BAX activation in mouse retinal ganglion cells occurs in two temporally and mechanistically distinct steps.

BACKGROUND: Pro-apoptotic BAX is a central mediator of retinal ganglion cell (RGC) death after optic nerve damage. BAX activation occurs in two stages including translocation of latent BAX to the mitochondrial outer membrane (MOM) and then permeabilization of the MOM to facilitate the release of apoptotic signaling molecules. As a critical component of RGC death, BAX is an attractive target for neuroprotective therapies and an understanding of the kinetics of BAX activation and the mechanisms controlling the two stages of this process in RGCs is potentially valuable in informing the development of a neuroprotective strategy. METHODS: The kinetics of BAX translocation were assessed by both static and live-cell imaging of a GFP-BAX fusion protein introduced into RGCs using AAV2-mediated gene transfer in mice. Activation of BAX was achieved using an acute optic nerve crush (ONC) protocol. Live-cell imaging of GFP-BAX was achieved using explants of mouse retina harvested 7 days after ONC. Kinetics of translocation in RGCs were compared to GFP-BAX translocation in 661W tissue culture cells. Permeabilization of GFP-BAX was assessed by staining with the 6A7 monoclonal antibody, which recognizes a conformational change in this protein after MOM insertion. Assessment of individual kinases associated with both stages of activation was made using small molecule inhibitors injected into the vitreous either independently or in concert with ONC surgery. The contribution of the Dual Leucine Zipper-JUN-N-Terminal Kinase cascade was evaluated using mice with a double conditional knock-out of both Mkk4 and Mkk7. RESULTS: ONC induces the translocation of GFP-BAX in RGCs at a slower rate and with less intracellular synchronicity than 661W cells, but exhibits less variability among mitochondrial foci within a single cell. GFP-BAX was also found to translocate in all compartments of an RGC including the dendritic arbor and axon. Approximately 6% of translocating RGCs exhibited retrotranslocation of BAX immediately following translocation. Unlike tissue culture cells, which exhibit simultaneous translocation and permeabilization, RGCs exhibited a significant delay between these two stages, similar to detached cells undergoing anoikis. Translocation, with minimal permeabilization could be induced in a subset of RGCs using an inhibitor of Focal Adhesion Kinase (PF573228). Permeabilization after ONC, in a majority of RGCs, could be inhibited with a broad spectrum kinase inhibitor (sunitinib) or a selective inhibitor for p38/MAPK14 (SB203580). Intervention of DLK-JNK axis signaling abrogated GFP-BAX translocation after ONC. CONCLUSIONS: A comparison between BAX activation kinetics in tissue culture cells and in cells of a complex tissue environment shows distinct differences indicating that caution should be used when translating findings from one condition to the other. RGCs exhibit both a delay between translocation and permeabilization and the ability for translocated BAX to be retrotranslocated, suggesting several stages at which intervention of the activation process could be exploited in the design of a therapeutic strategy.

BAX activation in mouse retinal ganglion cells occurs in two temporally and mechanistically distinct steps.

