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.

抽象的 帕金森氏病（PD）是第二大最常见的慢性进行性神经退行性疾病。 ep MIology和人口遗传学表明，零星PD（> 95％的病例）是由于复杂的相互作用而导致的 在遗传风险，衰老和环境因素之间。对遗传风险的详细理解是第一个 阐明这种复杂相互作用的步骤。基因组广泛的关联研究（GWAS）已经确定了数字 与78个基因组区域中存在的OUS风险变异（例如，单核苷酸多态性[SNP]） 发展PD的风险增加。但是，几乎没有关于这些SNP Mech-的见解。 无动于衷有助于PD的发展和发展。由于大多数功能性SNP都高度 富含非编码调节性DNA元件（例如盘式增强子），普遍的理论是顺式的理论 功能性非编码SNP对基因表达的作用作用在发育中起重要作用 复杂疾病。为了汇编大脑增强器中有问题的SNP列表，我们整合了GWAS-IDEN- 具有表观基因组数据识别脑特异性增强子和基因表达数据集的鉴定PD风险变体 在原发性脑组织中。该分析表明，在小胶质细胞特异性的en-中，多个候选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HACER元素。 GPNMB是类型1 跨膜蛋白已知已在PD患者的黑质中进行更新。鲜为人知 - 在其分子功能以及GPNMB的失调中构造了PD的失调。我们的初步分析 鉴定出HPSC衍生的小胶质细胞中GPNMB基因缺失后细胞转录组变化的SI 与CNS注射相关的多个关键基因表达水平的改变（即NLRP2， NLRP12）。此外，还有令人信服的证据表明GPNMB在自噬中起作用 Agy，Mitophagy和CMA）。根据以前的文献和我们的初步数据，我推测顺式动作 - PD风险相关序列变体在小胶质细胞特异性GPNMB增强子中的影响会导致IN- 创建了GPNMB表达，导致自噬功能障碍，并最终导致激活 小胶质细胞中的炎症途径，这有助于在PD中观察到的神经退行性。测试这个 假设该项目的目的是：（1）确定此上游增强子中存在的因果风险变体 GPNMB并表征了分子机制，使因果风险变异障碍GPNMB的分子机制 使用CRISPR/CAS9风险变体编辑的HPSC在小胶质细胞中的表达，（2）表征功能效应 与小胶质细胞激活相关的小胶质细胞注入和神经退行器的增益和损失 与ATION相关的诱导剂，（3）表征GPNMB对自噬的功能效应，并确定是否是否 自噬是GPNMB影响注射的一种中间机制。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型联系的有价值的见解 tion。