Figure 4.

AAV-Mediated secGAA Expression Restores Histology and Partially Reverses Autophagy Block in Muscle

(A–E) 9-month-old mice were injected with an AAV8-secGAA (secGAA-Gaa^−/−) vector at a dose of 2 × 10¹² vg/kg. Gaa^+/+ (PBS-Gaa^+/+) and Gaa^−/− (PBS-Gaa^−/−) mice injected with PBS served as controls in the study. Animals were followed for 9 months after treatment (n = 3/4 per cohort). (A) Representative images of hematoxylin and eosin (H&E) staining of diaphragm, heart, and triceps. Scale bar, 10 μm. (B) Western blot analysis of triceps lysates using anti-p62/sequestosome-1 (SQSTM1) monoclonal antibody. An anti-GAPDH antibody was used as a loading control. (C) Western blot analysis of triceps lysates using anti-Lamp1 polyclonal and anti-Beclin1 polyclonal antibodies. An anti-GAPDH antibody was used as a loading control. (D) Western blot analysis of triceps lysates using anti-LC3b monoclonal antibody. An anti-GAPDH antibody was used as a loading control. (E) Quantification of p62 levels normalized to GAPDH levels. (F) Quantification of Lamp1 levels normalized to GAPDH levels. (G) Quantification of Beclin1 levels normalized to GAPDH levels. (H) Quantification of LC3b-I levels normalized to GAPDH levels. (I) Electron microscopy analysis of tibialis anterior. Arrows indicate enlarged lysosomes. Scale bars, 2 μm. (J) Quantification of lysosome length from the sections in electron microscopy. Statistical analyses: (E–H) one-way ANOVA with Tukey’s post hoc. ∗p < 0.05; ∗∗p < 0.01. (J) unpaired t test. ∗p < 0.0001. In all graphs, error bars represent the standard deviation of the mean.