Alpha-Galactosidase A: a novel target for reducing alpha-synuclein toxicity

α-半乳糖苷酶 A：降低 α-突触核蛋白毒性的新靶点

• 批准号: 9318583
• 负责人: John J Shacka
• 金额: $ 18.38万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9318583
• 关键词: Address; Affect; Age; Alpha-galactosidase; American; Attenuated; Autophagocytosis; Autopsy; Biological Availability; Biological Models; Brain; Brain Diseases; Cells; Clinical; Data; Development; Disease; Dose; Enzymes; Exhibits; Fabry Disease; Fostering; Functional disorder; Future; Glycosphingolipids; Goals; Health; Human; In Vitro; Investigation; Knowledge; Link; Lysosomes; Mediating; Metabolism; Modeling; Mus; Mutation; Neurons; Outcome; Parkinson Disease; Parkinsonian Disorders; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Pharmacology; Play; Proteins; Reporting; Research; Role; Specimen; Substantia nigra structure; System; Testing; Therapeutic; Toxic effect; Transgenic Mice; Transgenic Organisms; Tyrosine 3-Monooxygenase; Wild Type Mouse; alpha synuclein; effective therapy; enzyme replacement therapy; epidemiology study; gene therapy; improved; neuroblastoma cell; neuron loss; neuropathology; neurotoxic; neurotoxicity; novel; novel therapeutics; overexpression; pre-clinical; pre-clinical research; preclinical study; response; success; synucleinopathy; therapeutic development; tool

PROJECT SUMMARY The pathological accumulation of alpha-synuclein (α-syn) is believed to play a major role in Parkinson's disease (PD) pathogenesis. The autophagy-lysosome pathway (ALP) provides for the high-capacity clearance of α-syn and its dysfunction is well-documented in PD. Inhibiting the ALP has been shown to induce α-syn accumulation. Conversely, excess α-syn has been shown to inhibit the ALP. Because the lysosome is critical for α-syn clearance we believe its continued investigation will further delineate mechanisms of PD pathogenesis and foster development of PD therapeutics. Alpha-Galactosidase A (α-Gal A) is a soluble lysosomal enzyme, with mutations causing the rare lysosomal disorder Fabry disease. While it is unknown if α- syn accumulates in Fabry patients, our analysis of postmortem PD brains indicates a decrease in α-Gal A activity specific to specimens with increased α-syn pathology. Our preliminary data also indicate reduced α-Gal A activity in neuroblastoma cells following the conditional over-expression of α-syn. Together with our report of α-syn pathology and altered ALP markers in α-Gal A-deficient mouse brain, these findings suggest a strong link between α-Gal A deficiency and α-syn accumulation. However, whether α-Gal A deficiency exacerbates the neurotoxic potential of α-syn is unknown. Increasing α-Gal A activity via enzyme replacement therapy (ERT) is clinically approved therapy for Fabry disease. Because ERT has limited CNS bioavailability, there is a critical gap in understanding its potential for treating PD. To help bridge this gap we developed novel research tools to increase α-Gal A activity in neuronal systems, including its dose-responsive increase in neuronal cells via ERT, and transgenic mice that exhibit two-fold increases in α-Gal A brain activity. Our preliminary data in neuroblastoma cells shows that α-Gal A ERT enhances the clearance of over-expressed α-syn. However, whether increasing α-Gal A activity attenuates α-syn-associated neurotoxicity has not been tested. Taken together, we hypothesize that α-syn-associated neurotoxicity is exacerbated by α-Gal A deficiency and is attenuated by increasing α-Gal A activity. In Aim 1 we will determine if α-Gal A-deficiency in primary neuron cultures exacerbates neurotoxicity resulting from the exogenous addition of α-syn pre-formed fibrils (PFFs) in a manner concomitant with ALP disruption. We will also determine if α-Gal A–deficient mice exhibit exacerbated loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra following AAV2-mediated over- expression of human wild-type α-syn. In Aim 2 we will determine if α-syn PFF-mediated neurotoxicity in primary neuron cultures is attenuated by α-Gal A ERT or the transgenic over-expression of α-Gal A and if this protection is regulated by the ALP. We will also determine if α-Gal A over-expressing mice exhibit a reduction in TH-positive neuron loss resulting from AAV2-α-syn. If our hypothesis is correct, it would suggest that α-Gal A deficiency regulates α-syn pathogenesis, a mechanism worthy of future investigation, and would accelerate the development of therapeutics for PD that act by increasing CNS α-Gal A activity.

项目概要 α-突触核蛋白 (α-syn) 的病理性积累被认为在帕金森病中起重要作用 自噬-溶酶体途径 (ALP) 提供高容量清除。 α-syn 及其功能障碍在 PD 中已被充分证明，抑制 ALP 可以诱导 α-syn。 离线积累，过量的 α-syn 已被证明会抑制 ALP，因为溶酶体至关重要。 对于 α-syn 清除，我们相信其持续研究将进一步阐明 PD 机制 α-半乳糖苷酶 A (α-Gal A) 是一种可溶性药物，其发病机制和促进其发展。 溶酶体酶，其突变会导致罕见的溶酶体疾病法布里病，但尚不清楚是否是α-。 Syn 在 Fabry 患者体内积聚，我们对死后 PD 大脑的分析表明 α-Gal A 减少 α-syn 病理学增加的标本特有的活性我们的初步数据也表明 α-Gal 减少。 α-syn 条件性过度表达后神经母细胞瘤细胞的活动以及我们的报告。 α-syn 病理学和 α-Gal A 缺陷小鼠大脑中 ALP 标记的改变，这些发现表明， α-Gal A 缺乏与 α-syn 积累之间的联系 然而，α-Gal A 缺乏是否会恶化。 通过酶替代疗法增加 α-Gal A 活性的神经毒性潜力尚不清楚。 (ERT) 是临床批准的法布里病治疗方法，因为 ERT 的中枢神经系统生物利用度有限，因此存在一定的局限性。 为了帮助弥合这一差距，我们开展了新的研究。 增加神经元系统中 α-Gal A 活性的工具，包括其在神经元细胞中的剂量反应性增加 通过 ERT，转基因小鼠的 α-Gal A 大脑活动增加了两倍。 神经母细胞瘤细胞显示 α-Gal A ERT 增强了过度表达的 α-syn 的清除。 尚未测试增加 α-Gal A 活性是否会减弱 α-syn 相关的神经毒性。 总之，我们认为 α-syn 相关的神经毒性因 α-Gal A 缺乏而加剧，并且是 通过增加 α-Gal A 活性来减弱。在目标 1 中，我们将确定初级神经元是否缺乏 α-Gal A。 培养物中外源添加 α-syn 预形成原纤维 (PFF) 会加剧神经毒性 我们还将确定 α-Gal A 缺陷小鼠是否表现出表现。 AAV2 介导的过度激活后黑质中酪氨酸羟化酶 (TH) 阳性神经元的丧失 人类野生型 α-syn 的表达 在目标 2 中，我们将确定 α-syn PFF 是否介导神经毒性。 原代神经元培养物会因 α-Gal A ERT 或 α-Gal A 转基因过度表达而减弱，如果这 我们还将确定 α-Gal A 过度表达的小鼠是否表现出减少。 在 AAV2-α-syn 导致的 TH 阳性神经元损失中，如果我们的假设是正确的，则表明 α-Gal。 缺陷调节 α-syn 发病机制，这是一个值得未来研究的机制，并将加速 通过增加 CNS α-Gal A 活性来治疗 PD 的疗法的开发。