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

项目摘要 α-突触核蛋白（α-syn）的病理性积累被认为在帕金森病中起主要作用。 疾病（PD）发病机制。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中有很好的记录。抑制ALP已被证明可诱导α-syn 积累相反，过量的α-syn已显示出抑制ALP。因为溶酶体是关键 对于α-syn清除，我们相信其继续研究将进一步阐明PD的机制 发病机制和促进PD治疗学的发展。α-半乳糖苷酶A（α-Gal A）是一种可溶性的 溶酶体酶，突变导致罕见的溶酶体疾病法布里病。虽然不知道α- 虽然法布瑞氏病患者体内有syn积累，但我们对死后PD脑组织的分析表明α-Gal A减少， 特异于α-syn病理学增加的标本的活性。我们的初步数据还表明， α-syn条件性过表达后神经母细胞瘤细胞中的A活性。连同我们的报告， 在α-Gal A缺陷小鼠脑中，α-syn病理学和改变的ALP标志物，这些发现表明， α-Gal A缺乏和α-syn积累之间的联系。然而，α-Gal A缺乏是否加剧了 α-syn的神经毒性潜力尚不清楚。通过酶替代疗法增加α-Gal A活性 (ERT)是临床上批准的法布里病治疗方法。由于ERT的CNS生物利用度有限， 在了解其治疗PD的潜力方面存在关键差距。为了帮助弥合这一差距，我们开发了新的研究， 增加神经系统中α-Gal A活性的工具，包括其在神经细胞中的剂量响应性增加 和表现出α-Gal A脑活性增加两倍的转基因小鼠。我们的初步数据， 在神经母细胞瘤细胞中的结果显示，α-Gal A ERT增强了过表达的α-syn的清除。然而，在这方面， α-Gal A活性的增加是否减弱α-syn-associated神经毒性还没有被测试。采取 总之，我们假设α-Gal A缺乏会加重α-syn-associated神经毒性， 通过增加α-Gal A活性而减弱。在目的1中，我们将确定在初级神经元中是否存在α-Gal A-缺乏， 培养物加剧了外源性添加α-syn预形成纤维（PFF）引起的神经毒性， 与ALP破坏同时发生。我们还将确定α-Gal A缺陷小鼠是否表现出加重的 在AAV 2介导的过度表达后，黑质中酪氨酸羟化酶（TH）阳性神经元的丢失， 人野生型α-syn的表达。在目标2中，我们将确定α-syn PFF介导的神经毒性是否在 原代神经元培养物被α-Gal A ERT或α-Gal A的转基因过表达减弱，如果这 保护由ALP管理。我们还将确定α-Gal A过表达小鼠是否表现出降低 在由AAV 2-α-syn引起的TH阳性神经元损失中。如果我们的假设是正确的，那么α-Gal 缺乏调节α-syn发病机制，这是一个值得进一步研究的机制， 开发通过增加CNS α-Gal A活性起作用的PD治疗剂。

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.