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: 10194629
• 负责人: Joseph R Mazzulli
• 金额: $ 61.56万
• 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/10194629
• 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.

神经退行性疾病包括路易体痴呆 (LBD) 和帕金森病 (PD) 其特征是 a-突触核蛋白 (a-syn) 聚集，但下游毒性事件会导致细胞死亡 不被理解。遗传学表明，蛋白质组功能障碍是突触核蛋白病的一个突出特征 和病理学。为了全面了解 PD 中蛋白质组的变化，我们进行了 定量蛋白质组学研究，鉴定在患者来源的 iPSC 神经元中聚集的蛋白质作为 a-syn积累的结果。通过将表达 A53T a-syn 的 iPSC 神经元与同基因的 iPSC 神经元进行比较 校正线后，我们发现聚集的蛋白质类别具有显着的选择性。 具体来说，我们发现 RNA 结合蛋白 NONO 和 SFPQ 的溶解度从 洗涤剂可溶状态转变为不溶状态。 NONO 和 SFPQ 是发挥关键作用的多功能核蛋白 在转录调控、RNA 剪接和调节轴突引导的基因 RNA 编辑中发挥作用。他们是 细胞核中称为副斑点的无膜子室的核心成分，并含有朊病毒 就像允许在生理条件下相分离的低复杂性域一样。副散斑发生在 神经元培养物和大脑体内的神经元被认为在调节稳态应激中发挥着关键作用 暂时隔离转录因子和 RNA 以阻止翻译。一旦压力消退， 副斑点通常会溶解，基因表达恢复正常。然而我们发现NONO SFPQ 和 SFPQ 在患者 iPSC 神经元和 LBD 患者大脑中不可逆地形成病理聚集体。这个效果 与 a-syn 积累特别相关，并且不会因一般细胞应激而发生。机械论 iPSC 神经元的研究表明，NONO/SFPQ 聚集体的形成与其丧失相关性有关。 功能，导致神经突退化。我们发现 SFPQ 转录靶标 ADAR3 介导 RNA编辑在患者神经元中几乎完全耗尽。在此，我们建议研究一下其机制 a-syn 积累如何导致细胞核和下游异常 NONO/SFPQ 聚集 病理生理学。鉴于它们在 RNA 剪接和编辑中的作用，我们建议同时采用靶向和无偏 识别 RNA 编辑变化的方法，包括 RNA-seq、用于检查 RNA 的外显子连接微阵列 剪接和 ChIP-seq 来检测 SFPQ 转录活性的变化。这些表型将相互关联 具有不同的 a-syn 和神经变性聚集形式。最后，我们将尝试拯救既定的 通过促进可溶性 NONO/SFPQ 功能来改变患者 iPSC 神经元的表型。我们的初步研究有 确定了突触核蛋白病的一种新的致病途径，我们将通过检查来扩展这些发现 基因失调和RNA加工的机制。我们将提供RNA编辑的第一个描述和 患者 iPSC 的剪接变化可能会揭示新的疾病机制和治疗策略。