Histone Deacetylase Activity Regulates Apaf-1 and Caspase 3 Expression in the Developing Mouse Retina

Abstract

purpose. Apoptosis is a form of programmed cell death essential for both tissue development and maintenance of tissue homeostasis. Apoptosis protease activating factor (Apaf)-1 and caspase 3 are downregulated in the retina during postnatal development. The decreased expression of these genes is potentially a critical survival strategy adopted to protect against accidental cell death. The purpose of this study was to investigate the transcriptional mechanism involved in the downregulation of Apaf-1 and caspase 3.

methods. SDS-polyacrylamide gel electrophoresis and semiquantitative PCR were used to examine Apaf-1 and caspase 3 expression levels during development. TdT-mediated dUTP nick-end labeling (TUNEL) and DNA laddering were used to identify cells undergoing apoptosis.

results. A decrease in expression of Apaf-1 and caspase 3 during retinal development correlated with a decreased susceptibility to an apoptotic stimulus. Furthermore, treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), resulted in widespread hyperacetylation in the retina, coinciding with transcriptional activation of Apaf-1 and caspase 3 and subsequent induction of apoptosis in postnatal day (P)5 and P15 retinas. However, inhibition of HDAC activity is not sufficient to induce apoptosis in the mature retina (P60).

conclusions. Overall, these results elicit the conclusion that downregulation of Apaf-1 and caspase 3 in the developing retina correlates with a decreased susceptibility to apoptotic stimuli and ensures the survival of the retina. Furthermore, the authors propose that, in the early postnatal retina, HDAC activity governs the transcriptional regulation of these genes. Upregulation of Apaf-1 and caspase 3 coincides with an induction of apoptosis. In the mature retina transcriptional activation of these genes or induction of apoptosis was not observed.

Apoptosis is a physiological process that is essential for both tissue development and maintenance of homeostasis. The cellular demolition that is achieved via this programmed cell death pathway is largely performed by a family of cysteine proteases called caspases. One major pathway to caspase activation involves the formation of a multiprotein complex known as the apoptosome. This pathway is activated by death stimuli that induce the release of mitochondrial cytochrome c into the cytosol. Once in the cytosol, it is free to bind Apaf-1 leading to the assembly of the apoptosome. Procaspase-9 is recruited to Apaf-1 by binding to an exposed CARD domain in the Apaf-1 protein. Mature caspase-9 remains attached to the apoptosome where it recruits and activates downstream caspases, such as caspase 3, resulting in the controlled demise of the cell. ¹  

Apaf-1 is a central protein of the intrinsic apoptotic pathway and is the core molecule in the formation of the apoptosome. Indeed Apaf-1-deficient cells are resistant to a variety of apoptotic insults. However, mice deficient in Apaf-1 exhibit reduced neuronal apoptosis; severe developmental defects, including enlarged brains; and embryonic mortality. ^{2 3} However, it is worth noting that some Apaf-1-knockout mice survive until adulthood, and apoptosis in postmitotic neurons lacking Apaf-1 can proceed normally. ^{4 5} These results suggest that in contrast to neuronal precursors in which Apaf-1 appears to play an essential role in developmental apoptosis, Apaf-1 and apoptosome activation may be dispensable for apoptosis of postmitotic neurons. In addition, evidence supports a role for caspase-3 in development. Caspase-3 mutant mice are born at a lower frequency than their littermates and have cerebral overgrowth, retinal hyperplasia, disorganized cell deployment, and a high level of perinatal death. ⁶ In summary, the pivotal role of caspase activation and Apaf-1 expression in the apoptotic pathway, especially in development, was evident from the observed phenotypes when knockouts were generated. Both caspase-9 ^{6 7} and Apaf-1 knockouts ^{2 3} die perinatally, whereas caspase-3 knockouts ⁶ die within weeks of birth. 

Previous results from this laboratory have demonstrated the downregulation in expression of key apoptotic modulators including Apaf-1 and caspase 3 in the retina during postnatal development. ⁸ This observation raises several interesting questions relating to the role of Apaf-1 and caspase 3 during developmental and degenerating processes. Neurons are postmitotic cells, and the body must put systems in place to try to prevent the death of these cells, since they cannot be replaced. One very effective way of achieving this is to downregulate the caspase-mediated cell death pathway, which has been described in the literature as being operational in prenatal development, particularly in the brain. ⁹ How this is achieved in the retina is as yet to be determined. In this study, we address the question of how these apoptotic genes are regulated in the developing retina. 

