BACKGROUND Haploinsufficiency in the Gaucher disease GBA gene, which encodes the lysosomal glucocerebrosidase GBA, and ageing represent major risk factors for developing Parkinson's disease (PD). Recently, more than fifty other lysosomal storage disorder gene variants have been identified in PD, implicating lysosomal dysfunction more broadly as a key risk factor for PD. Despite the evidence of multiple lysosomal genetic risks, it remains unclear how sphingolipid hydrolase activities, other than GBA, are altered with ageing or in PD. Moreover, it is not fully known if levels of glycosphingolipid substrates for these enzymes change in vulnerable brain regions of PD. Finally, little is known about the levels of complex gangliosides in substantia nigra which may play a significant role in ageing and PD. METHODS To study sphingolipid hydrolase activities and glycosphingolipid expression in ageing and in PD, two independent cohorts of human substantia nigra tissues were obtained. Fluorescent 4-methylumbelliferone assays were used to determine multiple enzyme activities. The lysosomal GBA and non-lysosomal GBA2 activities were distinguished using the inhibitor NB-DGJ. Sensitive and quantitative normal-phase HPLC was performed to study glycosphingolipid levels. In addition, glycosphingolipid levels in cerebrospinal fluid and serum were analysed as possible biomarkers for PD. RESULTS The present study demonstrates, in two independent cohorts of human post-mortem substantia nigra, that sporadic PD is associated with deficiencies in multiple lysosomal hydrolases (e.g. α-galactosidase and β-hexosaminidase), in addition to reduced GBA and GBA2 activities and concomitant glycosphingolipid substrate accumulation. Furthermore, the data show significant reductions in levels of complex gangliosides (e.g. GM1a) in substantia nigra, CSF and serum in ageing, PD, and REM sleep behaviour disorder, which is a strong predictor of PD. CONCLUSIONS These findings conclusively demonstrate reductions in GBA activity in the parkinsonian midbrain, and for the first time, reductions in the activity of several other sphingolipid hydrolases. Furthermore, significant reductions were seen in complex gangliosides in PD and ageing. The diminished activities of these lysosomal hydrolases, the glycosphingolipid substrate accumulation, and the reduced levels of complex gangliosides are likely major contributors to the primary development of the pathology seen in PD and related disorders with age.

中文翻译：

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帕金森病中鞘脂水解酶活性降低、底物积累和神经节苷脂下降。

背景技术编码溶酶体葡糖脑苷脂酶GBA的戈谢病GBA基因的单倍体不足和衰老代表了发展为帕金森病(PD)的主要风险因素。最近，在 PD 中发现了超过 50 种其他溶酶体贮积症基因变异，这表明溶酶体功能障碍更广泛地是 PD 的关键风险因素。尽管存在多种溶酶体遗传风险的证据，但仍不清楚除 GBA 之外的鞘脂水解酶活性如何随着衰老或 PD 发生变化。此外，尚不完全清楚这些酶的鞘糖脂底物水平是否会在 PD 的脆弱大脑区域发生变化。最后，关于黑质中可能在衰老和 PD 中起重要作用的复杂神经节苷脂的水平知之甚少。方法 为了研究衰老和 PD 中的鞘脂水解酶活性和鞘糖脂表达，获得了两个独立的人类黑质组织队列。荧光 4-甲基伞形酮测定用于确定多种酶活性。使用抑制剂 NB-DGJ 区分溶酶体 GBA 和非溶酶体 GBA2 活性。进行灵敏和定量的正相 HPLC 以研究鞘糖脂水平。此外，脑脊液和血清中的鞘糖脂水平被分析为 PD 的可能生物标志物。结果 本研究表明，在两个独立的人类死后黑质队列中，散发性 PD 与多种溶酶体水解酶（例如 α-半乳糖苷酶和 β-氨基己糖苷酶）的缺乏有关，除了减少 GBA 和 GBA2 活性以及伴随的鞘糖脂底物积累。此外，数据显示，在衰老、PD 和 REM 睡眠行为障碍中，黑质、CSF 和血清中的复杂神经节苷脂（例如 GM1a）水平显着降低，这是 PD 的强预测因子。结论 这些发现最终证明了帕金森病中脑 GBA 活性的降低，并且首次证明了其他几种鞘脂水解酶的活性降低。此外，在 PD 和衰老中的复杂神经节苷脂显着减少。这些溶酶体水解酶的活性降低，鞘糖脂底物积累，