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

基本信息

批准号：
10650320
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
项目类别：
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/10650320
关键词：
Axon Binding Brain Buffers Cell Death Cell Nucleus Cells Cellular Stress ChIP-seq Data Dementia with Lewy Bodies Detergents Development 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 Lewy Bodies Lewy Body Dementia Link Liquid substance Maintenance Measures Mediating Membrane Methods Midbrain structure Mitochondria Modeling Morphology Mutation Nerve Degeneration Neurites Neurodegenerative Disorders Neurons Nuclear Nuclear Proteins Nuclear RNA Nucleoplasm Parkinson Disease Parkinson&apos s Dementia Pathogenicity Pathologic Pathology Pathway interactions Patients Phase Phase Transition Phenotype Physiological Play Predisposition Process Proteins Proteome Proteomics RNA RNA Editing RNA Processing RNA Splicing RNA-Binding Proteins RNA-specific adenosine deaminase 3 Regulation Role Solubility Stress Synapses Therapeutic Transcriptional Regulation Translations Untranslated RNA Virulence Factors Work age related neurodegeneration alpha synuclein axon guidance functional restoration gene correction 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 purification protein transport proteostasis scaffold synucleinopathy trafficking transcription factor transcriptome sequencing

项目摘要

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Lewy身体痴呆（LBD）和帕金森氏病（PD）以a-突触核蛋白（A-Syn）的聚集为特征，但是导致细胞死亡的下游有毒事件不了解。蛋白质组功能障碍是突触核苷的突出特征，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，我们执行了一个定量蛋白质组学研究以鉴定患者衍生的IPSC-神经元中汇总的蛋白质作为一种 A-syn积累的结果。通过比较表达A53T A-Syn的IPSC神经元与等源性经过校正的线，我们在聚集的蛋白质类别中发现了显着的选择性。具体而言，我们发现RNA结合蛋白Nono和SFPQ经历了急剧的溶解度从洗涤剂固体进入不溶性状态。 NONO和SFPQ是多功能的核蛋白，可发挥关键在调节轴突引导的基因的转录调控，RNA剪接和RNA编辑中的作用。他们是核中无膜子室的核心成分称为paraspecckle，并包含prion- 就像允许在生理条件下分离相分离的低复杂性域。帕皮克斯出现在神经元文化和大脑体内的体内，被认为在调节体内压力方面起着关键作用瞬时隔离转录因子和RNA，以防止翻译。一旦压力为subides， paraspeccckles通常溶解，基因表达恢复正常。但是，我们发现Nono SFPQ在患者IPSC-神经元和LBD患者大脑中不可逆转地形成病理聚集体。这个效果与A-Syn积累特别相关，并且与一般的细胞应激不会发生。机理在IPSC-神经元中的研究表明，Nono/SFPQ聚集体的形成与其丢失有关功能，导致神经退行性。我们发现SFPQ转录目标，介导的ADAR3 RNA编辑几乎完全耗尽了患者神经元。在这里，我们建议检查 A-Syn积累如何导致核和下游的异常NONO/SFPQ聚集病理生理学。鉴于它们在RNA剪接和编辑中的作用，我们建议采用目标和公正识别RNA编辑变化的方法，包括RNA-seq，外显子连接微阵列检查RNA 剪接和chip-seq检测SFPQ转录活性的变化。这些表型将相关具有不同的A-syn和神经变性的汇总形式。最后，我们将尝试营救已建立的通过促进固体功能NONO/SFPQ，患者IPSC-神经元中的表型。我们的初步研究鉴定出一种新的致病性途径，我们将通过检查这些发现来扩展这些发现基因失调和RNA处理的机制。我们将提供RNA编辑的第一个描述，患者IPSC的剪接变化，这可能会发现新颖的疾病机制和治疗策略。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Dysregulation of the autophagic-lysosomal pathway in Gaucher and Parkinson's disease.

DOI：
10.1016/j.nbd.2018.03.008
发表时间：
2019-03
期刊：
Neurobiology of disease
影响因子：
6.1
作者：
Pitcairn C;Wani WY;Mazzulli JR
通讯作者：
Mazzulli JR

Rescue of α-synuclein aggregation in Parkinson's patient neurons by synergistic enhancement of ER proteostasis and protein trafficking.

DOI：
10.1016/j.neuron.2021.10.032
发表时间：
2022-02-02
期刊：
Neuron
影响因子：
16.2
作者：
Stojkovska I;Wani WY;Zunke F;Belur NR;Pavlenko EA;Mwenda N;Sharma K;Francelle L;Mazzulli JR
通讯作者：
Mazzulli JR