Abstract
Purpose: :
To examined the expression and activity of Calpain and PARP during photoreceptor cell death in transgenic rats with P23H and S334ter rhodopsin mutations.
Methods: :
Retinas of P23H-1, S334ter-3 and CD rats were collected at different developmental ages (PN0 - PN30). Retinas were examined by conventional histological techniques, TUNEL staining, immunohistochemistry and immunoblotting using specific antibodies for PAR, PARP and Calpastatin. Monitoring of calpain activity at the cellular level was investigated with an enzymatic in situ assay (Paquet-Durand et al., 2006) using the calpain specific fluorescent dye CMAC, t-BOC-Leu-Met on unfixed cryosections. For the PARP enzyme activity assays, we used a technique adapted by Paquet-Durand et al. (2007) using a PARP reaction mixture on unfixed cryosections.
Results: :
In S334ter-3 rats many photoreceptors and few cells in the inner retina were positive for TUNEL assay during the whole development. In P23H-1 rats only some photoreceptors were positive for TUNEL assay however, positive cells remains detectable as late in the development as PN30. PAR immunostaining showed numerous positive cells in the outer retina of both transgenic rats, their quantity however being higher in the S334ter-3. No positive cells were detected in the CD retina. We observed PARP activity only in a subset of cell bodies in P23H-1 and S334ter-3 photoreceptors, but never in the CD retinas. Contradictory to previous studies made in rd1 mouse, PARP immunostaining was found to be increased in both transgenic models when compared to CD rats. Calpastatin expression was reduced in both, P23H-1 and S334ter-3 retinas, whereas in their photoreceptors, calpain activity was considerably increased.
Conclusions: :
These results coincide with previous studies using other animal models for retinitis pigmentosa, indicate that activation of calpains and PARP could be a general mechanism involved in photoreceptor cell death, which elevates the possibility of using calpain and/or PARP inhibitors as therapeutic agents to prevent or delay photoreceptor degeneration.
Keywords: apoptosis/cell death • photoreceptors • retinal degenerations: cell biology