Modulation of OPRM1 alternative splicing by morphine and HIV-1 Nef

吗啡和 HIV-1 Nef 对 OPRM1 选择性剪接的调节

• 批准号: 10654016
• 负责人: SULIE L. CHANG
• 金额: $ 34.54万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654016
• 关键词: Alternative Splicing; Antisense Oligonucleotides; Autopsy; Behavior; Binding; Biochemical; Brain; Brain region; C-terminal; Cell Culture Techniques; Cells; Chronic; Clinical; Consensus; Cyclic AMP; Data; Development; Drug abuse; Elements; Event; Exons; Exposure to; Family; Functional disorder; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Gene Activation; HIV; HIV Envelope Protein gp120; HIV Infections; HIV-1; HIV/AIDS; Heroin; Human; Illicit Drugs; In Vitro; Inbred F344 Rats; Incidence; Individual; Mediating; Messenger RNA; Modeling; Molecular; Morphine; Morphine Dependence; Muscarinic M1 Receptor; N-terminal; Neurocognitive; Neuroglia; Neurons; Opiate Addiction; Opioid Receptor; Oral; Pathologic; Pathway interactions; Peripheral Blood Mononuclear Cell; Persons; Phosphotransferases; Physical Dependence; Physical assessment; Physiological; Protein Isoforms; Protein Kinase; RNA; RNA Splicing; Rattus; Recombinants; Regulation; Reporting; Research; Rewards; Role; Self Administration; Signal Pathway; Signal Transduction; Spinal Cord; Tail; Tissues; Transcript; Viral; Withdrawal; brain tissue; exosome; extracellular vesicles; genetic regulatory protein; in vivo; mRNA Precursor; member; nef Protein; novel; opioid abuse; opioid use; response; secretory protein

PROJECT SUMMARY This R01 application is in response to PAS-18-915 entitled “HIV/AIDS High Priority Drug Abuse Research”. Our project will investigate the molecular and functional interactions between HIV and morphine in regulation of alternative pre-mRNA splicing of the opioid receptor M1 (OPRM1) with implications for enhanced opioid dependence observed in the people with HIV (PWH). Clinically used opioids, such as morphine, as well as illicit drugs, such as heroin, activate OPRM1 that is a member of the G protein-coupled receptor (GPCR) family. OPRM1 pre-mRNA undergoes extensive alternative splicing events. To date, 21 isoforms of the human OPRM1 with alternative C-terminal and/or N-terminal regions and 17 isoforms of the rat OPRM1, have been identified. Given the importance of these regions in G protein-coupled receptor (GPCR) signaling, differential regulation of OPRM1 isoforms would have functional consequences. However, characterization of OPRM1 signaling is generalized, and only one isoform (MOR1) has been extensively studied. Our preliminary data suggest that expression of splicing regulatory protein SRSF1 and alternative splicing of MOR-1X is preferentially induced in neuronal cells exposed to morphine. Interestingly, our results also revealed that alternative splicing and expression of MOR-1X isoform is induced in postmortem brain tissues obtained from the PWH. These results suggested that HIV and morphine may impact OPRM1 alternative splicing and synergistically induce MOR-1X isoform expression. The mutually exclusive exon X of OPRM1 pre-mRNA is incorporated into the mature mRNA transcript following exon 3 in the MOR-1X mRNA transcript. This insertion results in substantially longer C-terminal tail having new motifs potentially binding with several cellular kinases that is unique to the MOR-1X isoform. We recently reported that glial cells infected with HIV-1 release Nef protein captured within the extracellular vesicles (Nef-EVs) which are readily taken up by neurons. We further assessed possible role of Nef-EVs and two other HIV secretory proteins, Tat and gp120, in alternative splicing of MOR-1X. Interestingly, while recombinant Tat and gp120 had no visible effects, treatment of neurons with Nef-EVs caused a comparable induction in MOR-1X alternative splicing as did treatment with morphine. Our preliminary results also revealed that co-treatment of neurons with Nef-EVs and morphine synergistically induced MOR-1X alternative splicing. Additionally, alternative splicing of MOR-1X was induced in brain regions involved in the reward pathways of the F344 rat that is the control strain of HIV-1Tg rat that has an additive induction with the exposure to morphine. Taken all together, we hypothesize that HIV-1 contributes to the increased rate of opioid dependence in the PWH by amplifying the rate of MOR-1X alternative splicing induced by morphine. Our proposed studies will reveal a novel synergistic interaction between morphine and HIV on alternative splicing of OPRM1 pre-mRNA leading to preferential expression of MOR-1X isoform with implications in physical dependence of morphine.

