Enhanced oxidative stress-regulated by cholesterol promotes necroptosis in Alzheimer's disease

Abstract

How do neurons die in Alzheimer¿s disease (AD)? The answer is not clear. Caspases, primary effectors during apoptosis, are activated in AD brains; however, apoptotic morphology is not evident. Furthermore, the progress of the disease that lasts for decades seems incompatible with an apoptotic death program. A recent study offers compelling evidence of active necroptosis, a regulated inflammatory cell death, in post-mortem AD brains, but still, the events that trigger or regulate necroptosis in AD are not fully known. It has been reported that oxidative stress may be determinant as to whether a cell initiates necroptosis; it is, therefore, likely that mitochondrial dysfunction, observed in the early stages of AD, might favor cells to undergo necroptosis. Previous studies from our group have shown that high intracellular cholesterol levels deplete the mitochondrial pool of GSH, thus sensitizing neurons against amyloid-beta (A¿)-induced mitochondrial oxidative stress. The present study is aimed to evaluate whether cholesterol can regulate the necroptotic pathway using both APP-PSEN1 mice that overexpress SREBF2 and cholesterol-enriched SH-SY5Y cells. Methods: Cholesterol enrichment was assessed by incubating the cells with soluble cholesterol:methyl-cyclodextrin complex (50 ¿g/ml) for 1 h followed by 4 h recovery. To induce necroptosis, cells were treated for 24 h with TLQ [TNF¿ (10 ng/ml) plus the SMAC mimetic (LCL-161, 10 ¿M) and the pan-caspase inhibitor qVD-OPH (10 ¿M)]. Results: In the brains of APP-PSEN1-SREBF2 mice, we found an up-regulated expression of the necroptosis-related proteins RIPK3 and MLKL, which together with RIPK1 accumulated in the urea-soluble protein fraction, indicative of necrosome assembly. Moreover, western blot analyses of brain extracts from triple transgenic mice also showed high levels of c-FLIP, the natural inhibitor of caspase-8, further pointing to a cholesterol-regulated engagement of the necroptotic pathway. In SH-SY5Y cells, cholesterol enrichment resulted in enhanced cell death after exposure to TLQ, which was prevented by RIPK1 and RIPK3 inhibitors (Necrostatin and GSK¿872, respectively). Similar protection against TLQ-induced necroptosis was achieved when mitochondrial GSH levels were recovered by GSH ethyl ester. Intriguingly, cholesterol also regulated the intracellular localization of RIPK3 and MLKL. Confocal microscopy showed an increased presence of nuclear RIPK3 and MLKL in cholesterol-enriched cells, recently described as a requirement for the subsequent necrosome formation in the cytosol. Overall, these findings indicate that high intracellular cholesterol levels compromise neuronal viability, promoting necroptosis and subsequent necroptosis, a pro-inflammatory type of cell death, which ultimately may contribute to chronic neuroinflammation in AD.