Abstract
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.
Highlights
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 [1,2,3]
The data presented in this study confirm our previous findings that caspase-2 is an important regulator of RGC apoptosis induced after ON crush (ONC) [7]
We report that caspase-2 is 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
Summary
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 [1,2,3]. Inhibition of apoptosis by neurotrophic factor administration [11], overexpression of Bcl-2 [12,13] and inhibition of caspase-1 and -3 [14,15] and caspase-6 and -8 [16] using pharmacological inhibitors all reduced the number of dying RGC after ON transection and ONC. Only caspase-6 and -8 inhibitors have yielded limited RGC axon regeneration after ON axotomy [16]. We report that a serially injected cell permeable pharmacological inhibitor of PLOS ONE | www.plosone.org z-VDVAD Rescues RGC from Death 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
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