Reversible histone acetylation and deacetylation are epigenetic phenomena that have a crucial role in the modulation of chromatin structure and, more important, in gene expression. Chromosome structure alteration, particularly chromatin remodeling, has an important role in transcriptional regulation. ^{10 11 12} Acetylation status has been shown to effect transcriptional activation in eukaryotic cells ¹⁰ where acetylated histones are associated with transcriptional activation. ¹¹ Furthermore, it has been reported that histone deacetylase (HDAC) activity controls selective gene transcription during neuronal apoptosis. ¹³  

Here, we have investigated the possibility of HDAC-mediated transcriptional repression of Apaf-1 and caspase 3 in the developing mouse retina. Results showed that acetylation status is indeed an important factor in determining transcriptional activity of these genes in the retina. Furthermore, we found that treatment with the HDAC inhibitor trichostatin A (TSA) reverses this transcriptional repression and induces re-expression of the downregulated genes. However, the mature retina appears to be resistant to TSA-induced re-expression and apoptosis, indicating the presence of an additional control mechanism to ensure survival of the adult retina. 

Materials and Methods

Animal Treatment

All experiments were performed in accordance with the ARVO Statement for Use of Animals in Ophthalmic and Vision Research. C57BL/6 mice were obtained from Harlan UK (Bicester, UK). 

Western Blot Analysis

Total protein from either whole retinas or retinal explants was obtained by lysing in RIPA buffer (50 mM Tris-HCl [pH 7.4], 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM sodium orthovanadate, and 1 mM sodium fluoride) containing antipain (1 μg/mL), aprotinin (1 μg/mL), chymostatin (1 μg/mL), leupeptin (0.1 μg/mL), pepstatin (1 μg/mL), and phenylmethylsulfonyl fluoride (PMSF; 0.1 mM). The total amount of protein in each sample was determined by protein assay (Bio-Rad, Hemel Hempstead, UK) using bovine serum albumin as a standard. Between 30 and 40 μg of total protein was electrophoresed on polyacrylamide gels followed by transfer to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) and incubated overnight with the appropriate antibodies. Apaf-1 (Apotech 13f11 APO-20A-06), anti-acetylated H4 (Upstate Biotechnology, Lake Placid, NY), caspase 3 (both pro- and cleaved forms; 9226; Cell Signaling Technology, Beverly, MA) caspase 3 (9661s, cleaved form only; Cell Signaling Technology), and tubulin (T5168; Sigma-Aldrich, Poole, UK). It should be noted that the caspase 3 antibody that detects the procaspase 3 and cleaved form (9226; Cell Signaling) failed to detect cleaved caspase 3 at P15. Therefore we used an antibody specific for the cleaved caspase 3 fragment (9661s; Cell Signaling Technology). 

Membrane development was achieved using enhanced chemiluminescence (ECL; GE Healthcare, Buckinghamshire, UK). 

Retinal Explants

C57BL/6 mice at P5, P10, P15, and P60 were decapitated and the eyes removed. Cleaning with 70% ethanol was followed by incubation in basal medium supplemented with proteinase K (Sigma-Aldrich) at 37°C for 15 minutes. The anterior segment, vitreous body, and sclera were removed and the retina mounted on nitrocellulose inserts (Millicell; Millipore, Billerica, MA) photoreceptor-side down. Explants were cultured without RPE in 1.2 mL of R16 medium (gift from Theo van Veen, Department of Ophthalmology, Wallenberg Retina Centre, Lund University, Lund, Sweden) without FCS. Retinal explants were treated with 100 nM TSA in dimethyl sulfoxide (DMSO) for 20 hours or exposed to UV light for 7 minutes followed by a 24-hour recovery. Explants were then fixed in neutral-buffered formalin followed by cryoprotection in 25% sucrose and TUNEL. 

TUNEL of Fragmented DNA

DNA strand breaks in photoreceptor nuclei were detected by TUNEL. Retinal explants were fixed in 10% buffered formalin for 24 hours and cryoprotected in 25% sucrose overnight. Cryosections were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 2 minutes on ice then washed three times in PBS. The explant sections were then incubated in 50 μL of reaction buffer containing 2.5 mM CoCl₂, 0.1 U/mL TdT (Promega, Madison, WI) in a 0.1 M Na cacodylate (pH 7.0) buffer and 0.75 nM fluorescein-12-dUTP (Roche Diagnostics). The sections were then incubated at 37°C for 1 hour in a humid chamber. After several washes in PBS, the sections were mounted (MoWiol; Calbiochem, La Jolla, CA) and viewed under a fluorescence microscope with a fluorescein isothiocyanate filter. Sections were counterstained with Hoechst. 

