Time course study of Aβ formation and neurite outgrowth disruption in differentiated human neuroblastoma cells exposed to H2O2: protective role of autophagy

Abstract

Here, we tried to elucidate the possible role of autophagy against H2O2 and Amyloid beta (Aβ) induced neurotoxicity using retinoic acid differentiated SH-SY5Y cells. We found that H2O2 disrupted neurite outgrowth concomitant with production of Aβ. Furthermore, we showed that H2O2 could increase the apoptotic factors such as Bax/Bcl-2 ratio, caspase-3 level, and PARP activity in a time course manner. These findings were confirmed by acridine orange/ethidium bromide and Hoechst staining. In addition, we observed that H2O2 led to conversion of LC3 protein from LC3I to LC3II and an increase in autophagy flux. Autophagy factors including LC3B, Atg7, and Atg12 increased and reached their highest level after 2h of insulting and then dropped to a lower level. Our results showed that autophagy could internalize and degrade intra- and extracellular Aβ after 3h treatment with H2O2. However, the remaining amount of Aβ accelerated morphological atrophy and, as a result, increased neuronal death (apoptosis). Inhibition of autophagy influx, using 3-methyl-adenine, increased intra- and extracellular levels of Aβ, providing more proof for a protective role of autophagy against oxidative stress. Further studies can shed light on the important role of autophagy by finding new pathways involved in Aβ degeneration.

Keywords: 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium a bromide; 3-methyladenine; 3MA; AD; AO/EB; Alzheimer’s disease; Amyloid-β; Atg; Autophagy; Aβ; ECL; FBS; H(2)O(2); LC3B; MDC; MTT; Monodansylcadaverine; Neurite outgrowth; PARP; PCD; Poly ADP-ribose polymerase; RA; ROS; SH-SY5Y cells; acridine orange/ethidium bromide; autophagy related-gene; electrochemiluminescence; fetal bovine serum; hydrogen peroxide; microtubule-associated protein light chain 3B; programmed cell death; reactive oxygen species; retinoic acid.