(poly pyrimidine monitor binding protein 2), binds the internal ribosome entry site in and gene expression was analysed by qPCR (Fig 5A)
(poly pyrimidine monitor binding protein 2), binds the internal ribosome entry site in and gene expression was analysed by qPCR (Fig 5A). cell-like markers, CD44 and vimentin, suggesting HIF-2 molecular mechanisms could potentially promote regression of neuronal-like cells to a stem-like state and trigger neuronal recovery following ischaemic insult. Our findings suggest the HIF-2 pathway predominates over HIF-1 signalling in neuronal-like cells following acute hypoxia. Introduction Blockage of cerebral arteries starves neurons of oxygen and glucose, triggering a cascade of events leading to irreversible cell death . The tissue surrounding the blockage is Ferroquine usually partially perfused, therefore neurons here are vulnerable, yet salvageable, and must adapt to survive, to prevent further loss Bmp4 of neuronal tissue . The hypoxia inducible factors (HIFs) are grasp regulators of oxygen homeostasis and critical for adaptation to hypoxic insult . The HIF alpha subunit exist as three isoforms; HIF-1, -2 and -3. HIF-1 and 2 are structurally comparable and share common transcriptional targets, including and [4,5]. HIF-1 and -2 also regulate distinct subsets of genes and elicit different cellular fates. regulates and expression to maintain metabolism, and can activate to trigger apoptosis, whilst promotes angiogenesis, cell division and tissue regeneration by regulating the expression of and the stem cell marker, increased ischaemic damage, infarct volume and mortality following transient cerebral artery occlusion , whilst indirect induction of mediated adaptation may be neuroprotective. However, neuronal-specific knockdown of and expression was shown to decrease infarct size and improve neuronal survival in the early acute stages of middle cerebral artery occlusion , suggesting signalling could contribute to stroke-associated damage. and display temporal differences in signalling; appears to be involved in adaptation to acute hypoxia whilst mediates adaptation to chronic hypoxic stress . The timescale of HIF signalling may therefore be critical for effective recovery from stroke. Indeed, whilst ablation of neuronal and is Ferroquine reported to be beneficial in the hours following stroke, loss of and correlated with increased apoptosis and reduced sensorimotor function in later stages . This may be due to the importance of angiogenesis in stroke-associated neurogenesis [14,15] and key role in tuning signalling and angiogenesis [5,16]. These studies highlight the intricacies of HIF signalling, and their potential importance in neuroprotection and recovery from stroke damage. Adaptation is usually central to neuronal recovery and stroke repair, however therapies promoting neuronal repair and regeneration are currently lacking. Fully understanding the adaptive mechanisms brought on in response to stroke is essential to develop novel therapeutics to enhance neuronal repair and regeneration, and limit the damage and disability associated with stroke . In this study, neuronal cell lines were used as a model to study the molecular changes occurring in response to acute hypoxic stress. We observed preferential activation of HIF-2 dependant adaptive mechanisms in neuronal-like cells in response to acute hypoxia and an absence of HIF-1 dependant signalling. We also observed increased expression of neural progenitor stem cell-like markers, thought to be transcriptionally regulated by HIF-2. Together, these findings underscore the importance of HIF-2 signalling in neuronal adaptation following acute hypoxic stress and highlight the potential for neuronal repair and regeneration. Experimental procedures Cell culture PC12, NT2 and MCF7 cell lines were obtained from the American Type Culture Collection (ATCC). NT2 and MCF7 cells were maintained in Dulbeccos Modified Eagles Media (DMEM, Gibco) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS, Sigma) Ferroquine and 1% (v/v) penicillin-streptomycin (Sigma). PC12 culture media was also supplemented with 5% (v/v) heat-inactivated horse serum (Sigma). Cells were produced at 37C in 5% (v/v) CO2 atmosphere under high humidity. PC12 and NT2 differentiation PC12 cells were cultured on poly-L-lysine (0.1 mg/mL, Sigma) coated 6-well plates at a density of 2×106 cells/well. After 24 hours, media was replaced with differentiation media (200 nM nerve growth factor (NGF; Sigma, Cat no: N0513), 1% (v/v) horse serum (Sigma) and 1% (v/v) penicillin-streptomycin in DMEM), and replenished every 2C3 days. NT2 cells were differentiated into a neuronal population by the method by Pleasure software (Biogazelle). Expression of target genes were analysed relative.