Reverse Transcription–Polymerase Chain Reaction Analysis

Semiquantitative PCR was performed on total RNA extracted from P6, P10, P15, and P60 whole retinas and P5, P15, and P60 retinal explants after treatment with 100 nM TSA for 20 hours. Total RNA was isolated (TRIzol Reagent; Invitrogen, Little Chalfont, UK) according to the protocol suggested by the manufacturers. The product was then reverse transcribed to cDNA by using M-MLV reverse transcriptase and oligo(dT) (Promega). To ensure linear signals, all products were optimized for both cycle number product amplification and equal loading, by using tubulin as a control. 

Primers were as follows: tubulin forward, 5′-TCGTATCCACTTCCCTCTGG-3′, tubulin reverse, 5′-AGCTTGGGGTCTCTGTCAAA-3′; Apaf-1 forward 5′-AGCTGATGGGAAGACACTGATTTC-3′, Apaf-1 reverse, 5′-CTGGAGATGACAATGGAGAAA-3′; and caspase 3 forward, 5′-ATTCAGGCCTGCCGGGGTAC-3′, caspase 3 reverse, 5′-AGTTCTTTCGTGAGCATGGA-3′. 

Histone Extraction

Retinal explants were suspended and snap-frozen three times in reticulocyte standard buffer (RSB; 10 mM Tris-HCl [pH 7.4], 10 mM NaCl, and 3 mM MgCl₂) and then centrifuged. The resultant pellet was suspended in 20 μL of RSB and 0.5% NP40, then placed on ice for 10 minutes. After centrifugation, the nuclei were suspended in 10 μL of 5 mM MgCl₂ and 0.8 M HCl. Histones were then extracted by placing samples on ice for 20 minutes and isolated by removing the supernatant after centrifugation. TCA (25%) was then added to precipitate the histones. The precipitated histones were washed in ice-cold acetone, air dried, and suspended in sterile ddH₂O. Total protein samples were then separated on 15% SDS-polyacrylamide gels and processed to observe acetylated H4 histones. 

DNA Laddering

Retinal DNA was isolated after phenol-chloroform extraction. Briefly, explants treated with 100 nM TSA for 20 hours or left untreated, were placed in 150 μL of lysis buffer (20 mM EDTA, 100 mM Tris, and 0.8% sodium lauryl sarcosinate) containing proteinase K (20μg/mL) and incubated at 50°C for 18 hours, vortexing occasionally. The retinal DNA was then extracted by the phenol-chloroform method, precipitated by ethanol and the pellet redissolved in Tris-EDTA. Total RNase-treated DNA was visualized by including ethidium bromide (0.5 μg/mL) in the agarose and observed by illumination on a 302 nm UV transilluminator. The molecular size standard was a 100-bp ladder. 

Statistical Analysis

Data are given as mean ± SD. Statistical significance was evaluated by Student’s t-test for comparisons between groups. Differences were considered significant for P < 0.05. 

Results

Downregulation of Apaf-1 and Caspase 3 in the Developing Retina and Differential Sensitivity to Cell Death

In our recent studies, we observed the downregulation in expression of Apaf-1 and caspase 3 protein in the retina during postnatal development. ⁸ In this study, we wished to determine whether this was a transcriptional event. As can be seen in Figure 1 , both Apaf-1 and caspase 3 levels were dramatically downregulated at both the protein (Fig. 1A)and mRNA levels (Fig. 1B)as the retina developed. The observed downregulation of these key apoptotic genes correlated with the inability of various apoptotic stimuli to induce death in the mature retina. This is illustrated in Figure 2using ultraviolet (UV) irradiation as a death stimulus which has been reported to induce DNA damage and trigger the intrinsic mitochondrial pathway. ⁷ Retinal explants were exposed to UV irradiation for 7 minutes and apoptotic cells detected by TUNEL after 24 hours. We observed a decrease in TUNEL-positive cells between P10 and P15, correlating with both protein and mRNA expression levels (Figs. 1) . Furthermore, the mature P60 (Fig. 2C)retina appeared to be resistant to this stimulus as indicated by the lack of TUNEL-labeling correlating with the diminished levels of Apaf-1 and caspase 3. 

