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

项目摘要 基因组的稳定性与衰老相关的疾病密切相关，如神经退行性疾病和癌症。 有趣的是，强有力的证据表明这两种疾病之间存在意想不到的联系，因为流行病学 研究发现，帕金森病（PD）患者患黑色素瘤的风险增加。 此外，这种关系是双向的;被诊断患有黑色素瘤的个体患黑色素瘤的风险增加。 发展PD。尽管这些临床关联已经得到了很好的证实，但细胞和分子途径 这些疾病之间的联系知之甚少。PD是第二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有关， 破坏性的运动和协调缺陷其标志性病理发现是存在聚集的 患者大脑黑质中的α-突触核蛋白（αSyn）形式。最近的研究还发现， αSyn在黑色素瘤发生和转移中的作用以前未被认识，突出了αSyn在 中枢神经系统以外的区域。几项研究表明黑色素瘤细胞过度表达 αSyn，这对促进细胞增殖和生长很重要;然而，αSyn的潜在作用 黑素生成的机制是未知的我们之前已经证明αSyn在DNA双链中很重要， 链断裂（DSB）修复，我的新研究表明αSyn在核仁中发挥重要作用。 此外，核仁内的基因组不稳定性与几种与细胞凋亡相关的疾病有关。 老化，并已显示诱导细胞衰老。因此，根据我的初步数据和这些 根据已发表的研究结果，我假设αSyn的上调在细胞中起着关键作用， DSB修复，限制衰老，从而有助于整体细胞存活。这一假设将通过 两个目标：首先，我将确定αSyn在核仁DSB修复中的作用（Aim 1），其次，确定 αSyn在细胞衰老抑制和黑色素瘤生长和转移潜力中的作用（目的2）。我会完成 这些目标通过体外和体内方法实现。使用人类黑色素瘤细胞系， 表达αSyn，SK-Mel 28，我将研究αSyn在核仁中的定位和功能， 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， 细胞衰老抑制是否改变。然后，我将不仅重新引入野生型αSyn， 神经退行性变相关的变体。检测αSyn是否在黑色素瘤生长和转移中发挥作用 通过核仁DSB和体内衰老抑制，我将培育一个αSyn KO自发性黑色素瘤 小鼠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的 DSB修复和衰老抑制，延迟原发性肿瘤生长，并减少转移。实现这些 通过引入基因组不稳定性和细胞衰老， 这些概念有助于我们理解PD和黑素瘤之间的相互作用。

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.