物复合体。与两者的交互 对CIC蛋白的稳定性至关重要。虽然ATXN1/1L-CIC复合体是生存所必需的，但杂合性丢失 CIC由严重的截断突变导致患者智力残疾。此外，我们最近发现 ATXN1-CIC功能受损可能与AD病理有关。当我们在小鼠中敲除ATXN1时，它会导致 -gt；由于稳定性降低而导致CIC蛋白减少50%，并激活转录级联反应 增加−分泌酶(BACE1)，这是产生致病性淀粉样β蛋白(A)的关键酶。这 最终导致加速的AD病理。ATXN1-CIC复合体功能丧失与功能增强的关系 在BACE1介导的AD病理中，导致CIC突变导致部分缺失的整体假说 ATXN1-CIC复合体的作用将加强AD的病理。我们发现了一种罕见的杂合子错义 变异体CICP36L，仅在LOAD患者中出现，而在对照组中不出现。CICP36L位于 高度保守的ATXN1结合结构域。为了确定该突变对ATXN1-CIC相互作用的影响，即 在细胞中表达标记的CICP36L，进行免疫共沉淀，发现CICP36L的相互作用 与CICWT相比，ATXN1减少了约60%。这表明CICP36L可能导致CIC降低 稳定性和降低ATXN1-CIC的复合体功能。因此，我假设CICP36L变体导致 ATXN1-CIC复合体功能的部分丧失，并通过上调ATXN1-CIC的表达来加强AD的病理 BACE1.为了理解生理相关系统中的功能结果，我生成了CicP36L 用CRISPR/Cas9敲入小鼠，并证明CICP36L降低了P0脑裂解物中CIC的稳定性， 支持CICP36L可导致ATXN1-CIC复合体功能部分丧失的假说。在这 我将进一步确定CICP36L变异体(Aim1)的体内功能后果，并确定 CICP36L变异对AD病理的影响(AIM2)。在研究结束时，我们将确定CICP36L是否 AD风险变种。这项工作将强调寻找全基因组范围内与AD相关的罕见变异的必要性 关联研究可能会遗漏并可能确定一种机制，即基因中不那么严重的突变是如何 这会导致智力残疾，可能会导致AD病理。对这些变体的识别可能会揭示 先前未知的通路在AD发病机制中起重要作用，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.