Fig. 5: Age-associated cerebellar neurodegeneration in Atg4d^−/− mice.

A Representative H&E images from 15-month-old WT and Atg4d^−/− mice hippocampus, vestibular nuclei, cerebral cortex, and cerebellum. Arrowheads label Purkinje cells (PC) bodies. Scale bars: 50 μm. B–C Representative images and quantification of histological analysis in 2- (B) and 15- (C) month-old WT and Atg4d^−/− mice cerebella. Up, representative H&E images. Scale bar 400 μm. Bars label gray matter thickness. Middle up, representative images for calbindin IHC. Scale bar: 20 μm. Middle bottom, IHC against Glial Fibrillary Acidic Protein (GFAP). Scale bar: 40 μm. Bottom, IHC against Neuronal Nuclei (NeuN). Scale bar: 20 μm. D Representative transmission electron microscopy (TEM) images showing the ultrastructure of WT and Atg4d^−/− mice cerebellar PCs. Scale bar, 1 μm. Autophagosomes (1), abnormal nuclear envelope invaginations (2) and swollen radial processes (3) in Atg4d^−/− PCs are shown in insets. E Results from Rotarod analyses of 2- and 15-month-old WT and Atg4d^−/− mice. Asterisks show significant differences when areas under the curve are compared to 2-month-old WT group. F Representative limb postures responding to tail suspension in 15-month-old WT and Atg4d^−/− mice. G Quantification of forelimb angles against body axes in the tail suspension test. H Representative images for raised-beam test in 15-month-old WT and Atg4d^−/− mice. I Performance on the raised-beam test of 2- and 15-month-old mice measured by the time mice spend before reaching the end of the beam. J Representative paw placement records of 15-month-old WT and Atg4d^−/− mice after footprint pattern analysis. K Quantification of stride length of WT and Atg4d^−/− mice. Data are presented as mean and SD of at least 5 mice per group. L Results from grip strength analyses of 2- and 15-month-old WT and Atg4d^−/− mice. Graph represents the time mice were able to hold themselves to a grid when inverted in the inverted-cling grip test. M Representative immunoblots of endogenous ATG8-like proteins in the cerebellum from WT and Atg4d^−/− mice. N Representative images of immunofluorescence analysis of endogenous mATG8 proteins in WT and Atg4d^−/− cerebellar PCs. O Quantification of the data shown in (N). Bars represent mean ± SD. N = 6, 2-month-old mice per genotype (B). N = 7, 15-month-old mice per genotype (C); N > 11, 2-month-old mice and N = 10, 15-month-old mice per genotype (E, G); N = 17, 2-month-old mice and N = 10, 15-month-old mice per genotype (I); N = 12, 2-month-old mice and N = 10, 15-month-old mice per genotype (K); N = 8, 2-month-old mice and N = 10, 15-month-old mice per genotype (L); N = 4 mice per genotype (M); N > 85 cells per condition (b). *P < 0.05, 2-tailed unpaired Student’s t test (B, C, and O) and two-way ANOVA followed by Dunnett´s post hoc test (E, G, I, K and L).