Purkinje cell degeneration in pcd mice isaccompanied by activation of the autophagy pathway. (A-C) Fluorescence microscopy of cerebellar sections from GFP-LC3 transgenic mice crossed with pcd5Jhomozygous mice. (A) At 28 days of age, wild-type mice transgenic for GFP-LC3 display a diffuse pattern of GFP staining. (B) GFP-LC3 - pcd5Jhomozygous mice, however, exhibit GFP puncta (arrows) in the cell bodies of their Purkinje cell neurons at 28 days of age. (C) These GFP-LC3 puncta are especially obvious at higher magnification, and can be seen in dendritic arbors as well as the cell body (arrows). GFP-LC3 = green; DAPI = blue. Scale bars correspond to 20 μM. (D) Western blot analysis of cerebellar protein lysates subjected to subcellular fractionation was also performed on protein samples from 28 day-old mice to assess autophagy activation. Both mitochondrial and cytosolic fractions from pcd5Jhomozygous mice showed a pronounced LC3-I to LC3-II shift. This increased LC3-II/LC3- I ratio is consistent with marked activation of the autophagy pathway in pcd5Jhomozygous mice. Note that visualized LC3 bands represent endogenous LC3 isoforms. Equivalent loading and the success of the fractionation were confirmed by re-probing the immunoblot with Cox-IV and SOD1 antibodies (not shown).