Quantitative data in Figure 3clearly illustrate the correlation between the downregulation of Apaf-1 and caspase 3 and cell death, as detected by TUNEL-positive staining. After UV treatment, the number of dead cells per field of view at P10 (162.2 ± 14.2) and P15 (63 ± 9) increased significantly (P < 0.05) over control counts (39.17 ± 3.65 and 26.5 ± 3.7, respectively). However, in contrast, at P60, UV-treated explants showed no significant increase (P > 0.05) in the number of dead cells (3.17 ± 2.13) compared with the control (3.3 ± 2.5). Furthermore, there was a significant decrease (P < 0.05) in the number of TUNEL-positive cells between P10 (162.2 ±14.2) and P15 (63 ± 9), indicating a differential susceptibility to a mitochondrial-targeting apoptotic stimulus. 

TSA Induction of Histone Hyperacetylation in the Retina and Re-expression of Apaf-1 and Caspase 3

It has been reported that the expression of Apaf-1 and caspase 3 can be regulated by HDAC activity. ^{13 14} Thus far, we have shown downregulation of Apaf-1 and caspase 3 during development. We therefore sought to determine whether HDAC activity played a role in the repression of these genes. 

First, we sought to determine whether treatment with TSA, a specific and potent inhibitor of HDAC activity when used at nanomolar range, would result in hyperacetylation of histones in the retina. We isolated histones from untreated P5, P15, and P60 retinal explants or explants that had been treated with 2.5 or 100 nM TSA for 20 hours (this time point and the concentrations were chosen based on published data ¹³ ) and probed with an antibody that detects the acetylated form of H4. At P5, Apaf-1 and caspase 3 were highly expressed compared with P15 and P60 when developmental apoptosis is no longer occurring and these proteins are downregulated (Fig. 1) . We initially examined the acetylation status of H4 to verify that TSA induced hyperacetylation. As can be seen from Figure 4 , isolated histones from TSA-treated explants exhibited an extensive dose-dependent acetylation of H4 compared with control nontreated histones. Included in this figure are also ponceau-S-stained blots of isolated histones for equal loading. 

We wanted to determine whether Apaf-1 and caspase 3 are also upregulated after inhibition of HDAC activity, as previously described for other neuronal systems. ¹⁵ Other inhibitors of HDAC activity such as suberoylanilide hydroxamic acid (SAHA) and depsipeptide have also been shown to induce upregulation of Apaf-1 and caspase 3 in other systems. ¹⁴ Total RNA was isolated from control and TSA-treated retinal explants, reverse-transcribed and subjected to semiquantitative polymerase chain reaction with gene-specific primers to Apaf-1 and caspase 3. We observed a clear induction of Apaf-1 and caspase 3 transcripts at P5 and P15 after TSA treatment. However, mRNA levels of Apaf-1 and caspase 3 remained unchanged at P60 (Fig. 5A) . 

Western blot analysis of total retinal lysates in Figures 5B and 5Cshowed that Apaf-1 and procaspase 3 were also upregulated at the protein level in P5 and P15 explants as a result of TSA treatment. However, protein expression levels in P60 explants (Fig. 5E)suggested a complete resistance to re-expression of Apaf-1 and procaspase 3 in accordance with unchanged mRNA expression levels between control and TSA-treated explants (Fig. 5A) . Furthermore, in P5 explants, we observe the appearance of a caspase-3 cleavage product (p20) with only low levels of this active fragment present at P15 (Fig. 5D) . A possible explanation for this is that after treatment with TSA, P5 retinal explants display far greater death than P15 explants. Alternatively, it may that the low level of cleaved caspase 3 present at P15 may explain the observed reduced death. Therefore, as can be seen from Figure 5B , at P5 the pro form of caspase 3 does not appear to be upregulated as there is significant cleavage to the p20 fragment. However, processing of procaspase 3 to the cleaved fragment at P15 is extremely low (Fig. 5D)when compared to P5 and therefore we observe an upregulation of procaspase 3 after treatment with TSA (Fig. 5C) . 

