School of Pharmacy, De Montfort University, The Gateway, Leicester LE1 9BH, UK.
European Molecular Biology Laboratory, Myerhofstraße 1, 69117 Heidelberg, Germany.
Neurobiol Dis. 2019 Jul;127:242-252. doi: 10.1016/j.nbd.2019.03.005. Epub 2019 Mar 12.
Niemann-Pick type C disease (NPCD) is a neurodegenerative disease associated with increases in cellular cholesterol and glycolipids and most commonly caused by defective NPC1, a late endosomal protein. Using ratiometric probes we find that NPCD cells show increased endolysosomal pH. In addition U18666A, an inhibitor of NPC1, was found to increase endolysosomal pH, and the number, size and heterogeneity of endolysosomal vesicles. NPCD fibroblasts and cells treated with U18666A also show disrupted targeting of fluorescent lipid BODIPY-LacCer to high pH vesicles. Inhibiting non-lysosomal glucocerebrosidase (GBA2) reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking in NPCD fibroblasts. GBA2 KO cells also show decreased endolysosomal pH. NPCD fibroblasts also show increased expression of a key subunit of the lysosomal proton pump vATPase on GBA2 inhibition. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic-facing GlcCer, which are reduced in NPC disease.
尼曼-匹克 C 型病（NPCD）是一种与细胞胆固醇和糖脂增加相关的神经退行性疾病，最常见的病因是晚期内体蛋白 NPC1 的缺陷。我们使用比率探针发现，NPCD 细胞表现出内溶酶体 pH 值升高。此外，NPC1 的抑制剂 U18666A 被发现增加内溶酶体 pH 值以及内溶酶体囊泡的数量、大小和异质性。NPCD 成纤维细胞和用 U18666A 处理的细胞也显示出荧光脂质 BODIPY-LacCer 向高 pH 囊泡的靶向作用被破坏。抑制非溶酶体β-葡萄糖脑苷脂酶（GBA2）可逆转内溶酶体 pH 值的升高，并恢复 NPCD 成纤维细胞中破坏的 BODIPY-LacCer 转运。GBA2 KO 细胞也显示出内溶酶体 pH 值降低。NPCD 成纤维细胞在 GBA2 抑制时还表现出溶酶体质子泵 vATPase 的关键亚基表达增加。这些结果与以下模型一致：内溶酶体 pH 值和 BODIPY-LacCer 的高尔基体靶向都依赖于细胞溶质面 GlcCer 的适当水平，而 NPC 疾病中 GlcCer 的水平降低。