Background Pro-apoptotic BAX is a central mediator of retinal ganglion cell (RGC) death after optic nerve damage. BAX activation occurs in two stages including translocation of latent BAX to the mitochondrial outer membrane (MOM) and then permeabilization of the MOM to facilitate the release of apoptotic signaling molecules. As a critical component of RGC death, BAX is an attractive target for neuroprotective therapies and an understanding of the kinetics of BAX activation and the mechanisms controlling the two stages of this process in RGCs is potentially valuable in informing the development of a neuroprotective strategy. Methods The kinetics of BAX translocation were assessed by both static and live-cell imaging of a GFP-BAX fusion protein introduced into RGCs using AAV2-mediated gene transfer in mice. Activation of BAX was achieved using an acute optic nerve crush (ONC) protocol. Live-cell imaging of GFP-BAX was achieved using explants of mouse retina harvested 7 days after ONC. Kinetics of translocation in RGCs were compared to GFP-BAX translocation in 661W tissue culture cells. Permeabilization of GFP-BAX was assessed by staining with the 6A7 monoclonal antibody, which recognizes a conformational change in this protein after MOM insertion. Assessment of individual kinases associated with both stages of activation was made using small molecule inhibitors injected into the vitreous either independently or in concert with ONC surgery. The contribution of the Dual Leucine Zipper-JUN-N-Terminal Kinase cascade was evaluated using mice with a double conditional knock-out of both Mkk4 and Mkk7 . Results ONC induces the translocation of GFP-BAX in RGCs at a slower rate and with less intracellular synchronicity than 661W cells, but exhibits less variability among mitochondrial foci within a single cell. GFP-BAX was also found to translocate in all compartments of an RGC including the dendritic arbor and axon. Approximately 6% of translocating RGCs exhibited retrotranslocation of BAX immediately following translocation. Unlike tissue culture cells, which exhibit simultaneous translocation and permeabilization, RGCs exhibited a significant delay between these two stages, similar to detached cells undergoing anoikis. Translocation, with minimal permeabilization could be induced in a subset of RGCs using an inhibitor of Focal Adhesion Kinase (PF573228). Permeabilization after ONC, in a majority of RGCs, could be inhibited with a broad spectrum kinase inhibitor (sunitinib) or a selective inhibitor for p38/MAPK14 (SB203580). Intervention of DLK-JNK axis signaling abrogated GFP-BAX translocation after ONC. Conclusions A comparison between BAX activation kinetics in tissue culture cells and in cells of a complex tissue environment shows distinct differences indicating that caution should be used when translating findings from one condition to the other. RGCs exhibit both a delay between translocation and permeabilization and the ability for translocated BAX to be retrotranslocated, suggesting several stages at which intervention of the activation process could be exploited in the design of a therapeutic strategy.

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.

Targeting HDAC3 in the DBA/2J spontaneous mouse model of glaucoma.

High intraocular pressure (IOP) is the most common risk factor associated with glaucoma in humans. While lowering IOP is effective at reducing the rate of retinal ganglion cell (RGC) loss, to date, no treatment exists to directly preserve these cells affected by damage to the optic nerve. Recently, histone deacetylase-3 (HDAC3) has become a potential therapeutic target because it plays an important role in the early nuclear atrophic events that precede RGC death. Conditional knockout or inhibition of HDAC3 prevents histone deacetylation, heterochromatin formation, apoptosis, and eventual RGC loss following acute optic nerve injury. Using these approaches to repress HDAC3 activity, we tested whether targeting HDAC3 protects RGCs from ganglion cell-specific BRN3A expression loss, total somatic cell loss, and optic nerve degeneration in the DBA/2J mouse model of spontaneous glaucoma. Targeted ablation of Hdac3 activity did not protect RGCs from axonal degeneration or somatic cell death in the DBA/2J mouse model of glaucoma. However, inhibition of HDAC3 activity using RGFP966 conferred mild protection against somatic cell loss in the ganglion cell layer in aged DBA/2J mice. Further experimentation is necessary to determine whether other class I HDACs may serve as potential therapeutic targets in chronic models of glaucoma.

Targeting HDAC3 Activity with RGFP966 Protects Against Retinal Ganglion Cell Nuclear Atrophy and Apoptosis After Optic Nerve Injury.

