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(全球2000万患者)的5%-10%，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生和进展。在这一点上，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这一过程中发挥关键作用。重要的是，我们报道了钙非依赖性磷脂酶A2β(iPLA2β)，它主要分布在胰岛的β细胞中，是β细胞在内质网应激(如促炎细胞因子和高血糖)诱导下的细胞凋亡的关键参与者。IPLA2β催化膜磷脂中的sn-2脂肪酰基取代基水解生成花生四烯酸，花生四烯酸可被代谢成二十烷基类化合物(即iPLA2β衍生的脂类(IDL))。我们发现，在T1D啮齿动物模型中，特定的IDL与糖尿病的发生有关，抑制或基因消融iPLA2β可促进这些IDL的减少，导致β细胞量的保存和T1D的改善。从机制上讲，内质网应激和细胞凋亡被促进β-细胞存活的Bcl2家族成员Mcl1和Bclx(L)等因素抑制。许多这样的凋亡因子通过选择性核糖核酸剪接(AS)来产生具有不同作用的剪接变体(例如，mcl1s和bclx(S)亚型)。例如，我们证明了在β细胞凋亡过程中，由于Bcl一x5‘剪接点(5’SS)选择的改变而产生了Bcl一x(S)核糖核酸，从而导致了抗凋亡的Bcl一x(S)蛋白的丢失。我们还发现，半胱氨酸天冬氨酸氨基转移酶-9的AS和对促凋亡变异体的RAGE与iPLA2β的表达和β细胞脂质体区的差异有关。我们的初步研究揭示：(A)候选的人胰岛β细胞来源的IDL调控AS，(B)[Bclx(L)/x(S)的比率与NOD胰岛β细胞中iPLA2RNAi的表达和T1D的发病率负相关]和(C)通过扩展剪接研究，通过深度测序的生物统计学分析，这些IDL调控的额外AS事件[(RUVBL1)]。这些发现促使我们评估β细胞中IDL介导的AS在T1D发育中的作用。具体地说，我们建议探索IDL导致β细胞凋亡事件的假设，以及这些脂质指纹可以作为T1D发病/进展的早期预测因子。从机制上讲，我们假设IDL调节为不利于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β细胞和巨噬细胞条件NOD.iPLA2β-KO小鼠、人IPSC来源的β细胞和人胰岛来]：SA1：确定连接内质网应激、细胞死亡和T1D发育的β细胞脂体和IDL；SA2：确定是否可以通过调节IDL诱导的特异性来提高β细胞的存活；SA3：描述IDL调节AS的分子机制。总体而言，我们假设，对这些IDL及其作用机制的更全面的了解将导致新的策略来保护β细胞的活性，并防止T1D的发生/进展。