Fig. 5
Dopaminergic autophagy begins in axons and spheroids. Top-left images show the somata of a nigral DAergic neuron with immunoreactivity for LC3 (autophagosomes) and Lamp1 (lysosomes). Top-right images show Lamp 2 (lysosomes) immunoreactivity (e) in a process of a striatal astrocyte (cyan in d) but not in a nearby spheroid (green in d). Bottom images were obtained in the MFB. g LC3 immunoreactivity in the degenerating axon shown in f. i An example of LC3 immunoreactivity found in a degenerating axon (h) that does not present Lamp1 immunoreactivity. k–m present Lamp1 immunoreactivity (m) in an astrocyte but not in a nearby spheroid (k). n shows the mean ± standard error of the density of the TH, LC3 and Lamp1 immunoreactivity (normalized as a percentage of the mean value of the extracellular medium) computed for MFB spheroids (n = 120; *P < 0.001). o1, p1 Two examples of DAergic spheroids that present a diffuse and generalized accumulation of Vmat2 (o2) and APP (p2) together with a puncta-like distribution of LC3 immunoreactivity. q, r Electron microscopy images of a DAergic spheroid delimited with yellow dotted lines (q), which presented flocculated areas (F) and high density areas with TH and DAT immunoreactivity, and contained damaged mitochondria (Mit) and autophagosomes (Aph). s1 An example of a degenerating thick axon which accumulated mitochondria (s2) and began to produce autophagosomes (s3). s4–s6 3D images of a degenerating axon with some of their mitochondria near autophagosomes (white arrows), a fact also observed in spheroids. s7 Distribution of mitochondria (YFP fluorescence in the red line) and autophagosomes (puncta of LC3 fluorescence in the purple line) inside a typical DAergic spheroid (TH fluorescence in the green line). However, mitochondria began to break apart (t2) within the spheroids (t1), and not all mitochondria showed a clear link to autophagosomes at this time (t3–t6)