Figure 5.
SOD1 is crucial for preserving lysosomal function and integrity. (A) NAG assays show that compared with NC 293T cells, NAG activity is significantly decreased in siSOD1 or siTP53INP1 cells. Double KD of SOD1 and TP53INP1 (DKD) does not result in further reduction. Quantification is presented as mean ± SEM (n = 6). ****, P < 0.0001. (B and C) Magic Red assays show that the Magic Red intensity upon starvation is significantly decreased in siSOD1 or siTP53INP1 293T cells (B). Quantification of Magic Red intensity is presented as mean ± SEM (NC, n = 19; siSOD1, n = 18; siTP53INP1, n = 19) (C). ****, P < 0.0001. Bars: 5 μm. (D) NAG assays show that compared with NC 293T cells, NAG activity is significantly reduced in siTP53INP1 cells, and this reduction is rescued by the re-expression of WT RNAi-resistant SNAP-TP53INP1r, but not SNAP-TP53INP1(ΔLIR)r or SNAP-TP53INP1(ΔC)r. Quantification is presented as mean ± SEM (n = 3). ****, P < 0.0001. (E) NAG assays show that compared with WT mice, NAG activity is significantly decreased in brain vesicles from SOD1(G93A) mice. Quantification is presented as mean ± SEM (n = 6). ****, P < 0.0001. (F and G) Confocal images of fed (Ctrl) and starved (Strv) 293T cells treated with 0, 400 and 800 μM LLoMe for 30 min and immunoblotted with CHMP4B antibody (F). Quantification of CHMP4B puncta is presented as mean ± SEM (0 μM Ctrl, n = 20; 0 μM Strv, n = 20; 400 μM Ctrl, n = 20; 400 μM Strv, n = 20; 800 μM Ctrl, n = 20; 800 μM Strv, n = 20) (G). ****, P < 0.0001. Bars: 5 μm. (H and I) Confocal images of starved (Strv) 293T cells treated with 200 μM LLoMe for 30 min and immunoblotted with CHMP4B antibody. Compared with NC cells, CHMP4B-positive puncta are present in siSOD1-treated cells after starvation (H). Quantification of CHMP4B puncta is presented as mean ± SEM (NC, n = 20; siSOD1, n = 20; siTP53INP1, n = 20) (I). ****, P < 0.0001. Bars: 5 μm. LLoMe, L-leucyl-L-leucine methyl ester. (J) The schematic model illustrates how starvation triggers the translocation of cytosolic SOD1 to lysosomes via autophagy, facilitated by the autophagy receptor TP53INP1. Within lysosomes, SOD1 eliminates ROS to preserve the functionality and structural integrity of these organelles. In situations where SOD1 is deficient, elevated ROS levels within lysosomes can disrupt lysosomal homeostasis. IM, isolation membrane; ROS, reactive oxidative species.

SOD1 is crucial for preserving lysosomal function and integrity. (A) NAG assays show that compared with NC 293T cells, NAG activity is significantly decreased in siSOD1 or siTP53INP1 cells. Double KD of SOD1 and TP53INP1 (DKD) does not result in further reduction. Quantification is presented as mean ± SEM (n = 6). ****, P < 0.0001. (B and C) Magic Red assays show that the Magic Red intensity upon starvation is significantly decreased in siSOD1 or siTP53INP1 293T cells (B). Quantification of Magic Red intensity is presented as mean ± SEM (NC, n = 19; siSOD1, n = 18; siTP53INP1, n = 19) (C). ****, P < 0.0001. Bars: 5 μm. (D) NAG assays show that compared with NC 293T cells, NAG activity is significantly reduced in siTP53INP1 cells, and this reduction is rescued by the re-expression of WT RNAi-resistant SNAP-TP53INP1r, but not SNAP-TP53INP1(ΔLIR)r or SNAP-TP53INP1(ΔC)r. Quantification is presented as mean ± SEM (n = 3). ****, P < 0.0001. (E) NAG assays show that compared with WT mice, NAG activity is significantly decreased in brain vesicles from SOD1(G93A) mice. Quantification is presented as mean ± SEM (n = 6). ****, P < 0.0001. (F and G) Confocal images of fed (Ctrl) and starved (Strv) 293T cells treated with 0, 400 and 800 μM LLoMe for 30 min and immunoblotted with CHMP4B antibody (F). Quantification of CHMP4B puncta is presented as mean ± SEM (0 μM Ctrl, n = 20; 0 μM Strv, n = 20; 400 μM Ctrl, n = 20; 400 μM Strv, n = 20; 800 μM Ctrl, n = 20; 800 μM Strv, n = 20) (G). ****, P < 0.0001. Bars: 5 μm. (H and I) Confocal images of starved (Strv) 293T cells treated with 200 μM LLoMe for 30 min and immunoblotted with CHMP4B antibody. Compared with NC cells, CHMP4B-positive puncta are present in siSOD1-treated cells after starvation (H). Quantification of CHMP4B puncta is presented as mean ± SEM (NC, n = 20; siSOD1, n = 20; siTP53INP1, n = 20) (I). ****, P < 0.0001. Bars: 5 μm. LLoMe, L-leucyl-L-leucine methyl ester. (J) The schematic model illustrates how starvation triggers the translocation of cytosolic SOD1 to lysosomes via autophagy, facilitated by the autophagy receptor TP53INP1. Within lysosomes, SOD1 eliminates ROS to preserve the functionality and structural integrity of these organelles. In situations where SOD1 is deficient, elevated ROS levels within lysosomes can disrupt lysosomal homeostasis. IM, isolation membrane; ROS, reactive oxidative species.

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