RNA:RNA binding protein complexes in neurons and SIV encephalitis

RNA：神经元和 SIV 脑炎中的 RNA 结合蛋白复合物

• 批准号: 8937093
• 负责人: Kelly L Jordan-Sciutto
• 金额: $ 23.31万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8937093
• 关键词: AIDS Dementia Complex; Acquired Immunodeficiency Syndrome; Affect; Age; Animals; Anti-Retroviral Agents; Ataxia; Atrophic; Axon; Binding; Binding Sites; Bioinformatics; Cell Cycle Regulation; Cellular Structures; Chronic; Cognitive; Communication; Control Animal; Defect; Dendrites; Development; Disease; Distal; Encephalitis; Excitatory Neurotoxins; Fragile X Syndrome; Frontotemporal Dementia; Future; Gene Expression; Gene Proteins; Global Change; HIV; HIV encephalitis; HIV-associated neurocognitive disorder; Hereditary Disease; Impaired cognition; In Vitro; Infiltration; Inflammation; Link; Macaca; Maintenance; Mediating; Messenger RNA; Modeling; Natural Immunity; Nervous System Physiology; Nervous system structure; Neurons; Neurotoxins; Parkinson Disease; Pathologic; Patients; Polyribosomes; Primates; Process; Progressive Disease; Protein Binding; Protein Footprinting; Proteins; Proteomics; RNA; RNA Sequences; RNA Splicing; RNA Stability; RNA-Binding Proteins; Rattus; Reactive Oxygen Species; Regulation; Research Personnel; Ribonucleases; SIV; SIV encephalitis; Site; Synapses; Synaptic plasticity; Therapeutic; Tissues; Translations; Viral; antiretroviral therapy; cytokine; deep sequencing; experience; high risk; in vitro Model; in vivo Model; insight; interest; macrophage; metabolomics; myelination; nervous system disorder; neuroinflammation; neuron loss; neurotoxicity; nonhuman primate; novel; prevent; protein complex; public health relevance; research study; response; synaptogenesis; therapeutic target; transcriptome sequencing; transcriptomics

 DESCRIPTION (provided by applicant): HIV-Associated Neurocognitive Disorders (HAND) continue to affect ~50% of HIV(+) patients1-4, despite suppression of HIV replication with antiretroviral compounds1-4. With antiretroviral therapy, HAND is now a chronic, progressing disease leading to a spectrum of neurologic disorders. In more severe forms of disease, such as HIV-Associated Dementia (HAD), there is neuronal loss, but in milder forms of disease, synaptic damage is the more salient pathologic change8-14. Both neuronal loss and synaptic simplification are associated with macrophage infiltration and neuroinflammation 15-18, which are believed to be mediated by release of excitotoxins, reactive oxygen species (ROS), cytokines and viral proteins10,15,19-42. Transcriptomic, Proteomic, and Metabolomic approaches have identified several classes of genes and proteins that are dysregulated in HAND, including those regulating innate immunity, inflammasome formation, cell cycle regulation, myelination and synaptic development and function43-51. As synaptic damage correlates best with HAND progression, we are most interested in understanding these changes; however, the mechanisms underlying synaptodendritic damage remain elusive. A particular hurdle to understanding the contribution of gene expression in neurons is the presence of polyribosome tracts in the distal axons and dendrites of neurons where translation is regulated locally to provide rapid changes in gene expression resulting in synaptic plasticity. Local translation, splicing, and stability of RNA as wll as delivery of RNA to these sites require RNA binding proteins (RBPs). It is now clear that RNA regulation by RBPs contributes significantly to regulation of synapse formation, maintenance, and turnover as evidenced by the growing number of genetic disorders of the nervous system with defects in RBPs including Fragile X Syndrome, Frontotemporal Dementia, Spinocerabellar Ataxia, Spinomuscular Atrophy, and Parkinson disease53-55. In order to gain insight into the synaptic changes observed in neurons responding to HIV-associated neurotoxins in HAND, we propose to use a novel, high yield approach, protein interaction profile-sequencing (PIP-Seq), to identify global changes in RNA sequences bound by RBPs in neurons exposed to HIV-associated neurotoxins in an in vitro model of disease, and in the CNS of primates in an in vivo model of lentiviral encephalitis. PIP-Seq employs a novel RNase-mediated protein footprinting strategy in which all unbound RNA is digested by RNAses and RNA sequences protected by RBPs are subjected to deep sequencing. Combining information gained from PIP-seq with bioinformatics approaches, this high risk, high yield approach will provide insights into: the RBP-bound RNA sequences in neurons at baseline, the changes in RBP-bound RNA sequences in neurons responding to HIV-associated neurotoxins, the region of RNA molecules bound by RBP, the conservation of the bound sites across vertebrate species, the overrepresented biologic processes in which differentially RBP-bound RNAs function, the ability to distinguish putative SNPs linked to changes in RBP activity, and the means to identify known and novel RBPs that regulate RNAs linked to HIV-associated changes. Finally, identified RBP-bound RNA sequences will be compared with previous transcriptomic and proteomic results to gain insight into the mechanisms underlying changes in observed gene expression in HAND. This novel, high risk, high yield approach stands to provide valuable insight into changes in neuronal responses in HAND and in diseases beyond, and will provide the underpinnings for studies to increase both our understanding of basic neuronal function and how disease processes impact that function.