TSA-Induced Apoptosis through Upregulation of Apaf-1 and Caspase 3 Expression

We sought to determine whether TSA-induced hyperacetylation and resultant upregulation of Apaf-1- and caspase 3-induced cell death as previously reported in other neuronal systems. ¹³ TUNEL analysis was performed on P5, P15, and P60 explants treated with 100 nM TSA. Treatment led to extensive TUNEL-positive labeling in both P5 and P15 explants (Fig. 6A vii-x)with less death observed at P15 in agreement with the low levels of active caspase 3 detected (Fig. 5D) . However, P60 samples displayed only background levels of DNA fragmentation (Fig. 6A xi, xii)indicating that TSA treatment was unable to activate cell death in the mature retina. 

To verify that the death detected was apoptosis, we looked for the presence of a DNA ladder. After 20 hours of treatment with 100 nM TSA a typical DNA ladder was observed in both P5 and P15 (Fig. 6B)explants compared with control explants consistent with the observed processing of caspase 3 to the active form. However, there was a notable absence of DNA degradation and ladder formation in genomic DNA isolated from P60 TSA-treated explants. Therefore, we can conclude that after TSA treatment apoptosis can be induced in P5 and P15 retinal explants; however, at P60 the retina appears to be completely resistant. 

Discussion

Programmed cell death clearly plays a critical role in the sculpting of the central nervous system (CNS) as evidenced by embryonic lethality/gross brain malformations in mice where key apoptotic modulators have been genetically eliminated. The retina as part of the nervous system is no different and retinal cells produced in excess during genesis are eliminated by a series of apoptotic waves resulting in the development of a multilayered structure of terminally differentiated cells. Once development is complete, the default apoptotic pathway must be shut down as adult neurons need to survive the life of the organism. ¹⁶ One way of achieving this is to downregulate proapoptotic genes central to the apoptotic cascade. This is exemplified during brain maturation where apoptosis is suppressed through the downregulation of Apaf-1 and caspase 3. However, after traumatic brain injury, there is a coordinated reactivation of Apaf-1 that results in death by apoptosis. ⁹  

In this study we have investigated the novel mechanism by which Apaf-1 and caspase 3 are developmentally downregulated in the retina. Apaf-1 function can be controlled at both the transcriptional level and via several posttranslational mechanisms. At the protein level, heat shock protein family members can effect expression through protein modification. ¹⁷ Furthermore, subcellular localization can play a role in Apaf-1 activity. ¹⁸ The regulation of caspase 3 is also highly complex. HDAC activity plays a role in the activation of caspase-3, as apoptosis was induced in cultured rat cerebellar granule neurons and mouse Neuro-2a neuroblastoma cells after treatment with the HDACi TSA and sodium butyrate. ¹⁹ Both of these inhibitors were found to induce neuronal apoptosis as defined by morphology and caspase-3 activation similar to other neuronal systems investigated. ¹⁴ However, evidence also exists for the posttranscriptional regulation of caspase-3 in rat and mouse adult skeletal muscle. ²⁰ Caspase 3 in skeletal muscle is uniquely regulated at the posttranscriptional level compared with other tissues, possibly as a mechanism to prevent accidental cell death. 

In this study, we have shown that expression of Apaf-1 and caspase 3 are controlled at the transcriptional level during retinal development (Fig. 1B) . We propose that this is an important mechanism for the maintenance of the differentiated postmitotic retina. Previously, we have shown that reduced Apaf-1 levels correlate with decreased susceptibility to cytochrome c–dependent caspase activation. ⁸ In this study we extended these results to show that downregulation of Apaf-1 and caspase 3 correlates with the inability of an apoptotic stimulus to induce death in the mature retina (Fig. 2) . We found that after exposure to UV irradiation, the number of apoptotic cells as detected by TUNEL decreased with the age of the explant (Fig. 3)correlating with expression levels of Apaf-1 and caspase 3 (Figs. 1A 1B) . However, the mature P60 (Fig. 1C)retina appeared to be resistant to this stimulus and other mitochondrial-targeting stimuli tested ²¹ ; therefore, we can conclude that there is a clear correlation between the mature retinas resistance to apoptotic stimuli and the downregulation of Apaf-1 and caspase 3 (Figs. 2 3) . It is worth noting that decreased expression of Apaf-1 and caspase 3 is probably not the only survival strategy used by the mature retina. We have recently reported on the downregulation of proapoptotic Bcl-2 proteins during retinal development, an event that will potentially protect from both caspase-dependent and -independent cell death mediated by factors released from the intermembrane space. ²¹  

The tight packaging of genomic DNA into chromatin is necessary as it facilitates storage in the nucleus. However, this can also prove problematic as this tightly wound form of DNA can prevent access to transcriptional activating proteins. Chromosomal remodeling, such as chromatin modification, is an important mechanism in the control of gene activation and propagation of epigenetic information. A body of experimental data now suggests that HDAC activity may play a role in the activation of apoptotic genes. ^{15 22} One particular report focuses on the ability of TSA, an HDAC inhibitor to induce apoptosis in neurons. ¹³ This occurs through hyperacetylation and subsequent activation of apoptotic genes such as Apaf-1 and caspase 3. 

