Pathways and critical regulators of early beta-cell dysfunction in type 1 diabetes

项目摘要/摘要 1型糖尿病(T1D)是一种复杂的自身免疫性疾病。最近的临床研究表明，胰岛β细胞 功能障碍(例如，第一时相胰岛素释放下降)在T1D临床发作前几年就会发生。 不幸的是，我们对早期β细胞功能障碍的机制知之甚少，尽管 这种理解对于确定可能阻止疾病的新的治疗靶点的重要性 发展。这个项目的长期目标是对早期的β细胞有一个详细的机制了解。 通过临床组织标本和T1D的高级原位分子表征 功能研究。我们的研究计划利用了在两种情况下观察到的显著的供体内胰岛异质性 糖尿病前期和新发的T1D受试者。我们的假设是，这种供体内胰岛的异质性反映了 进行性β细胞功能障碍的多个阶段，每个胰岛作为一群细胞对其独特的 微环境。识别每个阶段的分子特征提供了一个新的澄清机会 T1D早期β细胞功能障碍的机制为了确定早期β各阶段的分子决定因素- 细胞炎症和应激，蛋白质组学特征被认为是关键，因为表达的 蛋白质组与表型直接相关。我们将首先对单个细胞进行超灵敏蛋白质组学分析 用激光显微切割法从多个自身抗体阳性(AAB)供者中分离出胰岛。我们还将进行 表达Defined的池化胰岛亚群(例如，每个池10-20个胰岛部分)的深度蛋白质组学分析 免疫组织化学(IHC)标记识别早期β细胞功能障碍的关键调控途径。我们的 最近开发的创新纳米POTS(一锅纳米液滴处理)使这种方法成为可能 痕量样本)技术，用于单岛蛋白质组学和深层蛋白质组分析。具体地说，在目标1中，我们将 对症状前多个AAB供者进行单胰岛蛋白质组学分析以确定分子特征 代谢、分泌和应激途径中的进行性β细胞功能障碍。在目标2中，我们将重点关注 确定哪些应激和炎症途径与早期β细胞炎症关系最密切 通过深层蛋白质组图谱比较已知IHC标记所定义的胰岛亚群。目标3将 通过评估它们在细胞存活中的作用来探索已识别的调节因子的功能意义， 细胞凋亡与分泌功能。整个蛋白质组学数据将与转录组学和其他 为功能研究确定候选调节剂的实验数据。影响声明：我们预料到了这一点 该项目将通过建立首个关于胰岛机制的分子资源来推动该领域的发展 异质性和早期β细胞功能障碍在T1D进展中的作用，并展示了一条有效的途径 合理选择治疗靶点，以推动未来的预防策略。

Project Summary/Abstract Type 1 diabetes (T1D) is a complex autoimmune disease. Recent clinical studies show that pancreatic beta-cell dysfunction (e.g., a decline of first phase insulin release) occurs several years before the clinical onset of T1D. Unfortunately, we have little knowledge of mechanisms underlying early beta-cell dysfunction despite the essentiality of such understanding for identifying novel therapeutic targets that could potentially arrest disease development. The long-term goal of this project is to gain a detailed mechanistic understanding of early beta-cell dysfunction in T1D through advanced in situ molecular characterization of clinical tissue specimens and functional studies. Our research plan exploits the substantial intra-donor islet heterogeneity observed in both pre-diabetic and recent-onset T1D subjects. Our hypothesis is that such intra-donor islet heterogeneity reflects multiple stages of progressive β-cell dysfunction, with each islet responding as a cohort of cells to its unique microenvironment. Identifying the molecular signatures of each stage presents a novel opportunity to elucidate mechanisms of early β-cell dysfunction in T1D. To identify the molecular determinants of each stage of early β- cell inflammation and stress, proteomics characterization is considered as critical because the expressed proteome is directly connected to the phenotype. We will first conduct ultrasensitive proteomics analysis of single islets isolated by laser microdissection from multiple autoantibody positive (AAb+) donors. We will also conduct deep proteomics profiling of pooled islet subpopulations (e.g., 10-20 islet sections per pool) expressing defined immunohistochemical (IHC) markers to identify crucial regulatory pathways of early β-cell dysfunction. Our approach is enabled by the recently developed innovative nanoPOTS (Nanodroplet Processing in One-pot for Trace Samples) technology for single islet proteomics and deep proteome profiling. Specifically, in Aim 1 we will pursue single islet proteomics profiling of presymptomatic multiple AAb+ donors to identify molecular signatures of progressive β-cell dysfunction in the metabolic, secretory, and stress pathways. In Aim 2, we will focus on determining which stress and inflammatory pathways are most tightly associated with early β-cell inflammation through deep proteome profiling comparing islet subpopulations defined by known IHC markers. Aim 3 will explore the functional significance of identified regulators through assessments of their roles in cell survival, apoptosis and secretory function. The overall proteomics data will be integrated with transcriptomics and other experimental data to identify candidate regulators for functional studies. Statement of Impact: We anticipate this project will advance the field by establishing first-of-its-kind molecular resource on the mechanisms of islet heterogeneity and early β-cell dysfunction in the progression to T1D and demonstrate an effective path towards rational selection of therapeutic targets that can drive future preventive strategies.