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

基本信息

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
项目类别：
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&apos s Disease Alzheimer&apos s disease pathology Alzheimer&apos s disease patient Alzheimer&apos 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

项目摘要

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.

项目概要阿尔茨海默病 (AD) 是最常见的神经退行性疾病，影响超过 4400 万人虽然全球 95% 以上的 AD 病例为晚发性 AD (LOAD)，但其确切的致病机制。由于 AD 是一种高度遗传性疾病，因此 LOAD 仍不清楚，因此识别与 LOAD 相关的风险变异将有助于识别 LOAD 相关的风险变异。对于了解 AD 的发病机制和确定治疗靶点至关重要。本研究的目的是确定 CAPICUA (CIC) 基因是否存在部分功能丧失突变，该基因完全丧失了功能。功能丧失会导致智力障碍，并可能导致 AD。与 Ataxin1 (ATXN1) 或其旁系同源物 Ataxn1 样 (ATXN1L) 形成共阻遏物复合物。虽然 ATXN1/1L-CIC 复合物对于生存至关重要，但杂合性缺失对于 CIC 蛋白稳定性至关重要。另外，我们最近发现CIC通过严重截短突变导致患者智力障碍。 ATXN1-CIC 功能受损与 AD 病理有关。当我们在小鼠中敲除 ATXN1 时，会导致 AD 病理。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 (BACE1)，这是产生致病性β淀粉样蛋白 (Aβ) 的关键酶。最终导致 AD 病理加速 ATXN1-CIC 复合体功能丧失和增加之间的联系。 BACE1 介导的 AD 病理学导致了总体假设，即 CIC 突变导致部分丧失 ATXN1-CIC 复合体功能将增强 AD 病理学我们发现了一种罕见的杂合错义。变体 CICP36L 仅出现在 LOAD 患者中，但未出现在对照组中。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，我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 降低了约 60%，这表明 CICP36L 可能导致 CIC 降低。稳定性和降低 ATXN1-CIC 复合功能因此，我认为 CICP36L 变体会导致。 ATXN1-CIC 复合物功能部分丧失，并通过上调增强 AD 病理学 BACE1。为了了解生理相关系统中的功能结果，我生成了 CicP36L。使用 CRISPR/Cas9 敲入小鼠并证明 CICP36L CIC 在 P0 脑裂解液中的稳定性，支持 CICP36L 可能导致 ATXN1-CIC 复合体功能部分丧失的假设。建议，我将进一步确定 CICP36L 变体（Aim1）的体内功能后果并确定 CICP36L 变异对 AD 病理学的影响 (Aim2) 在研究结束时，我们将确定 CICP36L 是否是。这项工作将强调寻找在全基因组范围内与 AD 相关的罕见变异的必要性。关联研究可能会错过并可能确定基因中不太严重的突变如何发生的机制导致智力障碍的基因可能会导致 AD 病理学，对此类变异的识别可能会揭示这一点。以前未知的途径在 AD 发病机制中很重要，并导致新疗法的开发。