Severing the axons of retinal ganglion cells (RGC) by crushing the optic nerve (ONC) causes the majority of RGC to degenerate and die, primarily by apoptosis. We showed recently that after ONC in adult rats, caspase-2 activation occurred specifically in RGC while no localisation of caspase-3 was observed in ganglion cells but in cells of the inner nuclear layer. We further showed that inhibition of caspase-2 using a single injection of stably modified siRNA to caspase-2 protected almost all RGC from death at 7 days, offering significant protection for up to 1 month after ONC. In the present study, we confirmed that cleaved caspase-2 was localised and activated in RGC (and occasional neurons in the inner nuclear layer), while TUNEL+ RGC were also observed after ONC. We then investigated if suppression of caspase-2 using serial intravitreal injections of the pharmacological inhibitor z-VDVAD-fmk (z-VDVAD) protected RGC from death for 15 days after ONC. Treatment of eyes with z-VDVAD suppressed cleaved caspase-2 activation by >85% at 3–4 days after ONC. Increasing concentrations of z-VDVAD protected greater numbers of RGC from death at 15 days after ONC, up to a maximum of 60% using 4000 ng/ml of z-VDVAD, compared to PBS treated controls. The 15-day treatment with 4000 ng/ml of z-VDVAD after ONC suppressed levels of cleaved caspase-2 but no significant changes in levels of cleaved caspase-3, -6, -7 or -8 were detected. Although suppression of caspase-2 protected 60% of RGC from death, RGC axon regeneration was not promoted. These results suggest that caspase-2 specifically mediates death of RGC after ONC and that suppression of caspase-2 may be a useful therapeutic strategy to enhance RGC survival not only after axotomy but also in diseases where RGC death occurs such as glaucoma and optic neuritis.
Citation: Vigneswara V, Berry M, Logan A, Ahmed Z (2012) Pharmacological Inhibition of Caspase-2 Protects Axotomised Retinal Ganglion Cells from Apoptosis in Adult Rats. PLoS ONE 7(12): e53473. doi:10.1371/journal.pone.0053473
Editor: Kin-Sang Cho, Schepens Eye Research Institute, Harvard Medical School, United States of America
Received: September 25, 2012; Accepted: November 30, 2012; Published: December 28, 2012
Copyright: © 2012 Vigneswara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by a grant from the Wellcome Trust, grant no: 092539/Z/10/Z to ZA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Injury to the optic nerve (ON) triggers progressive death of retinal ganglion cells (RGC), the severity of which is dependent upon the type of lesion and its distance from the eye , , . For example, intraorbital ON transection and ON crush (ONC) both trigger 70–75% RGC loss within 7 days after injury , , ,  and by 28 days, 80–90% RGC are lost, primarily by apoptosis , , . RGC apoptosis, however, is recognised as a limiting factor to the regenerative potential of RGC axons. Therefore, treatments to block RGC apoptosis have been studied extensively. For example, inhibition of apoptosis by neurotrophic factor administration , overexpression of Bcl-2 ,  and inhibition of caspase-1 and -3 ,  and caspase-6 and -8  using pharmacological inhibitors all reduced the number of dying RGC after ON transection and ONC. To date, only caspase-6 and -8 inhibitors have yielded limited RGC axon regeneration after ON axotomy .
Apoptosis is orchestrated by caspases, cysteine-rich proteases capable of targeting proteins that play critical roles in DNA replication , , DNA repair , cell survival signalling  and the regulation of proteins that control cytoskeletal re-organisation and cellular disassembly , . There are two groups of caspases: initiator (caspase-2, -8, -9, and -10) and effector caspases (caspase-3, -6 and -7) the former are activated by either death receptor activation, or the release of cytochrome-c from mitochondria, which activate effector caspases through proteolytic processing of pro-caspases, culminating in cleavage of structural proteins and eventual death , , , .
One of the most highly conserved caspases is caspase-2, which acts as both an initiator and an executioner depending on the apoptotic stimuli , , , . Caspase-2 deficient neurons are resistant to apoptosis by β-amyloid ,  while activation of caspase-2 mediates apoptosis of hippocampal neurons after transient global ischemia . Caspase-2 is also expressed in the RGC of ischaemic retinae  and the neuroprotective effect of brain-derived neurotrophic factor (BDNF) is associated with reduced caspase-2 . We have shown unequivocally that, 7 days after ONC, caspase-2 is specifically activated in RGC and that inhibition of caspase-2 by stably-modified siRNA protects 98% of RGC from death at 7 days after ONC and significant RGC protection lasted for at least 30 days , , . Here, we report that a serially injected cell permeable pharmacological inhibitor of caspase-2 protects 60% of RGC from apoptotic death 15 days after ONC but does not promote RGC axon regeneration. Our results suggest that caspase-2 is an important executioner molecule in RGC apoptosis.
Materials and Methods
This study was carried out in strict accordance to the Animals Scientific Procedures Act, 1986 and all procedures were licensed and approved by the UK Home Office. The protocols and experiments were also approved by the University of Birmingham Ethical Review Sub-Committee. Animals were kept in environmentally controlled animal facilities at the University of Birmingham. All surgery was performed under inhalation anaesthesia using 5% Isofluorane (IsoFlo, Abbott Animal Health, North Chicago, IL, USA) induction and 2% for maintenance. Every effort was made to minimise animal suffering.
ON crush (ONC)
The ON of adult female 200–250 g Spraque-Dawley rats (Charles River, Margate, UK) was exposed through a supraorbital approach and crushed bilaterally within the orbit, 2 mm from the eye, using forceps as described previously , , , , , .
