Edinburgh Napier University

  Stroke is a leading cause of death and disability worldwide. Blockage, or occlusion, of cerebral arteries causes irreversible neuronal damage as disrupted blood flow starves neurones of oxygen and glucose. The hypoxia inducible factors (HIFs) are master regulators of oxygen homeostasis and critical for adaptation to hypoxic insult. Although HIF-1α and HIF-2 α share some common gene targets, they also promote specific adaptations to hypoxia. Differentiated PC12 and NT2 cells have been extensively used as a model to study the molecular changes associated with neurological pathologies, such as stroke. In this study, differentiated PC12 and NT2 cells were exposed to hypoxia for 4-24 hours in a hypoxic modular chamber before gene and protein expression was analysed by qPCR and immunoblotting. In order to validate the model, we characterised the in vitro changes associated with differentiation into neuronal-like cells, observing morphological, transcript and protein changes that revealed a neuronal-like phenotype. Following hypoxia, induction of the HIF-1α transcript or protein expression was not detected. Curiously, preferential activation of HIF-2α transcription and protein expression was detected. Increased expression of the neural progenitor stem cell-like markers, thought to be transcriptionally regulated by HIF-2α, were also observed. Furthermore, hypoxia caused loss of neuronal characteristics in the differentiated cells, as seen by a decrease in the expression of the neuronal markers, and loss of neurite number and extension. Our data shows the HIF-2α pathway predominates over the HIF-1α pathway in neuronal-like cell adaptation to hypoxia, and suggests such adaption could promote regression to neural progenitor stem-cells and thus, potentially proliferative states. This is highly significant as it shows neuronal cells possess molecular mechanisms which could trigger recovery following ischaemic insult. By completely understanding such adaptive mechanism and translating these results to in vivo models, it could represent a novel therapeutic approach to stimulate recovery after stroke.