Lysosome vacuolation disrupts the completion of autophagy during norephedrine exposure in SH-SY5Y human neuroblastoma cells

In our current study, we examined the mechanism underlying neuronal cell injuries caused by norephedrine in SH-SY5Y human neuroblastoma cells. Norephedrine was found to induce cytoplasmic vacuolation and a resultant loss of cell viability. In the cells treated with norephedrine also, an autophagic marker LC3 was converted to its LC3-II activated form, suggesting the induction of autophagy. In cells transfected with RFP-LC3 and GFP-LAMP1, a punctate patterning of LC3 expression and colocalization of LAMP1 with the formed vacuoles were observed, highlighting the lysosomal nature of the vacuoles and their association with autophagosomes. An autophagic flux assay using tfLC3 (mRFP-GFP-LC3) indicated the formation of autophagosomes and autolysosomes by norephedrine stimulation at an early timepoint (∼3 h). However, at a later timepoint (∼6 h), both the dilation of autolysosomes/lysosomes and the neutralization of the vacuolar pH were also observed. These results thus indicate that norephedrine induces autophagy at an early timepoint and cell death with lysosomal dysfunction and autophagy disruption at a later timepoint.

We examined the toxic effects of norephedrine on cells which was found to induce vacuolation.
► Norephedrine also increased the lysosomal pH and inhibited the maturation of lysosomal protease.
► Norephedrine additionally induced an autophagic protective response at an early timepoint.
► At a later, norephedrine induced cell death with lysosomal dysfunction and autophagy disruption.