Neuroligins and Neuroligin-Neurexin Interactions in Islet Beta Cell Function

描述（由申请人提供）：开发用于β细胞替代治疗的葡萄糖反应性、胰岛素分泌细胞将需要更好地了解胰岛功能和β细胞分化的分子决定因素。增加对调节胰岛素分泌机制的了解对于确定治疗2型糖尿病的新药物靶点也是必要的。指导这一建议的长期目标是确定神经元突触蛋白和神经递质机制在胰岛素分泌、细胞旁分泌信号传导和胰岛活动协调中的作用。本应用的目的是确定突触细胞表面粘附分子的一个独特子集在β细胞功能中的作用：那些能够诱导突触形成和抑制或兴奋性神经传递所必需的机械组装的分子。在新的初步数据的指导下，本应用侧重于这些粘附蛋白的两个关键家族，神经素和它们的主要结合伙伴，神经素。核心假设是，神经素和神经素之间的细胞外相互作用有助于驱动细胞功能成熟，包括胞外机制的组装，并负责细胞-细胞接触诱导胰岛素分泌的增强。为了实现这一应用的目标，有三个特定的目标：1)确定正常β细胞分泌功能所必需的神经素和神经素亚型及其在胰岛素胞外分泌中的作用；2)确定神经素和神经素影响β细胞功能的机制；3)检测神经素和神经rexin表达变化在2型糖尿病相关β细胞功能障碍中的作用。现有的神经素敲除小鼠和RNA干扰将被用来评估神经素和神经素同种异构体表达减少对胰岛素分泌的影响。神经素和神经素-神经素相互作用在细胞功能中的作用将通过INS-1和分离的大鼠胰岛细胞与表达神经素的HEK293细胞共培养，通过免疫组织化学和免疫沉淀实验，通过胰岛浸润和细胞内钙的监测来分析。糖毒性条件下培养的INS-1细胞和Zucker糖尿病脂肪大鼠胰岛将被用作2型糖尿病β细胞功能障碍的模型。这项提议的工作是创新的，因为它源于神经素和神经素在细胞表面共表达并影响胰岛素分泌的新观察，因为它将与突触功能领域的领先实验室合作进行。这项研究具有重要意义，因为它有望推进和扩大我们对突触细胞表面分子和细胞外蛋白相互作用在胰岛素分泌和细胞功能成熟中的作用的理解。公共卫生相关性：拟议的工作有望使使用β细胞表面蛋白作为治疗糖尿病相关β细胞功能障碍的药理学靶点的策略得以发展，并作为细胞成分增强工程细胞与真实细胞的生理相似性。它还可以帮助鉴定功能性β细胞的有用细胞表面标记物。因此，它有望对1型和2型糖尿病的治疗方法的开发具有直接意义，并且对PA-07-056有反应。

DESCRIPTION (provided by applicant): The development of glucose-responsive, insulin-secreting cells for beta cell replacement therapy will require a better understanding of the molecular determinants of pancreatic islet function and beta-cell differentiation. Increased knowledge of the mechanisms that mediate insulin secretion is also necessary for the identification of new pharmaceutical targets for the treatment of type 2 diabetes. Guiding this proposal is the long-term goal of determining the role of neuronal synaptic proteins and neurotransmitter mechanisms in insulin secretion, in ¿-cell paracrine signaling, and in the coordination of islet activity. The objective of the present application is to identify the role in beta-cell function of a distinct subset of synaptic cell-surface adhesion molecules: those capable of inducing synapse formation and assembly of the machinery necessary for inhibitory or excitatory neurotransmission. Guided by novel preliminary data, this application focuses on two key families of these adhesion proteins, the neuroligins and their major binding partners, the neurexins. The central hypothesis is that extracellular interactions between neuroligins and neurexins help drive ¿-cell functional maturation, including assembly of the exocytic machinery, and are responsible for the enhancement of insulin secretion induced by ¿-cell-¿ cell contact. To attain the objective of this application, three specific aims are pursued: 1) Identify the neuroligin and neurexin isoforms that are necessary for normal beta-cell secretory function and their role in insulin exocytosis; 2) Identify the mechanisms whereby the neuroligins and neurexins influence beta-cell function; and 3) Test the role of changes in neuroligin and neurexin expression in type 2 diabetes-associated beta-cell dysfunction. Existing neuroligin knockout mice and RNA interference will be used to assess the effect on insulin secretion of reduced expression of neuroligin and neurexin isoforms. The role of neuroligins and neuroligin-neurexin interactions in ¿-cell function will be analyzed using cocultures of INS-1 and dissociated rat-islet cells with neuroligin-expressing HEK293 cells, by immunohistochemical and immunoprecipitation experiments, and by use islet perifusion and monitoring of intracellular calcium. INS-1 cells cultured under glucotoxic conditions and islets from Zucker diabetes fatty rats will be used as models of beta cell dysfunction in type 2 diabetes. The proposed work is innovative because it stems from the novel observation that neuroligins and neurexins are coexpressed on the ¿ cell surface and influence insulin secretion and because it will be carried out in collaboration with a laboratory that is a leader in the field of synaptic function. The proposed research is significant because it is expected to advance and expand our understanding of the role of synaptic cell-surface molecules and extracellular protein interactions in insulin secretion and ¿-cell functional maturation. PUBLIC HEALTH RELEVANCE: The proposed work is expected to enable the development of strategies to use beta cell surface proteins as pharmacological targets for the treatment of diabetes-associated beta-cell dysfunction and as cellular components to enhance the physiological resemblance of engineered ¿ cells to true ¿ cells. It may also assist in the identification of useful cell-surface markers of functional beta cells. It is thus expected to be of direct significance for the development of therapies for type 1 and type 2 diabetes and is responsive to PA-07-056.