USHERIN--FUNCTION, EXPRESSION, AND ROLE IN PATHOGENESIS

Usher综合征IIa型可能是综合征性耳聋最常见的遗传原因。这种常染色体隐性遗传疾病的纯合子患有先天性感音神经性听力损失，并伴有进行性视网膜色素变性。通过该项目的努力，最近鉴定了Usher IIA型基因，并发现其编码一种新型细胞外基质分子，我们称之为usherin。在此，我们发现usherin是一种分泌性分子，定位于耳蜗螺旋神经节细胞体和神经束膜周围的基质，以及视网膜感光细胞周围的感光细胞间基质。在这项提案中，我们将探索这种新蛋白质的功能特性，并研究它的缺乏如何导致观察到的病理。我们将采用多种方法来确定它的生化特性以及它与哪些蛋白质相互作用，检查基质相互作用和与整合素的潜在相互作用。为此，我们将小鼠和人的全长cDNA克隆到诱导型和非诱导型表达系统中。我们已经使用合成肽抗原开发了人和小鼠抗体，并且正在使用更大的融合肽开发另外的抗血清。基因敲除小鼠模型正在开发中，该模型将用于检查与内耳病理学相关的结构和分子变化。由于该基因的表达仅限于重要的耳蜗和视网膜细胞类型，我们克隆了一部分启动子，并在Y79感光细胞中表达。将对该构建体进行缺失诱变以鉴定最小启动子，并进行迁移率变动测定以测试特异性核因子的存在。将产生从启动子表达绿色荧光蛋白的转基因动物，以评估克隆片段是否含有适当调节和特异性所需的所有元件。

Usher syndrome type IIa is possibly the most common genetic cause of syndromic deafness. Homozygotes for this autosomal recessive disorder suffer congenital sensorineural hearing loss with progressive retinitis pigmentosa. Through the efforts of this program, the gene for Usher type IIA was recently identified and found to encode a novel extracellular matrix molecule which we refer to as usherin. Herein we show that usherin is a secreted molecule that localizes to the matrix surroundi8ng the spiral ganglion cell bodies and the perineurium in the cochlea, and to the inter-photoreceptor cell matrix surround the photoreceptor cells in the retina. In this proposal we will explore the functional properties of this novel protein and investigate how its absence leads to the observed pathologies. We will employ a number of approaches to determine its biochemical properties as well as what proteins it interacts with, examining both matrix interactions and potential interactions with integrins. Towards this end, we have cloned the full length cDNAs for both mouse and human into both inducible and non-inducible expression systems. We have developed both human and mouse antibodies using synthetic peptide antigens, and are developing additional antisera using larger fusion peptides. A gene knockout mouse model is under development which will be used to examine the structural and molecular changes associated with the inner ear pathology. Since expression of the gene is restricted to important cochlear and retinal cell types, we have cloned a portion of the promoter and expressed it in Y79 photoreceptor cells. This construct will be subjected to deletion mutagenesis to identify the minimal promoter and mobility shift assays to test for the existence of specific nuclear factors. A transgenic animal will be produced expressing green fluorescent protein from the promoter to assess whether the cloned fragment contains all of the elements necessary for appropriate regulation and specificity.