The cell membrane permeable caspase-2 inhibitor, z-V-D-(OMe)-V-A-D(OMe)-fluromethylkeone (z-VDVAD) (R&D Systems, Abingdon, Oxford, UK), was dissolved in sterile DMSO (Sigma, Poole, UK) and further diluted in sterile PBS before intravitreal injection in a final volume of 5 µl. Control animals (n = 6 rats/treatment (12 eyes/treatment)) were intravitreally injected with PBS (containing the same diluted amount of DMSO as z-VDVAD) (vehicle) at the same time-points as z-VDVAD, whilst treated rats (n = 6 rats/treatment (12 eyes/treatment)) received either 400 ng/ml, 1000 ng/ml, 2000 ng/ml, 4000 ng/ml or 5000 ng/ml of z-VDVAD immediately after ONC (day 0), and intravitreal injections repeated at 4, 8 and 12 d after ONC. The rationale for doses of 400 ng/ml and 4000 ng/ml were based on previously published data using caspase-3 inhibitors . Animals survived for 15 d, after which the retinae were dissected out for retinal wholemounts and protein extraction for western blotting, while whole eyes were removed for immunohistochemistry. None of the animals developed cataracts, confirming that the lens had not been injured either during surgery or after subsequent intravitreal injections.
Determining the optimal dosing regime
To determine the frequency of intravitreal z-VDVAD injections, we used the same dose of a pharmacological inhibitor to caspase-3 and the same 3–4 d injection schedule after ONC, reported by Kermer et al. (1998)  that caused RGC survival. We therefore chose to intravitreally inject 4000 ng/ml immediately after ONC and killed animals at 2, 3 and 4 days by overdose of CO2 (n = 3 rats/treatment (6 retinae/treatment)). Eyes were enucleated, retinae harvested and snap frozen until required for extraction of total proteins and western blotting as described later.
At 13 d after ONC, 2 µl of 4% FluoroGold (FG, Cambridge Bioscience, Cambridge, UK), were prepared from a solid stock and injected into the proximal ON segment mid-way between the lamina cribrosa and the site of ONC. Animals were killed and eyes were enucleated 48 h later and retinae were immersion-fixed in 4% formaldehyde (TAAB Laboratories, Aldermaston, UK) for 30 min, and flattened onto a Superfrost Plus microscope slides (VWR International, Lutterworth, UK), after dividing the retinae with 4 equidistant radial cuts to give 4 equally sized quadrants attached together at the optic disc. Retinal wholemounts were dried onto glass slides and mounted in Vectamount (Vector Laboratories, Peterborough, UK). Samples were randomised and blinded by a second investigator and photographs were captured using a Zeiss fluorescent microscope equipped with a digital camera in Axiovision 4 (all from Zeiss, Hertfordshire, UK). The number of FG-labelled RGC was counted using automated particle counting software in ImagePro Version 6.0 (Media Cybernetics, Bethesda, USA) from photographs of 12 rectangular areas (0.36×0.24 mm), 3 from each quadrant, placed at radial distances from the centre of the optic disc of the inner (1/6 eccentricity), midperiphery (1/2 eccentricity) or outer retina (5/6 eccentricity). RGC densities were summed together and averaged over the entire retina and expressed as mean RGC densities/mm2 for each treatment (n = 6 rats/treatment (12 retinae/treatment)).
Tissue preparation and sectioning
After intracardiac perfusion with 4% formaldehyde, eyes and ON were removed, immersion-fixed in 4% formaldehyde (TAAB) for 2 h, washed for 10 min in 10 mM phosphate buffered saline (PBS), and immersed in 10% and 20% sucrose (Sigma) each for 2 h and finally immersed in 30% sucrose overnight. Eyes and ON were embedded in OCT mounting medium (Raymond A Lamb Ltd) and 15 µm thick parasaggital sections of eyes and longitudinal sections of ON were cut on a cryostat (Bright Instruments, Huntingdon, UK), adhered onto glass slides and stored at −20°C until required.
Double immunohistochemistry for cleaved caspase-2 (C-CASP2) and βIII-tubulin was performed on sections of retina as described by us previously . Briefly, sections were washed in PBS, non-specific binding blocked with PBS containing 3% BSA and 0.05% Tween 20 for 20 min before incubation with rabbit anti-C-CASP2 (Abcam, Cambridge, UK; 1:200 dilution) and monoclonal anti-βIII-tubulin (Sigma, Poole, UK; 1:200 dilution) primary antibodies overnight at 4°C. Sections were then washed in ×3 changes of PBS, incubated with appropriate Alexa Fluor 488 and Texas Red-labelled secondary antibodies for 1 h at room temperature, washed, mounted using Vectashield mounting medium with DAPI (Vector Laboratories) and examined under an Axioplan-2 epi-fluorescent microscope (Zeiss).
GAP-43 immunohistochemistry was performed on ON sections (n = 9 rats/treatment; (i.e. 18 ON)) to detect RGC axon regeneration at 15 d after ONC using a sheep polyclonal anti-GAP-43 antibody (donated by Professor Larry Benowitz, Harvard Medical School, Boston, USA) as described previously .
Protein extraction and Western blotting
At 15 d after ONC, a total of 3 rats/treatment group were killed and retinae (n = 3 rats/treatment (6 retinae/treatment)) were pooled prior to protein extraction in cell lysis buffer and processed for Western blotting as previously described . Experiments were then repeated×2 using a further 3 rats/treatment/experiment and retinae (n = 6 retinae/treatment/experiment) were again pooled and proteins extracted prior to western blot analysis. Western blots were probed overnight at 4°C with antibodies against: Goat anti-human cleaved caspase-2 (directed against the p12 fragment) (C-CASP2) and rabbit anti-human cleaved caspase-8 (p20 fragment) (C-CASP8) (both from Santa Cruz Biotechnology, CA, USA); Rabbit anti-cleaved caspase-3 (C-CASP3), rabbit anti-cleaved caspase-6 (C-CASP6) and rabbit anti-cleaved caspase-7 (C-CASP7) (all from Cell Signalling Technology, Danvers, MA, USA). Relevant bands were detected with an appropriate HRP-labelled secondary antibody (GE Healthcare, Buckinghamshire, UK) and detected using an enhanced chemiluminescence system (ECL) (GE Healthcare).