PURPOSE: HDAC3 regulates nuclear atrophy as an early response to axonal injury in retinal ganglion cells (RGCs) following optic nerve crush (ONC). Since conditional knockout of Hdac3 prevents nuclear atrophy post ONC, HDAC3 selective inhibition with RGFP966 through localized and systemic dosing of RGFP966 is necessary for application to acute and chronic models of optic nerve injury. METHODS: C57BL/6 mice were injected intravitreally with 1-10 µM RGFP966 immediately following ONC, and retinas were analyzed at 5, 7, and 14 days for metrics of nuclear atrophy and cell loss. Mice were similarly assessed after intraperitoneal (IP) injections with RGFP966 doses of 2-10 mg/kg, and eyes were harvested at 5, 14, and 28 days after ONC. H&E and BrdU staining were used to analyze toxicity to off-target tissues after 14 days of daily treatment with RGFP966. RESULTS: A single intravitreal injection of RGFP966 prevented histone deacetylation, heterochromatin formation, apoptosis, and DNA damage at 5 and 7 days post ONC. After IP injection, RGFP966 bioavailability in the retina reached peak concentration within 1 h after injection and then rapidly declined. A single IP injection of 2-10 mg/kg RGFP966, significantly prevented histone deacetylation. Repeated IP injections of 2 mg/kg RGFP966 over the course of 2 and 4 weeks post ONC prevented RGC loss. There were no significant toxic or antiproliferative effects to off-target tissues in mice treated daily for 14 days with RGFP966. CONCLUSION: Inhibition of HDAC3 activity with systemic dosing of RGFP966 prevents apoptosis-related histone deacetylation and attenuates RGC loss after acute optic nerve injury.

AAV2-Mediated Transduction of the Mouse Retina After Optic Nerve Injury.

Purpose: Gene therapy of retinal ganglion cells (RGCs) has promise as a powerful therapeutic for the rescue and regeneration of these cells after optic nerve damage. However, early after damage, RGCs undergo atrophic changes, including gene silencing. It is not known if these changes will deleteriously affect transduction and transgene expression, or if the therapeutic protein can influence reactivation of the endogenous genome. Methods: Double-transgenic mice carrying a Rosa26-(LoxP)-tdTomato reporter, and a mutant allele for the proapoptotic Bax gene were reared. The Bax mutant blocks apoptosis, but RGCs still exhibit nuclear atrophy and gene silencing. At times ranging from 1 hour to 4 weeks after optic nerve crush (ONC), eyes received an intravitreal injection of AAV2 virus carrying the Cre recombinase. Successful transduction was monitored by expression of the tdTomato reporter. Immunostaining was used to localize tdTomato expression in select cell types. Results: Successful transduction of RGCs was achieved at all time points after ONC using AAV2 expressing Cre from the phosphoglycerate kinase (Pgk) promoter, but not the CMV promoter. ONC promoted an increase in the transduction of cell types in the inner nuclear layer, including Müller cells and rod bipolar neurons. There was minimal evidence of transduction of amacrine cells and astrocytes in the inner retina or optic nerve. Conclusions: Damaged RGCs can be transduced and at least some endogenous genes can be subsequently activated. Optic nerve damage may change retinal architecture to allow greater penetration of an AAV2 virus to transduce several additional cell types in the inner nuclear layer.

Live-cell imaging to measure BAX recruitment kinetics to mitochondria during apoptosis.

The pro-apoptotic BCL2 gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous BAK or BAX genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies.

BAX to basics: How the BCL2 gene family controls the death of retinal ganglion cells.

Retinal ganglion cell (RGC) death is the principal consequence of injury to the optic nerve. For several decades, we have understood that the RGC death process was executed by apoptosis, suggesting that there may be ways to therapeutically intervene in this cell death program and provide a more direct treatment to the cells and tissues affected in diseases like glaucoma. A major part of this endeavor has been to elucidate the molecular biological pathways active in RGCs from the point of axonal injury to the point of irreversible cell death. A major component of this process is the complex interaction of members of the BCL2 gene family. Three distinct family members of proteins orchestrate the most critical junction in the apoptotic program of RGCs, culminating in the activation of pro-apoptotic BAX. Once active, BAX causes irreparable damage to mitochondria, while precipitating downstream events that finish off a dying ganglion cell. This review is divided into two major parts. First, we summarize the extent of knowledge of how BCL2 gene family proteins interact to facilitate the activation and function of BAX. This area of investigation has rapidly changed over the last few years and has yielded a dramatically different mechanistic understanding of how the intrinsic apoptotic program is run in mammalian cells. Second, we provided a comprehensive analysis of nearly two decades of investigation of the role of BAX in the process of RGC death, much of which has provided many important insights into the overall pathophysiology of diseases like glaucoma.

