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（Lewy body Dementia，LBD）和帕金森病（Parkinson's disease，PD）在内的神经退行性疾病是一种常见的神经退行性疾病。 其特征在于α-突触核蛋白（α-syn）的聚集，然而导致细胞死亡的下游毒性事件 不被理解。蛋白质组功能障碍是突触核蛋白病的一个突出特征，如遗传学所示 和病理为了全面了解蛋白质组在PD中的变化，我们进行了一项研究。 定量蛋白质组学研究，以鉴定在患者来源的iPSC神经元中聚集的蛋白质， a-syn积累的结果。通过比较表达A53 T a-syn的iPSC神经元与表达A53 T a-syn的同基因iPSC神经元， 通过校正谱线，我们发现聚集的蛋白质类别具有显著的选择性。 具体来说，我们发现RNA结合蛋白NONO和SFPQ经历了戏剧性的溶解度变化， 洗涤剂溶解成不溶状态。NONO和SFPQ是发挥关键作用的多功能核蛋白 在调节轴突导向的基因的转录调节、RNA剪接和RNA编辑中的作用。他们是 核心成分的无膜亚区室在细胞核称为paraspeckle，并含有朊病毒- 例如在生理条件下允许相分离的低复杂度结构域。Paraspeckles发生在 神经元培养物和体内脑中，并且被认为在调节稳态应激中发挥关键作用， 瞬时隔离转录因子和RNA以防止翻译。一旦压力消退， 旁斑正常溶解，基因表达恢复正常。然而，我们发现NONO 和SFPQ在患者iPSC-神经元和LBD患者脑中不可逆地形成病理聚集体。这种效果 特别与α-syn积累相关，并且不发生于一般细胞应激。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功能丧失有关。 功能，导致神经突变性。我们发现SFPQ的转录靶点ADAR 3介导了 RNA编辑在患者神经元中几乎完全耗尽。在这里，我们建议检查的机制， α-syn积累如何导致细胞核和下游的异常NONO/SFPQ聚集 病理生理学鉴于它们在RNA剪接和编辑中的作用，我们建议采用靶向和无偏的方法， 识别RNA编辑变化的方法，包括RNA-seq，外显子连接微阵列，以检查RNA 剪接和ChIP-seq以检测SFPQ转录活性的变化。这些表型将与 具有不同的聚集形式的α-syn和神经变性。最后，我们将尝试拯救建立 通过促进可溶性功能NONO/SFPQ，在患者iPSC-神经元中观察表型。我们的初步研究表明 在突触核蛋白病中发现了一种新的致病途径，我们将通过检查 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， 在患者iPSC中的剪接变化，这可能揭示新的疾病机制和治疗策略。