Increased Susceptibility and Intrinsic Apoptotic Signaling in Neurons by Induced HDAC3 Expression.

Purpose: Inhibition or targeted deletion of histone deacetylase 3 (HDAC3) is neuroprotective in a variety neurodegenerative conditions, including retinal ganglion cells (RGCs) after acute optic nerve damage. Consistent with this, induced HDAC3 expression in cultured cells shows selective toxicity to neurons. Despite an established role for HDAC3 in neuronal pathology, little is known regarding the mechanism of this pathology. Methods: Induced expression of an HDAC3-mCherry fusion protein in mouse RGCs was accomplished by transduction with AAV2/2-Pgk-HDAC3-mCherry. Increased susceptibility to optic nerve damage in HDAC3-mCherry expressing RGCs was evaluated in transduced mice that received acute optic nerve crush surgery. Expression of HDAC3-FLAG or HDAC3-mCherry was induced by nucleofection or transfection of plasmids into differentiated or undifferentiated 661W tissue culture cells. Immunostaining for cleaved caspase 3, localization of a GFP-BAX fusion protein, and quantitative RT-PCR was used to evaluate HDAC3-induced damage. Results: Induced expression of exogenous HDAC3 in RGCs by viral-mediated gene transfer resulted in modest levels of cell death but significantly increased the sensitivity of these neurons to axonal damage. Undifferentiated 661W retinal precursor cells were resilient to induced HDAC3 expression, but after differentiation, HDAC3 induced GFP-BAX recruitment to the mitochondria and BAX/BAK dependent activation of caspase 3. This was accompanied by an increase in accumulation of transcripts for the JNK2/3 kinases and the p53-regulated BH3-only gene Bbc3/Puma. Cell cycle arrest of undifferentiated 661W cells did not increase their sensitivity to HDAC3 expression. Conclusions: Collectively, these results indicate that HDAC3-induced toxicity to neurons is mediated by the intrinsic apoptotic pathway.

Tools and resources for analyzing gene expression changes in glaucomatous neurodegeneration.

Evaluating gene expression changes presents one of the most powerful interrogative approaches to study the molecular, biochemical, and cellular pathways associated with glaucomatous disease pathology. Technologies to study gene expression profiles in glaucoma are wide ranging. Qualitative techniques provide the power of localizing expression changes to individual cells, but are not robust to evaluate differences in expression changes. Alternatively, quantitative changes provide a high level of stringency to quantify changes in gene expression. Additionally, advances in high throughput analysis and bioinformatics have dramatically improved the number of individual genes that can be evaluated in a single experiment, while dramatically reducing amounts of input tissue/starting material. Together, gene expression profiling and proteomics have yielded new insights on the roles of neuroinflammation, the complement cascade, and metabolic shutdown as important players in the pathology of the optic nerve head and retina in this disease.

Histone deacetylase 3 (HDAC3) plays an important role in retinal ganglion cell death after acute optic nerve injury.

BACKGROUND: Optic nerve damage initiates a series of early atrophic events in retinal ganglion cells (RGCs) that precede the BAX-dependent committed step of the intrinsic apoptotic program. Nuclear atrophy, including global histone deacetylation, heterochromatin formation, shrinkage and collapse of nuclear structure, and the silencing of normal gene expression, comprise an important obstacle to overcome in therapeutic approaches to preserve neuronal function. Several studies have implicated histone deacetylases (HDACs) in the early stages of neuronal cell death, including RGCs. Importantly, these neurons exhibit nuclear translocation of HDAC3 shortly after optic nerve damage. Additionally, HDAC3 activity has been reported to be selectively toxic to neurons. RESULTS: RGC-specific conditional knockout of Hdac3 was achieved by transducing the RGCs of Hdac3fl/fl mice with an adeno-associated virus serotype 2 carrying CRE recombinase and GFP (AAV2-Cre/GFP). Controls included similar viral transduction of Rosa26fl/fl reporter mice. Optic nerve crush (ONC) was then performed on eyes. The ablation of Hdac3 in RGCs resulted in significant amelioration of characteristics of ONC-induced nuclear atrophy such as H4 deacetylation, heterochromatin formation, and the loss of nuclear structure. RGC death was also significantly reduced. Interestingly, loss of Hdac3 expression did not lead to protection against RGC-specific gene silencing after ONC, although this effect was achieved using the broad spectrum inhibitor, Trichostatin A. CONCLUSION: Although other HDACs may be responsible for gene expression changes in RGCs, our results indicate a critical role for HDAC3 in nuclear atrophy in RGC apoptosis following axonal injury. This study provides a framework for studying the roles of other prevalent retinal HDACs in neuronal death as a result of axonal injury.