Retinal glial responses to optic nerve crush are attenuated in Bax-deficient mice and modulated by purinergic signaling pathways.

BACKGROUND: Retinal ganglion cell (RGC) soma death is a consequence of optic nerve damage, including in optic neuropathies like glaucoma. The activation of the innate immune network in the retina after nerve damage has been linked to RGC pathology. Since the eye is immune privileged, innate immune functions are the responsibility of the glia, specifically the microglia, astrocytes, and Müller cells that populate the retina. Glial activation, leading to the production of inflammatory cytokines, is a hallmark feature of retinal injury resulting from optic nerve damage and purported to elicit secondary degeneration of RGC somas. METHODS: A mouse model of optic nerve crush (ONC) was used to study retinal glial activation responses. RGC apoptosis was blocked using Bax-deficient mice. Glial activation responses were monitored by quantitative PCR and immunofluorescent labeling in retinal sections of activation markers. ATP signaling pathways were interrogated using P2X receptor agonists and antagonists and Pannexin 1 (Panx1)-deficient mice with RGC-specific deletion. RESULTS: ONC induced activation of both macroglia and microglia in the retina, and both these responses were dramatically muted if RGC death was blocked by deletion of the Bax gene. Macroglial, but not microglial, activation was modulated by purinergic receptor activation. Release of ATP after optic nerve damage was not mediated by PANX1 channels in RGCs. CONCLUSIONS: RGC death in response to ONC plays a principal stimulatory role in the retinal glial activation response.

Role of HDACs in optic nerve damage-induced nuclear atrophy of retinal ganglion cells.

Optic neuropathies are characterized by retinal ganglion cell (RGC) death, resulting in the loss of vision. In glaucoma, the most common optic neuropathy, RGC death is initiated by axonal damage, and can be modeled by inducing acute axonal trauma through procedures such as optic nerve crush (ONC) or optic nerve axotomy. One of the early events of RGC death is nuclear atrophy, and is comprised of RGC-specific gene silencing, histone deacetylation, heterochromatin formation, and nuclear shrinkage. These early events appear to be principally regulated by epigenetic mechanisms involving histone deacetylation. Class I histone deacetylases HDACs 1, 2, and 3 are known to play important roles in the process of early nuclear atrophy in RGCs, and studies using both inhibitors and genetic ablation of Hdacs also reveal a critical role in the cell death process. Select inhibitors, such as those being developed for cancer therapy, may also provide a viable secondary treatment option for optic neuropathies.

Tumor necrosis factor alpha has an early protective effect on retinal ganglion cells after optic nerve crush.

BACKGROUND: Glaucoma is an optic neuropathy that is characterized by the loss of retinal ganglion cells (RGCs) initiated by damage to axons in the optic nerve. The degeneration and death of RGCs has been thought to occur in two waves. The first is axogenic, caused by direct insult to the axon. The second is somatic, and is thought to be caused by the production of inflammatory cytokines from the activated retinal innate immune cells. One of the cytokines consistently linked to glaucoma and RGC damage has been TNFα. Despite strong evidence implicating this protein in neurodegeneration, a direct injection of TNFα does not mimic the rapid loss of RGCs observed after acute optic nerve trauma or exposure to excitotoxins. This suggests that our understanding of TNFα signaling is incomplete. METHODS: RGC death was induced by optic nerve crush in mice. The role of TNFα in this process was examined by quantitative PCR of Tnfα gene expression, and quantification of cell loss in Tnfα (-/-) mice or in wild-type animals receiving an intraocular injection of exongenous TNFα either before or after crush. Signaling pathways downstream of TNFα were examined by immunolabeling for JUN protein accumulation or activation of EGFP expression in NFκB reporter mice. RESULTS: Optic nerve crush caused a modest increase in Tnfα gene expression, with kinetics similar to the activation of both macroglia and microglia. A pre-injection of TNFα attenuated ganglion cell loss after crush, while ganglion cell loss was more severe in Tnfα (-/-) mice. Conversely, over the long term, a single exposure to TNFα induced extrinsic apoptosis in RGCs. Müller cells responded to exogenous TNFα by accumulating JUN and activating NFκB. CONCLUSION: Early after optic nerve crush, TNFα appears to have a protective role for RGCs, which may be mediated through Müller cells.

