The role of Nxf in neurogenesis

Nxf, also known as LE-PAS or NPAS4, is a brain specific transcription factor of the basic helix-loop-helix PAS family. Members of this family are typically involved in developmental processes or cellular response to environmental stresses. Nxf is expressed throughout the limbic system, in particular, the dentate gyrus and CA1 regions of the hippocampus. The hippocampus is a site of continued neurogenesis throughout adulthood and an area of the brain in which neural stem cells persist after developmental programmes have been completed. Nxf expression in the hippocampus is up-regulated following treatment with pentylenetetrazol, a gamma- aminobutyric acid antagonistic chemiconvulsant that induces generalised seizure. It is also transiently up- regulated in cortical neurons stimulated by cortical spreading depression, a mechanism which provides protection from subsequent ischaemic insult. Animal models of neuropathy have shown that both seizure and ischaemic stroke lead to increased neurogenesis within the hippocampus. These findings suggest a role for Nxf in the repair mechanisms responsible for the production of new neurons in the hippocampus in response to insult. We hypothesise that Nxf is involved in neuronal differentiation of endogenous neural/progenitor cells. We have used two in vitro model systems of stem cell differentiation to investigate the role of Nxf in neurogenesis: (1) Differentiation of mouse embryonic stem cells (mESCs) to neural fates, and (2) Derivation of neurons from adherent primary cultures of mouse adult neural stem cells (mANSCs). Preliminary data supports the hypothesis that Nxf is involved in neuronal differentiation. Neurons staining positive for neurofilament medium chain were obtained by culturing mESCs in suspension as embryoid bodies with four days of exposure to retinoic acid (RA), whereas cultures not exposed to RA experienced spontaneous differentiation resulting in predominantly mesodermal derivatives. Levels of Nxf expression were 10-fold greater in RA treated cultures than those not treated with RA which suggests that the increase in Nxf is specific to neuronal differentiation. Currently, we are investigating whether these results are recapitulated by mANSC differentiation. Adherent cultures of mANSCs can be directed to preferentially differentiate into neurons, astrocytes or oligodendrocytes. We aim to use this system to investigate in further detail which differentiation pathways Nxf is associated with and whether they are specific to certain subpopulations of neurons.