title: Inhibition of autophagy with bafilomycin and chloroquine decreases mitochondrial quality and bioenergetic function in primary neurons.
abstract: Autophagy is an important cell recycling program responsible for the clearance of damaged or long-lived proteins and organelles. Pharmacological modulators of this pathway have been extensively utilized in a wide range of basic research and pre-clinical studies. Bafilomycin A1 and chloroquine are commonly used compounds that inhibit autophagy by targeting the lysosomes but through distinct mechanisms. Since it is now clear that mitochondrial quality control, particularly in neurons, is dependent on autophagy, it is important to determine whether these compounds modify cellular bioenergetics. To address this, we cultured primary rat cortical neurons from E18 embryos and used the Seahorse XF96 analyzer and a targeted metabolomics approach to measure the effects of bafilomycin A1 and chloroquine on bioenergetics and metabolism. We found that both bafilomycin and chloroquine could significantly increase the autophagosome marker LC3-II and inhibit key parameters of mitochondrial function, and increase mtDNA damage. Furthermore, we observed significant alterations in TCA cycle intermediates, particularly those downstream of citrate synthase and those linked to glutaminolysis. Taken together, these data demonstrate a significant impact of bafilomycin and chloroquine on cellular bioenergetics and metabolism consistent with decreased mitochondrial quality associated with inhibition of autophagy.
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Matthew
authors.initials: M
authors.lastname: Redmann
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Gloria A
authors.initials: GA
authors.lastname: Benavides
authors.affiliation: Targeted Metabolomics & Proteomics Laboratory, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Taylor F
authors.initials: TF
authors.lastname: Berryhill
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Willayat Y
authors.initials: WY
authors.lastname: Wani
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Xiaosen
authors.initials: X
authors.lastname: Ouyang
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Johnson
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Saranya
authors.initials: S
authors.lastname: Ravi
authors.affiliation: Targeted Metabolomics & Proteomics Laboratory, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Stephen
authors.initials: S
authors.lastname: Barnes
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors.forename: Victor M
authors.initials: VM
authors.lastname: Darley-Usmar
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States; VA Medical Center, University of Alabama at Birmingham, Birmingham, AL 35294, United States. Electronic address: jianhuazhang@uabmc.edu.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001343:Autophagy / Q000235:genetics*; D002738:Chloroquine / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects; D004272:DNA, Mitochondrial / Q000187:drug effects; D004734:Energy Metabolism / Q000235:genetics; D008247:Lysosomes / Q000187:drug effects / Q000235:genetics; D018942:Macrolides / Q000494:pharmacology; D055432:Metabolomics / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D051381:Rats
chemical_list: D002738:Chloroquine; D004272:DNA, Mitochondrial; D018942:Macrolides; D008869:Microtubule-Associated Proteins; C526109:Anxa3 protein, rat; C040929:bafilomycin A1