WIPI2 PHOSPHORYLATION MEDIATES EARLY INTRA-AXONAL AUTOPHAGY DYSFUNCTION IN ALZHEIMER’S DISEASE

The pathological accumulation of proteins observed in the Alzheimer’s disease (AD) patients’ brains suggests early dysregulation of autophagy. Autophagy is a constitutive pathway essential for neuronal homeostasis and synaptic maintenance and is tightly regulated by proteins controlling autophagosome biogenesis, including WD-repeat proteins interacting with phosphoinositides (WIPI) proteins and the Autophagy-Related 16 Like 1 (ATG16L1) protein. In neurons, autophagosomes are constitutively generated in distal axons through a conserved mechanism required for proper synaptic function. Here, we investigated how amyloid-β impair intra-axonal autophagy during early stages of AD. Using 3xTg-AD mice, we detected a decrease in WIPI2 phosphorylation at Ser413 in the hippocampus of three-month-old female mice. Consistently, the ratio of phosphorylated (p-)WIPI2 to total WIPI2 was also reduced, indicating early alterations in autophagy-related signaling. In primary cultures of rat hippocampal neurons, we found that short exposure to Aβ oligomers (AβO) induces fast alterations in p-WIPI2, total WIPI2, LC3, and ATG16L1 levels within distal axons and synaptic sites, suggesting impaired local autophagosome biogenesis. Indeed, AβO also altered autophagic flux and autophagosome transport dynamics along the axon. Given the role of WIPI2 phosphorylation in autophagosome formation, we investigated upstream signaling mechanisms and found that AβO-induced autophagy dysregulation involves N-methyl-D-aspartate receptor (NMDAR) activation and Calcium/calmodulin-dependent protein kinase II (CaMKII) signaling. Modulation of WIPI2 phosphorylation with phospho-mimetic and phospho-null constructs further supports a direct contribution of WIPI2 phosphorylation dysregulation to synaptic and axonal dysfunction. Together, these findings identify early defects in axonal autophagy as a contributing mechanism to synaptic vulnerability in AD.