title: SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages.
abstract: We previously observed that SNAPIN, which is an adaptor protein in the SNARE core complex, was highly expressed in rheumatoid arthritis synovial tissue macrophages, but its role in macrophages and autoimmunity is unknown. To identify SNAPIN's role in these cells, we employed siRNA to silence the expression of SNAPIN in primary human macrophages. Silencing SNAPIN resulted in swollen lysosomes with impaired CTSD (cathepsin D) activation, although total CTSD was not reduced. Neither endosome cargo delivery nor lysosomal fusion with endosomes or autophagosomes was inhibited following the forced silencing of SNAPIN. The acidification of lysosomes and accumulation of autolysosomes in SNAPIN-silenced cells was inhibited, resulting in incomplete lysosomal hydrolysis and impaired macroautophagy/autophagy flux. Mechanistic studies employing ratiometric color fluorescence on living cells demonstrated that the reduction of SNAPIN resulted in a modest reduction of H+pump activity; however, the more critical mechanism was a lysosomal proton leak. Overall, our results demonstrate that SNAPIN is critical in the maintenance of healthy lysosomes and autophagy through its role in lysosome acidification and autophagosome maturation in macrophages largely through preventing proton leak. These observations suggest an important role for SNAPIN and autophagy in the homeostasis of macrophages, particularly long-lived tissue resident macrophages.
authors.affiliation: a Division of Rheumatology , Feinberg School of Medicine, Northwestern University , Chicago , IL , USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Shi
authors.affiliation: a Division of Rheumatology , Feinberg School of Medicine, Northwestern University , Chicago , IL , USA.
authors.forename: Qi-Quan
authors.initials: QQ
authors.lastname: Huang
authors.affiliation: a Division of Rheumatology , Feinberg School of Medicine, Northwestern University , Chicago , IL , USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Birkett
authors.affiliation: a Division of Rheumatology , Feinberg School of Medicine, Northwestern University , Chicago , IL , USA.
authors.forename: Renee
authors.initials: R
authors.lastname: Doyle
authors.affiliation: a Division of Rheumatology , Feinberg School of Medicine, Northwestern University , Chicago , IL , USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Dorfleutner
authors.affiliation: a Division of Rheumatology , Feinberg School of Medicine, Northwestern University , Chicago , IL , USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Stehlik
authors.affiliation: b Department of Cell and Molecular Biology , Feinberg School of Medicine, Northwestern University , Chicago , IL , USA.
authors.forename: Congcong
authors.initials: C
authors.lastname: He
authors.affiliation: a Division of Rheumatology , Feinberg School of Medicine, Northwestern University , Chicago , IL , USA.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Pope
mesh_terms: D000143:Acids / Q000378:metabolism*; D000071182:Autophagosomes / Q000378:metabolism* / Q000648:ultrastructure; D001343:Autophagy; D002402:Cathepsin D / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure; D004789:Enzyme Activation; D020868:Gene Silencing; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D008264:Macrophages / Q000378:metabolism* / Q000648:ultrastructure; D008561:Membrane Fusion; D011522:Protons; D034741:RNA, Small Interfering / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism / Q000648:ultrastructure; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D000143:Acids; D011522:Protons; D034741:RNA, Small Interfering; C118519:SNAPIN protein, human; D033921:Vesicular Transport Proteins; D002402:Cathepsin D
keywords: CTSD; LC3; SNAPIN; acidification; autophagy; endosome; lysosome; macrophage; proton leak; vacuolar-type H+-ATPase