Mechanisms of gene regulation and RNA processing in synucleinopathies

突触核蛋白病中的基因调控和 RNA 加工机制

• 批准号: 10447768
• 负责人: Joseph R Mazzulli
• 金额: $ 52.19万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10447768
• 关键词: Axon; Binding; Brain; Buffers; Cell Death; Cell Nucleus; Cells; Cellular Stress; ChIP-seq; Data; Dementia; Dementia with Lewy Bodies; Detergents; Disease; Enzymes; Event; Exons; Functional disorder; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Health; Human; Inclusion Bodies; Induced pluripotent stem cell derived neurons; Knock-out; Lead; Lewy Bodies; Lewy Body Dementia; Link; Liquid substance; Measures; Mediating; Methods; Midbrain structure; Mitochondria; Modeling; Morphology; Mutation; Nerve Degeneration; Neurites; Neurodegenerative Disorders; Neurons; Nuclear; Nuclear Proteins; Nuclear RNA; Parkinson Disease; Parkinson' s Dementia; Pathogenicity; Pathologic; Pathology; Pathway interactions; Patients; Phase; Phase Transition; Phenotype; Physiological; Play; Process; Proteins; Proteome; Proteomics; RNA; RNA Editing; RNA Processing; RNA Splicing; RNA-Binding Proteins; RNA-specific adenosine deaminase 3; Regulation; Role; Seeds; Solubility; Stress; Synapses; Therapeutic; Transcriptional Regulation; Translations; Untranslated RNA; Virulence Factors; Work; age related neurodegeneration; alpha synuclein; axon guidance; in vitro Model; in vivo; induced pluripotent stem cell; knock-down; mouse model; neurotoxicity; novel; novel therapeutics; prevent; prion-like; protein aggregation; protein degradation; protein transport; proteostasis; scaffold; synucleinopathy; trafficking; transcription factor; transcriptome sequencing

Neurodegenerative disorders including Lewy body Dementia (LBD) and Parkinson’s disease (PD) are characterized by aggregation of a-synuclein (a-syn), however the downstream toxic events that lead to cell death are not understood. Proteome dysfunction is a prominent feature of synucleinopathies, as indicated by genetics and pathology. To gain a comprehensive understanding of how the proteome changes in PD, we performed a quantitative proteomic study to identify proteins that aggregate in patient derived iPSC-neurons as a consequence of a-syn accumulation. By comparing iPSC neurons expressing A53T a-syn with isogenic corrected lines, we discovered a remarkable level of selectivity in the classes of proteins that aggregate. Specifically, we found that RNA binding proteins NONO and SFPQ undergo dramatic solubility shifts from detergent soluble into the insoluble state. NONO and SFPQ are multifunctional nuclear proteins that play critical roles in transcription regulation, RNA splicing, and RNA editing of genes that regulate axon guidance. They are core components of a membraneless sub-compartment in the nucleus called the paraspeckle, and contain prion- like low complexity domains that permit phase separation under physiological conditions. Paraspeckles occur in neuronal cultures and in vivo in the brain, and are thought to play key roles in regulating homeostatic stress by transiently sequestering transcription factors and RNAs to prevent translation. Once stress subsides, paraspeckles normally dissolve and gene expression returns to normal. However, we have found that NONO and SFPQ irreversibly form pathological aggregates in patient iPSC-neurons and LBD patient brain. This effect is specifically associated with a-syn accumulation, and does not occur with general cellular stress. Mechanistic studies in iPSC-neurons suggest that formation of NONO/SFPQ aggregates is associated with loss of their functions, resulting in neurite degeneration. We find that the SFPQ transcriptional target, ADAR3 that mediates RNA editing, is nearly completely depleted in patient neurons. Here, we propose to examine the mechanism of how a-syn accumulation leads to aberrant NONO/SFPQ aggregation in the nucleus and downstream pathophysiology. Given their role in RNA splicing and editing, we propose to employ both targeted and unbiased methods to identify changes in RNA editing including RNA-seq, exon-junction microarrays to examine RNA splicing, and ChIP-seq to detect changes in SFPQ transcriptional activity. These phenotypes will be correlated with distinct aggregated forms of a-syn and neurodegeneration. Finally, we will attempt to rescue established phenotypes in patient iPSC-neurons by promoting soluble, function NONO/SFPQ. Our preliminary studies have identified a novel pathogenic pathway in synucleinopathies, and we will extend these findings by examining the mechanisms of gene dysregulation and RNA processing. We will provide the first description of RNA editing and splicing changes in patient iPSCs, which may uncover novel disease mechanisms and therapeutic strategies.

包括路易体痴呆(LBD)和帕金森病(PD)在内的神经退行性疾病 以a-突触核蛋白(a-syn)聚集为特征，然而导致细胞死亡的下游毒性事件 是不被理解的。蛋白质组功能障碍是共核病的一个显著特征，遗传学表明。 和病理学。为了全面了解蛋白质组在帕金森病中的变化，我们进行了 定量蛋白质组学研究鉴定聚集在患者来源的IPSC神经元中的蛋白质 A-syn累积的结果。通过比较表达A53T a-syn的IPSC神经元和同基因的 校正线条后，我们发现聚集的蛋白质类别具有显著的选择性。 具体地说，我们发现RNA结合蛋白NONO和SFPQ经历了戏剧性的溶解度变化 洗涤剂可溶于不溶状态。NONO和SFPQ是一种多功能核蛋白，在核中起着至关重要的作用 在转录调节、RNA剪接和调节轴突引导的基因的RNA编辑中的作用。他们是 核内无膜性小室的核心成分，称为副核团，含有普里恩- 例如在生理条件下允许相分离的低复杂性结构域。Paraspeckles出现在 在脑内的神经元培养和活体中，被认为在调节动态平衡应激中发挥关键作用 暂时隔离转录因子和RNA以阻止翻译。一旦压力消退， 副颗粒通常会溶解，基因表达也会恢复正常。然而，我们发现NONO 和SFPQ在患者IPSC神经元和LBD患者脑内不可逆转地形成病理性聚集体。这一效果 与a-syn的积累特别相关，一般的细胞胁迫不会发生这种情况。机械论 对IPSC神经元的研究表明，NONO/SFPQ聚集体的形成与它们的丢失有关 功能，导致轴突变性。我们发现SFPQ的转录靶标ADAR3介导了 在患者的神经元中，RNA编辑几乎完全耗尽。在这里，我们建议研究一下 α-SYN积聚如何导致NONO/SFPQ在细胞核及其下游的异常聚集 病理生理学。鉴于它们在RNA剪接和编辑中的作用，我们建议同时使用有针对性的和无偏见的 识别RNA编辑变化的方法，包括检测RNA的rna-seq、外显子连接微阵列 剪接，以及ChIP-SEQ检测SFPQ转录活性的变化。这些表型将是相关的 伴有不同聚集形式的a-syn和神经退行性变。最后，我们将尝试拯救已经建立的 通过促进可溶性、功能NONO/SFPQ促进患者IPSC神经元的表型。我们的初步研究已经 发现了一种新的联核病致病途径，我们将通过研究 基因失调和RNA加工的机制。我们将提供有关RNA编辑的第一个描述 患者ipscs的剪接变化，这可能揭示新的疾病机制和治疗策略。