Exploring the role of protein farnesylation in the regulation of SNARE protein ykt6 in synucleinopathy models

摘要 突触核蛋白病，如帕金森病（PD），是一组无法治愈的神经退行性疾病 其特征在于聚集的α-突触核蛋白（α-syn）的积累。蛋白质运输的干扰， 细胞降解途径如溶酶体与这些疾病的发病机制有关。 PD最强的遗传风险因素是溶酶体葡萄糖脑苷脂酶的功能缺失突变 （GCase），其降解葡糖神经酰胺（GluCer）。我们以前的工作表明，GCase活性的丧失和 GluCer积累直接诱导神经元中的α-syn聚集和细胞死亡。这表明溶酶体 功能障碍在PD发病机制中起着重要作用，增强溶酶体功能可能会减少 同步聚合。我们还发现，当a-syn聚集在神经元的细胞体上时， 内质网（ER）和高尔基体之间的溶酶体水解酶，从而减少溶酶体 功能因此，增强ER-高尔基体运输的方法可以改善溶酶体水解酶靶向， 功能，并增强病理性a-syn聚集体的清除。然而，不存在治疗靶点， 能够增强运输或溶酶体活性。我们建议探索一种潜在的 称为ykt 6的新型治疗靶点，其作为可溶性NSF附着蛋白受体（SNARE）发挥作用， 增强内质网和高尔基体之间的蛋白质运输，从而改善溶酶体的成熟 酶和水解酶活性。先前的工作表明，ykt 6的同源SNARE结合受以下因素调节： 法尼基化，我们将研究如何法尼基化的ykt 6影响其能力，以控制ER-高尔基体运输 使用来自诱导多能干细胞（iPSC）模型和等基因校正的PD患者来源的神经元 对照初步数据表明，抑制ykt 6的法尼基化增强了溶酶体的运输。 水解酶并改善溶酶体功能。此外，法尼基转移酶的药理学抑制 使用脑渗透剂，临床验证的小分子抑制剂可以减少病理性的，不溶性的α-syn， 以YKT 6依赖的方式对细胞系进行抑制。我们建议研究法尼基介导的调节机制 YKT 6功能的降低与其增强溶酶体功能和减少病理性α-syn的能力有关。因为这些 研究是探索性的，它们旨在评估ykt 6作为治疗靶点的未来潜力。 突触核蛋白病的机制，重点是调节其ER-高尔基体运输活动。我们预计这些 研究，导致新的假说，调节ER-高尔基体运输和溶酶体活性，并可能 细胞中的其他囊泡运输途径，可能会在未来的提案中进行审查。目前没有 已知的方法来增强水解酶运输和溶酶体功能，我们的研究可能有助于验证 YKT 6作为可药物化的靶点，以增强这些过程并减少病理形式的α-syn。我们的研究可能 由于法尼基转移酶抑制剂在临床上作为癌症的治疗药物得到了很好的发展， 治疗，导致重新利用这些药物治疗PD的可能性。

Abstract Synucleinopathies, such as Parkinson's disease (PD), are a group of incurable neurodegenerative diseases characterized by the accumulation of aggregated alpha-synuclein (a-syn). Disturbances in protein trafficking and cellular degradation pathways, such as lysosomes, have been implicated in the pathogenesis of these disorders. The strongest genetic risk factors for PD are loss-of-function mutations in lysosomal glucocerebrosidase (GCase), that degrades glucosylceramide (GluCer). Our previous work showed that loss of GCase activity and GluCer accumulation directly induces a-syn aggregation and cell death in neurons. This indicates that lysosomal dysfunction plays an important role in PD pathogenesis, and that enhancing lysosomal function may reduce a- syn aggregation. We also showed that a-syn, when aggregated at the cell body of neurons, inhibits the trafficking of lysosomal hydrolases between the endoplasmic reticulum (ER) and the Golgi, thereby reducing lysosomal function. Therefore, methods to enhance ER-Golgi trafficking may improve lysosomal hydrolase targeting and function, and enhance the clearance of pathological a-syn aggregates. However no therapeutic targets exist that are capable of enhancing trafficking or lysosomal activity. We propose to explore the mechanism of a potential novel therapeutic target called ykt6 that functions as a Soluble NSF Attachment protein REceptor (SNARE) to enhance protein trafficking between the ER and the Golgi, therefore improving the maturation of lysosomal enzymes and hydrolase activity. Previous work has shown that cognate SNARE binding of ykt6 is regulated by farnesylation, and we will examine how farnesylation of ykt6 influences its ability to control ER-Golgi trafficking using PD patient-derived neurons from induced pluripotent stem cell (iPSC) models and isogenic corrected controls. Preliminary data indicate that inhibiting the farnesylation of ykt6 enhances the trafficking of lysosomal hydrolases and improves lysosomal function. Furthermore, pharmacological inhibition of farnesyltransferase using a brain-penetrant, clinically validated small-molecule inhibitor can reduce pathological, insoluble a-syn in cell lines in a ykt6-dependent manner. We propose to examine how the farnesyl-mediated regulatory mechanism of ykt6 function is related to its ability to enhance lysosomal function and reduce pathological a-syn. As these studies are exploratory, they are aimed to assess the future potential of ykt6 as a therapeutic target for the synucleinopathies by focusing on the mechanism that regulates its ER-Golgi trafficking activity. We expect these studies to lead to novel hypothesis for regulation of ER-Golgi trafficking and lysosomal activity, and possibly other vesicular trafficking pathways in the cell, that may be examined in future proposals. There are currently no known methods to enhance hydrolase trafficking and lysosomal function, and our studies may help to validate ykt6 as a druggable target to enhance these processes and reduce pathological forms of a-syn. Our studies may be rapidly translated into therapies since farnesyltransferase inhibitors are well-developed in the clinic as cancer therapies, leading to the possibility of repurposing these agents for the treatment of PD.