Neuroprotective Effects of Necrostatin-1 Against Oxidative Stress-Induced Cell Damage: an Involvement of Cathepsin D Inhibition

Abstract

Necroptosis, a recently discovered form of non-apoptotic programmed cell death, can be implicated in many pathological conditions including neuronal cell death. Moreover, an inhibition of this process by necrostatin-1 (Nec-1) has been shown to be neuroprotective in in vitro and in vivo models of cerebral ischemia. However, the involvement of this type of cell death in oxidative stress-induced neuronal cell damage is less recognized. Therefore, we tested the effects of Nec-1, an inhibitor of necroptosis, in the model of hydrogen peroxide (H₂O₂)-induced cell damage in human neuroblastoma SH-SY5Y and murine hippocampal HT-22 cell lines. The data showed that Nec-1 (10-40 μM) attenuated the cell death induced by H₂O₂ in undifferentiated (UN-) and neuronal differentiated (RA-) SH-SY5Y cells with a higher efficacy in the former cell type. Moreover, Nec-1 partially reduced cell damage induced by 6-hydroxydopamine in UN- and RA-SH-SY5Y cells. The protective effect of Nec-1 was of similar magnitude as the effect of a caspase-3 inhibitor in both cell phenotypes and this effect were not potentiated after combined treatment. Furthermore, the non-specific apoptosis and necroptosis inhibitor curcumin augmented the beneficial effect of Nec-1 against H₂O₂-evoked cell damage albeit only in RA-SH-SY5Y cells. Next, it was found that the mechanisms of neuroprotective effect of Nec-1 against H₂O₂-induced cell damage in SH-SY5Y cells involved the inhibition of lysosomal protease, cathepsin D, but not caspase-3 or calpain activities. In HT-22 cells, Nec-1 was protective in two models of oxidative stress (H₂O₂ and glutamate) and that effect was blocked by a caspase inhibitor. Our data showed neuroprotective effects of the necroptosis inhibitor, Nec-1, against oxidative stress-induced cell damage and pointed to involvement of cathepsin D inhibition in the mechanism of its action. Moreover, a cell type-specific interplay between necroptosis and apoptosis has been demonstrated.

Keywords: Caspase-3; Glutamate; HT-22 cells; Hydrogen peroxide; Pepstatin A; SH-SY5Y cells.