Delayed activation of caspase-3 in response to cytochrome c release in axotomized retinal ganglion cells

Transection of the optic nerve results in delayed death of axotomized retinal ganglion cells (RGCs). While activation of caspase-3 and -9 has been implicated in axotomized RGC death, the upstream pathways involved in the activation of these caspases remain unknown. Since cytochrome c release from the mitochondria can lead to activation of casapse-9 and subsequently caspase-3, we hypothesize that cytochrome c release is involved in the initiation of apoptosis in axotomized RGCs. In adult hamsters, incidence of cytochrome c release, activation of caspase-3 and nuclear fragmentation were quantified at different time-points following optic nerve transection using immunohistochemistry. We found that cytochrome c release was observed at 1 day post-axotomy (dpa). Cytochrome c immunoreactivity localized entirely to the ganglion cell layer (GCL) and almost exclusively to Fluoro-Gold-labeled RGCs. This suggests that the cytochrome c release observed is injury-related. Activation of caspase-3 and nuclear fragmentation were first observed at 3 dpa. The activation of caspase-3 coincided with the beginning of axotomized RGC loss and was again observed only in the GCL. Our data suggest that cytochrome c release may be implicated in the activation of caspase-3 in axotomized RGCs. Activation of caspase-3 was delayed by two days after cytochrome c release. The delayed activation of caspase-3 suggests that the apoptotic cascade initiated after axotomy may be halted at a site downstream of cytochrome c release but upstream of caspase-3 activation. This observation may account for the delayed death of RGCs after axotomy in adult hamsters. Supported by An award from the University of Hong Kon