Role of SUSD4 in Retinal Development and Function

描述（由申请人提供）：Sushi结构域通常参与蛋白质-蛋白质相互作用。Sushi domain containing protein 4（SUSD 4）是一种由4个连续的Sushi结构域组成的细胞表面蛋白，在不同物种间高度保守（人与小鼠之间> 95%）。在初步研究中，我们克隆，表达，纯化和重折叠人SUSD 4的寿司结构域。利用复性的重组SUSD 4蛋白，我们在鸡中产生了抗体。我们发现SUSD 4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节。在体外功能测定中，重组SUSD 4蛋白增强补体激活的替代途径，但不是经典途径，并且在体内研究中，其中SUSD 4表达被Morpholinos敲低的斑马鱼表现出发育和运动障碍，包括异常的眼睛发育。此外，先前的临床研究已经将SUSD 4基因的缺失与Fryn综合征和自闭症联系起来，其中许多患者表现出异常的视网膜电图（自闭症）并发展出包括无眼症和视网膜发育不良（Fryns综合征）的眼部异常。所有这些数据结合在一起，有力地证明SUSD 4可能整体参与视网膜发育和/或功能。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SUSD 4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特异性cDNA噬菌体展示系统鉴定SUSD 4的结合伴侣。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SUSD 4在视网膜发育和/或功能中的潜在作用。在目标2中，我们将直接研究SUSD 4在小鼠视网膜发育中的作用。我们首先通过开发loxp位点侧翼SUSD 4基因靶向小鼠，然后将该小鼠与感光细胞特异性Cre重组酶转基因小鼠杂交，产生感光细胞特异性SUSD 4敲除小鼠。我们将评估光感受器的组织学和功能，在所得的光感受器特异性SUSD 4缺陷小鼠。在目标3中，我们将研究SUSD 4在视网膜功能中的作用。我们将通过局部引入Cre重组酶在Aim 2中开发的成年SUSD 4条件靶向小鼠的发育视网膜中表达SUSD 4基因，然后使用Aim 2中描述的相同测定法检查感光细胞结构和功能。 这些研究将为SUSD 4的作用提供见解，SUSD 4是一种可能与神经和视觉功能密切相关的基因。SUSD 4结合伴侣的筛选/验证和条件性SUSD 4敲除的发展将为研究SUSD 4的生理作用开辟新的途径，SUSD 4是一种与自闭症和Fryns综合征发病机制有关的新基因。 公共卫生相关性：SUSD 4是一个功能未知的基因，可能与自闭症和Fryns综合征有关，患者会出现视网膜疾病。在本申请中，我们将研究SUSD 4在视网膜发育和功能中的作用。这些结果将为SUSD 4的生理作用提供见解，并有助于开发自闭症和Fryns综合征的新诊断和治疗策略。

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.