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%（全球超过 2000 万患者），预计到 2050 年，T1D 患者将增加两倍。因此，必须了解导致 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作和进展。在这方面，T1D 是 β 细胞自身免疫破坏的结果，内质网应激和细胞因子在此过程中发挥着关键作用。重要的是，我们报道了 Ca2+ 独立的磷脂酶 A2β (iPLA2β) 在胰岛中主要位于 β 细胞中，是响应诱导内质网应激的刺激（例如促炎细胞因子 (CTK) 和高血糖）的 β 细胞凋亡的关键参与者。 iPLA2β 催化膜磷脂中 sn-2 脂肪酰基取代基的水解，释放花生四烯酸，花生四烯酸可代谢为类二十烷酸（即 iPLA2β 衍生脂质 (iDL)）。我们发现，在 T1D 啮齿动物模型中，特定的 iDL 与糖尿病的发病相关，并且 iPLA2β 的抑制或基因消除可促进这些 iDL 的减少，从而导致 β 细胞质量的保存和 T1D 的改善。 从机制上讲，MCL-1 和 Bcl-x(L)、Bcl-2 家族成员等促进 β 细胞存活的因子可抑制 ER 应激和细胞凋亡。诸如此类的许多细胞凋亡因子会经历选择性 RNA 剪接 (AS)，以产生具有相反作用的剪接变体（例如，MCL-1S 和 Bcl-x(s) 亚型）。例如，我们证明，由于 Bcl-x 5' 剪接位点 (5'SS) 选择发生变化并生成 Bcl-x(s) RNA，抗凋亡 Bcl-x(L) 蛋白从经历凋亡的 β 细胞中丢失。我们还发现 caspase-9 和 RAGE 对促凋亡变体的 AS 与 iPLA2β 表达和 β 细胞脂质组差异相关。我们的初步研究揭示了 (a) 调节 AS 的候选人胰岛 β 细胞衍生的 iDL，(b) [Bcl-x(L)/x(s) 比率与 NOD 胰岛 β 细胞中 iPLA2β 表达和 T1D 发病率呈负相关，以及] (c) 通过深度 RNA 测序的生物统计分析进行扩展剪接体研究，其他 AS 事件 [(RUVBL1)] 受这些 iDL 监管。这些发现促使我们评估 iDL 介导的 AS 在 β 细胞中在 T1D 发展中的作用。具体来说，我们建议探索以下假设：iDL 导致 β 细胞凋亡事件，并且这些脂质指纹可作为 T1D 发病/进展的早期预测因子。从机制上讲，我们假设 iDL 调节 AS 不利于 β 细胞中抗凋亡异构体的产生。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.iPLA2β-KO 小鼠、人类 iPSC 衍生的 β 细胞和人类胰岛的胰岛]： SA1：确定与 ER 应激、β 细胞死亡和 T1D 发展相关的 β 细胞脂质组和 iDL； SA2：确定是否可以通过调节 iDL 诱导的特定 AS 来增强 β 细胞存活率； SA3：描述 iDL 调节 AS 的分子机制。总体而言，我们认为对这些 iDL 及其作用机制的更全面的了解将带来保护 β 细胞活力并防止 T1D 发作/进展的新策略。