GAD65 release in autoimmune diabetes

描述(由申请人提供)： 本申请是应NIDDK K08/K23获奖者小额助学金计划(PAR-01-066)的申请而提交的。如果获奖，这笔R03奖助金将在申请人的K08奖(DK02944)的最后两年提供补充资金。额外资金将提供关键资源，如技术援助，这将增强申请人争取进一步赠款支助的能力，并将促进向完全独立的过渡。这项申请提出了两条研究路线，这将建立在和扩展正在进行的胰岛谷氨酸脱羧酶(GAD)细胞生物学研究的基础上。我发现，令人惊讶的是，囊泡型GABA转运体VIAAT在大鼠β细胞和人类胰岛中缺失。这表明，有一种尚未确定的GABA转运蛋白，我推测，它与GAD65特异地相互作用。在杰伊·海内克博士和斯坦利·菲尔兹博士的实验室的帮助下，我将寻求识别与GAD65相互作用的β细胞蛋白，这些蛋白可能因此影响GAD65的贩运，甚至可能影响其从受伤的胰岛中排出。二线研究的动力是检测GAD65在体内释放的重要性，以及我成功开发出一种灵敏的、原型的血液GAD65检测方法。对心肌和肝脏等组织的损害可以通过血清蛋白排放检测来检测和监测。相比之下，代谢测试只显示胰岛在事后受到了广泛的损害。我推测，GAD65的血浆检测将提供一种手段来检测和监测糖尿病和无症状胰腺炎患者以及自身免疫性糖尿病动物模型中正在进行的β细胞损伤。这样的测试对于各种应用都是非常有价值的，包括研究胰岛损伤和自身免疫的发病机制，帮助确定预防性治疗的候选者，以及监测移植的胰岛的排斥反应。我的目标是完成GAD65高灵敏度血浆检测方法的开发和验证，并对对照组和糖尿病受试者以及糖尿病发病前后BB大鼠的血浆GAD65水平进行初步分析。这些研究产生的数据将构成进一步研究GAD65细胞内转运的基础，以及监测GAD65放电的时间进程、触发因素和临床应用的体内研究。

DESCRIPTION (provided by applicant): This application is submitted in response to an invitation to apply for funds under the Small Grant Program for NIDDK K08/K23 Recipients (PAR-01-066). If awarded, this RO3 grant would provide supplemental funding during the final two years of the applicant's K08 award (DK02944). The additional funding would provide crucial resources, such as technical assistance, that would enhance the applicant's ability to compete for further grant support and would facilitate the transition to full independence. This application proposes two lines of investigation which will build upon and extend ongoing studies of the cell biology of glutamic acid decarboxylase (GAD) in the pancreatic islets. I have found that, surprisingly, the vesicular GABA transporter VIAAT is absent from rat beta cells and human islets. This suggests that there is an as yet unidentified GABA transporter which, I hypothesize, interacts specifically with GAD65. With assistance from the laboratories of Drs. Jay Heinecke and Stanley Fields, I will seek to identify beta-cell proteins that interact with GAD65 and that may therefore influence its trafficking and perhaps its discharge from injured islets. The impetus for the second line of investigation is the importance of detecting GAD65 release in vivo together with my success in developing a sensitive, prototype, blood GAD65 assay. Damage to tissues such as myocardium and liver can be detected and monitored by serum assays for discharged proteins. In contrast, metabolic tests reveal only extensive islet damage after the fact. I hypothesize that a plasma assay for GAD65 will provide a means to detect and monitor ongoing beta-cell damage in individuals with diabetes and asymptomatic insulitis and in animal models of autoimmune diabetes. Such a test would be invaluable for a variety of applications, including investigating the pathogenesis of islet injury and autoimmunity, helping identify candidates for preventative therapies and monitoring transplanted islets for rejection. My goal is to complete the development and validation of a highly sensitive, plasma assay for GAD65 and to conduct a preliminary analysis of plasma GAD65 levels in control and diabetic subjects and in BB rats prior to and after the onset of diabetes. Data generated by these studies will form the basis of further investigations of the intracellular trafficking of GAD65 as well as of in vivo studies of the time-course, triggers and clinical utility of monitoring GAD65 discharge.