Fig. 5: Autophagic flux disruption in VMP1^cKO mice.

A IFC staining for p62 in the SNc was performed by using the antibody against p62 (green) together with TH (red). The nuclei were labeled with DAPI (Blue). Scale bar, 250 μm. Scale bar of the high-magnification images, 10 μm. B The proportion of TH⁺ cells with p62 puncta (>1 μm²) was quantified (N = 3 mice per genotype). C IFC staining for LC3 in the SNc was performed by using the antibody against LC3 (Green) together with TH (Red). The nuclei were labeled with DAPI (Blue). Scale bar, 250 μm. Scale bar of the high-magnification images, 10 μm. D The proportion of TH⁺ cells with LC3 puncta (>0.5 μm²) was quantified (N = 3 mice per genotype). E Double-label immunofluorescence of LC3 (green) and LAMP1 (red) in the mDAergic neurons. Scale bar, 10 μm. F The number of LAMP1⁺ puncta (>0.1 μm²) per TH⁺ neuron (N = 3 mice per genotype). G Quantification of the area of LC3⁺ puncta per TH⁺ neuron (N = 3 mice per genotype). H The proportion of LC3⁺ puncta co-localized with LAMP1 was quantified (N = 3 mice per genotype). I WB analysis for p62, LC3, LAMP1 and Beclin1 protein expression levels in the midbrain and striatum. GAPDH as an internal reference. J–M Quantification of p62, LC3II, LAMP1 and Beclin1 relative to GAPDH was shown, respectively (N = 3 mice per genotype). The representative TEM images of the AP (N), Ly (P), as well as AVd and LVSs (R) in cells. Scale bar, 0.5 μm. The quantification of the number of AP per cell section (O), the diameters of Ly (Q), and the number of LVSs per cell section (S) was shown. B, D, F, G, H, O, Q, S were analyzed by using Student’s t-test. J–M were analyzed by using two-way ANOVA followed by Sidak’s multiple comparisons test. Data were represented as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001. mid midbrain, stri striatum, Ly lysosome, AP autophagosome, AVd degradative autophagic vacuoles, LVSs large vacuolar-like structures.