Western blots were probed with a rabbit monoclonal antibody to GAPDH (Cell Signalling Technology, Danvers, MA, USA) at 1:1000 dilution and used as a loading control. Blots were stripped and re-probed as required.
Biotin-VAD-fmk trapping assay of active caspase
The caspase trapping assay which employs the bVAD (biotin-Val-Ala-DL-Asp-fluoromethylketone(biotin-VAD-fmk)) probe is the best way to determine whether caspases are active after a death stimuli. bVAD is an irreversible pan-caspase inhibitor and binds irreversibly to all activated caspases. Once bVAD binds irreversibly it inhibits that specific caspase and blocks downstream events, and as the bVAD is biotinylated, it can be isolated on streptavidin agarose along with any active caspases that are bound to it. We therefore used the biotin-VAD-fmk assay was used to detect the presence of active caspase-2 as described previously , ,  but with modifications for our in vivo experiments. Briefly, 200 nmol of b-VAD-fmk (MP Biomedical, UK) was diluted in 5 µl of sterile saline and intravitreally injected 24 hr prior to ONC (to capture any caspases being activated by ONC) while control animals received 5 µl of sterile saline prior to ONC. bVAD treated animals were killed at 1, 4 and 7 d after ONC whilst control animals were killed at 4 d (since our previous experiment demonstrated optimal C-CASP2 immunohistochemistry at this time-point). Eyes were enucleated and retinae were immediately homogenised in ice-cold CHAPS buffer (150 mM KCl, 50 mM HEPES and 0.1% CHAPS (pH 7.4)) supplemented with protease inhibitor cocktail (Sigma). Samples were incubated on ice for 10 min prior to centrifugation at 13000× g for 10 mins at 4°C and the supernatants were collected and boiled for 5 min. Streptavidin-agarose beads were then added to the boiled supernatant and incubated overnight at 4°C with constant agitation. Beads were spun at 7500× g for 5 min at 4°C, washed ×5 in PBS, resuspended in 2× Laemmli buffer and boiled for 5 min. The beads were centrifuged at 15000× g for 10 min, supernatant collected and supplemented with 5% 2-mercaptoethanol and samples were resolved on 12% SDS-PAGE gels and analysed by western blotting as described above using an affinity purified goat polyclonal anti-C-CASP2 antibody (Santa Cruz Biotechnology). Blots were stripped and re-probed with antibodies against total CASP-2 (Santa Cruz Biotechnology) and GAPDH (Cell Signalling Technology).
Western blots were quantified by densitometry as described by us previously , , . Briefly, blots were scanned into Adobe Photoshop and TIFF files were analysed in ScionImage (version 4.0.2, Scion Corp, Maryland, USA) using the built-in gel plotting macros. The integrated density of each band in each lane was calculated from 3 separate blots.
The significance of differences between sample means were calculated using GraphPad Prism (GraphPad Software Inc., Version 4.0, CA, San Diego, USA) by one-way analysis of variance (ANOVA) followed by post-hoc testing with Dunnett's method.
ONC induced caspase-2 activation and TUNEL in RGC
We have previously shown that C-CASP2 is localised in RGC within 5 hr after ONC and is present in a significant number of RGC after 24 hr . Here we confirmed by immunohistochemistry that C-CASP2 was absent in βIII-tubulin+ RGC in intact (uninjured) animals (Figure 1A), but was specifically localised in RGC and present in abundant numbers of RGC at 4 d after ONC (Fig. 1A). The localisation of C-CASP2 paralleled the presence of TUNEL+ nuclei in βIII-tubulin+ RGC in ONC animals (Figure 1B), indicating RGC apoptosis. The b-VAD caspase trapping assay captured increasing amounts of CASP2 activity and demonstrated 2-, 3- and 6-fold higher levels of CASP2 at 1 d, 4 d and 7 d, respectively, when compared to controls (Figure 1C and D). Other caspases (e.g. CASP3 or CASP7 (data not shown)) were not captured. These results demonstrate that CASP2 is localised and specifically activated in RGC after ONC.
(A) Immunohistochemistry to show the absence of C-CASP2 (red) reactivity in βIII-tubulin+ (green) RGC in intact controls, while 4 days after ONC the majority of βIII-tubulin+ RGC (green) were C-CASP2+ (red; arrowheads). (B) βIII-tubulin+ RGC in intact controls were also negative for TUNEL staining while some TUNEL+ (arrowheads) RGC were present at 4 d after ONC. Some RGC were also negative for TUNEL staining at 4 d after ONC (arrow). (C) Active CASP2 is induced by ONC and its levels increase over the 7 d time period and (D) densitometry to quantify the relative CASP2 activity compared to controls. GCL = ganglion cell layer. Scale bars in A and B = 50 µm.
Determining the frequency of z-VDVAD administration
After ONC and immediate intravitreal injection of 4000 ng/ml of z-VDVAD, western blotting detected reduced levels of C-CASP2, of approximately 50% (P<0.0001, ANOVA), at 2 d after ONC compared to ONC+vehicle treated eyes (Figure 2A and B). The levels of C-CASP2 decreased significantly by >85% at 3 days (P<0.0001, ANOVA), remaining significantly low for 4 days (P<0.0001, ANOVA) but without further reductions from day 3 (Figure 2A and B). Based on these results, every 4th day was selected for repeated intravitreal injections of z-VDVAD for the duration of the 15 d experimental time period to minimise the number of intravitreal injections required prior to harvesting of tissues.
