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

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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.