Alleviation of a polyglucosan storage disorder by enhancement of autophagic glycogen catabolism (2021)
Figure 6

Figure 6. LAMP1-KD and 144DG11 enhance autophagic flux.

(A) Left panel, Autophagic flux, determined by the extent of lysosomal inhibitors-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II/LC3I), is increased by 144DG11 (50µM, 24h). Right panel, 144DG11-mediated increase in autophagic flux, demonstrated by enhanced degradation of the autophagy substrate p62. Shown are representative immunoblots (grouped from different gels) and densitometric quantifications +/-s.d. (for LC3II/LC3I, n=4 biological replicates, *p<0.0023; for p62, n=5 biological replicates, *p<0.0099; two-tailed t-test).

(B) Left panel, TEM images of liver tissue from vehicle or 144DG11-treated Gbeys/ys mice. High-magnification (right, scale bars=200 nm) and low-magnification (left, scale bars=1 µm) images show higher levels of glycogen/polyglucosan in lysosomes and cytosol, respectively. Right panel, quantification (+/- s.d) of lysosomal glycogen particles (n=3 biological replicates, *p<0.03, two-tailed t-test). G, Glycogen/polyglucosan; L, Lysosomes; M, Mitochondria.

(C) 144DG11 reduces LC3 and p62 in mouse liver, but not muscle (n=3 biological replicates, *p<0.04 for LC3, **p<0.0007 for p62; two-tailed t-test, SEM). Scale bars, 10 µm

(D) LAMP1-knockdown increases autophagic flux, which is further facilitated by 144DG11 (see densitometric quantification +/-SEM (n=3 biological replicates, *, p<0.1; **, p<0.05; ***, p<0.01 (two-tailed t-tests)). Immunoblots show LAMP1, LC3II/I, and actin in LAMP1 knocked-down and control APBD fibroblasts treated or not with 144DG11 and lysosomal inhibitors (LI).

(E) LAMP1-KD and 144DG11 treatment cause lysosomal acidification. Upper panel, flow cytometry results showing that 144DG11 slightly increased acidification (yellow to blue median fluorescence ratio (Y/B)) in control, GFP-transduced, APBD fibroblasts (Y/B(GFP/144DG11)>Y/B(GFP), p<0.12), but significantly acidified LAMP1-KD, GFP-shLAMP1-transduced, APBD fibroblasts (Y/B(LAMP1-KD/144DG11)>(Y/B(LAMP1-KD), p<0.03). LAMP1-KD itself led to the most significant acidification (Y/B (LAMP1-KD)>Y/B(GFP), p<0.007). n=3, two-tailed t-tests. Middle panel Lysosensor staining of the corresponding cells. Yellow fluorescence intensity correlates with acidification. Lower panel, PAS (glycogen) staining of the corresponding cells. Scale bars, 50 µm.

(F) ATP production rates (Figure 4B) +/- SEM in serum-starved (-S) and non-starved (+S) LAMP1-KD and GFP (Control) cells, untreated (UT), or treated for 24 h (CHR, chronic) or on assay (acute) with 144DG11. See text (144DG11 enhances LAMP1 knockdown-induced autolysosomal degradation and catabolism of glycogen, third paragraph) for results of statistical analysis (multiple t-tests, One Way ANOVA with Sidak's post-hoc corrections, n=3 biological replicates).

(G) 144DG11 reduces lysosomal (LAMP1 positive) area in liver, but not muscle, of Gbeys/ys mice (n=3 biological replicates, *p<0.01, two-tailed t-test , SEM).Scale bars, 5 µm for liver (upper left panel), and 10 µm for muscle (lower left panel). Right panel shows quantification of the left panel.