Disruptions of endoplasmic reticulum and mitochondria prime mTOR suppression in low molecular weight Aβ-induced autophagy

Abstract

BACKGROUND: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder which pathological hallmarks associate with the deposition of insoluble beta-amyloid (Aβ) plaques in various regions of the brain. Reports suggested that various soluble Aβ species can induce neurotoxicity and are capable in inducing autophagy, the type II programmed cell death, with elusive mechanism of activation. METHODS: Primary cultures of rat embryonic cortical neurons were prepared and transfected with LC3-, DFCP-1-, endoplasmic reticulum (ER) retention signal (KDEL)-, and mitochondrial retention signal expressing plasmids. Neurons were treated with low molecular weight (LMW) Aβ. Protein lysates were collected afterwards for Western-blot analysis. Autophagosome, omegosome, lysosome and the morphology of the ER and mitochondria were examined by confocal microscopy. Quantitative data was analyzed by one way analysis of variance (ANOVA) followed by Student Newman Keul test according to the statistical program SigmaStat® (Jandel Scientific) to compare the level significance. A p-value less than 0.05 was regarded as significant, at *p < 0.05. RESULTS: In the present study, we demonstrated that low molecular weight Aβ induced autophagic vacuole and omegosome formation which showed partial colocalization in cortical neurons. Aβ induced AMP-activated protein kinase (AMPK) but did not cause significant suppression on mTOR and its downstream target p70 S6 kinase (p70S6K) simultaneously. On the other hand, low MW Aβ caused morphological damages in both ER and mitochondria at early time points. Organelle damage coincided with up-regulations in autophagy and lysosomal machinery. CONCLUSIONS: Our results suggest that low MW Aβ first confers its toxicity on the intracellular organelles by causing structural damages to initiate autophagy. Dysregulated cellular metabolism due to mitochondrial damage activates AMPK, which suppresses mTOR at later time points to exert synergistic effects on autophagy regulation.