 描述（由申请人提供）：尽管抗逆转录病毒化合物可抑制 HIV 复制1-4，但 HIV 相关神经认知障碍 (HAND) 继续影响约 50% 的 HIV(+) 患者1-4。通过抗逆转录病毒治疗，手足口病现已成为一种慢性、进展性疾病，导致一系列神经系统疾病。在更严重的疾病中，例如 HIV 相关痴呆 (HAD)，会出现神经元损失，但在较轻的疾病中，突触损伤是更显着的病理变化 8-14。神经元损失和突触简化均与巨噬细胞浸润和神经炎症有关 15-18，这被认为是由兴奋毒素、活性氧 (ROS)、细胞因子和病毒蛋白的释放介导的 10,15,19-42。转录组学、蛋白质组学和代谢组学方法已经鉴定出 HAND 中失调的几类基因和蛋白质，包括调节先天免疫、炎性体形成、细胞周期调节、髓鞘形成和突触发育和功能的基因和蛋白质43-51。由于突触损伤与 HAND 进展最相关，因此我们最感兴趣的是了解这些变化；然而，突触树突损伤的机制仍然难以捉摸。了解神经元中基因表达的贡献的一个特殊障碍是神经元远端轴突和树突中存在多核糖体束，其中翻译受到局部调节，以提供基因表达的快速变化，从而导致突触可塑性。 RNA 的局部翻译、剪接和稳定性以及将 RNA 递送到这些位点都需要 RNA 结合蛋白 (RBP)。现在已经清楚，RBP 对 RNA 的调节对突触形成、维持和周转的调节有显着贡献，越来越多的 RBP 缺陷神经系统遗传性疾病就证明了这一点，包括脆性 X 综合征、额颞叶痴呆、脊髓小脑共济失调、脊髓肌肉萎缩和帕金森病 53-55。为了深入了解 HAND 中对 HIV 相关神经毒素作出反应的神经元中观察到的突触变化，我们建议使用一种新颖、高产量的方法，即蛋白质相互作用谱测序 (PIP-Seq)，以识别体外疾病模型中暴露于 HIV 相关神经毒素的神经元中以及在体外疾病模型中暴露于 HIV 相关神经毒素的神经元中 RBP 结合的 RNA 序列的整体变化。 慢病毒脑炎的体内模型。 PIP-Seq 采用新型 RNase 介导的蛋白质足迹策略，其中所有未结合的 RNA 均被 RNAses 消化，并且受 RBP 保护的 RNA 序列进行深度测序。将 PIP-seq 获得的信息与生物信息学方法相结合，这种高风险、高产量的方法将提供以下方面的见解：基线时神经元中 RBP 结合的 RNA 序列、响应 HIV 相关神经毒素的神经元中 RBP 结合的 RNA 序列的变化、RBP 结合的 RNA 分子的区域、脊椎动物物种中结合位点的保守性、 过度代表的生物过程中，RBP 结合的 RNA 功能存在差异，区分与 RBP 活性变化相关的推定 SNP 的能力，以及识别调节与 HIV 相关变化相关的 RNA 的已知和新型 RBP 的方法。最后，将鉴定出的 RBP 结合 RNA 序列与之前的转录组和蛋白质组结果进行比较，以深入了解 HAND 中观察到的基因表达变化的机制。这种新颖、高风险、高收益的方法将为了解 HAND 和其他疾病中神经元反应的变化提供有价值的见解，并将为研究提供基础，以增加我们对基本神经元功能以及疾病过程如何影响该功能的理解。