Herein we have described a role for the acetylation status in the transcriptional activity of Apaf-1 and caspase 3 in the developing retina. First, when treated with TSA we observed dose-dependent hyperacetylation of histones isolated from various age retinal explants (Fig. 4) . A link between HDAC levels and transcriptional activity has been established. ¹⁰ In addition, in our study mRNA and protein levels of Apaf-1 and caspase 3 were upregulated in P5 and P15 retinal explants after treatment with TSA (Figs. 5A 5B 5C 5D) . Activation of these genes in P5 and P15 explants is therefore likely to be due to the inhibition of HDAC activity, resulting in hyperacetylation and transcriptional activation. Whether this acetylation occurs on histones, nonhistone proteins located at responsive elements or whether it is elevated acetylation at promoters is currently under investigation. One possible mechanism is that TSA activates E2F-1, which then may act on the promoters of target genes. ^{23 24} The precise mechanism in place has yet to be elucidated. We also found that in addition to an increase in levels of Apaf-1 and caspase 3 TSA was capable of inducing cell death in both P5 and P15 explants (Fig. 6) . However, in contrast, mature P60 explants did not display an increase in the expression of Apaf-1 and caspase 3 (Figs. 5A 5E)and were resistant to TSA-induced apoptosis (Fig. 6) . Work in our laboratory has found P60 retinas to be extremely resistant to a wide variety of apoptotic stimuli and stresses that target the mitochondria. ²¹ Unlike early postnatal explants, it is likely that mature retinas have a secondary mechanism in addition to HDAC activity to prevent reactivation of apoptotic genes, affording them protection from cell death. In this system, hyperacetylation is not enough per se to result in a transcriptional upregulation of proapoptotic genes and induction of apoptosis. The nature of this secondary mechanism that affords an extra defense from death is presently unknown. However, a possible role for the recruitment of enzymes that alter the methylation status of the relevant promoters cannot be ruled out and is currently under investigation. Many promoters contain CpG islands, stretches of DNA exceeding 200 bp having more than 50% C+G content and a CpG frequency of at least 0.6 of that expected on the basis of C+G content of the region. The CpG status—that is, the methylation status of these regions—is a key factor in the transcriptional regulation of many genes. ²⁵ We can conclude from these results that HDAC activity plays a role in the transcriptional repression of apoptotic genes at P5 and P15. However at P60, inhibition of HDAC activity alone is not sufficient to induce re-expression and cell death, indicating that there may be a secondary defense mechanism present in the adult retina. 

 Supported by Science Foundation Ireland.

 Submitted for publication October 24, 2005; revised March 15, 2006; accepted May 12, 2006.

 Disclosure: D.M. Wallace , None; M. Donova n, None; T.G. Cotter, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Apaf-1 and caspase 3 were downregulated during development. (A) Total protein from various age retinas was isolated and separated on SDS-PAGE gels and probed with an antibodies specific to Apaf-1 and caspase 3. Both Apaf-1 and caspase 3 levels decreased dramatically during development in the retina, particularly at P30 and P60. (B) Semiquantitative analysis of mRNA expression of Apaf-1 and caspase 3 in the developing retina. Total retinal RNA was isolated from P5-P60 mice, reverse transcribed, and subjected to semiquantitative PCR using gene-specific primers to Apaf-1, caspase 3, and tubulin. mRNA levels correlated with protein expression levels. Tubulin was used as a loading control.

Figure 2.

 

Downregulation of Apaf-1 and caspase 3 correlated with decreased susceptibility to an apoptotic stimulus. Retinal explants (P10, P15, and P60) were exposed to ultraviolet light (UV) irradiation for 7 minutes and, after a 24-hour recovery, control and UV-treated explants were sectioned. Apoptosis was detected after treatment by TUNEL labeling. As can be seen P10 and P15 (A, B) explants were susceptible to the mitochondrial-targeting apoptotic stimulus as shown by the presence of TUNEL-positive cells compared with control. However, mature, P60 (C) explants appeared to be completely resistant, with minimal TUNEL-positive cells present. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Figure 3.