(A) Representative western blot to show that 4000 ng/ml z-VDVAD treatment caused optimal suppression of C-CASP2 (>80%) by 3 d after ONC. (B) Densitometry confirmed 50% reduction of C-CASP2 by 2 d after ONC+z-VDVAD treatment compared to ONC alone, which increased to 80% at 3 d after ONC+z-VDVAD treatment with no further reduction by 4 d. GAPDH was used as a loading control. n = 6 rats/treatment (12 eyes/treatment).
z-VDVAD promoted RGC survival after ONC
To determine whether inhibition of caspase-2 protected RGC from apoptosis for 15 d after ONC, we injected z-VDVAD intravitreally every 4th day and compared results with control vehicle treated eyes. Very few FG+ RGC were observed in retinal wholemounts from ONC+vehicle injected eyes (Figure 3A and G) compared to those treated with increasing concentrations of z-VDVAD (400 ng/ml to 5000 ng/ml) (Figure 3B–F and G), where progressively more FG+ RGC were present up to a concentration of 4000 ng/ml (Figure 3E and G) (P<0.0001, ANOVA). There was no increase in FG+ RGC using the highest concentration of z-VDVAD (5000 ng/ml) (Figure 3F and G) compared to that observed at 4000 ng/ml RGC (Figure 3E and G). No changes in cell morphology, nor soma size were observed in retinal wholemounts after treatment with z-VDVAD, with all RGC morphology appearing healthy and as diverse soma sizes as that observed in controls without z-VDVAD treatment (Figure 3A–F: insets) The average density of FG+ RGC in ONC+vehicle treated eyes were 293±34 RGC/mm2 (Figure 3A). Intravitreal delivery of 400 ng/ml of z-VDVAD increased RGC survival to 590±56 RGC/mm2, while the optimum concentration of 4000 ng/ml z-VDVAD increased survival to 1430±30 RGC/mm2 (Figure 3G). Therefore, 4000 ng/ml of z-VDVAD protected 60% of RGC from death after 15 d compared to uninjured, intact controls (Figure 3G).
FG+ RGC in flat mounted retina and high power inserts after (A) ONC+vehicle, (B) ONC+400 ng/ml z-VDVAD, (C) 1000 ng/ml and ONC+1000 ng/ml z-VDVAD, (D) ONC+2000 ng/ml z-VDVAD, (E) ONC+4000 ng/ml z-VDVAD, (F) ONC+5000 ng/ml z-VDVAD at 15 d after ONC. (G) Quantitation of surviving RGC density (±SEM) at 15 d after ONC and treatment with z-VDVAD. Scale bars in A–F = 50 µm, and in insets = 25 µm.
z-VDVAD reduces caspase-2 activation in RGC of the ganglion cell layer
We investigated if intravitreal z-VDVAD injection maintained the suppression of cleaved caspase-2 (C-CASP2) in sections of retinae by immunohistochemistry at 15 d. After ONC+vehicle treatment (Figure 4A and B), C-CASP2 was localised in most ganglion cells (GCL) and cells of the inner nuclear layer (INL). However, treatment with 4000 ng/ml of z-VDVAD (Figure 4C and D) almost completely suppressed C-CASP2 immunohistochemistry. High power magnifications of the GCL confirmed C-CASP2 immunolocalisation in cells of the GCL after ONC+vehicle treatment (Figure 4E and F) while little or no immunolocalisation for C-CASP2 was observed in the cells of the GCL of eyes treated with 4000 ng/ml z-VDVAD (Figure 4G and H).
(A, B) C-CASP2 is highly up regulated in ONC+vehicle treated in the GCL and INL cells. (C, D) C-CASP2 immunoreactivity (red) was barely detectable in the GCL after treatment with 4000 ng/ml z-VDVAD. (E–H) High power magnification of the GCL showed C-CASP2 immunoreactivity (red) in RGC after ONC+vehicle treatment (arrowheads) whilst treatment with z-VDVAD suppressed immunoreactivity in the GCL. Double immunohistochemistry showing that βIII-tubulin+ RGC (I) were also C-CASP2+ (J) (arrowheads) after ONC+vehicle treatment while treatment with ONC+4000 ng/ml z-VDVAD suppressed C-CASP2 levels in RGC (L–N). GCL = ganglion cell layer, INL = inner nuclear layer; ONL = outer nuclear layer. Scale bars in A–D, 100 µm, scale bars in E–N, 50 µm.
Co-localisation of C-CASP2 with βIII-tubulin+ confirmed that βIII-tubulin+ RGC showed specific C-CASP2 activation in RGC after ONC+vehicle (Figure 4I–K) but little or no C-CASP2 activation in βIII-tubulin+ RGC after treatment with 4000 ng/ml z-VDVAD (Figure 4L–N).
Only C-CASP2 levels are suppressed by z-VDVAD
Using western blots of retinal lysates, we found that ONC+vehicle treatment not only upregulated pro-caspase-2 (Pro-CASP2) (P<0.001, ANOVA) in the retina but also significantly upregulated the levels of C-CASP2 (P<0.0001, ANOVA), particularly the p12 fragment recognised by our which our antibody, as detected by the relative band size (Figure 5A) but also after quantification by densitometry (Figure 5B). However, ONC+4000 ng/ml of z-VDVAD treatment significantly reduced C-CASP2 and the integrated density of the C-CASP2 p12 fragment (P<0.0001, ANOVA) (Figure 5A and B).
