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（T1D）约占糖尿病的5-10％（全球> 2000万患者），预计到2050年，T1D患者预计将三倍。因此，必须了解有助于T1D进化的机制，因此可以开发改善治疗以防止和延迟该疾病的发作和进展。在这方面，T1D是β细胞自身免疫性破坏的结果，而ER应力和细胞因子在此过程中起着关键作用。重要的是，我们报道说，Ca2+独立的磷脂酶A2β（IPLA2β）在胰岛中主要定位于β细胞中，是参与β-细胞凋亡的关键，以响应刺激诱导ER应激的刺激（例如，促炎性细胞因子（ctks）和超肽）。 IPLA2β催化SN-2脂肪酰基亚tus剂量从膜磷脂中催化的水解，以释放花生四烯酸，可以将其代谢为eicosanoids（即IPLA2β衍生的脂质（IDLS））。我们发现，在T1D的啮齿动物模型中，精选的IDL与糖尿病的发作有关，并且IPLA2β的抑制或遗传消融会促进这些IDL中的下降，从而保存β细胞质量和T1D的改善。从机械上讲，诸如MCL-1和Bcl-X（L），BCL-2家族成员等因素抑制了ER应激和凋亡，这些因素促进了β细胞存活。许多凋亡因素（例如这些因素）都会经历替代的RNA剪接（AS），以产生具有对比作用（例如MCL-1S和BCL-X（S）同工型）的剪接变体。例如，我们证明了抗凋亡BCL-X（L）蛋白因BCl-X 5'剪接位点（5’SS）选择并产生Bcl-X（S）RNA而导致凋亡的β细胞损失。我们还发现，从caspase-9和迈向促凋亡变体的愤怒中，与IPLA2β的表达和β细胞脂肪组的差异相关。我们的初步研究揭示了（a）候选人胰岛β细胞衍生的IDL，该IDL的调节为（b）[Bcl-X（L）/X（S）与NODβ细胞中IPLA2β表达的BCl-X（l）/X（S）成反比，通过扩展的跨度研究（C）通过扩展的specteronic as sequection（c）通过额外的研究（c）进行了额外的研究（c）。由这些IDL监管。这些发现激发了我们评估IDL介导的作用，如β细胞在T1D开发中的作用。具体而言，我们建议探讨IDL有助于β细胞中凋亡事件的假设，并且这些脂质指纹是T1D发作/进展的早期预测指标。从机械上讲，我们假设IDL会调节细胞中抗凋亡同工型的产生。为了询问我们的假设，[我们将利用来自β细胞和巨噬细胞条件点头的小胰岛。 SA2：确定是否可以通过IDL诱导的特异性调制来增强β细胞存活； SA3：描述IDL调制为的分子机制。总体而言，我们指出，对这些IDL及其作用机制的更完整了解将导致保持β细胞生存能力并防止T1D的发作/进展的新型策略。