ATP6V0C knockdown in neuroblastoma cells alters autophagy-lysosome pathway function and metabolism of proteins that accumulate in neurodegenerative disease.

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.

Low-dose bafilomycin attenuates neuronal cell death associated with autophagy-lysosome pathway dysfunction.

We have shown previously that the plecomacrolide antibiotics bafilomycin A1 and B1 significantly attenuate cerebellar granule neuron death resulting from agents that disrupt lysosome function. To further characterize bafilomycin-mediated cytoprotection, we examined its ability to attenuate the death of naïve and differentiated neuronal SH-SY5Y human neuroblastoma cells from agents that induce lysosome dysfunction in vitro, and from in vivo dopaminergic neuron death in C. elegans. Low-dose bafilomycin significantly attenuated SH-SY5Y cell death resulting from treatment with chloroquine, hydroxychloroquine amodiaquine and staurosporine. Bafilomycin also attenuated the chloroquine-induced reduction in processing of cathepsin D, the principal lysosomal aspartic acid protease, to its mature 'active' form. Chloroquine induced autophagic vacuole accumulation and inhibited autophagic flux, effects that were attenuated upon treatment with bafilomycin and were associated with a significant decrease in chloroquine-induced accumulation of detergent-insoluble alpha-synuclein oligomers. In addition, bafilomycin significantly and dose-dependently attenuated dopaminergic neuron death in C. elegans resulting from in vivo over-expression of human wild-type alpha-synuclein. Together, our findings suggest that low-dose bafilomycin is cytoprotective in part through its maintenance of the autophagy-lysosome pathway, and underscores its therapeutic potential for treating Parkinson's disease and other neurodegenerative diseases that exhibit disruption of protein degradation pathways and accumulation of toxic protein species.