Dissecting GWAS Identified Risk Variants in Parkinson's Disease – Functional Role of GPNMB in the Pathogenesis of PD

剖析 GWAS 确定的帕金森病风险变异 — GPNMB 在帕金森病发病机制中的功能作用

• 批准号: 10680117
• 负责人: Riana Lo Bu
• 金额: $ 4.77万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680117
• 关键词: Affect; Age; Aging; Autophagocytosis; Autophagosome; Binding; Brain; CRISPR/Cas technology; Chromatin Structure; Chronic; Complex; DNA; Data; Data Set; Development; Disease; Disease susceptibility; Distal; Electrophoretic Mobility Shift Assay; Elements; Enhancers; Environmental Risk Factor; Epidemiology; Etiology; Functional disorder; Gene Deletion; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic Enhancer Element; Genetic Risk; Genetic Transcription; Genomic Segment; Glycoproteins; Impairment; Individual; Inflammasome; Inflammation; Inflammatory; Integral Membrane Protein; Link; Literature; Macrophage; Mediating; Membrane; Microglia; Molecular; Molecular Chaperones; Nerve Degeneration; Neurodegenerative Disorders; Nonmetastatic; Parkinson Disease; Pathogenesis; Pathway interactions; Patients; Phagocytes; Phagocytosis; Play; Population Genetics; Probability; Proteins; Quantitative Reverse Transcriptase PCR; Regulatory Element; Reverse Transcriptase Polymerase Chain Reaction; Risk; Role; Single Nucleotide Polymorphism; Stress; Substantia nigra structure; System; Testing; Transcriptional Regulation; Untranslated RNA; Up-Regulation; Upstream Enhancer; Variant; Work; brain tissue; cytokine; disorder risk; epigenomics; gain of function; genetic risk factor; genome editing; genome wide association study; genomic locus; glial activation; human pluripotent stem cell; inhibitor; insight; loss of function; lysosome membrane; melanoma; migration; neuroinflammation; new therapeutic target; novel; protein B; protein expression; risk variant; sporadic Parkinson' s Disease; theories; transcription factor; transcriptome; transcriptome sequencing; zinc finger nuclease

ABSTRACT Parkinson’s Disease (PD) is the second most common chronic progressive neurodegenerative disease. Epide- miology and population genetics suggest that sporadic PD (>95% of cases) results from a complex interaction between genetic risk, aging, and environmental factors. A detailed understanding of the genetic risk is the first step to elucidating this complex interaction. Genome wide association studies (GWAS) have identified numer- ous risk variants (e.g., single nucleotide polymorphisms [SNPs]) present in 78 genomic regions associated with an increased risk of developing PD. However, there is little insight regarding which and how these SNPs mech- anistically contribute to the development and progression of PD. Since most of the functional SNPs are highly enriched in non-coding regulatory DNA elements such as distal enhancers, the prevailing theory is that cis- acting effects of the functional non-coding SNPs on gene expression play a significant role in the development of complex diseases. To compile a list of probable causal SNPs in brain enhancers, we integrated GWAS-iden- tified PD-risk variants with epigenomic data identifying brain-specific enhancers and gene expression datasets in primary brain tissue. This analysis revealed multiple candidate PD-risk variants in a microglia-specific en- hancer element in the glycoprotein nonmetastatic melanoma protein B (GPNMB) locus. GPNMB is a type 1 transmembrane protein known to be upregulated in the substantia nigra of PD patients. Very little is known re- garding its molecular function and how the dysregulation of GPNMB contributes to PD. Our preliminary analy- sis of changes in the cellular transcriptome after GPNMB gene deletion in hPSC-derived microglia identified alterations in expression levels of multiple key genes associated with CNS inflammation (i.e. NLRP2, NLRP12). In addition, there is compelling evidence indicating a role for GPNMB in autophagy (macroautoph- agy, mitophagy, and CMA). Based on previous literature and our preliminary data, I speculate that the cis-act- ing effect of a PD-risk associated sequence variant in a microglia-specific GPNMB enhancer leads to in- creased GPNMB expression, resulting in autophagy dysregulation and ultimately culminating in the activation of inflammatory pathways in microglia which contribute to the neurodegeneration observed in PD. To test this hypothesis this project aims to: (1) to identify the causal risk variant present in this upstream enhancer of GPNMB and characterize the molecular mechanisms by which the causal risk variant dysregulates GPNMB expression in microglia using CRISPR/Cas9-risk variant edited hPSCs, (2) characterize the functional effects of gain and loss of GPNMB on microglial inflammation associated with microglial activation and neurodegener- ation associated-inducers, and (3) characterize the functional effect of GPNMB on autophagy and determine if autophagy is an intermediate mechanism by which GPNMB influences inflammation. This work will provide in- valuable insight into the novel link between non-coding PD risk variants, GPNMB, autophagy, and inflamma- tion.

摘要 帕金森病（Parkinson's Disease，PD）是第二常见的慢性进行性神经退行性疾病。Epide- 骨髓学和群体遗传学表明，散发性PD（>95%的病例）是由复杂的相互作用引起的， 遗传风险、衰老和环境因素之间的联系。详细了解遗传风险是第一位的 进一步阐明这种复杂的相互作用。全基因组关联研究（GWAS）已经确定了许多 我们的风险变体（例如，单核苷酸多态性[SNP]）存在于78个与 患PD的风险增加。然而，关于这些SNPs是哪些以及如何作用的了解很少。 有助于PD的发展和进展。由于大多数功能性SNPs都是高度保守的， 富含非编码调控DNA元件，如远端增强子，流行的理论是顺式- 功能性非编码SNPs对基因表达的作用在发育中起重要作用 复杂的疾病。为了编制一份大脑增强剂中可能的因果SNP列表，我们整合了GWAS-iden- 用鉴定脑特异性增强子和基因表达数据集的表观基因组数据确定PD风险变体 在初级脑组织中。该分析揭示了小胶质细胞特异性基因中的多种候选PD风险变体， 糖蛋白非转移性黑色素瘤蛋白B（GPNMB）基因座中的促癌元件。GPNMB是类型1 已知在PD患者的黑质中上调的跨膜蛋白。我们知之甚少， 了解GPNMB的分子功能以及GPNMB的调节异常如何参与PD。我们的初步分析- 在鉴定的hPSC衍生的小胶质细胞中GPNMB基因缺失后细胞转录组的变化 与CNS炎症相关的多个关键基因（即NLRP 2， NLRP 12）。此外，有令人信服的证据表明GPNMB在自噬（macroautophy）中的作用。 agy、mitophagy和CMA）。根据以前的文献和我们的初步数据，我推测，顺式-act- PD风险相关序列变体在小胶质细胞特异性GPNMB增强子中的作用导致 GPNMB表达增加，导致自噬失调，并最终在激活 小胶质细胞中的炎症通路，这有助于在PD中观察到的神经变性。为了验证这一 假设本项目旨在：（1）确定存在于该上游增强子中的因果风险变体， GPNMB和表征的分子机制，其中因果风险变异失调GPNMB 使用CRISPR/Cas9风险变体编辑的hPSC在小胶质细胞中表达，（2）表征功能效应 GPNMB的获得和损失对小胶质细胞炎症的影响与小胶质细胞活化和神经变性有关， （3）表征GPNMB对自噬的功能作用，并确定是否 自噬是GPNMB影响炎症的中间机制。这项工作将提供- 对非编码PD风险变体，GPNMB，自噬和炎症之间的新联系的有价值的见解， 是的。