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

项目摘要 本R 01申请是对PAS-18-915“HIV/AIDS高优先药物滥用研究”的回应。 我们的项目将研究HIV和吗啡之间的分子和功能相互作用， 阿片受体M1（OPRM 1）的前体mRNA选择性剪接与增强阿片 艾滋病毒感染者（PWH）的依赖性。临床使用的阿片类药物，如吗啡，以及 非法药物，如海洛因，激活OPRM 1，它是G蛋白偶联受体（GPCR）的成员 家人OPRM 1前mRNA经历广泛的选择性剪接事件。到目前为止，人类的21种亚型 具有交替的C-末端和/或N-末端区域的OPRM 1和大鼠OPRM 1的17种同种型，已经被发现。 鉴定考虑到这些区域在G蛋白偶联受体（GPCR）信号传导中的重要性，差异表达是可能的。 OPRM 1同种型的调节将具有功能性后果。然而，OPRM 1的表征 信号传导是普遍的，只有一种同种型（MOR 1）被广泛研究。我们的初步数据 提示剪接调节蛋白SRSF 1表达和莫尔-1X的选择性剪接， 优先在暴露于吗啡的神经元细胞中诱导。有趣的是，我们的结果还显示， 莫尔-1X同种型的选择性剪接和表达在死后脑组织中被诱导，所述死后脑组织获自 威尔斯亲王医院这些结果表明，HIV和吗啡可能影响OPRM 1的选择性剪接， 协同诱导莫尔-1X同种型表达。OPRM 1前体mRNA的互斥外显子X是 在莫尔-1X mRNA转录物中，在外显子3之后掺入成熟mRNA转录物中。该插入 导致具有潜在地与几种细胞激酶结合的新基序的显著更长的C-末端尾部 这是莫尔-1X同种型所特有的。我们最近报道了感染HIV-1的神经胶质细胞释放Nef 在细胞外囊泡（Nef-EV）内捕获的蛋白质，其容易被神经元摄取。我们进一步 评估了Nef-EV和其他两种HIV分泌蛋白达特和gp 120在选择性剪接中的可能作用 的莫尔-1X。有趣的是，虽然重组达特和gp 120没有明显的作用，但用 Nef-EV在莫尔-1X选择性剪接中引起的诱导与吗啡治疗相当。我们 初步结果还显示，用Nef-EV和吗啡协同处理神经元， 诱导莫尔-1X选择性剪接。此外，在脑区域中诱导了莫尔-1X的选择性剪接 参与F344大鼠的奖励途径，F344大鼠是HIV-1 Tg大鼠的对照品系， 吗啡诱导综上所述，我们假设HIV-1有助于 通过放大诱导的莫尔-1X选择性剪接的速率， 吗啡。我们提出的研究将揭示吗啡和艾滋病毒之间的一种新的协同作用， OPRM 1前体mRNA的选择性剪接导致莫尔-1X同种型的优先表达， 对吗啡身体依赖的影响。

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.