ATP6V0C knockdown in neuroblastoma cells alters autophagy-lysosome pathway function and metabolism of proteins that accumulate in neurodegenerative disease.
ATP6V0C knockdown in neuroblastoma cells alters autophagy-lysosome pathway function and metabolism of proteins that accumulate in neurodegenerative disease.
复制标题
DOI：
10.1371/journal.pone.0093257
复制
发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Shacka JJ
中科院分区：
文献类型：
--
作者：
Mangieri LR;Mader BJ;Thomas CE;Taylor CA;Luker AM;Tse TE;Huisingh C;Shacka JJ
ATP6V0C是空泡ATPase的巴菲霉素A1结合亚基，是一种关键调节囊泡酸化的酶复合体。我们和其他人之前已经证明，巴菲尔霉素A1调节细胞活力、自噬通量和在神经退行性疾病中积累的蛋白质的代谢。为了确定ATP6V0C在自噬-溶酶体途径功能中的重要性，将分化为神经元表型的SH-SY5Y人神经母细胞瘤细胞用非靶向或ATP6V0C siRNA进行核修饰，复苏后分别用赋形剂或巴菲霉素A1(0.3-100 nM)处理48h，用定量RT-PCR和Lysostracker Red染色显著降低ATP6V0C基因的表达。ATP6V0C基因敲除显著增加微管相关蛋白轻链3-II(Lc3-II)、α-突触核蛋白高分子量物种和APP C末端片段的基础水平，并抑制自噬通量。敲除后增强的Lc3和LAMP-1共定位表明，自噬通量受到抑制的部分原因是溶酶体的降解，而不是囊泡融合的障碍。ATP6V0C基因的敲除也使细胞对自噬底物的积累和用1 NM巴菲霉素A1处理后的轴突长度缩短敏感，该浓度在非靶标对照细胞中不会产生这种变化。用3 NM巴菲霉素A1处理后，轴突长度缩短和碘化丙啶阳性死亡细胞的百分比也显著增加。综上所述，这些结果表明ATP6V0C在维持结构性和应激诱导的ALP功能方面发挥了作用，特别是在与年龄相关的神经退行性疾病中积累的底物的代谢，可能有助于疾病的发病。
ATP6V0C is the bafilomycin A1-binding subunit of vacuolar ATPase, an enzyme complex that critically regulates vesicular acidification. We and others have shown previously that bafilomycin A1 regulates cell viability, autophagic flux and metabolism of proteins that accumulate in neurodegenerative disease. To determine the importance of ATP6V0C for autophagy-lysosome pathway function, SH-SY5Y human neuroblastoma cells differentiated to a neuronal phenotype were nucleofected with non-target or ATP6V0C siRNA and following recovery were treated with either vehicle or bafilomycin A1 (0.3–100 nM) for 48 h. ATP6V0C knockdown was validated by quantitative RT-PCR and by a significant decrease in Lysostracker Red staining. ATP6V0C knockdown significantly increased basal levels of microtubule-associated protein light chain 3-II (LC3-II), α-synuclein high molecular weight species and APP C-terminal fragments, and inhibited autophagic flux. Enhanced LC3 and LAMP-1 co-localization following knockdown suggests that autophagic flux was inhibited in part due to lysosomal degradation and not by a block in vesicular fusion. Knockdown of ATP6V0C also sensitized cells to the accumulation of autophagy substrates and a reduction in neurite length following treatment with 1 nM bafilomycin A1, a concentration that did not produce such alterations in non-target control cells. Reduced neurite length and the percentage of propidium iodide-positive dead cells were also significantly greater following treatment with 3 nM bafilomycin A1. Together these results indicate a role for ATP6V0C in maintaining constitutive and stress-induced ALP function, in particular the metabolism of substrates that accumulate in age-related neurodegenerative disease and may contribute to disease pathogenesis.
登录
查看更多内容
影响因子：
15.1
作者：
Liu QY;Lei JX;Sikorska M;Liu R
通讯作者：
Liu R
影响因子：
2.5
作者：
Byun, Yu Jeong;Lee, Seong-Beom;Jeong, Seong-Whan
通讯作者：
Jeong, Seong-Whan
影响因子：
64.5
作者：
Mazzulli JR;Xu YH;Sun Y;Knight AL;McLean PJ;Caldwell GA;Sidransky E;Grabowski GA;Krainc D
通讯作者：
Krainc D
DOI：
10.1083/jcb.200212004
发表时间：
2003-07-21
期刊：
The Journal of cell biology
影响因子：
--
作者：
Bayer MJ;Reese C;Buhler S;Peters C;Mayer A
通讯作者：
Mayer A
影响因子：
16.2
作者：
Di Giovanni, Jerome;Boudkkazi, Sami;El Far, Oussama
通讯作者：
El Far, Oussama