(A) Pro-CASP2 levels rose after ONC and remained high even after treatment with 4000 ng/ml z-VDVAD. The level of the C-CASP2 p12 fragment also rose after ONC but was significantly decreased after treatment with z-VDVAD. (A) and (B) Levels of Pro-CASP2 and C-CASP2 (p12) reflected these changes and showed that the C-CASP2 fragment was 40% lower in retinae treated with 4000 ng/ml z-VDVAD than those treated with vehicle alone. C-CASP3 and C-CASP6 levels increased after ONC+vehicle treatment but these levels remained unchanged after ONC+4000 ng/ml z-VDVAD treatment. C-CASP7 and C-CASP8 levels were much lower and did not change after ONC+vehicle, or ONC+4000 ng/ml z-VDVAD treatments. (B) and (C) Densitometry reflected these observed changes in Pro-CASP2, C-CASP2, C-CASP3, C-CASP6, C-CASP7 and C-CASP8. GAPDH was used as a loading control.
Since Pro-CASP2 can be activated by cleaved caspase-3 (C-CASP3) we probed our western blots against antibodies to C-CASP and detected the p17/19 processed fragment of C-CASP3 (Figure 5A) which was upregulated after ONC+vehicle treatment (P<0.001, ANOVA) (Figure 5A and C). However, these raised levels of C-CASP3 p17/p19 fragment were unaffected by 4000 ng/ml z-VDVAD treatment. We then probed our blots with antibodies against cleaved caspase-6 (C-CASP6), -7 (C-CASP7) and -8 (C-CASP8) to investigate if z-VDVAD non-specifically attenuated other caspases implicated in RGC survival after ONC and failed to observe any significant changes after treatments in C-CASP3, C-CASP6, C-CASP7, or C-CASP8 (Figure 5A–C).
Inhibition of caspase-2 does not promote RGC axon regeneration after ONC
To investigate if suppression of caspase-2 by z-VDVAD enhanced RGC axon regeneration, we stained longitudinal sections of ON with antibodies against GAP-43 to detect regenerating axons. In ONC+vehicle treated rats, there were few surviving axons in the proximal ON segment between lesion site and eye at 15 d while no axons extended across the lesion into the distal ON segment (Figure 6A). However, in sections of ON treated with ONC+4000 ng/ml z-VDVAD greater numbers of GAP-43+ RGC axons were seen in the proximal ON segment, reflecting RGC survival, but no axons traversed the crush site to enter the distal ON segment (Figure 6B). Taken together, these data showed that caspase-2 inhibition promotes significant RGC survival but not axon regeneration.
(A) GAP-43 immunostaining in longitudinal sections of ON from eyes at 15 d after ONC, treated with ONC+vehicle showed few GAP-43+ axons in the proximal ON stump (P) and none leaving the lesion site (arrow) and invading the distal (D) ON segment. (B) Numerous GAP-43+ axons were present in the proximal segment of the transected ON after treatment with 4000 ng/ml z-VDVAD but once again no axons left the crush site (arrow) and invaded into the distal ON segment. The retina (not shown) is towards the left hand side of the image. Scale bars in (A) and (B) = 100 µm.
The data presented in this study confirm our previous findings that caspase-2 is an important regulator of RGC apoptosis induced after ONC . We report that caspase-2 is specifically activated in RGC and that significant RGC survival is promoted after pharmacological inhibition of caspase-2 with z-VDVAD, but no RGC axon regeneration occurs. These results complement those of our previous study with a gene silencing approach , demonstrating that caspase-2 is a key orchestrator of RGC apoptosis.
Caspase-mediated RGC apoptosis
Caspase-3, -6, -8 and -9 activities all play a role in RGC apoptosis after ON injury , , . Using inhibitors besides caspase-2, RGC neuroprotection ranged from 30–60% at 15 d after ONC , , , with caspase-8 inhibitors supporting the survival of nearly 60% RGC compared to intact controls . Inhibition of caspase-2 using a chemically stabilised siRNA at 7 d after ONC rescued 98% RGC from apoptosis . In the current study, pharmacological inhibition of caspase-2 with repeated delivery of z-VDVAD over a 15 d period after ONC protected 60% RGC from apoptosis, corroborating our earlier study that caspase-2 is an important regulator of RGC apoptosis . We have not established the long-term efficacy of z-VDVAD on RGC neuroprotection, however, the advantage of z-VDVAD is that it is an already available, small molecule inhibitor that may be used immediately in clinical trials. Our results are similar to those of Monnier et al. (2011)  who reported similar levels of RGC rescue at 14 d after ONC and ON transection with a similar intravitreal delivery regime, using a caspase-8 and caspase-6 inhibitor, respectively.
Although caspase-3 inhibition rescued ~35% RGC from death , we localised C-CASP3 not in RGC but in neurons of the INL , suggesting that caspase-3 was not a direct regulator of RGC apoptosis. In the current study, the observed elevated levels of C-CASP3 after ONC were not attenuated by z-VDVAD, suggesting that caspase-3 was not activated as a downstream effector molecule by caspase-2. Moreover, C-CASP6, C-CASP7 and C-CASP8 levels were unchanged after ONC and were also unaffected by z-VDVAD caspase-2 inhibitor suggesting that, although caspase-3 and -7 can be inhibited by the VDVAD substrates , C-CASP3 and C-CASP7 were not affected in this instance. We therefore, attribute the RGC protective effect of z-VDVAD to specific inhibition of caspase-2. To our knowledge this is the first study that demonstrates significant RGC protection using the pharmacological z-VDVAD inhibitor and suggests that despite these inhibitors showing some promiscuity, z-VDVAD in this case specifically inhibited caspase-2.