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.

Spink2 modulates apoptotic susceptibility and is a candidate gene in the Rgcs1 QTL that affects retinal ganglion cell death after optic nerve damage.

The Rgcs1 quantitative trait locus, on mouse chromosome 5, influences susceptibility of retinal ganglion cells to acute damage of the optic nerve. Normally resistant mice (DBA/2J) congenic for the susceptible allele from BALB/cByJ mice exhibit susceptibility to ganglion cells, not only in acute optic nerve crush, but also to chronic inherited glaucoma that is characteristic of the DBA/2J strain as they age. SNP mapping of this QTL has narrowed the region of interest to 1 Mb. In this region, a single gene (Spink2) is the most likely candidate for this effect. Spink2 is expressed in retinal ganglion cells and is increased after optic nerve damage. This gene is also polymorphic between resistant and susceptible strains, containing a single conserved amino acid change (threonine to serine) and a 220 bp deletion in intron 1 that may quantitatively alter endogenous expression levels between strains. Overexpression of the different variants of Spink2 in D407 tissue culture cells also increases their susceptibility to the apoptosis-inducing agent staurosporine in a manner consistent with the differential susceptibility between the DBA/2J and BALB/cByJ strains.

Evaluation of the percentage of ganglion cells in the ganglion cell layer of the rodent retina.

PURPOSE: Retinal ganglion cells comprise a percentage of the neurons actually residing in the ganglion cell layer (GCL) of the rodent retina. This estimate is useful to extrapolate ganglion cell loss in models of optic nerve disease, but the values reported in the literature are highly variable depending on the methods used to obtain them. METHODS: We tested three retrograde labeling methods and two immunostaining methods to calculate ganglion cell number in the mouse retina (C57BL/6). Additionally, a double-stain retrograde staining method was used to label rats (Long-Evans). The number of total neurons was estimated using a nuclear stain and selecting for nuclei that met specific criteria. Cholinergic amacrine cells were identified using transgenic mice expressing Tomato fluorescent protein. Total neurons and total ganglion cell numbers were measured in microscopic fields of 10(4) µm(2) to determine the percentage of neurons comprising ganglion cells in each field. RESULTS: Historical estimates of the percentage of ganglion cells in the mouse GCL range from 36.1% to 67.5% depending on the method used. Experimentally, retrograde labeling methods yielded a combined estimate of 50.3% in mice. A retrograde method also yielded a value of 50.21% for rat retinas. Immunolabeling estimates were higher at 64.8%. Immunolabeling may introduce overestimates, however, with non-specific labeling effects, or ectopic expression of antigens in neurons other than ganglion cells. CONCLUSIONS: Since immunolabeling methods may overestimate ganglion cell numbers, we conclude that 50%, which is consistently derived from retrograde labeling methods, is a reliable estimate of the ganglion cells in the neuronal population of the GCL.

Nuclear atrophy of retinal ganglion cells precedes the bax-dependent stage of apoptosis.

