From the Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada and.
the Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University Graz, 8010 Graz, Austria.
J Biol Chem. 2014 Jun 6;289(23):16278-89. doi: 10.1074/jbc.M114.559914. Epub 2014 Apr 30.
Niemann-Pick type C1 (NPC1) is a late endosomal transmembrane protein, which, together with NPC2 in the endosome lumen, mediates the transport of endosomal cholesterol to the plasma membrane and endoplasmic reticulum. Loss of function of NPC1 or NPC2 leads to cholesterol accumulation in late endosomes and causes neuronal dysfunction and neurodegeneration. Recent studies indicate that cholesterol also accumulates in mitochondria of NPC1-deficient cells and brain tissue and that NPC1 deficiency leads to alterations in mitochondrial function and energy metabolism. Here, we have investigated the effects of increased mitochondrial cholesterol levels on energy metabolism, using RNA interference to deplete Chinese hamster ovary cells of NPC1 alone or in combination with MLN64, which mediates endosomal cholesterol transport to mitochondria. Mitochondrial cholesterol levels were also altered by depletion of NPC2 in combination with the expression of NPC2 mutants. We found that the depletion of NPC1 increased lactate secretion, decreased glutamine-dependent mitochondrial respiration, and decreased ATP transport across mitochondrial membranes. These metabolic alterations did not occur when transport of endosomal cholesterol to mitochondria was blocked. In addition, the elevated mitochondrial cholesterol levels in NPC1-depleted cells and in NPC2-depleted cells expressing mutant NPC2 that allows endosomal cholesterol trafficking to mitochondria were associated with increased expression of the antioxidant response factor Nrf2. Antioxidant treatment not only prevented the increase in Nrf2 mRNA levels but also prevented the increased lactate secretion in NPC1-depleted cells. These results suggest that mitochondrial cholesterol accumulation can increase oxidative stress and in turn cause increased glycolysis to lactate and other metabolic alterations.
尼曼-匹克 C1 型（NPC1）是一种晚期内体跨膜蛋白，与内体腔中的 NPC2 一起，介导内体胆固醇向质膜和内质网的转运。NPC1 或 NPC2 的功能丧失导致晚期内体中胆固醇的积累，从而引起神经元功能障碍和神经退行性变。最近的研究表明，胆固醇也在 NPC1 缺陷细胞和脑组织的线粒体中积累，并且 NPC1 缺陷导致线粒体功能和能量代谢的改变。在这里，我们通过 RNA 干扰单独或与介导内体胆固醇向线粒体运输的 MLN64 一起耗尽中国仓鼠卵巢细胞中的 NPC1，研究了增加线粒体胆固醇水平对能量代谢的影响。还通过耗尽 NPC2 与表达 NPC2 突变体相结合来改变线粒体胆固醇水平。我们发现，NPC1 的耗竭增加了乳酸盐的分泌，减少了谷氨酰胺依赖性线粒体呼吸，并减少了线粒体膜之间的 ATP 转运。当阻断内体胆固醇向线粒体的运输时，这些代谢变化不会发生。此外，NPC1 耗尽细胞和表达允许内体胆固醇向线粒体运输的 NPC2 突变体的 NPC2 耗尽细胞中升高的线粒体胆固醇水平与抗氧化反应因子 Nrf2 的表达增加有关。抗氧化剂治疗不仅可以防止 Nrf2 mRNA 水平的增加，还可以防止 NPC1 耗尽细胞中乳酸盐分泌的增加。这些结果表明，线粒体胆固醇的积累会增加氧化应激，进而导致糖酵解增加为乳酸盐和其他代谢变化。