Although caspase-2 is structurally similar to an initiator caspase, its predicted cleavage specificity resembles that of the effector caspase-3 . Caspase-2 shows a multifaceted participation in apoptosis after a variety of cellular stresses that include DNA damage, stimulation of death receptors, heat shock, cytoskeletal disruption and oxidative stress , . In addition, caspase-2 deficient neurons are resistant to apoptosis induced by β-amyloid , , while activation of caspase-2 occurs in hippocampal neurons after transient global ischemia . Moreover, caspase-2 is expressed in the RGC of ischemic retinae and the reported neuroprotective effect of intravitreal BDNF administration was associated with diminished caspase-2 immunoreactivity , . Given this body of evidence for the involvement of caspase-2 in RGC apoptosis, the results of our previous  and current studies suggest that RGC death is orchestrated directly by caspase-2 after ON injury, a proposition that is supported by the observed absence of caspase-3 in RGC . Similarly, few RGC were immunopositive for C-CASP6 and C-CASP8 (VV and ZA, unpublished observations), while no significant changes in the levels of C-CASP6 and C-CASP8 were observed after z-VDVAD treatment. Furthermore, significant RGC protection was observed in our current study after caspase-2 inhibition with z-VDVAD treatment without significant reductions in other potential effector caspases such as caspase-3 and -7.
Caspase-2 inhibition does not promote RGC axon regeneration
Although there are early reports that caspase inhibition does not prevent axon degeneration , , recent evidence shows otherwise. For example, caspase-6 has been implicated as a downstream regulator of axon degeneration initiated by p75NTR and β-amyloid precursor protein and death receptor 6 , , while inhibition of caspase-6 and -8 promotes RGC axon regeneration after ON injury . This latter observation has been explained by demonstrations that caspase-6 predominantly degrades nuclear and cytoskeletal components and triggers microtubule destabilization , ,  which may contribute to the failure of RGC axon regeneration . Similarly, caspase-8 acts on molecules that regulate cytoskeletal dynamics, including p21-activated kinase (PAK)  and Rho-associated protein kinase (ROCK) , and may therefore contribute to the failure of RGC axon regeneration . However, we did not observe RGC axon regeneration after intravitreal delivery of z-VDVAD caspase-2 inhibitor, suggesting that caspase-2 does not interact with components of the signalling cascades in which caspase-6 and -8 regulates cytoskeletal dynamics.
In conclusion, our results demonstrate that caspase-2: (1), plays a critical role in RGC apoptosis and that inhibition of caspase-2 significantly rescues RGC from death; (2), triggers RGC apoptosis in a manner that is distinct to that elicited by caspase-6 or -8; and (3), protects RGC from apoptosis after visual trauma and injury, including glaucoma  and optic neuropathies . These results imply that targeting caspase-2 activation may be a useful therapeutic in the fight against diseases in which RGC are specifically lost, e.g. glaucoma, optic nerve injury and optic neuritis.
Conceived and designed the experiments: ZA MB AL. Performed the experiments: VV MB ZA. Analyzed the data: VV ZA. Wrote the paper: VV MB AL ZA.
- 1. Kermer P, Klocker N, Labes M, Thomsen S, Srinivasan A, et al. (1999) Activation of caspase-3 in axotomized rat retinal ganglion cells in vivo. FEBS Lett 453: 361–364. doi: 10.1016/s0014-5793(99)00747-4
- 2. Berkelaar M, Clarke DB, Wang YC, Bray GM, Aguayo AJ (1994) Axotomy Results in Delayed Death and Apoptosis of Retinal Ganglion-Cells in Adult-Rats. J Neurosci 14: 4368–4374.
- 3. Villegas-Perez MP, Vidal-Sanz M, Rasminsky M, Bray GM, Aguayo AJ (1993) Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats. J Neurobiol 24: 23–36. doi: 10.1002/neu.480240103
- 4. Agudo M, Perez-Marin MC, Lonngren U, Sobrado P, Conesa A, et al. (2008) Time course profiling of the retinal transcriptome after optic nerve transection and optic nerve crush. Mol Vis 14: 1050–1063.
- 5. Berry M, Carlile J, Hunter A (1996) Peripheral nerve explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve. J Neurocytol 25: 147–170. doi: 10.1007/bf02284793
- 6. Berry M, Carlile J, Hunter A, Tsang W, Rosustrel P, et al. (1999) Optic nerve regeneration after intravitreal peripheral nerve implants: trajectories of axons regrowing through the optic chiasm into the optic tracts. J Neurocytol 28: 721–741.
- 7. Ahmed Z, Kalinski H, Berry M, Almasieh M, Ashush H, et al. (2011) Ocular neuroprotection by siRNA targeting caspase-2. Cell Death Dis 2: e173. doi: 10.1038/cddis.2011.54
- 8. Garcia-Valenzuela E, Gorczyca W, Darzynkiewicz Z, Sharma SC (1994) Apoptosis in adult retinal ganglion cells after axotomy. J Neurobiol 25: 431–438. doi: 10.1002/neu.480250408
- 9. Rabacchi SA, Bonfanti L, Liu XH, Maffei L (1994) Apoptotic cell death induced by optic nerve lesion in the neonatal rat. J Neurosci 14: 5292–5301.
- 10. Isenmann S, Wahl C, Krajewski S, Reed JC, Bahr M (1997) Up-regulation of Bax protein in degenerating retinal ganglion cells precedes apoptotic cell death after optic nerve lesion in the rat. Eur J Neurosci 9: 1763–1772. doi: 10.1111/j.1460-9568.1997.tb01534.x
- 11. Koeberle PD, Ball AK (1998) Effects of GDNF on retinal ganglion cell survival following axotomy. Vision Res 38: 1505–1515. doi: 10.1016/s0042-6989(97)00364-7
- 12. Bonfanti L, Strettoi E, Chierzi S, Cenni MC, Liu XH, et al. (1996) Protection of retinal ganglion cells from natural and axotomy-induced cell death in neonatal transgenic mice overexpressing bcl-2. J Neurosci 16: 4186–4194.
