Abstract 550: TFEB Expression, Turnover, and Nuclear Localization are Altered in DBA/2 Mouse Macrophages Associated With Impaired Autolysosome Formation and Lipid Droplet Clearance

Abstract

In a previous study, we identified autolysosome formation as the limiting step for turnover of cholesterol esters in lipid droplets of macrophage foam cells from the atherosclerosis sensitive DBA/2 strain compared to the atherosclerosis resistant AKR mouse strain. As autophagosome formation was similar in these two strains, we wanted to evaluate the role of lysosomes in autolysosome formation in AKR and DBA/2 cells. For all our studies, we cultured AKR and DBA/2 bone marrow-derived macrophages with or without acetylated LDL (AcLDL) for 24h. Lysosome function and number, assessed by flow cytometry on Lysotracker red DND-99 (LyT) labeled cells, was not affected by AcLDL loading in AKR and DBA/2 cells. However, in both unloaded and loaded conditions, DBA/2 cells exhibited a 30 to 50% lower LyT intensity suggesting that they have intrinsically decreased lysosome number/function. Lysosomal degradation capacity was assayed by incubation with DQ-ovalbumin and we observed a 27% decrease in lysosome function in DBA/2 vs. AKR foam cells (p<0.01). The impairment of lysosomal degradation was confirmed by 44.8% decreased levels of cathepsin L mature form in DBA/2 vs. AKR foam cells (p<0.05). As the transcription factor TFEB is a key regulator for lysosome biogenesis and function, we studied this factor in our system. First, we analyzed TFEB protein expression by western blot. Upon cholesterol loading, TFEB was induced in AKR (48%, p<0.01) but not DBA/2 cells leading to a 45% higher TFEB levels in AKR vs. DBA/2 foam cells (p<0.05). A preliminary evaluation of TFEB turnover by western blot also revealed that, upon loading, TFEB half-life is twice as long in AKR vs. DBA/2 macrophages (10.1h and 4.8h, respectively). Finally, we investigated TFEB nuclear translocation by immunofluorescence. AKR and DBA/2 macrophages were labeled with an antibody against TFEB and the percentage of TFEB positive nuclei was assessed. After 24h AcLDL loading, only 26% of DBA/2 nuclei contained TFEB vs. 48% for AKR nuclei (p=0.0002 by Fisher’s exact test). In conclusion, we found that DBA/2 vs. AKR foam cells have altered TFEB processing that may explain the altered lysosome number and function. This may result in the autolysosome formation defect in the DBA/2 foam cells.