Much of the recent work of my group has focused on the molecular regulation of neural development, using the retina as a model system. Our initial work studying opsin expression was an extension of our early studies using antibody probes. We were the first group to map the opsin promoter and define the binding sites for key transcription factors. Following this we studied a number of other retinal genes, most notably the cyclic nucleotide gated channels. While studies of individual genes gave us important information, we wanted to take a broader approach and so we undertook a collaborative study to generate a large database of retinal est sequences from which we were able to perform an in silico analysis of retinal gene expression. By comparing our database with similar sets of data from other tissues we were able to categorize retinal genes into common, neural and presumptive rod genes. From our est libraries we prepared microarrays that allowed us to study expression of over 10,000 retinal genes. This developmental profile remains the most comprehensive description of retinal gene expression and demarcates a number of key milestones in retinal development. As well as being of broader use to the scientific community, these results have allowed us to home in on specific aspects of retinal development, particularly rod photoreceptor development. We have recently used mouse ES cells as a model system to recapitulate aspects of rod photoreceptor development and are defining molecular pathways responsible for sequential steps of progenitor restriction.
It is clear that changes in chromatin structure can modulate and even control retinal gene expression and development. Using ChIP-seq we have been able to show the changing patterns of gene expression correlate with changes in methylation of histone H3 in nucleosomes at the promoter and over the gene body. An important finding from this work was that rod photoreceptor specific genes have a specific epigenetic signature. The spacing between nucleosomes is critical for the efficient condensation of chromatin. We have shown that this process in rod photoreceptors is dependent on the linker histone isoform H1c. H1c promotes the increase in nucleosome repeat length necessary for chromatin condensation. Knockout mice lacking H1c show patches of cells in the photoreceptor layer that lack the normal condensed heterochromatin. In an extension of our findings we have used pharmacological inhibitors to block specific changes in histone methylation and acetylation and followed the changes in retinal development. Our conclusion from these studies is that histone modifying enzymes can control key developmental steps and their specificity relies on association with DNA-binding proteins that may act independently of more traditional transcription factors. In addition to helping define the molecular basis of normal retinal development, this work is helping understand ways in which the epigenome may be perturbed in retinal diseases and how drugs that modify the epigenome might be used to resort normal patterns of retinal gene expression.
We have also had a strong interest in the causes and potential therapies of neurodegenerative diseases. Mitochondrial uncoupling proteins are powerful agents for the control of reactive oxygen species production and subsequent cell death. In the Central Nervous System uncoupling protein 2 (UCP2) is the dominant member of this gene family. We have shown that when transfected into cells UCP2 provides protection from oxidative stress as mimicked by hydrogen peroxide. We have also shown that in vivo overexpression of UCP2 protects mice against a variety of insults including MPTP induced Parkinsonism, NMDA-induced excitotoxic neuronal death and drug-induced seizure mediated neuronal death. More recently we have also found that over expression of UCP2 can affect the normal process of developmental cell death that occurs in retinal ganglion cells. We have also shown that in primates, activation of UCP2 by dietary supplementation of cofactors can provide protect against MPTP induced Parkinsonism. This is strong evidence that manipulation of UCP2 levels and activity can be an important mechanism of combating human neurodegenerative disease.
We have studied a variety of neuroprotective factors in a range of disease models. For example, the IL-6 family of factors, primarily CNTF and LIF are able to block the transition from a retinal progenitor into a rod photoreceptor by activating STAT3. In the mature retina STAT3 is vital in pathways involved in neuroprotection and we have carried out a series of studies in vivo and with purified cell populations that show its importance for the survival of retinal ganglion cells subjected to a variety of excitotoxic and other stresses. can provide in Leukemia inhibitory factor (LIF) regulates the expression of UCP2 in a STAT3 dependent fashion and this can decrease mitochondrial ROS production.