- 13. Chierzi S, Cenni MC, Maffei L, Pizzorusso T, Porciatti V, et al. (1998) Protection of retinal ganglion cells and preservation of function after optic nerve lesion in bcl-2 transgenic mice. Vision Res 38: 1537–1543. doi: 10.1016/s0042-6989(97)00332-5
- 14. Lucius R, Sievers J (1997) YVAD protect post-natal retinal ganglion cells against axotomy-induced but not free radical-induced axonal degeneration in vitro. Brain Res Mol Brain Res 48: 181–184. doi: 10.1016/s0169-328x(97)00170-8
- 15. Kermer P, Klocker N, Labes M, Bahr M (1998) Inhibition of CPP32-like proteases rescues axotomized retinal ganglion cells from secondary cell death in vivo. J Neurosci 18: 4656–4662.
- 16. Monnier PP, D'Onofrio PM, Magharious M, Hollander AC, Tassew N, et al. (2011) Involvement of caspase-6 and caspase-8 in neuronal apoptosis and the regenerative failure of injured retinal ganglion cells. J Neurosci 31: 10494–10505. doi: 10.1523/jneurosci.0148-11.2011
- 17. Song Q, Lees-Miller SP, Kumar S, Zhang Z, Chan DW, et al. (1996) DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis. EMBO J 15: 3238–3246.
- 18. Casciola-Rosen LA, Miller DK, Anhalt GJ, Rosen A (1994) Specific cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein is a characteristic biochemical feature of apoptotic cell death. J Biol Chem 269: 30757–30760.
- 19. Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC (1994) Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371: 346–347. doi: 10.1038/371346a0
- 20. Leist M, Single B, Castoldi AF, Kuhnle S, Nicotera P (1997) Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med 185: 1481–1486. doi: 10.1084/jem.185.8.1481
- 21. Bokoch GM (1998) Caspase-mediated activation of PAK2 during apoptosis: proteolytic kinase activation as a general mechanism of apoptotic signal transduction? Cell Death Diff 5: 637–645. doi: 10.1038/sj.cdd.4400405
- 22. Porter AG, Ng P, Janicke RU (1997) Death substrates come alive. BioEssays 19: 501–507. doi: 10.1002/bies.950190609
- 23. Baumgartner R, Meder G, Briand C, Decock A, D'Arcy A, et al. (2009) The crystal structure of caspase-6, a selective effector of axonal degeneration. Biochem J 423: 429–439. doi: 10.1042/bj20090540
- 24. Inoue S, Browne G, Melino G, Cohen GM (2009) Ordering of caspases in cells undergoing apoptosis by the intrinsic pathway. Cell Death Diff 16: 1053–1061. doi: 10.1038/cdd.2009.29
- 25. Boyce M, Degterev A, Yuan J (2004) Caspases: an ancient cellular sword of Damocles. Cell Death Diff 11: 29–37. doi: 10.1038/sj.cdd.4401339
- 26. Degterev A, Boyce M, Yuan J (2003) A decade of caspases. Oncogene 22: 8543–8567. doi: 10.1038/sj.onc.1207107
- 27. Ho LH, Read SH, Dorstyn L, Lambrusco L, Kumar S (2008) Caspase-2 is required for cell death induced by cytoskeletal disruption. Oncogene 27: 3393–3404. doi: 10.1038/sj.onc.1211005
- 28. Tu S, McStay GP, Boucher LM, Mak T, Beere HM, et al. (2006) In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock-induced apoptosis. Nat Cell Biol 8: 72–77. doi: 10.1038/ncb1340
- 29. Sidi S, Sanda T, Kennedy RD, Hagen AT, Jette CA, et al. (2008) Chk1 suppresses a caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and caspase-3. Cell 133: 864–877. doi: 10.1016/j.cell.2008.03.037
- 30. Upton JP, Austgen K, Nishino M, Coakley KM, Hagen A, et al. (2008) Caspase-2 cleavage of BID is a critical apoptotic signal downstream of endoplasmic reticulum stress. Mol Cell Biol 28: 3943–3951. doi: 10.1128/mcb.00013-08
- 31. Troy CM, Rabacchi SA, Friedman WJ, Frappier TF, Brown K, et al. (2000) Caspase-2 mediates neuronal cell death induced by beta-amyloid. J Neurosci 20: 1386–1392.
- 32. Troy CM, Ribe EM (2008) Caspase-2: vestigial remnant or master regulator? Sci Signal 1: e42. doi: 10.1126/scisignal.138pe42
- 33. Niizuma K, Endo H, Nito C, Myer DJ, Kim GS, et al. (2008) The PIDDosome mediates delayed death of hippocampal CA1 neurons after transient global cerebral ischemia in rats. PNAS 105: 16368–16373. doi: 10.1073/pnas.0806222105
- 34. Singh M, Savitz SI, Hoque R, Gupta G, Roth S, et al. (2001) Cell-specific caspase expression by different neuronal phenotypes in transient retinal ischemia. J Neurochem 77: 466–475. doi: 10.1046/j.1471-4159.2001.00258.x
- 35. Kurokawa T, Katai N, Shibuki H, Kuroiwa S, Kurimoto Y, et al. (1999) BDNF diminishes caspase-2 but not c-Jun immunoreactivity of neurons in retinal ganglion cell layer after transient ischemia. Invest Ophthalmol Vis Sci 40: 3006–3011.
