The role of alpha-synuclein in nucleolar DNA double-strand break repair and cellular senescence

α-突触核蛋白在核仁 DNA 双链断裂修复和细胞衰老中的作用

• 批准号: 10677497
• 负责人: Moriah Rebecca Arnold
• 金额: $ 5.27万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677497
• 关键词: Affect; Age; Aging; Alzheimer' s Disease; Animals; Antibodies; Binding; Biogenesis; Biological Assay; Biotinylation; Brain; Breeding; Cell Aging; Cell Line; Cell Nucleolus; Cell Nucleus; Cell Proliferation; Cell Survival; Cells; Central Nervous System; Clinical; Complex; Cutaneous Melanoma; Cytoplasmic Inclusion; DNA; DNA Double Strand Break; DNA Repair; Data; Development; Diagnosis; Disease; Double Strand Break Repair; Electron Microscopy; Environmental Risk Factor; Epidemiology; Eukaryotic Cell; Genes; Genetic Transcription; Genome Stability; Genomic Instability; Goals; Growth; Histologic; Human; Human Cell Line; Image; Immunofluorescence Microscopy; Impairment; In Vitro; Incidence; Individual; Invaded; Knock-out; Knockout Mice; Label; Lewy Bodies; Lewy Body Dementia; Lewy Body Disease; Link; Malignant Neoplasms; Measures; Melanogenesis; Melanoma Cell; Metastatic Melanoma; Metastatic Neoplasm to Lymph Nodes; Methodology; Methods; Modeling; Molecular; Movement; Mus; Mutate; Mutation; Neoplasm Metastasis; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear; Parkinson Disease; Pathologic; Pathway interactions; Patients; Persons; Phenotype; Play; Presynaptic Terminals; Primary Neoplasm; Proteomics; Publishing; Regulation; Ribosomal DNA; Ribosomes; Risk; Role; SNCAIP gene; Skin Cancer; Substantia nigra structure; Techniques; Testing; Tissues; Up-Regulation; Variant; Work; alpha synuclein; cell growth; disorder risk; endonuclease; epidemiology study; gain of function; genetic risk factor; in vivo; insight; loss of function; melanoma; melanomagenesis; mouse model; mutant; new therapeutic target; novel; overexpression; repair function; repaired; senescence; tumor; tumor growth

PROJECT SUMMARY Genomic stability is very closely linked to diseases associated with aging, like neurodegeneration and cancer. Interestingly, strong evidence suggests unexpected links between these two diseases, since epidemiological studies have found that Parkinson’s Disease (PD) patients are at an increased risk of developing melanoma. Furthermore, this relationship is bidirectional; individuals diagnosed with melanoma are at an increased risk of developing PD. Although these clinical associations are well-established, the cellular and molecular pathways linking these diseases are poorly understood. PD is the second most common neurodegenerative disease associated with a complex combination of genetic and environmental risk factors, which manifests into devastating movement and coordination deficits. Its hallmark pathological finding is the presence of aggregated forms of alpha-synuclein (αSyn) in the substantia nigra of patient brains. Recent studies have also found a previously unrecognized role for αSyn in melanomagenesis and metastasis, highlighting the role of αSyn in regions outside of the central nervous system. Several studies have shown that melanoma cells overexpress αSyn and that this is important for promoting cell proliferation and growth; however, the underlying role of αSyn within melanogenesis is unknown. We have previously demonstrated that αSyn is important in DNA double- strand break (DSB) repair, and my new studies show an important role for αSyn within the nucleolus. Furthermore, genomic instability within the nucleolus has been associated with several diseases linked to cellular aging and has been shown to induce cellular senescence. Therefore, based on my preliminary data and these published findings, I hypothesize that the upregulation of αSyn plays a critical role in cells to facilitate nucleolar DSB repair, limit senescence, and thus contribute to overall cell survival. This hypothesis will be tested through two aims: first, I will determine the role of αSyn in nucleolar DSB repair (Aim 1) and next, determine the role of αSyn in cellular senescence inhibition and melanoma growth and metastatic potential (Aim 2). I will accomplish these goals through both in vitro and in vivo methodology. Using a human melanoma cell line that highly expresses αSyn, SK-Mel28, I will investigate the localization and function of αSyn in the nucleolus using various imaging and proteomic techniques. I will knockout αSyn to investigate loss-of-function nucleolar phenotypes and whether cellular senescence inhibition is altered. I will then reintroduce not only wildtype αSyn, but also four neurodegeneration-associated variants. To test whether αSyn plays a role in melanoma growth and metastasis through nucleolar DSB and senescence inhibition in vivo, I will breed an αSyn KO spontaneous melanoma mouse model. Through histological examination I will determine whether αSyn loss-of-function impairs nucleolar DSB repair and senescence inhibition, delays primary tumor growth, and diminishes metastasis. Achieving these goals will have a profound impact on the aging field by introducing genomic instability and cellular senescence concepts to our understanding of the cross-talk between PD and melanoma.

项目摘要 基因组稳定性与与衰老相关的疾病非常紧密，例如神经变性和癌症。 有趣的是，有力的证据表明这两种疾病之间存在意外联系，因为流行病学 研究发现，帕金森氏病（PD）患者患黑色素瘤的风险增加。 此外，这种关系是双向的。被诊断为黑色素瘤的人有增加的风险 开发PD。尽管这些临床关联是完善的，但细胞和分子途径 关联这些疾病的理解很少。 PD是第二大最常见的神经退行性疾病 与遗传和环境风险因素的复杂组合相关联，这表现为 毁灭性运动和协调不足。它的标志性病理发现是汇总的存在 患者大脑的底膜中α-核蛋白（αSyn）的形式。最近的研究还发现 以前在黑色素作和转移中αSyn的作用未被认可的作用，强调了αsyn在 中枢神经系统以外的地区。几项研究表明，黑色素瘤细胞过表达 αSyn，这对于促进细胞增殖和生长很重要。但是，αSyn的基本作用 黑色素发生尚不清楚。我们以前已经证明，αSyn在DNA双 - Strand Break（DSB）修复，而我的新研究表明，核仁中αSyn的重要作用。 此外，核仁内的基因组不稳定性与与细胞有关的几种疾病有关 衰老并已被证明会诱导细胞感应。因此，根据我的初步数据和这些 我假设发表的发现，αSyn的上调在细胞中起着至关重要的作用，以促进核 DSB修复，限制感应，从而有助于整体细胞存活。该假设将通过 两个目的：首先，我将确定αSyn在核仁DSB修复中的作用（AIM 1），然后确定 αSyn在细胞抑制和黑色素瘤生长和转移性潜力中（AIM 2）。我会完成的 这些目标是通过体外和体内方法论。使用人类黑色素瘤细胞系高度 表示αSyn，SK-MEL28，我将使用各种 成像和蛋白质组学技术。我将敲除αsyn以研究功能丧失的核仁表型和 细胞感应抑制是否改变。然后，我将不仅重新引入野生型αSyn，还要重新引入四个 神经变性相关的变体。测试αSyn是否在黑色素瘤生长和转移中起作用 通过核仁DSB和体内衰老抑制作用，我将繁殖αSynKO自发黑色素瘤 鼠标模型。通过组织学检查，我将确定αSyn功能丧失是否会损害核仁 DSB修复和抑制作用，延迟原发性肿瘤的生长并减少转移。实现这些 通过引入基因组不稳定性和细胞感应，目标将对衰老领域产生深远的影响 我们对PD和黑色素瘤之间的串扰的理解的概念。