Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
Ann Neurol. 2011 Jun;69(6):940-53. doi: 10.1002/ana.22400. Epub 2011 Apr 6.
Heterozygous mutations in the GBA1 gene elevate the risk of Parkinson disease and dementia with Lewy bodies; both disorders are characterized by misprocessing of α-synuclein (SNCA). A loss in lysosomal acid-β-glucosidase enzyme (GCase) activity due to biallelic GBA1 mutations underlies Gaucher disease. We explored mechanisms for the gene's association with increased synucleinopathy risk.
We analyzed the effects of wild-type (WT) and several GBA mutants on SNCA in cellular and in vivo models using biochemical and immunohistochemical protocols.
We observed that overexpression of all GBA mutants examined (N370S, L444P, D409H, D409V, E235A, and E340A) significantly raised human SNCA levels to 121 to 248% of vector control (p < 0.029) in neural MES23.5 and PC12 cells, but without altering GCase activity. Overexpression of WT GBA in neural and HEK293-SNCA cells increased GCase activity, as expected (ie, to 167% in MES-SNCA, 128% in PC12-SNCA, and 233% in HEK293-SNCA; p < 0.002), but had mixed effects on SNCA. Nevertheless, in HEK293-SNCA cells high GCase activity was associated with SNCA reduction by ≤32% (p = 0.009). Inhibition of cellular GCase activity (to 8-20% of WT; p < 0.0017) did not detectably alter SNCA levels. Mutant GBA-induced SNCA accumulation could be pharmacologically reversed in D409V-expressing PC12-SNCA cells by rapamycin, an autophagy-inducer (≤40%; 10μM; p < 0.02). Isofagomine, a GBA chaperone, showed a related trend. In mice expressing two D409Vgba knockin alleles without signs of Gaucher disease (residual GCase activity, ≥20%), we recorded an age-dependent rise of endogenous Snca in hippocampal membranes (125% vs WT at 52 weeks; p = 0.019). In young Gaucher disease mice (V394Lgba+/+//prosaposin[ps]-null//ps-transgene), which demonstrate neurological dysfunction after age 10 weeks (GCase activity, ≤10%), we recorded no significant change in endogenous Snca levels at 12 weeks of age. However, enhanced neuronal ubiquitin signals and axonal spheroid formation were already present. The latter changes were similar to those seen in three week-old cathepsin D-deficient mice.
Our results demonstrate that GBA mutants promote SNCA accumulation in a dose- and time-dependent manner, thereby identifying a biochemical link between GBA1 mutation carrier status and increased synucleinopathy risk. In cell culture models, this gain of toxic function effect can be mitigated by rapamycin. Loss in GCase activity did not immediately raise SNCA concentrations, but first led to neuronal ubiquitinopathy and axonal spheroids, a phenotype shared with other lysosomal storage disorders.
GBA1 基因的杂合突变会增加帕金森病和路易体痴呆的风险；这两种疾病的特征都是α-突触核蛋白（SNCA）的错误处理。由于双等位基因 GBA1 突变导致溶酶体酸性β-葡萄糖苷酶（GCase）活性丧失，导致戈谢病。我们探讨了该基因与增加突触核蛋白病风险相关的机制。
我们使用生化和免疫组织化学方案，在细胞和体内模型中分析了野生型（WT）和几种 GBA 突变体对 SNCA 的影响。
我们观察到，在所检查的所有 GBA 突变体（N370S、L444P、D409H、D409V、E235A 和 E340A）的过表达下，人 SNCA 水平显著升高，在神经 MES23.5 和 PC12 细胞中升高至载体对照的 121%至 248%（p < 0.029），但不改变 GCase 活性。在神经和 HEK293-SNCA 细胞中过表达 WT GBA 会增加 GCase 活性，如预期的那样（即在 MES-SNCA 中增加 167%，在 PC12-SNCA 中增加 128%，在 HEK293-SNCA 中增加 233%；p < 0.002），但对 SNCA 有混合影响。然而，在 HEK293-SNCA 细胞中，高 GCase 活性与 SNCA 减少 32%（p = 0.009）有关。细胞 GCase 活性的抑制（至 WT 的 8-20%；p < 0.0017）并没有明显改变 SNCA 水平。在 D409V 表达的 PC12-SNCA 细胞中，雷帕霉素，一种自噬诱导剂，可以逆转突变体 GBA 诱导的 SNCA 积累（≤40%；10μM；p < 0.02）。GBA 伴侣 Isofagomine 也表现出类似的趋势。在没有戈谢病迹象（残留 GCase 活性，≥20%）的表达两个 D409Vgba 敲入等位基因的小鼠中，我们记录到海马膜中内源性 Snca 随年龄的增长而上升（52 周时为 125%比 WT；p = 0.019）。在年轻的戈谢病小鼠（V394Lgba+/+//prosaposin[ps]-null//ps-transgene）中，在 10 周龄后表现出神经功能障碍（GCase 活性，≤10%），我们在 12 周龄时没有记录到内源性 Snca 水平的显著变化。然而，增强的神经元泛素信号和轴突球体形成已经存在。后一种变化与在 3 周大的组织蛋白酶 D 缺乏型小鼠中看到的变化相似。
我们的结果表明，GBA 突变体以剂量和时间依赖的方式促进 SNCA 积累，从而确定了 GBA1 突变携带者状态和增加的突触核蛋白病风险之间的生化联系。在细胞培养模型中，这种毒性功能获得效应可以通过雷帕霉素来缓解。GCase 活性的丧失不会立即升高 SNCA 浓度，但首先会导致神经元泛素病和轴突球体形成，这是一种与其他溶酶体贮积症共享的表型。
