Figure 2.

Induction of autophagy in the axonal dystrophic swellings of degenerating Purkinje cells in GFP–LC3/Lurcher mice. A, Representative confocal image shows GFP–LC3 puncta within the axonal dystrophic swellings of degenerating Purkinje cells (P12). Scale bar, 5 μm. B, Representative electron microscopic image shows an axonal dystrophic swelling of a degenerating Purkinje cell (P10). A number of autophagosomes (arrows), autolysosomes (arrowheads), and atrophic mitochondria (triangles) accumulated in this axonal dystrophic swelling. In addition, two synapses located on the axonal dystrophic swellings are labeled by red asterisks. Scale bar, 1 μm. The inset shows a representative image of wild-type mouse DCN. Two axons are labeled: 1, myelinated; 2, an axon terminal with two synapses (red asterisks). Scale bar, 0.15 μm. Notice that the size of the control axons is significantly smaller than that of the axonal dystrophic swellings. C, Representative immuno-EM image of an axonal dystrophic swelling (C1) of degenerating Purkinje cells (P10) and enlarged images of four autophagosomes/autolysosomes (C2–C5) selected from 18 immuno-EM images show that the gold particles coated with polyclonal anti-GFP antibody (1:5000) are primarily associated with autophagosomes. Scale bars: C1, 1 μm; C2–C5, 0.2 μm. D, Live imaging of GFP–LC3 fluorescence (green) and LysoTracker staining (red) of axonal dystrophic swellings of degenerating Purkinje cells in cultured cerebellar slice revealed partial colocalization of GFP–LC3 and LysoTracker (bottom row). Wild-type control (top row) shows low GFP–LC3 level and no labeling of LysoTracker. No GFP–LC3 puncta are observed. Scale bar, 5 μm.