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.
Department Pathology, Neuropathology Division, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
Leandra R
LR
Mangieri
Department Pathology, Neuropathology Division, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Birmingham Veterans Administration Medical Center, Birmingham, Alabama, United States of America.
Burton J
BJ
Mader
Department Pathology, Neuropathology Division, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
Cailin E
CE
Thomas
Department Pathology, Neuropathology Division, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
Charles A
CA
Taylor
Department Pathology, Neuropathology Division, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
Austin M
AM
Luker
Department Pathology, Neuropathology Division, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
Tonia E
TE
Tse
Department Ophthalmology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
Carrie
C
Huisingh
Department Pathology, Neuropathology Division, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Department Ophthalmology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
John J
JJ
Shacka
D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D001343:Autophagy / Q000502:physiology*; D045744:Cell Line, Tumor; D006801:Humans; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism / Q000502:physiology*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism* / Q000503:physiopathology; D019636:Neurodegenerative Diseases / Q000378:metabolism* / Q000503:physiopathology; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*; D051844:alpha-Synuclein / Q000378:metabolism; D052118:Lysosomal-Associated Membrane Protein 1
D016564:Amyloid beta-Protein Precursor; D051907:Lysosomal Membrane Proteins; D008869:Microtubule-Associated Proteins; D025262:Vacuolar Proton-Translocating ATPases; D051844:alpha-Synuclein; C545487:APP protein, human; C541389:ATP6V0C protein, human; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C477012:MAP1LC3A protein, human