Oligomeric β-amyloid (1-42) induces autophagy through early recruitment of omegasomes and autophagosomes to ER aggregation sites in primary neurons

Abstract

Background: The Autophagy Lysosomal Pathway (ALP) is a major cellular degradation pathway by which proteins and organelles process and remove dysfunctional proteins. Many neurological disorders are prominently linked with dysfunction in ALP, thereby propagating the aggregation of misfolded protein (e.g. β-Amyloid; Aβ). However, the underlying cellular pathomechanism of how Aβ modulates autophagy remains elusive. Method: To investigate this, a murine model of primary hippocampal neurons were transfected with different constructs such as GFP-KDEL, DsRed-KDEL, DsRed-Atg14L, DsRed-LC3, GFP-p62 and GFP-p62 L343A to allow confocal live-imaging of protein recruitment in ALP following timed Aβ-oligomer(1-42) treatment. Transfected cells were then fixed and analyzed through immunohistochemistry, Western blot and the Proximity Ligation Assay to study the colocalization between proteins important in both omegasome and autophagosome recruitment with the endoplasmic reticulum. Result: Here we report that Aβ1-42 oligomer (AβO) treatment increased AMPK-dependent recruitment of ULK-1 to the ER in early autophagy, independent of mTOR activation. Moreover, AβO treatment upregulated recruitment of components of the PI3K Complex III (i.e. Beclin-1, Atg14L and Vps34) to the ER, and subsequently induced increases in LC3 puncta distribution, autophagosome formation and accumulation within the endoplasmic reticulum. In parallel, p62, an autophagosome cargo protein was also found to translocate within ER aggregation sites following AβO treatment in both wildtype p62, and p62-L343A LIR mutant counterpart, suggesting roles of p62 not just in cargo shuttling, but omegasome maturation as well. Conclusion: Collectively, these data indicate that Aβ-induced autophagy begins at aggregation sites within the ER, and that Aβ primarily exerts cellular damage by upregulating autophagic machinery particularly in the ER during early autophagy.