iPLA2beta-mediated alternative splicing and beta-cell death in type 1 diabetes

iPLA2beta 介导的 1 型糖尿病中的选择性剪接和 β 细胞死亡

• 批准号: 10594556
• 负责人: CHARLES E. CHALFANT
• 金额: $ 47.13万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-10 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594556
• 关键词: 5' Splice Site; Ablation; Alternative Splicing; Amendment; Antisense RNA; Apoptosis; Apoptotic; Arachidonic Acids; BCL2 gene; Beta Cell; Biological Assay; Biometry; CASP9 gene; Cell Death; Cell Survival; Cells; Cellular Stress; Cessation of life; Development; Diabetes Mellitus; Disease Progression; Eicosanoids; Elements; Enzymes; Event; Evolution; Family member; Female; Fingerprint; Future; Generations; Genetic; Human; Hydrolysis; Hydroxyeicosatetraenoic Acids; Hyperglycemia; Inbred NOD Mice; Incidence; Inflammatory; Insulin-Dependent Diabetes Mellitus; Knockout Mice; Link; Lipids; MCL1 gene; Macrophage; Mediating; Membrane; Modeling; Molecular; Oligonucleotides; Participant; Patients; Phospholipase; Phospholipids; Play; Prediabetes syndrome; Process; Protein Isoforms; Proteomics; RNA; RNA Binding; RNA Splicing; Reporting; Rodent; Rodent Model; Role; Small Interfering RNA; Stimulus; Testing; Text; Variant; antagonist; autoimmune pathogenesis; bcl-xlong protein; cell type; cytokine; endoplasmic reticulum stress; improved; induced pluripotent stem cell; inhibitor; insulin dependent diabetes mellitus onset; interdisciplinary approach; islet; lipidome; liquid chromatography mass spectrometry; mRNA Precursor; novel; novel strategies; novel therapeutics; preservation; prevent; receptor; response; small hairpin RNA; transcriptome sequencing

Type 1 diabetes (T1D) accounts for approximately 5-10% of diabetes (> 20 million patients worldwide), and T1D patients are predicted to triple by 2050. It is therefore imperative to understand the mechanisms that contribute to T1D evolution, so that improved treatments can be developed to prevent and delay the onset and progression of the disease. In this regard, T1D is a consequence of autoimmune destruction of β-cells, and ER stress and cytokines play critical roles in this process. Importantly, we reported that the Ca2+-independent phospholipase A2β (iPLA2β), which in the islet is predominantly localized in β-cells, is a key participant in β-cell apoptosis in response to stimuli that induce ER stress (e.g., pro-inflammatory cytokines (CTKs) and hyperglycemia). The iPLA2β catalyzes hydrolysis of the sn-2 fatty acyl substituent from membrane phospholipids to liberate arachidonic acid, which can be metabolized to eicosanoids (i.e., iPLA2β-derived lipids (iDLs)). We find that select iDLs are associated with the onset of diabetes in rodent models of T1D, and that inhibition or genetic ablation of iPLA2β promotes decreases in these iDLs, leading to preservation of β-cell mass and T1D amelioration. Mechanistically, ER stress and apoptosis are suppressed by factors such as MCL-1 and Bcl-x(L), Bcl-2 family members that promote β-cell survival. Many apoptotic factors such as these undergo alternative RNA splicing (AS) to generate splice variants with contrasting roles (e.g., the MCL-1S and Bcl-x(s) isoforms). For example, we demonstrated that anti-apoptotic Bcl-x(L) protein is lost from β-cells undergoing apoptosis due to a shift in Bcl-x 5’ splice site (5’SS) selection and generating Bcl-x(s) RNA. We also find that AS of caspase-9 and RAGE towards pro-apoptotic variants correlates with iPLA2β expression and differences in the β-cell lipidome. Our preliminary studies reveal (a) candidate human islet β-cell-derived iDLs that regulate AS, (b) [ratio of Bcl-x(L)/x(s) inversely correlating with iPLA2β expression in NOD islet β-cells and T1D incidence and] (c) through expanded splicomic studies via biostatistical analyses of deep RNA sequencing, additional AS events [(RUVBL1)] regulated by these iDLs. These findings motivate us to assess the role of iDL-mediated AS in β-cells in T1D development. Specifically, we propose to explore the hypothesis that iDLs contribute to apoptotic events in β-cells and that these lipid fingerprints act as early predictors of T1D onset/progression. Mechanistically, we hypothesize that iDLs modulate AS dis-favoring the generation of anti-apoptotic isoforms in β-cells. To interrogate our hypotheses, [we will utilize islets from β-cell and macrophage conditional NOD.iPLA2β-KO mice, human iPSC-derived β-cells, and human islets to]: SA1: Determine the ?-cell lipidome and iDLs that link ER stress, β-cell death and T1D development; SA2: Determine whether β-cell survival can be enhanced by modulation of specific AS induced by iDLs; SA3: Delineate the molecular mechanisms through which iDLs modulate AS. Overall, we posit that a more complete understanding of these iDLs and their mechanisms of action will lead to novel strategies to preserve β-cell viability and prevent the onset/progression of T1D.

