Endoplasmic Reticulum Aggregation act as a Nucleation Site for Autophagosome Formation in an Amyloid-β model of Alzheimer’s Disease

Abstract

Background: Autophagy via formation of the autophagosome is a major cellular degradation pathway for long-lived proteins and organelles. Dysfunction in autophagic processes are prominently linked to neurodegenerative disorders associated with an aggregation of misfolded protein. However, the underlying cellular mechanisms of how amyloid-β (Aβ) promotes autophagy in Alzheimer’s Disease remains elusive. Method: To investigate this, primary culture rat hippocampal neurons were transfected with GFP-KDEL construct at DIV5 and treated with 15µM ABO at DIV7. Either an AMPKα activator (1mM AICAR) or inhibitor (10µM Dorsomorphin) was applied at timed 2, 8, 24h intervals. Cells were fixed and analysed using confocal imaging and biochemical data. Result: Aβ-induced autophagy was shown to begin from recruitment of ULK-1 via an AMPK-dependent pathway to the endoplasmic reticulum (ER). Pharmacological inhibition of AMPKα by Dorsomorphin in Aβ-treated primary hippocampal neurons demonstrated reduced recruitment of ULK-1 to the ER, while AMPKα activation by AICAR resulted in increased ULK-1 recruitment to the ER. Live cell confocal imaging demonstrated intracellular interactions between ULK-1 and the Atg14L-Beclin1-Vps34 complex. There were visible aggregation and ubiquitination on the ER promoting the formation of omegasomes and ultimately kick-starting the autophagic pathway. In parallel, p62 was also found to translocate to ER aggregation sites after Aβ treatment aside from its known roles in protein shuttling towards mature autophagosomes. Conclusion: This study demonstrates that Aβ-induced autophagy is an AMPK-dependent process and begins with recruitment and aggregation of important proteins such as ULK-1 and PI3K-III complex to the ER thereby pushing off the formation of mature autophagosomes.