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: 10554009
• 负责人: CHARLES E. CHALFANT
• 金额: $ 54.28万
• 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/10554009
• 关键词: 5' Splice Site; Ablation; Alternative Splicing; Antisense RNA; Apoptosis; Apoptotic; Arachidonic Acids; BCL2 gene; Beta Cell; Biological Assay; Biometry; CASP9 gene; Cell Death; Cell Survival; Cells; 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; Islet Cell; Knockout Mice; Lead; Link; Lipids; MCL1 gene; Mediating; Membrane; Modeling; Molecular; Oligonucleotides; Participant; Patients; Phospholipase; Phospholipase A2; 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; macrophage; 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 -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（T1 D）约占糖尿病的5-10%（全球> 2000万患者），预计到2050年T1 D患者将增加两倍。因此，必须了解有助于T1 D演变的机制，以便开发改进的治疗方法来预防和延迟疾病的发作和进展。在这方面，T1 D是β细胞的自身免疫破坏的结果，并且ER应激和细胞因子在该过程中起关键作用。重要的是，我们报告说，Ca 2+非依赖性磷脂酶A2β（iPLA 2 β）在胰岛中主要位于β细胞中，是响应诱导ER应激的刺激的β细胞凋亡的关键参与者（例如，促炎细胞因子（CTK）和高血糖症）。iPLA 2 β催化sn-2脂肪酰基取代基从膜磷脂水解以释放花生四烯酸，花生四烯酸可以代谢为类二十烷酸（即，iPLA 2 β衍生脂质（iDL））。我们发现，在T1 D啮齿动物模型中，选择的iDL与糖尿病的发作相关，并且iPLA 2 β的抑制或基因消融促进这些iDL的减少，从而导致β细胞群的保留和T1 D的改善。 在机制上，ER应激和细胞凋亡受到促进β细胞存活的因子如MCL-1和Bcl-x（L）、Bcl-2家族成员的抑制。许多凋亡因子如这些经历选择性RNA剪接（AS）以产生具有相反作用的剪接变体（例如，MCL-1 S和Bcl-x（s）同种型）。例如，我们证明了抗凋亡Bcl-x（L）蛋白由于Bcl-x 5'剪接位点（5' SS）选择的移位和产生Bcl-x（s）RNA而从经历凋亡的β细胞中丢失。我们还发现，caspase-9和RAGE对促凋亡变体的AS与iPLA 2 β表达和β细胞脂质体的差异相关。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）的比率与NOD胰岛β细胞中的iPLA 2 β表达和T1 D发病率负相关，和]（c）通过深度RNA测序的生物统计学分析的扩展剪接组学研究，由这些iDL调节的额外AS事件[（RUVBL 1）]。这些发现促使我们评估iDL介导的AS在β细胞中在T1 D发展中的作用。具体而言，我们建议探索iDL有助于β细胞中的凋亡事件以及这些脂质指纹作为T1 D发作/进展的早期预测因子的假设。从机制上讲，我们假设iDL调节AS不利于在细胞中产生抗凋亡亚型。为了质疑我们的假设，[我们将利用来自β细胞和巨噬细胞条件性NOD.iPLA2β-KO小鼠的胰岛、人iPSC衍生的β细胞和人胰岛]：SA 1：确定将ER应激、β细胞死亡和T1 D发展联系起来的β细胞脂质组和iDL; SA 2：确定β细胞存活是否可以通过调节iDL诱导的特异性AS来增强; SA 3：阐明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理解将导致保护β细胞活力和预防T1 D发作/进展的新策略。