Neuroligins and Neuroligin-Neurexin Interactions in Islet Beta Cell Function

描述(申请人提供)：开发葡萄糖反应、胰岛素分泌细胞用于β细胞替代治疗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的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、细胞旁分泌信号和胰岛活动协调中的作用的长期目标。本申请的目的是确定突触细胞表面黏附分子的一个独特的子集在β细胞功能中的作用：那些能够诱导突触形成和组装抑制性或兴奋性神经传递所需的机制的分子。在新的初步数据的指导下，该应用重点关注这些黏附蛋白的两个关键家族，神经连接蛋白及其主要结合伙伴Neurexins。中心假说是，神经连接蛋白和Neurexins之间的细胞外相互作用有助于推动细胞功能成熟，包括胞外机制的组装，并负责细胞-细胞接触诱导的胰岛素分泌增加。为了达到这一应用的目的，我们追求三个特定的目标：1)确定正常的β细胞分泌功能所必需的神经连接素和神经尿素素亚型及其在胰岛素排出中的作用；2)确定神经连接素和神经尿素素影响胰岛细胞功能的机制；以及3)测试神经连接素和神经尿素素表达的变化在2型糖尿病相关的胰岛细胞功能障碍中的作用。现有的神经连接蛋白基因敲除小鼠和RNA干扰将被用来评估神经连接蛋白和神经尿素异构体表达减少对胰岛素分泌的影响。通过INS-1和分离的大鼠胰岛细胞与表达神经连接蛋白的HEK293细胞共培养，通过免疫组织化学和免疫沉淀实验，通过胰岛灌流和细胞内钙监测，分析神经连接蛋白和神经连接蛋白-神经尿素素相互作用在细胞功能中的作用。在糖毒性条件下培养的INS-1细胞和Zucker糖尿病肥胖大鼠的胰岛将被用作2型糖尿病β细胞功能障碍的模型。这项拟议的工作具有创新性，因为它源于一项新的观察结果，即神经连接蛋白和Neurexins在细胞表面共表达并影响胰岛素的分泌，而且它将与突触功能领域的领先实验室合作进行。这项拟议的研究具有重要意义，因为它有望促进和扩大我们对突触细胞表面分子和细胞外蛋白质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：预计拟议的工作将有助于制定策略，将β细胞表面蛋白用作治疗糖尿病相关的β细胞功能障碍的药理靶点，并作为细胞成分，以增强工程细胞与真实细胞的生理相似性。它还可能有助于识别有功能的β细胞的有用的细胞表面标记。因此，它有望对开发1型和2型糖尿病的治疗方法具有直接意义，并对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.