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

项目总结 α-突触核蛋白(α-SYN)的病理性堆积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)提供了高容量的清除 α-SYN及其功能障碍在帕金森氏病中有很好的文献记载。抑制碱性磷酸酶可诱导α-SYN 积累。相反，过量的α-SYN已被证明抑制碱性磷酸酶。因为溶酶体是至关重要的 对于α-SYN的清除，我们相信它的继续调查将进一步揭示PD的机制 帕金森病的发病机制及促进其发展。α-半乳糖苷酶A(α-GalA)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。虽然目前还不清楚α是否- α在法布里患者体内积聚，我们对帕金森病死后大脑的分析表明SYN-GalA减少 α-SYN病理增高的标本的特异性活性。我们的初步数据也表明α-Gal减少 α-SYN条件性过表达后神经母细胞瘤细胞的活性。连同我们的报告 α-SYN病理和α-GalA缺陷小鼠脑内碱性磷酸酶标志物的改变，这些发现表明 α-GalA缺乏与α-SYN积累之间的联系。然而，α-GalA缺乏是否会加剧 α-SYN的神经毒性潜力尚不清楚。通过酶替代疗法提高α-GalA活性 (ERT)是临床批准的治疗Fabry病的方法。由于ERT的CNS生物利用度有限，因此存在 在了解其治疗帕金森病的潜力方面存在严重差距。为了帮助弥补这一差距，我们开展了新的研究 提高神经系统中α-GalA活性的工具，包括其在神经细胞中的剂量反应增加 通过ERT，以及α-GalA脑活动增加两倍的转基因小鼠。我们的初步数据在 神经母细胞瘤细胞显示α-GalA ERT增强了过表达的α-SYN的清除。然而， 增加α-GalA活性是否减轻α-SYN相关的神经毒性尚未得到测试。已被占用 总之，我们假设α-SYN相关的神经毒性因α-GalA缺乏而加剧，并且 通过增加α-GalA活性而减弱。在目标1中，我们将确定初级神经元中是否存在α-GalA缺乏 培养加剧由外源添加α-SYN预形成的纤维(PFF)引起的神经毒性 态度伴随着碱性磷酸酶的破坏。我们还将确定α-GalA缺陷小鼠是否表现出恶化 AAV2介导的过度损伤后黑质酪氨酸羟化酶阳性神经元的丢失 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 原代培养的神经元被α-GalA ERT或α-GalA转基因过表达减弱，如果这 保护是由碱性磷酸酶调节的。我们还将确定α-GalA过度表达的小鼠是否表现出减少 AAV2-α-SYN所致的TH阳性神经元丢失。如果我们的假设是正确的，这将表明α-Gal 一种缺陷调节α-SYN的发病机制，这是一个值得进一步研究的机制，并将加速 通过提高中枢神经系统α-GalA活性治疗帕金森病的研究进展。

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.