Autophagy is disrupted in a knock-in mouse model of juvenile neuronal ceroid lipofuscinosis
Autophagy is disrupted in a knock-in mouse model of juvenile neuronal ceroid lipofuscinosis
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DOI：
10.1074/jbc.m602180200
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发表时间：
2006-07-21
影响因子：
4.8
通讯作者：
Cotman, Susan L.
中科院分区：
文献类型：
--
作者：
Cao, Yi;Espinola, Janice A.;Cotman, Susan L.
幼年神经性蜡样脂褐质病是由CLN3编码的一种新型内体/溶酶体膜蛋白突变引起的。观察到线粒体atp酶亚基c蛋白在这种疾病中积累，表明自噬（一种调节线粒体更新的途径）可能被破坏。为了验证这一假设，我们检测了Cln3（Delta ex7/8）敲入小鼠和CbCln3（Delta ex7/8）小脑细胞的自噬途径，这是幼年神经元类脂褐质病的准确遗传模型。在纯合子敲入小鼠中，我们发现自噬标志物LC3-II升高，哺乳动物雷帕霉素靶蛋白下调。此外，纯合子敲入小鼠分离的自噬液泡和溶酶体的超微结构形态比野生型细胞器更不成熟，亚基c在自噬液泡中积累。有趣的是，我们还在正常衰老小鼠的自噬液泡中观察到亚单位c的积累。在进一步研究纯合子敲入小脑细胞的自噬途径后，我们发现，与野生型细胞相比，自噬刺激时lc3阳性囊泡发生改变，内吞和溶酶体染料的重叠减少。然而，令人惊讶的是，刺激自噬不会显著影响细胞存活，而抑制自噬会导致细胞死亡。综上所述，这些观察结果表明，自噬在幼年神经蜡样脂褐质病中被破坏，可能在自噬液泡成熟的水平上，自噬的激活可能是疾病过程中促进生存的反馈反应。
Juvenile neuronal ceroid lipofuscinosis is caused by mutation of a novel, endosomal/lysosomal membrane protein encoded by CLN3. The observation that the mitochondrial ATPase subunit c protein accumulates in this disease suggests that autophagy, a pathway that regulates mitochondrial turnover, may be disrupted. To test this hypothesis, we examined the autophagic pathway in Cln3(Delta ex7/8) knock-in mice and CbCln3(Delta ex7/8) cerebellar cells, accurate genetic models of juvenile neuronal ceroid lipofuscinosis. In homozygous knock-in mice, we found that the autophagy marker LC3-II was increased, and mammalian target of rapamycin was down-regulated. Moreover, isolated autophagic vacuoles and lysosomes from homozygous knock-in mice were less mature in their ultrastructural morphology than the wild-type organelles, and subunit c accumulated in autophagic vacuoles. Intriguingly, we also observed subunit c accumulation in autophagic vacuoles in normal aging mice. Upon further investigation of the autophagic pathway in homozygous knock-in cerebellar cells, we found that LC3-positive vesicles were altered and overlap of endocytic and lysosomal dyes was reduced when autophagy was stimulated, compared with wildtype cells. Surprisingly, however, stimulation of autophagy did not significantly impact cell survival, but inhibition of autophagy led to cell death. Together these observations suggest that autophagy is disrupted in juvenile neuronal ceroid lipofuscinosis, likely at the level of autophagic vacuolar maturation, and that activation of autophagy may be a prosurvival feedback response in the disease process.