The increase of the (?)IRE DMT1 isoform, which is unresponsive to post-transcriptional regulation by intracellular iron levels through the IRE-IRP system, was confirmed by immunocytochemistry in SK-N-SH cells after 4 h of OGD. Lys310 residue during ischemia. Chromatin immunoprecipitation analysis of the 1B/DMT1 promoter showed there was increased conversation with RelA and acetylation of H3 histone during OGD exposure of cortical neurons. Over-expression of wild-type RelA increased 1B/DMT1 promoter-luciferase activity, the (?)IRE DMT1 protein, as well as neuronal death. Expression of the acetylation-resistant RelA-K310R construct, which carried a mutation from lysine 310 to arginine, but not the acetyl-mimic mutant RelA-K310Q, down-regulated the 1B/DMT1 promoter, consequently offering neuroprotection. Our data showed that 1B/(?)IRE DMT1 expression and intracellular iron influx are early downstream responses to NF-B/RelA activation and acetylation during brain ischemia and contribute to the pathogenesis of stroke-induced neuronal damage. Introduction Cellular iron homeostasis is usually a finely regulated process that prevents cellular damage due to iron accumulation and the formation of free radicals through Fanapanel the Fenton reaction . The iron concentration in the brain increases with age and is much higher in the central nervous system of subjects affected by neurodegenerative diseases C. An important pathogenic role of iron has been suggested in Alzheimer’s, Parkinson’s and Huntington’s diseases, as significant iron accumulation was found in affected brain regions of patients . The relevance of neuronal cellular Fanapanel damage by increased iron levels was further resolved by and studies of iron and 6-hydroxydopamine (6-OHDA)-dependent neurodegeneration, respectively . Increased iron content, correlated with a reduced Fanapanel quantity of TH-positive neurons, was found in the substantia nigra (SN) of rats that had been overloaded with iron dextran. Significant neuroprotection was produced by deferoxamine (DFO), an iron chelator capable of permeating the bloodCbrain barrier, and more recent chelators in experimental models of Parkinson’s and Alzheimer’s diseases C, brain ischemia-reperfusion ,  and hemorrhage . Iron could be transported into mammalian cells as transferrin (Tf)-bound iron (TBI) via Tf receptor (TfR) mediated endocytosis or through the non-transferrin-bound iron (NTBI) pathway via divalent metal transporter-1 (DMT1). The role of TfR-mediated iron transport in neurodegeneration and ischemia is still controversial. TBI, TBI-binding sites and TfR expression are poorly correlated with the final steady-state distribution of iron . Moreover, the number of TBI-binding sites decreased in dopaminergic neurons of the SN of PD patients Aviptadil Acetate , , suggesting that this NTBI pathway is usually preferentially involved in the iron accumulation of PD brains. Conversely, both TfR and DMT1 were recently shown to increase in the ischemic cortex of rats subjected to middle cerebral artery occlusion (MCAO) . A significant consensus has emerged about the involvement of the NTBI pathway in neurodegenerative diseases, with iron accumulation mediated by DMT1 in specific brain areas . DMT1 is usually highly expressed in mammalian neuronal cells C,  and is present at a relevant concentration in the basal ganglia, caudate-putamen and substantia nigra pars reticulata . The mammalian DMT1 gene family (SLC11A2; Nramp2) is composed of integral membrane proteins with 10C12 putative membrane-spanning domains  subjected to alternative splicing. The 5 alternate splicing of exons 1A and 1B produces the 1A and 1B DMT1 mRNA Fanapanel isoforms, with 1A/DMT1 predominantly expressed in kidney and duodenum and 1B/DMT1 ubiquitously expressed in the peripheral organs and brain . The 3 splicing generates two isoforms with or without the iron responsive element (IRE) motif in the 3UTR, named (+)IRE or (?)IRE isoforms, respectively. These variants give rise to four DMT1 isoforms, all of which are active in ferrous iron transport. The two (+)IRE isoforms are post-transcriptionally regulated by the IRE/Iron Regulatory Protein (IRP) system which stabilize them in the absence of iron, while (?)IRE splice variants.