Calcium-induced Calpain Mediates Apoptosis via Caspase-3 in a Mouse Photoreceptor Cell Line

Abstract

The rd mouse, an accepted animal model for photoreceptor degeneration in retinitis pigmentosa, has a recessive mutation for the gene encoding the ␤-subunit of the cGMP phosphodiesterase. This mutation results in high levels of cGMP, which leaves an increased number of the cGMP-gated channels in the open state, thus allowing intracellular calcium (Ca 2؉ ) to rise to toxic levels, and rapid photoreceptor degeneration follows. To delineate the events in rd photoreceptor degeneration, we demonstrated an increase in calpain and caspase-3 activity, hypothesizing that Ca 2؉ -mediated apoptosis in photoreceptors is mediated by calpain, involving mitochondrial depolarization and caspase-3 activation. To examine this hypothesis further, a murine photoreceptor-derived cell line (661W) was treated with the Ca 2؉ ionophore A23187, cGMP-gated channel agonist 8-bromocGMP, or phosphodiesterase inhibitor isobutylmethylxanthine to mimic the increased Ca 2؉ influx seen in the rd photoreceptors. Ca 2؉ -induced cell death in 661W cells was found to be mediated by calpain and caspase-3 and could be completely inhibited by the calpain inhibitor SJA6017, implicating both calpain and caspases in the apoptotic process. The apoptotic events correlated in an SJA6017-inhibitable manner with bid cleavage, mitochondrial depolarization, cytochrome c release, and caspase-3 and -9 activation. We concluded that Ca 2؉ influx in the rd model of photoreceptor degeneration leads to the activation of the cysteine protease calpain, which executes apoptosis via modulation of caspase-3 activity.

Early alterations in mitochondrial reserve capacity; a means to predict subsequent photoreceptor cell death

“…The stressors generated by the effects of the gene mutation in the rd1 photoreceptor, calcium and oxidant stress, have been shown to result in mitochondria-dependent cell death (Sharma and Rohrer 2004, 2007). To examine whether short-term calcium or oxidant stress results in changes in mitochondrial reserve capacity, the 661W cells were exposed to calcium ionophore, A23187 (500 nM), or the oxidant, tBuOOH (50 µM), on the XF24 instrument for 30 min, after which the treated cells were exposed to FCCP (1 µM) to uncouple the mitochondrial membrane potential and thereby estimate mitochondrial reserve capacity.…”

“…Sustained, increased levels of intracellular calcium also cause mitochondrial stress due to activation of ETC flux, and mitochondrial swelling (Yoshida et al 1992). Eventually, elevated calcium leads to mitochondrial failure and apoptosis as has been shown in the rd1 model of retinitis pigmentosa (RP) (Fox et al 1999; Sharma and Rohrer 2004) as well as other neurodegenerative diseases (Zglinicki 2003) and perhaps aging (Beckman and Ames 1998; Brand 2000; Brand and Nicholls 2011). Loss of mitochondrial reserve capacity in response to elevated ROS levels has also been demonstrated with the Seahorse Biosciences extracellular flux instrument in cellular models of renal, cardiovascular, and neurodegenerative diseases (Dranka et al 2010, 2011), as well as in MERRF syndrome using isolated skin fibroblasts (Wu and Wei 2012).…”

“…The 661W cells, a mouse retina tumor-derived cell with cone-photoreceptor cell characteristics (Tan et al 2004), also display the Pasteur Effect when challenged with hypoxia or mitochondrial inhibitors (Winkler et al 2004a, b). We have shown that 661W cells treated with compounds to increase intracellular calcium or oxidative stress undergo rapid degeneration (Sharma and Rohrer 2004, 2007). …”

Multidestructive Pathways Triggered in Photoreceptor Cell Death of the RD Mouse as Determined through Gene Expression Profiling

“…Refs. 12-15), immunohistochemical analyses have been performed to describe changes in protein expression in the rd/rd retina (16,17), and calcium-induced cell death in a photoreceptor culture model has been studied to unravel the apoptotic pathway (64). However, a clear picture has yet to emerge that describes the apoptotic pathways involved in photoreceptor degeneration in the rd/rd mouse.…”

Ca2+/Calmodulin-dependent Protein Kinase IV Links Group I Metabotropic Glutamate Receptors to Fragile X Mental Retardation Protein in Cingulate Cortex

“…To further confirm the role of calcium in the activation of CaMKIV by Group I mGluRs, we then tested the effect of raising intracellular Ca 2ϩ by application of calcium ionophore A23187 (54,55)in ACC slices. We found that application of calcium ionophore A23187 (5 M, 15 min, and 30 min) could induce the phosphorylation of CaMKIV in ACC slices (169 Ϯ 8 and 157 Ϯ 9% of the control levels for A23187 at 15 and 30 min, respectively; p Ͻ 0.01, compared with control, n ϭ 4, Fig.…”