Silencing of Fem1cR3 gene expression in the DBA/2J mouse precedes retinal ganglion cell death and is associated with histone deacetylase activity.

Axonal degeneration and retinal ganglion cell body atrophy and death are the fundamental characteristics of glaucomatous optic neuropathies.1 The identification of intrinsic apoptosis in glaucomatous ganglion cell somas2 has led to the hypothesis that disruption of this program may be a viable target to prevent ganglion cell neurodegeneration and, combined with conventional pressure-lowering therapy, may yield more effective preservation of vision. Substantial focus, therefore, has been given to obtaining a better understanding of the temporal sequence of events being executed in apoptotic ganglion cells, particularly to the early events that may critically activate subsequent and irreversible steps in the apoptotic cascade.1,3 One of the early events associated with ganglion cell death is the silencing of normal gene expression. This phenomenon has been documented in both acute and chronic (experimental ocular hypertension) models of optic nerve disease.4–8 These studies have typically reported an exponential decay in the mRNA abundance of genes that are normally expressed in ganglion cells. In all studies to date, the loss of mRNAs occurs well in advance of a measurable loss of ganglion cell bodies. Our initial characterization of this phenomenon was a quantitative analysis of the transcript abundance for the Thy1 gene. Thy1 mRNA becomes depleted before cell loss in mouse models of optic nerve crush, and intravitreal injection of the glutamate analog, N-methyl-d-aspartate. Additionally Thy1 transcripts were depleted in advance of cell loss in a rat model of experimental glaucoma.4,5 Loss of this mRNA was also observed in Bax-deficient retinal ganglion cells after optic nerve crush, even though these cells were completely arrested in the apoptotic program. Similar kinetics of decay of other ganglion cell–specific genes, including Brn3b,9 Nrn1, and Sncg6,7 have now been reported. Collectively, these data indicate that downregulation, or the silencing of normal gene expression, is an early event in the apoptotic program, occurring before the point at which BAX is activated. The global nature of the effect suggests a coordinated mechanism that can simultaneously affect multiple actively expressed genes. One mechanism that could account for this effect is rapid modification of histones in the promoters of genes in an “open” conformation, particularly by histone deacetylation, which has been described as a principal epigenetic change that can rapidly silence active genes.10 Using the mouse optic nerve crush model to activate ganglion cell apoptosis, we observed that histones in the promoters of actively expressed genes were rapidly deacetylated, which was associated with the translocation of histone deacetylase 3 (HDAC3) from the cytoplasm to the nuclei of dying cells. Moreover, the downregulation of the Rosa3 ganglion cell reporter gene (Fem1cR3)11 could be attenuated in mice pretreated with the broad-spectrum HDAC inhibitor trichostatin A (TSA).8 Here, we report that DBA/2J mice, carrying the Fem1cR3 reporter gene, exhibit loss of expression of this gene before glaucomatous ganglion cell loss, similar to reports made for other genes in this mouse strain.7 This downregulation preceded the function of the proapoptotic BAX protein. Evaluation of dying cells in DBA/2J mice indicated both the nuclear accumulation of HDAC3 and deacetylation of histone H4, suggesting an epigenetic mechanism affecting gene transcription similar to that reported after acute optic nerve damage. Continuous treatment of mice with TSA was able to ameliorate the silencing of Fem1cR3, and therefore somewhat preserve retinal integrity. This effect was not translated to optic nerve axons, however, which still exhibited a similar level of damage compared with control treatment groups.