项目概要 此 R01 申请是对题为“HIV/AIDS 高优先级药物滥用研究”的 PAS-18-915 的回应。 我们的项目将研究艾滋病毒和吗啡之间的分子和功能相互作用，以调节 阿片受体 M1 (OPRM1) 的选择性前 mRNA 剪接对增强阿片类药物的影响 在艾滋病毒感染者 (PWH) 中观察到的依赖性 临床上使用的阿片类药物，例如吗啡以及吗啡。 海洛因等非法药物会激活 OPRM1，OPRM1 是 G 蛋白偶联受体 (GPCR) 的成员 OPRM1 前体 mRNA 经历了广泛的选择性剪接事件，迄今为止，人类有 21 种亚型。 具有替代 C 末端和/或 N 末端区域的 OPRM1 以及大鼠 OPRM1 的 17 种亚型，已被 鉴于这些区域在 G 蛋白偶联受体 (GPCR) 信号传导中的重要性，已确定差异。 OPRM1 同种型的调控会产生功能性影响，但是 OPRM1 的表征。 信号传导是广义的，我们仅主要研究了一种亚型（MOR1）。 表明剪接调节蛋白 SRSF1 的表达和 MOR-1X 的选择性剪接是 优先在暴露于吗啡的神经细胞中诱导，我们的结果还表明。 在死后脑组织中诱导 MOR-1X 同种型的选择性剪接和表达 这些结果表明 HIV 和吗啡可能影响 OPRM1 选择性剪接和 协同诱导 MOR-1X 同种型表达 OPRM1 前体 mRNA 的互斥外显子 X 是 并入 MOR-1X mRNA 转录本中外显子 3 后的成熟 mRNA 转录本中。 导致 C 端尾部明显更长，具有可能与多种细胞激酶结合的新基序 我们最近报道感染 HIV-1 的神经胶质细胞释放 Nef。 我们进一步研究了细胞外囊泡（Nef-EV）中捕获的蛋白质，该蛋白质很容易被神经元摄取。 评估了 Nef-EV 和另外两种 HIV 分泌蛋白 Tat 和 gp120 在选择性剪接中的可能作用 MOR-1X 提示，而重组 Tat 和 gp120 对神经元的治疗没有明显的影响。 Nef-EV 引起的 MOR-1X 选择性剪接的诱导效果与吗啡治疗的效果相当。 初步结果还表明，Nef-EV 和吗啡联合治疗神经元具有协同作用 诱导 MOR-1X 选择性剪接 此外，MOR-1X 选择性剪接在大脑区域被诱导。 参与 F344 大鼠的奖赏途径，F344 大鼠是 HIV-1Tg 大鼠的对照品系，具有添加剂 综上所述，我们认为 HIV-1 会导致这种情况。 通过放大 MOR-1X 选择性剪接诱导的比率来增加 PWH 中阿片类药物的依赖率 我们提出的研究将揭示吗啡和艾滋病毒之间的新型协同相互作用。 OPRM1 前体 mRNA 的选择性剪接导致 MOR-1X 亚型的优先表达 吗啡身体依赖性的影响。

项目成果

iPSC-derived three-dimensional brain organoid models and neurotropic viral infections.

• DOI: 10.1007/s13365-023-01133-3
• 发表时间: 2023-04
• 期刊: JOURNAL OF NEUROVIROLOGY
• 影响因子: 3.2
• 作者: Swingler, Michael;Donadoni, Martina;Bellizzi, Anna;Cakir, Senem;Sariyer, Ilker K.
• 通讯作者: Sariyer, Ilker K.

Modulation of OPRM1 Alternative Splicing by Morphine and HIV-1 Nef.

• DOI: 10.1007/s11481-021-10009-4
• 发表时间: 2022-06
• 期刊: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology
• 作者: Donadoni M;Huang W;Yarandi SS;Burdo TH;Chang SL;Sariyer IK
• 通讯作者: Sariyer IK