 

Number of TUNEL-positive cells in P10, P15, and P60 control and UV-treated retinal explants. The number of dead cells per field of view after UV treatment (UV) at P10 (162.2 ± 14.2) and P15 (63 ± 9) increased significantly (P < 0.05) over the control (UNT; 39.17 ± 3.65 and 26.5 ± 3.7, respectively). In contrast, at P60, UV treated explants showed no significant increase (P > 0.05) in the number of dead cells (3.17 ± 2.13) compared with the control (3.3 ± 2.5). Furthermore, there was a significant decrease (P < 0.05) in the number of TUNEL-positive cells between P10 (162.2 ± 14.26) and P15 (63 ± 9), indicating a differential susceptibility to a mitochondria-targeting apoptotic stimulus.

Figure 4.

 

TSA treatment caused histone hyperacetylation in the mouse retina. Histones were isolated from control and TSA-treated retinal explants (P5, P15, and P60), separated by SDS-PAGE (15%), and probed with an antibody specific for the acetylated form of H4. Treatment with 2.5 nM and 100 nM TSA for 20 hours caused extensive dose-dependent acetylation of H4 compared with the control. Bottom: ponceau S staining of isolated histones for equal loading. All results are representative of three individual experiments.

Figure 5.

 

TSA induced an upregulation of Apaf-1 and caspase 3 expression in P5 and P15 retinal explants, but P60 explants were resistant. Total RNA was isolated from P5, P15, and P60 retinal explants, reverse transcribed, and subjected to semiquantitative PCR with primers specific to Apaf-1, caspase 3, and tubulin, to measure mRNA expression after TSA treatment. mRNA transcripts of Apaf-1 and caspase 3 increased after TSA-treatment at P5 and P15; however, P60 levels remain unchanged (A). Total protein lysates from P5, P15, and P60 explants (B–E) were separated by SDS-PAGE and probed with antibodies specific for Apaf-1, caspase 3 (both the pro- and cleaved forms), and tubulin. HDAC inhibition led to an increase in Apaf-1 at both P5 and P15 (B, C). At P5, upregulation of procaspase 3 expression was not observed, but the active form of caspase 3 (p20) was detected (B). A clear upregulation of the pro form of caspase-3 was observed at P15 (C) with only low levels of active caspase 3 (p20) detected (D), P5 is included for comparative purposes, with the blot overexposed to aid in detecting the active fragment at P15. Apaf-1 and procaspase 3 protein expression levels remained unchanged in P60-treated samples (E). Tubulin was used as a loading control in all experiments, the results are representative of at least three independent experiments. Both DNA gels and SDS blots were overexposed for detection of P60 Apaf-1 and caspase 3 transcripts and protein levels.

Figure 6.

 

TSA induced apoptosis in P5 and P15 retinal explants but P60 is resistant. (A) Terminal dUTP nick end-labeling of fragmented DNA. Hoechst and TUNEL staining of control and TSA-treated P5, P15, and P60 explants. Control explants (A i–A vi) showed very little TUNEL-labeling. After treatment with 100 nM TSA for 20 hours, there was an induction of apoptosis, as measured by DNA fragmentation in both P5 and P15 explants (A vii–A x). P60 explants, however, appeared to be resistant to TSA-induced apoptosis, as there was minimal TUNEL positive cells present (A xii) compared with the control (A vi) (B) Control genomic DNA or treated with 100 nM TSA was isolated from P5, P15, and P60 retinal explants and separated on a 2% agarose gel stained with ethidium bromide. Treatment with TSA resulted in DNA internucleosomal cleavage, a hallmark of neuronal apoptosis in P5 and P15 retinal explants; however, internucleosomal cleavage was not observed at P60.

The authors thank Theo van Veen (Department of Ophthalmology, Wallenberg Retina Centre, Lund Unviersity) for the opportunity to learn retinal explantation techniques in his laboratory, Romeo Caffe (Department of Ophthalmology, Wallenberg Retina Centre, Lund University) for training and advice on the subject, and Francesca Doonan (Cell Development and Disease Laboratory, Department of Biochemistry, Biosciences Institute, University College Cork, Ireland) for many useful discussions and a critical reading of the manuscript.