Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, Royal Free Campus, London NW3 2PF, UK.
Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA.
Hum Mol Genet. 2023 Feb 19;32(5):773-789. doi: 10.1093/hmg/ddac233.
Sequence variants or mutations in the GBA gene are numerically the most important risk factor for Parkinson disease (PD). The GBA gene encodes for the lysosomal hydrolase enzyme, glucocerebrosidase (GCase). GBA mutations often reduce GCase activity and lead to the impairment of the autophagy-lysosomal pathway, which is important in the turnover of alpha-synuclein, accumulation of which is a key pathological hallmark of PD. Although the E326K variant is one of the most common GBA variants associated with PD, there is limited understanding of its biochemical effects. We have characterized homozygous and heterozygous E326K variants in human fibroblasts. We found that E326K variants did not cause a significant loss of GCase protein or activity, endoplasmic reticulum (ER) retention or ER stress, in contrast to the L444P GBA mutation. This was confirmed in human dopaminergic SH-SY5Y neuroblastoma cell lines overexpressing GCase with either E326K or L444P protein. Despite no loss of the GCase activity, a significant increase in insoluble alpha-synuclein aggregates in E326K and L444P mutants was observed. Notably, SH-SY5Y overexpressing E326K demonstrated a significant increase in the lipid droplet number under basal conditions, which was exacerbated following treatment with the fatty acid oleic acid. Similarly, a significant increase in lipid droplet formation following lipid loading was observed in heterozygous and homozygous E326K fibroblasts. In conclusion, the work presented here demonstrates that the E326K mutation behaves differently to the common loss of function GBA mutations; however, lipid dyshomeostasis and alpha-synuclein pathology are still evident.
GBA 基因中的序列变异或突变是帕金森病 (PD) 的最重要风险因素。GBA 基因编码溶酶体水解酶，葡萄糖脑苷脂酶 (GCase)。GBA 突变通常会降低 GCase 活性，并导致自噬溶酶体途径受损，该途径对α-突触核蛋白的周转很重要，其积累是 PD 的关键病理标志。尽管 E326K 变体是与 PD 相关的最常见 GBA 变体之一，但对其生化作用的了解有限。我们已经对人类成纤维细胞中的纯合和杂合 E326K 变体进行了特征描述。我们发现，与 L444P GBA 突变相反，E326K 变体不会导致 GCase 蛋白或活性、内质网 (ER) 保留或 ER 应激的显著丧失。这在过表达 GCase 的人类多巴胺能 SH-SY5Y 神经母细胞瘤细胞系中得到了证实，这些细胞系中表达的 E326K 或 L444P 蛋白。尽管 GCase 活性没有丧失，但在 E326K 和 L444P 突变体中观察到不溶性α-突触核蛋白聚集体的显著增加。值得注意的是，在基础条件下，过表达 E326K 的 SH-SY5Y 细胞的脂滴数量显著增加，在用脂肪酸油酸处理后加剧。同样，在杂合和纯合 E326K 成纤维细胞中，在脂质负荷后观察到脂质滴形成的显著增加。总之，这里介绍的工作表明，E326K 突变的行为与常见的功能丧失 GBA 突变不同；然而，脂质代谢失衡和α-突触核蛋白病理学仍然明显。
