Maistrenko A. Involvment of hypoxia-inducible factor in the molecular mechanisms of hippocampal cells neuroprotection

The study is focused on the molecular mechanisms mediating endogenous neuroprotection in CA1 and CA3 neurons of the hippocampus and a possibility to activate this meuroprotection with anoxic precondition (APC) and inhibition of HIF- prolylhydroxylase. We have shown the differential susceptibility of CA1 and CA3 hippocampal neurons to cell damage after oxygen-glucose derivation (OGD) in situ, particularly higher vulnerability of CA1 neurons and lower one for CA3 neurons, accompanied by the decayed level of both HIF-1? and HIF-3? subunit expression in CA1 neurons with none changes in CA3 neurons. APC (performed three times for 5-min duration) led to a recovery of HIF-1? and HIF-3? subunit expression in CA1 neurons those remained increased following subsequent OGD (as compared to OGD without APC). We also combined Ca2+ imaging in organotypic hippocampal slices with quantitative real time PCR analysis to investigate the ischemic impairments in intracellular Ca2+ regulation in CA1 and CA3 neurons and to establish the role of HIF-1? in alleviating of these changes in both neuronal populations. Our results demonstrate the differential vulnerability of CA1 and CA3 hippocampal neurons to the ischemic impairments of intracellular Ca2+ regulation. For instance, the depolarization-induced [Ca2+]i transients were decelerated (both fast and slow decay kinetics) and the intracellular Ca2+ store functioning was impaired specifically in CA1 neurons 4 h post-OGD, a time-point representing a massive delayed death of CA1 neurons induced by OGD. The OGD-induced Ca2+ store dysfunction in CA1 neurons was manifested as the decrease of Ca2+ release from the intracellular Ca2+ stores (both caffeine-sensitive and -insensitive) that resulted in the abolished contribution of Ca2+ stores to generation of [Ca2+]i transients during neuronal depolarization. Together this demonstares the impaired cytosolic Ca2+ sequestration following ischemia and suggests the affected Ca2+ pumping by Ca2+-ATPases (PMCA and SERCA). Indeed, our q-RT PCR analysis revealed the downregulation in both PMCA1 and PMCA2 mRNAs in CA1 area at 4 h post-OGD. In CA3 neurons, the depolarization-induced [Ca2+]i transients as well as Ca2+ release from the ER were not changed following OGD. Further, the PMCA1 and PMCA2 gene expression were unchanged in CA3 neuorns while the SERCA2b gene expression was upregulated in these neurons by ischemic conditions. Our findings of the cross-link between differently impaired intracellular Ca2+ regulation in CA1 and CA3 neurons and the altered Ca2+-ATPase gene expression indicate the heterogenosity of CA1- and CA3-neuron-specific responses to ischemia at both cellular and genetic levels. Ischemic upregulation of SERCA2b in CA3 neurons, evidenced by the increased gene expression and unaltered intracellular Ca2+ store function after OGD, argues for the role of SERCA2b upregulatino as a mechanism mediating, at least partially, less vulnerability of CA3 neurons to the ischemia-induced cytoplasmic Ca2+ overload and Ca2+-dependent excitotoxicity. We have also shown the capability of APC and stabilization of HIF-1? to modulate Ca2+-ATPase expression and to amend the intracellular Ca2+ regulation in CA1 and CA3 hippocampal neurons. Either APC or stabilization of HIF-1? arrested the ischemic dysfunction of intracellular Ca2+ stores in CA1 neurons, probably through a direct post-transcriptional control of gene coding SERCA2b, and alleviated the ischemic downregulation of PMCA1 and PMCA2 gene expression in these neurons. Thus, our results unveil, for the first time, the HIF-1?-mediated, CA1- and CA3- neuron-specific modulation of Ca2+-ATPase expression (both PMCA and SERCA) and provide basis for further investigations of the effects of APC and HIF-1? on the CA1- and CA3-neuron-specific tolerance to the ischemia-induced Ca2+-dependent excitotoxicity with the potential significance for implementation of HIF-1? stabilizing strategy to activate endogenous neuroprotection against cell damage in ischemic conditions.