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

评估阿尔茨海默病相关变异 CICP36L 的功能后果

• 批准号: 10464611
• 负责人: Hamin Lee
• 金额: $ 4.68万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10464611
• 关键词: Affect; Age of Onset; Alleles; Alzheimer' s Disease; Alzheimer' s disease pathology; Alzheimer' s disease patient; Alzheimer' s disease risk; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Behavioral Assay; Binding; Body Weight; Brain; CRISPR/Cas technology; Cells; Child; Co-Immunoprecipitations; Cognition; Complex; Development; Disease; ETV4 gene; Enzymes; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; HMG-Box Domains; Heritability; Hippocampus (Brain); Human; Impairment; Intellectual functioning disability; Knock-in Mouse; Knock-out; Knockout Mice; Late Onset Alzheimer Disease; Lead; Learning; Link; Measures; Mediating; Memory impairment; Mus; Mutation; Neurodegenerative Disorders; Outpatients; Pathogenesis; Pathogenicity; Pathologic; Pathology; Pathway interactions; Patients; Perinatal mortality demographics; Persons; Phenotype; Physiological; Proteins; Proteolysis; Repression; Repressor Proteins; Risk Factors; Role; Senile Plaques; Severities; Syndrome; System; Testing; Therapeutic; Transcription Repressor; Up-Regulation; Variant; Western Blotting; Wild Type Mouse; Work; amyloid precursor protein processing; behavioral phenotyping; beta secretase; beta-site APP cleaving enzyme 1; cohort; conditional knockout; exome; experimental study; genetic corepressor; genome wide association study; in silico; in vivo; insight; loss of function; loss of function mutation; mouse model; nervous system disorder; neuroinflammation; novel therapeutics; overexpression; paralogous gene; risk variant; secretase; therapeutic target

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.

项目摘要 阿尔茨海默氏病（AD）是影响超过4400万人的最常见神经退行性疾病 全世界。虽然超过95％的AD病例是迟到的AD（负载），但确切的致病机制 负载仍不清楚。由于AD是一种高度可遗传的疾病，因此确定与负载相关的风险变异 对于理解发病机理和识别AD的治疗靶标至关重要。总体目标 这项研究的是确定CAPICUA（CIC）中功能突变的部分丧失，该基因完整 功能丧失会导致智力障碍，可能导致AD。 CIC编码转录表示形式 与Ataxin1（ATXN1）或其旁系同源物（Ataxn1 Like（ATXN1L））形成共抑制剂复合物。与两者相互作用 对于CIC蛋白稳定性至关重要。虽然ATXN1/1L-CIC复合物对于生存至关重要，但杂合损失 CIC通过严重的截断突变导致智力障碍。此外，我们最近发现 受损的ATXN1-CIC功能与AD病理有关。当我们在小鼠中淘汰ATXN1时，它会导致 由于稳定性降低并激活转录级联，> 50％的CIC蛋白质降低 增加-分泌酶（BACE1），这是产生致病性淀粉样β（A）物种的关键酶。 最终导致了加速的AD病理学。 ATXN1-CIC复合功能的丢失与增加之间的联系 在BACE1介导的AD病理学中，总体假设CIC突变导致部分丧失 ATXN1-CIC复合功能将潜在AD病理。我们确定了一个罕见的杂合错过 变体CICP36L，仅出现在负载患者中，但不在对照中。 cicp36l位于 高度保守的ATXN1结合域。为了确定该突变对ATXN1-CIC相互作用的影响，I 在细胞中表达标记为CICP36L，进行了共免疫沉淀，并发现Cicp36l的相互作用 与CICWT相比，ATXN1的ATXN1降低了约60％。这表明CICP36L可能导致CIC下降 稳定性和降低的ATXN1-CIC复合功能。因此，我假设CICP36L变体导致 通过上调ATXN1-CIC复合物的功能部分损失和潜在的AD病理病理 Bace1。为了了解物理相关系统中的功能后果，我生成了Cicp36l 使用CRISPR/CAS9进行敲入小鼠，并证明CICP36L降低了P0脑裂解物中的CIC稳定性， 支持CICP36L可能导致ATXN1 -CIC复合函数的部分丧失的假设。在这个 提案，我将进一步确定CICP36L变体的体内功能后果（AIM1）并确定 CICP36L变体对AD病理学的影响（AIM2）。在研究结束时，我们将确定Cicp36L是否为 广告风险变体。这项工作将强调需要搜索全基因组的稀有广告相关变体 关联研究可能会错过并有可能确定基因中不太严重突变的机制 导致智力残疾可能导致AD病理学。这种变体的识别可能揭示 以前未知的途径在AD发病机理中很重要，并导致新治疗的发展。