The neurotoxicant PCB-95 by increasing the neuronal transcriptional repressor REST down-regulates caspase-8 and increases Ripk1, Ripk3 and MLKL expression determining necroptotic neuronal death

Our previous study showed that the environmental neurotoxicant non-dioxin-likepolychlorinated biphenyl (PCB)-95 increases RE1-silencing transcription factor(REST) expression, which is related to necrosis, but not apoptosis, of neurons.Meanwhile, necroptosis is a type of a programmed necrosis that is positivelyregulated by receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixedlineage kinase domain-like (MLKL) and negatively regulated by caspase-8. Here we evaluated whether necroptosis contributes to PCB-95-induced neuronal deaththrough REST up-regulation. Our results demonstrated that in cortical neuronsPCB-95 increased RIPK1, RIPK3, and MLKL expression and decreased caspase-8 at thegene and protein level. Furthermore, the RIPK1 inhibitor necrostatin-1 orsiRNA-mediated RIPK1, RIPK3 and MLKL expression knockdown significantly reducedPCB-95-induced neuronal death. Intriguingly, PCB-95-induced increases in RIPK1,RIPK3, MLKL expression and decreases in caspase-8 expression were reversed byknockdown of REST expression with a REST-specific siRNA (siREST). Notably, insilico analysis of the rat genome identified a REST consensus sequence in thecaspase-8 gene promoter (Casp8-RE1), but not the RIPK1, RIPK3 and MLKL promoters.Interestingly, in PCB-95-treated neurons, REST binding to the Casp8-RE1 sequence increased in parallel with a reduction in its promoter activity, whereas underthe same experimental conditions, transfection of siREST or mutation of theCasp8-RE1 sequence blocked PCB-95-induced caspase-8 reduction. Since RIPK1, RIPK3and MLKL rat genes showed no putative REST binding site, we assessed whether the transcription factor cAMP Responsive Element Binding Protein (CREB), which has a consensus sequence in all three genes, affected neuronal death. In neuronstreated with PCB-95, CREB protein expression decreased in parallel with areduction in binding to the RIPK1, RIPK3 and MLKL gene promoter sequence.Furthermore, CREB overexpression was associated with reduced promoter activity ofthe RIPK1, RIPK3 and MLKL genes. Collectively, these results indicate that PCB-95was associated with REST-induced necroptotic cell death by increasing RIPK1,RIPK3 and MLKL expression and reducing caspase-8 levels. In addition, since REST is involved in several neurological disorders, therapies that block REST-induced necroptosis could be a new strategy to revert the neurodetrimental effectsassociated to its overexpression.