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D. Sanges, A. Auricchio, V. Marigo; Molecular Analysis of P23h Photoeceptor Cell Death: Role of AIF, Caspase-12 and Calpains. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4519.
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Programmed cell death has a crucial role in the pathogenesis of Retinitis Pigmentosa (RP), a group of retinal degenerations that represents the largest cause of blindness in the developed world. An animal model for autosomal dominant RP (ADRP) is represented by a rat transgenic for a mutant form of rhodopsin with the proline to histidine substitution at codon 23 (P23H), that accounts for 12% of all RP cases in the US. Many studies have suggested that this mutation causes disease through the misfolding of the protein; however the connection between rhodopsin mislocalization and activation of cell death program is still elusive and this hampers the development of a cure for this blinding disease. Recently we reported that unfolded proteins lead to endoplasmatic reticulum (ER) stress and subsequently to activation of AIF and caspase-12 that could be blocked by calpain inhibitors (Sanges et al., Apoptosis 2006). This treatment is already able to arrest apoptosis in an animal model of autosomal recessive RP, the rd1 mouse, in which AIF and caspase-12 are activated (Sanges et al., PNAS, 2006).
We cultured photoreceptors derived from differentiated retinal stem cells obtained from P23H rats. Relative amounts of GADD153 and GRP-78 were calculated to evaluate ER stress. Cells were treated with calpain inhibitors and siRNAs targeting AIF, caspase-12 and calpains. Cells were finally analyzed by immunoflorescence.
We observed that endogenous rhodopsin accumulate in the ER of the P23H photoreceptors. Cells that showed rhodopsin aggregation spontaneously underwent apoptosis in vitro. Over-expression of GADD153 and GRP-78 indicated that the unfolding protein response system (UPR) was activated in P23H photoreceptor cells. We analyzed the possible activation of AIF and caspase-12 and we found that both factors translocated to the nucleus of the apoptotic cells. Most interestingly, by blocking calpain activity in vitro, we were able to abolish translocation of both factors and consequently apoptosis in the P23H photoreceptors.
This study provides the first evidence that P23H photoreceptors cell death is dependent on activation of AIF and caspase-12, and that calpains can be targeted to arrest activation of the specific apoptotic program. Moreover, thanks to the identification of common apoptotic pathways activated during retinal degeneration, it is possible to hypotize an unique treatment for different genetic form of RP.<br
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