Fig. 4

ATG16L1 accumulates in GAN^-/- cortical neurons and impairs the autophagy elongation conjugate. a MAP2 positive neurons, prepared from cortices of E15.5 GAN embryos were immunostained with ATG16L1 antibody after 4 and 15 div (days in vitro) ± proteasome inhibitor. Top panel shows an enlarge picture of the single perinuclear bundle of ATG16L1 found in the soma of GAN^-/- cells. Dashed line shows the nucleus. Scale bar: 10 µm for upper panel and 20 µm for bottom panel. b The effect of Gigaxonin in the conjugation of ATG12-ATG5 was evidenced in COS-7 cells. Transfected Flag-Gigaxonin degraded endogenous ATG16L1 but did not alter the formation of ATG12-ATG5 elongation complex, which migrates at 55 kDa. c ATG5 does not aggregate in Gigaxonin-null neurons. The visualisation of the docking of endogenous ATG5 to autophagosomes was hampered by the high density of LC3 dots in cortical neurons, but would reveal a decreased density around autophagic structures in the GAN^-/- neurons (inserts: circles drawn to highlight LC3-ATG5 proximity in individual dots). Scale bar: 10 µm