- 36. Ahmed Z, Dent RG, Leadbeater WE, Smith C, Berry M, et al. (2005) Matrix metalloproteases: degradation of the inhibitory environment of the transected optic nerve and the scar by regenerating axons. Mol Cell Neurosci 28: 64–78. doi: 10.1016/j.mcn.2004.08.013
- 37. Ahmed Z, Mazibrada G, Seabright RJ, Dent RG, Berry M, et al. (2006) TACE-induced cleavage of NgR and p75(NTR) in dorsal root ganglion cultures disinhibits outgrowth and promotes branching of neurites in the presence of inhibitory CNS myelin. FASEB J 20: 1939–1941. doi: 10.1096/fj.05-5339fje
- 38. Douglas MR, Morrison KC, Jacques SJ, Leadbeater WE, Gonzalez AM, et al. (2009) Off-target effects of epidermal growth factor receptor antagonists mediate retinal ganglion cell disinhibited axon growth. Brain 132: 3102–3121. doi: 10.1093/brain/awp240
- 39. Ahmed Z, Aslam M, Lorber B, Suggate EL, Berry M, et al. (2010) Optic nerve and vitreal inflammation are both RGC neuroprotective but only the latter is RGC axogenic. Neurobiol Dis 37: 441–454. doi: 10.1016/j.nbd.2009.10.024
- 40. Kermer P, Klocker N, Labes M, Bahr M (1998) Inhibition of CPP32-like proteases rescues axotomized retinal ganglion cells from secondary cell death in vivo. J Neurosci 18: 4656–4662.
- 41. Ribe EM, Jean YY, Goldstein RL, Manzl C, Stefanis L, et al. (2012) Neuronal caspase 2 activity and function requires RAIDD, but not PIDD. Biochem J 444: 591–599. doi: 10.1042/bj20111588
- 42. Akpan N, Serrano-Saiz E, Zacharia BE, Otten ML, Ducruet AF, et al. (2011) Intranasal delivery of caspase-9 inhibitor reduces caspase-6-dependent axon/neuron loss and improves neurological function after stroke. J Neurosci 31: 8894–8904. doi: 10.1523/jneurosci.0698-11.2011
- 43. Kermer P, Klocker N, Labes M, Bahr M (1998) CPP32-like proteases are major mediators of apoptosis in axotomized retinal ganglion cells in vivo. Eur J Neurosci 10: 202–202.
- 44. Weishaupt JH, Diem R, Kermer P, Krajewski S, Reed JC, et al. (2003) Contribution of caspase-8 to apoptosis of axotomized rat retinal ganglion cells in vivo. Neurobiol Dis 13: 124–135. doi: 10.1016/s0969-9961(03)00032-9
- 45. Chauvier D, Ankri S, Charriaut-Marlangue C, Casimir R, Jacotot E (2007) Broad-spectrum caspase inhibitors: from myth to reality? Cell Death Diff 14: 387–391. doi: 10.1038/sj.cdd.4402044
- 46. Werner AB, Tait SW, de Vries E, Eldering E, Borst J (2004) Requirement for aspartate-cleaved bid in apoptosis signaling by DNA-damaging anti-cancer regimens. J Biol Chem 279: 28771–28780. doi: 10.1074/jbc.m400268200
- 47. Niizuma K, Endo H, Nito C, Myer DJ, Kim GS, et al. (2008) The PIDDosome mediates delayed death of hippocampal CA1 neurons after transient global cerebral ischemia in rats. PNAS 105: 16368–16373. doi: 10.1073/pnas.0806222105
- 48. Kuida K, Zheng TS, Na S, Kuan C, Yang D, et al. (1996) Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384: 368–372. doi: 10.1038/384368a0
- 49. Finn JT, Weil M, Archer F, Siman R, Srinivasan A, et al. (2000) Evidence that Wallerian degeneration and localized axon degeneration induced by local neurotrophin deprivation do not involve caspases. J Neurosci 20: 1333–1341.
- 50. Park KJ, Grosso CA, Aubert I, Kaplan DR, Miller FD (2010) p75NTR-dependent, myelin-mediated axonal degeneration regulates neural connectivity in the adult brain. Nat Neurosci 13: 559–566. doi: 10.1038/nn.2513
- 51. Nikolaev A, McLaughlin T, O'Leary DD, Tessier-Lavigne M (2009) APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature 457: 981–989. doi: 10.1038/nature07767
- 52. Guo H, Albrecht S, Bourdeau M, Petzke T, Bergeron C, et al. (2004) Active caspase-6 and caspase-6-cleaved tau in neuropil threads, neuritic plaques, and neurofibrillary tangles of Alzheimer's disease. Am J Pathol 165: 523–531. doi: 10.1016/s0002-9440(10)63317-2
- 53. Koeberle PD, Tura A, Tassew NG, Schlichter LC, Monnier PP (2010) The repulsive guidance molecule, RGMa, promotes retinal ganglion cell survival in vitro and in vivo. Neurosci 169: 495–504. doi: 10.1016/j.neuroscience.2010.04.079
- 54. Fischer U, Stroh C, Schulze-Osthoff K (2006) Unique and overlapping substrate specificities of caspase-8 and caspase-10. Oncogene 25: 152–159. doi: 10.1038/sj.onc.1209015
- 55. Ehrenschwender M, Siegmund D, Wicovsky A, Kracht M, Dittrich-Breiholz O, et al. (2010) Mutant PIK3CA licenses TRAIL and CD95L to induce non-apoptotic caspase-8-mediated ROCK activation. Cell Death Diff 17: 1435–1447. doi: 10.1038/cdd.2010.36
- 56. Baltmr A, Duggan J, Nizari S, Salt TE, Cordeiro MF (2010) Neuroprotection in glaucoma - Is there a future role? Exp Eye Res 91: 554–566. doi: 10.1016/j.exer.2010.08.009
- 57. Bernstein SL, Johnson MA, Miller NR (2011) Nonarteritic anterior ischemic optic neuropathy (NAION) and its experimental models. Prog Retinal Eye Res 30: 167–187. doi: 10.1016/j.preteyeres.2011.02.003