Neuroligins and Neuroligin-Neurexin Interactions in Islet Beta Cell Function

描述（由申请方提供）：开发用于β细胞替代疗法的葡萄糖响应性胰岛素分泌细胞需要更好地了解胰岛功能和β细胞分化的分子决定因素。增加对介导胰岛素分泌的机制的了解对于鉴定用于治疗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、细胞旁分泌信号传导和胰岛活动协调中的作用的长期目标。本申请的目的是鉴定突触细胞表面粘附分子的不同子集在β细胞功能中的作用：能够诱导突触形成和抑制性或兴奋性神经传递所必需的机制的组装的那些。新的初步数据的指导下，本申请集中在两个关键的家庭，这些粘附蛋白，神经连接素和他们的主要结合伙伴，neurexins。核心假设是，神经连接素和神经毒素之间的细胞外相互作用有助于驱动细胞功能成熟，包括胞外机器的组装，并负责由细胞-细胞接触诱导的胰岛素分泌的增强。为了实现本申请的目的，追求三个具体目标：1）鉴定正常β细胞分泌功能所必需的神经连接素和neurexin同种型及其在胰岛素胞吐中的作用; 2）鉴定神经连接素和neurexin影响β细胞功能的机制;和3）测试神经连接素和neurexin表达的变化在2型糖尿病相关β细胞功能障碍中的作用。现有的神经连接素敲除小鼠和RNA干扰将用于评估神经连接素和neurexin同种型的表达减少对胰岛素分泌的影响。将使用INS-1和解离的大鼠胰岛细胞与表达神经连接素的HEK 293细胞的共培养物，通过免疫组织化学和免疫沉淀实验，并通过使用胰岛灌流和监测细胞内钙，分析神经连接素和神经连接素-神经连接素相互作用在胰岛细胞功能中的作用。在葡萄糖毒性条件下培养的INS-1细胞和来自Zucker糖尿病肥胖大鼠的胰岛将用作2型糖尿病中β细胞功能障碍的模型。拟议的工作是创新的，因为它源于新的观察，即neuroligins和neurexins在细胞表面共表达并影响胰岛素分泌，并且因为它将与突触功能领域的领导者实验室合作进行。这项研究意义重大，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。

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.