Role of XIRP2 in hair cell function and degeneration

描述(由申请者提供)：我们的长期目标是了解机械感觉发束的发育、维持和退化的机制。由于三分之一的已知耳聋基因编码发束蛋白，我们以前使用了基于质谱学的策略来表征发束蛋白质组。事实证明，这种方法是对传统遗传学策略的有力补充，导致发现了以前仅使用遗传策略无法检测到的新型耳聋基因。为了进一步分析，我们将重点放在了相应基因位置与未解决的人类耳聋基因重叠的蛋白质上。其中一种蛋白质是XIRP2(含有Xin肌动蛋白结合重复序列的蛋白质2)。在这个项目中，我们正在验证一种假设，即XIRP2是一种新的毛细胞蛋白，是立体纤毛和毛细胞/支持细胞连接长期稳定所必需的，其结果是XIRP2缺乏导致毛细胞退化和进行性听力损失。初步研究表明，毛细胞中存在不同的XIRP2剪接形式，在毛细胞中显示出不同的定位 立体纤毛和皮质周围分别粘连连接处。为了探讨XIRP2在听力功能中的作用，我们使用CRISPR(集群规则间隔短回文重复序列)/Cas技术产生了Xirp2基因功能性零突变的转基因小鼠，并发现了高频听力损失的证据。在特定的目标1中，我们将详细研究Xirp2基因缺失小鼠的听力损失表型。此外，我们将通过测试Xirp2基因缺失小鼠对噪音导致的听力损失的敏感性，来测试XIRP2缺失是否会使毛细胞对机械应力更加敏感。初步研究表明，立体纤毛和粘连连接具有不同的XIRP2亚型，这意味着任何一种毛细胞结构的缺陷都可能是Xirp2基因缺失小鼠听力损失的原因。因此，在目标2和3中，我们将使用Xirp2基因同型特异性缺失的转基因小鼠，专门研究它们在毛细胞退化和听力损失中的作用。我们的假设是，XIRP2是长期维持毛细胞结构所必需的，这与它在心肌中的已知作用很好地吻合，在心肌中，XIRP2参与了肌节Z线的维持。XIRP2的S在毛细胞中的作用可能不同于它在心脏中的功能，也可能比它在心脏中的功能更复杂：毛束含有一种新的异构体，具有非常不同的蛋白质结构域，这使得它实际上是一种未知的蛋白质。了解XIRP2在毛细胞退化中的作用对人类听力健康具有重要意义，这将有助于阐明由遗传和环境因素造成的微小缺陷可能损害毛细胞结构完整性的机制，这对于理解年龄和噪声导致听力损失的机制基础具有重要意义。最后，XIRP2基因的染色体位点与人类耳聋基因座DFNB27和DFNA16重叠，为鉴定两种耳聋表型的致病突变开辟了前景。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the mechanisms that underlie the development, maintenance and degeneration of the mechanosensory hair bundle. Motivated by the fact that a third of all known deafness genes encode hair bundle proteins, we previously used a mass spectrometry based strategy to characterize the hair bundle proteome. This approach proved to be a powerful complement to traditional genetics strategies, leading to the discovery of novel deafness genes previously undetected using genetic strategies alone. For further analysis, we focused on proteins for which the corresponding gene locations overlap with unresolved human deafness loci. One such protein is XIRP2 (for xin actin-binding repeat containing protein 2). In this project, we are testing the hypothesis that XIRP2 is a novel hair cell protein required for long-term stability of stereocilia and the hair cell/supporting cell junctions, with the consequence that XIRP2 deficiency causes hair cell degeneration and progressive hearing loss. Preliminary studies demonstrated that different XIRP2 splice forms are present in the hair cell, displaying distinct localizations in the stereocilia and pericuticular adherens junctions, respectively. To explore the role of XIRP2 for hearing function, we used the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas technology to generate transgenic mice with a functional null mutation in the Xirp2 gene and found evidence for high-frequency hearing loss. In Specific Aim 1, we will investigate the hearing loss phenotype in Xirp2 null mice in detail. Furthermore, we will test whether XIRP2 deficiency renders the hair cell more sensitive to mechanical stress, by testing the sensitivity of Xirp2 null mice to noise-induced hearing loss. Preliminary studies suggested that the stereocilia and the adherens junctions harbor distinct XIRP2 isoforms, implying that defects in either hair cell structure could underlie the observed hearing loss in Xirp2 null mice. n Aim 2 and 3, we will therefore use transgenic mice with isoform specific deletions in the Xirp2 gene, to specifically address their contribution to hair cell degeneration and hearing loss. Our hypothesis that XIRP2 is required for long-term maintenance of hair cell structures dovetails well with its known role in cardiac muscle, where it is involved in the maintenance of the sarcomeric Z-line. XIRP2's role in the hair cell is likely to be distinct from and more complex than its functon in the heart: the hair bundle harbors a novel isoform with vastly different protein domain structure, rendering it practically an unknown protein. Significant for human hearing health, understanding the role of XIRP2 in hair cell degeneration is expected to shed light on mechanisms by which subtle defects caused by genetic and environmental factors can compromise the structural integrity of hair cell structures, significant for understanding the mechanistic basis of age and noise-induced hearing loss. Finally, the chromosomal locus of the XIRP2 gene overlaps with the human deafness loci DFNB27 and DFNA16, opening up the prospect of identifying the causative mutation for two human deafness phenotypes.