Role of SUSD4 in Retinal Development and Function

描述（由申请人提供）：寿司结构域通常涉及蛋白质-蛋白质相互作用。SUSD4 （Sushi domain containing protein 4）是一种由四个连续的Sushi结构域组成的假想细胞表面蛋白，在不同物种间具有高度的保守性（人与小鼠间的保守性约为95%）。在初步研究中，我们克隆、表达、纯化和折叠了人类SUSD4的sushi结构域。利用重组的SUSD4蛋白，我们在鸡体内培养了抗体。我们发现SUSD4仅在小鼠的眼睛、大脑、脊髓和睾丸中可检测到，并且在眼睛中其表达仅限于光感受器外段。在体外功能测试中，重组SUSD4蛋白增强了补体激活的替代途径，而不是经典途径。在体内研究中，被Morpholinos敲低SUSD4表达的斑马鱼表现出发育和运动障碍，包括眼睛发育异常。此外，以往的临床研究将SUSD4基因缺失与Fryn's综合征和自闭症联系起来，许多患者表现出视网膜电图异常（自闭症），并出现包括眼失和视网膜发育不良（Fryns综合征）在内的眼部异常。所有这些数据加在一起，有力地表明SUSD4可能与视网膜发育和/或功能有关。为了验证这一假设，我们将结合3个实验室的专业知识，研究SUSD4在视网膜发育和功能中的作用。在目标1中，我们将使用新开发的眼睛特异性cDNA噬菌体展示系统识别SUSD4的结合伙伴。在此之后，我们将通过生物信息学分析鉴定的结合蛋白，以探索SUSD4在视网膜发育和/或功能中的潜在作用。在Aim 2中，我们将直接研究SUSD4在小鼠视网膜发育中的作用。我们首先通过培养loxp位点侧的SUSD4基因靶向小鼠来产生光感受器特异性SUSD4敲除小鼠，然后将其与光感受器特异性Cre重组酶转基因小鼠杂交。我们将在光感受器特异性SUSD4缺陷小鼠中评估光感受器的组织学和功能。在Aim 3中，我们将研究SUSD4在视网膜功能中的作用。我们将通过局部引入cre -重组酶，灭活将在Aim 2中发育的成年SUSD4条件靶向小鼠的已发育视网膜中的SUSD4基因，然后使用Aim 2中描述的相同检测方法检查光感受器结构和功能。这些研究将提供对SUSD4的作用的深入了解，SUSD4是一种可能全面参与神经和视觉功能的基因。SUSD4结合伙伴的筛选/验证和条件性SUSD4基因敲除的开发将为研究SUSD4的生理作用开辟新的途径，SUSD4是一个与自闭症和Fryns综合征发病机制有关的新基因。

DESCRIPTION (provided by applicant): Sushi domains are commonly involved in protein-protein interactions. Sushi domain containing protein 4 (SUSD4) was a hypothetical cell surface protein composed of four continuous Sushi domains, and is highly conserved among different species ( > 95% between human and mouse). In pilot studies we cloned, expressed, purified and refolded the sushi domains of human SUSD4. Using the refolded recombinant SUSD4 protein, we raised antibodies in chickens. We found that SUSD4 is only detectable in murine eyes, brains, spinal cords and testes, and in eyes its expression is restricted to photoreceptor outer segments. In vitro function assays, recombinant SUSD4 protein augments the alternative but not the classical pathway of complement activation, and in vivo studies zebrafish in which the SUSD4 expression is knocked down by Morpholinos exhibit developmental and locomotive disorders including abnormal eye development. In addition, previous clinical studies have connected deletions of SUSD4 gene with Fryn's syndrome and Autism in which many patients demonstrate abnormal electroretinograms (autism) and develop ocular abnormalities including anophthalmia and retinal dysplasia (Fryns syndrome). All these data, taken together, strongly argue that SUSD4 could be integrally involved in retinal development and/or function. To validate this hypothesis, we will study the role of SUSD4 in retinal development and function by combining expertise from 3 laboratories. In Aim 1, we will identify binding partners of SUSD4 using a newly developed, eye specific cDNA phage display system. After this, we will analyze the identified binding proteins by bioinformatics to explore potential roles of SUSD4 in retinal development and/or functions. In Aim 2, we will directly study the role of SUSD4 in retinal development in mice. We first will generate a photoreceptor-specific SUSD4 knockout mouse by developing a loxp sites-flanked SUSD4 gene targeted mouse, and then crossing this mouse with a photoreceptor specific Cre recombinase transgenic mouse. We will assess photoreceptors histologically and functionally in the resultant photoreceptor-specific SUSD4 deficient mice. In Aim 3, we will study the role of SUSD4 in retinal function. We will inactivate SUSD4 gene in developed retinas of adult SUSD4 conditional targeted mice that will be developed in Aim 2 by locally introducing Cre-recombinase, then examine photoreceptor structure and function using the same assays described in Aim 2. These studies will provide insights into the role of SUSD4, a gene that could be integrally involved in neurological and visual functions. The screening/validation of SUSD4 binding partners and the development of conditional SUSD4 knockouts will open new avenues of research on physiological roles of SUSD4, a new gene which has been implicated in the pathogenesis of autism and Fryns syndrome. PUBLIC HEALTH RELEVANCE: SUSD4 is a gene with unknown function and could be integrally involved in autism and Fryns syndrome in which patients develop retinal disorders. In this application, we will investigate the role of SUSD4 in retinal development and function. These results would provide insights into physiological roles of SUSD4 and could help to develop novel diagnostic and therapeutic strategies for autism and Fryns syndrome.