Background/Purpose:  Our recent data indicates that Snapin, a SNAP associated protein, is critical for maintaining healthy autophagy and monocytes to macrophage (MFs) differentiation which requires functional autophagy. Snapin and autophagosomes are increased in MFs from the joints of patients with rheumatoid arthritis.  Reduction of Snapin hindered the maturation of autophagosomes, resulting in autolysosome accumulation and delayed bacterial clearance in macrophages. Failure to digest the sequestered cargos in these vacuoles might have resulted from deficient capacity of lysosomal hydrolysis. Here, we examined cathepsin D activity, a major hydrolase in lysosomes, in macrophages following the forced reduction of Snapin.

Methods:   The reduction of Snapin in primary human MFs was performed using siRNA, while in the J774A murine MF cell line it was reduced by infection with a lentiviral vector expressing Snapin shRNA. Cell fractions enriched with lysosomes were isolated using a density gradient separation method. The protein levels of non-active pro-form and active cathepsin D were detected by Western blot analysis. The active cathepsin D in cells was also detected by staining with bodipy-pepstatin A which binds to catalytic portion of cathepsin D.  Autophagosomes were identified by the accumulation of LC3 punctae. Acidification of autophagosomes was assessed by transfection of tandem fluorescent LC3 plasmid expressing a tandem red (not pH dependent) and green (quenched at low pH) fluorescence-tagged LC3. Results:  Following the reduction of Snapin, bodipy-pepstatin A staining was greatly reduced in lysosomes in primary human MFs, suggesting a reduction of active cathepsin D.  The forced reduction of Snapin in MFs resulted in 30% reduction in the active form of cathepsin D, identified by immunoblot analysis, in whole cell lysates, compared to control MFs, while the pro-form of cathepsin D was slightly increased. Cathepsin D extracted from lysosome enriched fractions from Snapin reduced J774A MF cells showed a 40% reduction in the active form, but no reduction of the pro-form, indicating the cathepsin D activation was disturbed, rather than the delivery of hydrolases to lysosomes. Interestingly, cathepsin B activation was unchanged in both whole cell lysates and in lysosome enriched fractions.  Cathepsin D is activated in a low pH environment. Disrupting the acidification process in lysosomes or in autophagosomes will reduce cathepsin D activation. The forced reduction of Snapin in HEK293 cells increased green LC3 punctae, indicative of an increased pH in the autophagosomes/autolysosomes. 

Conclusion: Snapin in macrophages is necessary for cathepsin D activation in lysosomes and autophagosomes, preventing the maturation of autolysosomes, by limiting the degradation of cargo within the autophagosomes.  Snapin may contribute to the pathogenesis of rheumatoid arthritis by maintaining healthy autophagy and monocyte to MF differentiation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/snapin-is-critical-for-cathepsin-d-activation-and-the-normal-lysosomal-function/