1型糖尿病（T1D）约占糖尿病的5-10%（全球约2000万患者），预计到2050年，T1D患者将增加两倍。因此，有必要了解促进T1D演变的机制，以便开发改进的治疗方法来预防和延缓疾病的发生和进展。在这方面，T1D是自身免疫破坏β细胞的结果，内质网应激和细胞因子在这一过程中起关键作用。重要的是，我们报道了Ca2+独立磷脂酶A2β (iPLA2β)，在胰岛中主要定位于β细胞，是β细胞凋亡的关键参与者，以响应刺激诱导内质网应激（例如，促炎细胞因子（CTKs）和高血糖）。iPLA2β催化膜磷脂的sn-2脂肪酸取代基水解，释放花生四烯酸，花生四烯酸可代谢为类二十烷酸（即iPLA2β衍生的脂质（iDLs））。我们发现，在T1D啮齿类动物模型中，选择性idl与糖尿病发病有关，抑制或基因消融iPLA2β可促进这些idl的减少，从而保持β细胞质量和T1D改善。机制上，内质网应激和细胞凋亡受到MCL-1和Bcl-x(L)等因子的抑制，Bcl-2家族成员促进β-细胞存活。许多凋亡因子（如这些）经历选择性RNA剪接（as）以产生具有不同作用的剪接变异体（例如，MCL-1S和Bcl-x(s)亚型）。例如，我们证明抗凋亡的Bcl-x(L)蛋白从发生凋亡的β细胞中丢失，这是由于Bcl-x 5 ‘剪接位点（5 ’ ss）选择的改变和生成Bcl-x(s) RNA。我们还发现caspase-9和RAGE对促凋亡变异体的抑制作用与iPLA2β表达和β细胞脂质组的差异相关。我们的初步研究揭示(a)候选人类胰岛β细胞衍生的idl调节AS， (b) [Bcl-x(L)/x(s)的比例与NOD胰岛β细胞中iPLA2β的表达和T1D发病率呈负相关](c)通过深度RNA测序的生物统计学分析，通过扩展剪接学研究，这些idl调节了其他AS事件[(RUVBL1)]。这些发现促使我们评估idl介导的AS在β细胞T1D发展中的作用。具体来说，我们建议探索idl有助于β细胞凋亡事件的假设，以及这些脂质指纹作为T1D发病/进展的早期预测因子。在机制上，我们假设idl调节AS不利于β细胞中抗凋亡同种异构体的产生。为了验证我们的假设，我们将利用来自β细胞和巨噬细胞条件NOD的胰岛。iPLA2β-KO小鼠，人ipsc衍生的β-细胞和人胰岛[:SA1]：测定？-细胞脂质组和idl与内质网应激、β-细胞死亡和T1D发生有关；SA2：观察idl诱导的特异性AS是否能提高β细胞的存活率；SA3：描述idl调节AS的分子机制。总之，我们认为对这些idl及其作用机制的更全面了解将导致新的策略来保持β细胞活力和防止T1D的发生/进展。