Fig. 7

Progressive degeneration of GAN^-/- neurons and model of action of Gigaxonin. a Immunostaining with Doublecortine (Dbx) and MAP2 identified neurons from control and GAN^-/- mice after 3 hours, 5, 15 and 20 div. Scale bar: 25 µm. b Neurodegeneration of GAN^-/- neurons was evidenced from 15 div onwards, as measured by the decreased proportion of neurons (as expressed by a MAP2 to DAPI ratio), relatively to the 3 h time point (Dbx to Dapi ratio). WT and mutant cells are represented in black and red circles, respectively. n = 3 independent experiments with triplicate measures (161–821 DAPI-positive cells counted per measure). Individual measures and means ± SEM are represented; **P < 0.01 and *P < 0.05 values at 15 and 20 div, respectively using the two-way ANOVA test (Bonferroni post-hoc test). c Schematic model of Gigaxonin action. Top panel: Gigaxonin controls the steady-state level of ATG16L1, by interacting with its C-terminal WD40 domain and promoting its K48 poly-ubiquitination and clearance by the proteasome and the autophagy pathways. ATG16L1 binds to the ATG12-ATG5 elongation conjugate and targets it to the nascent autophagic membrane, through its interaction with WIPI2. Thus, the E3 ligase activity of the ATG12-ATG5-ATG16 complex lipidates LC3 onto the membranes, allowing the elongation of the phagophore. The mature autophagosome sequesters cytosolic materials, including p62 bound cargo, which are degraded upon fusion to the lysosome. Thus, by controlling ATG16L1 steady-state, the Gigaxonin-E3 ligase promotes autophagosome production and ensures a normal autophagic flux within cells. In a physiological context, autophagosome biogenesis occurs at the neurite tip but also in the soma of primary neurons. Bottom panel: Gigaxonin depletion in primary neurons induces aggregation of ATG16L1, without impairing the formation of the ATG12-ATG5 conjugate. We propose that the impairment in LC3 lipidation observed in GAN^-/- neurons is due to a defect in anchoring ATG12-ATG5 to the autophagic membranes. Overall, Gigaxonin depletion alters autophagosome synthesis and causes an abnormal accumulation of the main autophagy receptor p62, hence impairing the autophagic flux. ATG16L1 and P62 accumulation are localised within the soma, opening interesting perspectives on the study of autophagy compartimentalisation within neurons, and on its role in human neuropathy, as exemplified in GAN. (Image templates coming from Servier Medical Art (https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fsmart.servier.com%2F&data=02%7C01%7Caurora.scrivo%40einstein.yu.edu%7Cc44f29f2622d461b35a508d654507f0e%7C04c70eb48f2648079934e02e89266ad0%7C1%7C0%7C636789102036800397&sdata=zpNk9F6HX1iwcI%2Fdm5MXXUj3yRUvykH64lwEWuH1J8U%3D&reserved=0)