PURPOSE: Retinal ganglion cells atrophy during the execution of the intrinsic apoptotic program. This process, which has been termed the apoptotic volume decrease (AVD) in other cell types, has not been well-characterized in ganglion cells. METHODS: Acute optic nerve crush was used to examine neuronal atrophy in the ganglion cell layer in wild-type and Bax-deficient mice. Nuclear size was measured from retinal wholemounts. Heterochromatin formation was assessed using transmission electron microscopy, whereas histone H4 acetylation was monitored using immunofluoresence. Ganglion cell and retinal transcript abundance was measured using quantitative PCR. RESULTS: Nuclear and soma sizes linearly correlated in both control and damaged retinas. Cells in wild-type mice exhibited nuclear atrophy within 1 day after optic nerve damage. Three days after crush, nuclear atrophy was restricted to ganglion cells identified by retrograde labeling, while amacrine cells also exhibited some atrophy by 5 days. Similar kinetics of nuclear atrophy were observed in cells deficient for the essential proapoptotic gene Bax. Bax-deficient cells also exhibited other nuclear changes common in wild-type cells, including the deacetylation of histones, formation of heterochromatin, and the silencing of ganglion cell-specific gene expression. CONCLUSIONS: Retinal ganglion cell somas and nuclei undergo the AVD in response to optic nerve damage. Atrophy is rapid and precedes the Bax-dependent committed step of the intrinsic apoptotic pathway.

Regulated Expression of Chromobox Homolog 5 Revealed in Tumors of Apc(Min) (/+) ROSA11 Gene Trap Mice.

The gene-trap lacZ reporter insertion, ROSA11, in the Cbx5 mouse gene illuminates the regulatory complexity of this locus in Apc(Min) (/+) mice. The insertion site of the ß-Geo gene-trap element lies in the 24-kb intron proximal to the coding region of Cbx5. Transcript analysis indicates that two promoters for Cbx5 flank this insertion site. Heterozygotes for the insertion express lacZ widely in fetal tissues but show limited expression in adult tissues. In the intestine, strong expression is limited to proliferative zones of crypts and tumors. Homozygotes for ROSA11, found at a lower than Mendelian frequency, express reduced levels of the coding region transcript in normal tissues, using a downstream promoter. Analysis via real-time polymerase chain reaction indicates that the upstream promoter is the dominant promoter in normal epithelium and tumors. Bioinformatic analysis of the Cbx5 locus indicates that WNT and its target transcription factor MYC can establish a feedback loop that may play a role in regulating the self-renewal of the normal intestinal epithelium and its tumors.

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.

Mouse models of retinal ganglion cell death and glaucoma.

Once considered too difficult to use for glaucoma studies, mice are now becoming a powerful tool in the research of the molecular and pathological events associated with this disease. Often adapting technologies first developed in rats, ganglion cell death in mice can be induced using acute models and chronic models of experimental glaucoma. Similarly, elevated IOP has been reported in transgenic animals carrying defects in targeted genes. Also, one group of mice, from the DBA/2 line of inbred animals, develops a spontaneous optic neuropathy with many features of human glaucoma that is associated with IOP elevation caused by an anterior chamber pigmentary disease. The advent of mice for glaucoma research is already having a significant impact on our understanding of this disease, principally because of the access to genetic manipulation technology and genetics already well established for these animals.

Involvement of the Bcl2 gene family in the signaling and control of retinal ganglion cell death.

Retinal ganglion cell death by apoptosis is a well-established outcome in the glaucomatous pathology of the retina. Extensive research into the molecular events underlying this process show us that members of the Bcl2 gene family play a critical role in the activation and control of ganglion cell death. Perhaps the most critical molecule at play is the pro-apoptotic protein BAX. Without BAX, ganglion cell somas appear to survive an optic nerve insult indefinitely. Once BAX is activated, however, the cell death program reaches an irreversible point, where the process cannot be blocked. Interacting with BAX are other members of this larger gene family, including the anti-apoptotic protein BCL-X, and several members of the BH3-only proteins that serve as sensors and activators of the cell death program. A hypothetical model of how all these molecules interact in glaucoma is presented.