Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Department of Neurology, Baltimore, MD, USA.
Mol Neurodegener. 2018 Jan 8;13(1):1. doi: 10.1186/s13024-017-0233-5.
Mutations in glucocerebrosidase (GBA) cause Gaucher disease (GD) and increase the risk of developing Parkinson's disease (PD) and Dementia with Lewy Bodies (DLB). Since both genetic and environmental factors contribute to the pathogenesis of sporadic PD, we investigated the susceptibility of nigrostriatal dopamine (DA) neurons in L444P GBA heterozygous knock-in (GBA ) mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a selective dopaminergic mitochondrial neurotoxin.
We used GBA mice, α-synuclein knockout (SNCA ) mice at 8 months of age, and adeno-associated virus (AAV)-human GBA overexpression to investigate the rescue effect of DA neuronal loss and susceptibility by MPTP. Mitochondrial morphology and functional assay were used to identify mitochondrial defects in GBA mice. Motor behavioral test, immunohistochemistry, and HPLC were performed to measure dopaminergic degeneration by MPTP and investigate the relationship between GBA mutation and α-synuclein. Mitochondrial immunostaining, qPCR, and Western blot were also used to study the effects of α-synuclein knockout or GBA overexpression on MPTP-induced mitochondrial defects and susceptibility.
L444P GBA heterozygous mutation reduced GBA protein levels, enzymatic activity and a concomitant accumulation of α-synuclein in the midbrain of GBA mice. Furthermore, the deficiency resulted in defects in mitochondria of cortical neurons cultured from GBA mice. Notably, treatment with MPTP resulted in a significant loss of dopaminergic neurons and striatal dopaminergic fibers in GBA mice compared to wild type (WT) mice. Levels of striatal DA and its metabolites were more depleted in the striatum of GBA mice. Behavioral deficits, neuroinflammation, and mitochondrial defects were more exacerbated in GBA mice after MPTP treatment. Importantly, MPTP induced PD-like symptoms were significantly improved by knockout of α-synuclein or augmentation of GBA via AAV5-hGBA injection in both WT and GBA mice. Intriguingly, the degree of reduction in MPTP induced PD-like symptoms in GBA α-synuclein (SNCA) mice was nearly equal to that in SNCA mice after MPTP treatment.
Our results suggest that GBA deficiency due to L444P GBA heterozygous mutation and the accompanying accumulation of α-synuclein render DA neurons more susceptible to MPTP intoxication. Thus, GBA and α-synuclein play dual physiological roles in the survival of DA neurons in response to the mitochondrial dopaminergic neurotoxin, MPTP.
葡萄糖脑苷脂酶 (GBA) 的突变导致戈谢病 (GD)，并增加帕金森病 (PD) 和路易体痴呆 (DLB) 的发病风险。由于遗传和环境因素都有助于散发性 PD 的发病机制，我们研究了 L444P GBA 杂合子敲入 (GBA) 小鼠黑质纹状体多巴胺 (DA) 神经元对 1-甲基-4-苯基-1,2,3,6-四氢吡啶 (MPTP) 的易感性，MPTP 是一种选择性的多巴胺能线粒体神经毒素。
我们使用 GBA 小鼠、α-突触核蛋白敲除 (SNCA) 小鼠 8 个月大以及腺相关病毒 (AAV)-人 GBA 过表达来研究 DA 神经元丢失和 MPTP 易感性的挽救作用。使用线粒体形态和功能测定来鉴定 GBA 小鼠中的线粒体缺陷。进行运动行为测试、免疫组织化学和 HPLC 来测量 MPTP 引起的多巴胺能退行性变，并研究 GBA 突变与 α-突触核蛋白之间的关系。还使用线粒体免疫染色、qPCR 和 Western blot 来研究 α-突触核蛋白敲除或 GBA 过表达对 MPTP 诱导的线粒体缺陷和易感性的影响。
L444P GBA 杂合突变降低了 GBA 蛋白水平、酶活性以及 GBA 小鼠中脑的 α-突触核蛋白的积累。此外，该缺陷导致 GBA 小鼠皮质神经元培养物中线粒体的缺陷。值得注意的是，与野生型 (WT) 小鼠相比，MPTP 处理导致 GBA 小鼠中多巴胺能神经元和纹状体多巴胺能纤维明显丢失。GBA 小鼠纹状体中的多巴胺及其代谢物水平消耗更多。在 GBA 小鼠中，MPTP 处理后行为缺陷、神经炎症和线粒体缺陷更为加剧。重要的是，通过 AAV5-hGBA 注射在 WT 和 GBA 小鼠中敲除 α-突触核蛋白或增强 GBA 可显著改善 MPTP 诱导的 PD 样症状。有趣的是，在 GBA 小鼠中，MPTP 诱导的 PD 样症状的减轻程度几乎与 SNCA 小鼠在 MPTP 处理后相同。
我们的结果表明，由于 L444P GBA 杂合突变导致的 GBA 缺陷和随之而来的 α-突触核蛋白的积累使 DA 神经元更容易受到 MPTP 中毒的影响。因此，GBA 和 α-突触核蛋白在 DA 神经元对线粒体多巴胺能神经毒素 MPTP 的生存中发挥双重生理作用。
