Figure 2.

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 pcd^5J homozygous mice. (A) At 28 days of age, wild-type mice transgenic for GFP-LC3 display a diffuse pattern of GFP staining. (B) GFP-LC3 - pcd^5J homozygous 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 pcd^5J homozygous 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 pcd^5J homozygous 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).