Abstract
Abstract: :
Purpose:Alpha-fodrin, a known caspase target, is a neuronal cytoskeleton protein. Its proteolysis is suggested to contribute to structural rearrangements including membrane blebbing during apoptosis. It is cleaved by caspase-3 during apoptosis and co-localizes with the formation of neurofibrillary tangles in Alzheimer's disease. We sought to determine whether α-fodrin is cleaved by caspase-3 in retinas of COH rats and whether this process is reduced by adeno-associated virus (AAV)-induced retinal ganglion cell (RGC) expression of X-linked inhibitor of apoptosis protein (XIAP), a potent caspase-3 inhibitor. Methods: Ocular hypertension was induced unilaterally in eyes of brown Norway rats by limbal injection of hypertonic saline (2.0 M). Protein from COH and paired control retinas (n=5 each) were electrophoresed on a 10% Tris-HCl gel. Western immunoblotting was performed using a mouse monoclonal antibody to full-length α-fodrin. Densitometry of COH and control bands with subsequent paired t-tests were performed to determine significant differences in caspase-3 specific α-fodrin cleavage between COH and control retinas. In a similar experiment, COH was induced unilaterally in eyes pre-treated with an intravitreal injection of 2 µl of AAV-XIAP (n=4). Western immunoblots of retinal protein were performed in an identical fashion. Results: Caspase-3 cleavage of α-fodrin yields a specific protein fragment of 120 kDa. Densitometry of COH retina immunoblots showed significantly more cleavage of α-fodrin into 120 kDa fragments than control retinas (p<0.01, paired t-test). In addition, inhibition of retinal caspase-3 activity with XIAP gene therapy reduced the levels of the 120 kDa α-fodrin fragment compared to paired control retinas. Conclusion: Our results confirm our previous finding that caspase-3 is activated in the retina in a COH rat model of glaucoma, and that it cleaves α-fodrin, thereby contributing to the pathophysiology of glaucoma. This process parallels the pathology seen in other chronic neurodegenerations such as Alzheimer's disease, in which neurons undergo chronic caspase activation, slow build-up of cleavage products, and a delayed course of apoptosis. If caspase activation in glaucoma leads to protracted rather than rapid apoptosis of RGCs, a much longer therapeutic window exists for inhibition of apoptosis with caspase inhibitors such as XIAP.
Keywords: 323 apoptosis/cell death • 415 ganglion cells • 489 neuroprotection