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

PURPOSE: Downregulation of normal gene expression in dying retinal ganglion cells has been documented in both acute and chronic models of optic nerve disease. The authors examined the mechanism and timing of this phenomenon in DBA/2J mice, using genetically modified substrains of this inbred line. METHODS: DBA/2J mice, doubly congenic for the Bax mutant allele and the ganglion cell reporter gene Fem1c(Rosa3) (R3), were evaluated to elucidate the timing of loss of normal gene expression during the apoptotic process. The localization of histone deacetylase 3 (HDAC3) and nuclear histone H4 acetylation were examined by immunofluorescence in dying cells. The role of HDACs in gene silencing during glaucoma was interrogated using the global HDAC inhibitor trichostatin A (TSA). RESULTS: Silencing of the R3 allele occurred in Bax(-/-) ganglion cells, indicating that this process preceded the committed step of the intrinsic apoptotic pathway. Weekly TSA treatment, between the ages of 6 and 10 months, was able to attenuate the loss of R3 expression in the retina, but had no effect on optic nerve degeneration. Dying cells in aging DBA/2J mice exhibited nuclear localization of HDAC3 and a decrease in the level of H4 acetylation. CONCLUSIONS: Retinal ganglion cells exhibit a loss of normal gene expression as an early (pre-BAX involvement) part of their apoptotic program during glaucomatous degeneration. This process can be ameliorated, but not completely blocked, using HDAC inhibitors. Epigenetic changes to active chromatin, such as deacetylation, may be mediated by HDAC3 in dying neurons.

Histone H4 deacetylation plays a critical role in early gene silencing during neuronal apoptosis.

BACKGROUND: Silencing of normal gene expression occurs early in the apoptosis of neurons, well before the cell is committed to the death pathway, and has been extensively characterized in injured retinal ganglion cells. The causative mechanism of this widespread change in gene expression is unknown. We investigated whether an epigenetic change in active chromatin, specifically histone H4 deacetylation, was an underlying mechanism of gene silencing in apoptotic retinal ganglion cells (RGCs) following an acute injury to the optic nerve. RESULTS: Histone deacetylase 3 (HDAC3) translocates to the nuclei of dying cells shortly after lesion of the optic nerve and is associated with an increase in nuclear HDAC activity and widespread histone deacetylation. H4 in promoters of representative genes was rapidly and indiscriminately deacetylated, regardless of the gene examined. As apoptosis progressed, H4 of silenced genes remained deacetylated, while H4 of newly activated genes regained, or even increased, its acetylated state. Inhibition of retinal HDAC activity with trichostatin A (TSA) was able to both preserve the expression of a representative RGC-specific gene and attenuate cell loss in response to optic nerve damage. CONCLUSIONS: These data indicate that histone deacetylation plays a central role in transcriptional dysregulation in dying RGCs. The data also suggests that HDAC3, in particular, may feature heavily in apoptotic gene silencing.

Decrease of cone opsin mRNA in experimental ocular hypertension.

PURPOSE: This study was designed to test the hypothesis that photoreceptors are adversely affected in glaucoma. As a measure of this effect, we examined the levels of rod opsin, and red/green and blue cone opsin mRNAs in monkeys with experimental ocular hypertension and glaucoma and in human eyes from donors with diagnosed glaucoma. METHODS: Experimental ocular hypertension was induced in one eye of 19 cynomolgous and 2 rhesus monkeys by laser ablation of the trabecular meshwork. In 15 monkeys, the elevated IOP was reduced by trabeculectomy. When the animals had experienced prolonged elevations of IOP (128 to 260 days), they were killed and the eyes enucleated. Fresh retinal tissue from the macula, inferotemporal retina (mid-peripheral), and far peripheral regions were harvested from some animals using a 3 mm trephine. The remaining retinas from these monkeys, and whole retinas from other animals were fixed. RNA isolated from each trephined sample was used for RNase Protection Analysis or real time PCR analysis to quantify opsin mRNA levels from different photoreceptor cell types. Fixed tissue was used for in situ hybridization studies. Human donor eyes (7 glaucoma and 4 control) were obtained from eye banks. All human specimens were used for in situ hybridization studies. RESULTS: Quantitative mRNA analysis and in situ hybridization studies both showed a reduction in the expression of red/green and blue cone opsin mRNAs in 6 monkey eyes with chronic ocular hypertension, relative to the contralateral eye. No loss of rod opsin mRNA was observed. The principal reduction occurred in cells of the mid-peripheral retina, a region of retina that often shows early and progressive damage in humans with glaucoma. In monkeys with ocular hypertension followed by trabeculectomy, there was a similar decrease in cone opsin mRNAs, but only in six out of fifteen (40%) of the monkeys. The decrease in these animals was correlated with a significantly elevated IOP at some time during the 2 weeks prior to euthanization and not with the extent of glaucomatous damage. Of the 7 human eyes with diagnosed glaucoma that were examined, 5 showed a decrease of cone opsin mRNA in the mid-peripheral retina, whereas none of the 4 normal eyes examined showed a decrease. CONCLUSIONS: Ocular hypertension leading to glaucoma also affects the outer retina, particularly the cone photoreceptors. We speculate that these cells become stressed leading to a disruption in the expression of normal genes, such as that encoding opsin. There is some evidence that this effect is reversible, when IOP levels are reduced.