Elsevier

Brain Research

Volume 1256, 23 February 2009, Pages 190-204
Brain Research

Research Report
Selective blockade of CaMKII-α inhibits NMDA-induced caspase-3-dependent cell death but does not arrest PARP-1 activation or loss of plasma membrane selectivity in rat retinal neurons

https://doi.org/10.1016/j.brainres.2008.12.051Get rights and content

Abstract

Calcium/calmodulin-dependent protein kinase II-α (CaMKII-α) has been implicated in a number of receptor mediated events in neurons. Pharmacological blockade of CaMKII-α has been shown to prevent phosphorylation of NMDA-R2A and R2B receptor subunits, suggesting that this enzyme may be linked to receptor trafficking of glutamate receptors and serve as a regulatory protein for neuronal cell death. In the retina, inhibition of CaMKII-α has been reported to be neuroprotective against NMDA-induced cell death by preventing the activation of the caspase-3 dependent pathway. However, the effects of CaMKII-α blockade on the caspase-3 independent, PARP-1 dependent and the non-programmed cell death pathways have not previously been investigated. In the present study, blockade of CaMKII-α with the highly specific antagonist myristoylated autocamtide-2-related inhibitory peptide (AIP) was used in a rat in vivo model of retinal toxicity to compare the effects of on NMDA-induced caspase-3-dependent, PARP-1 dependent and the non-programmed (necrosis) cell death pathways. Results confirmed that AIP fully attenuates caspase-3 activation for at least 8 h following NMDA insult and also significantly improves retinal ganglion cell survival. However, this blockade had little effect on reducing the loss of plasma membrane selectivity (LPMS, e.g. necrosis) in cells located in the ganglion cell and inner nuclear layers and did not alter NMDA-induced PARP-1 hyperactivation, or prevent TUNEL labeling following a moderate NMDA-insult. These findings support a specific role for CaMKII-α in mediating the caspase-3 dependent cell death pathway and provide evidence that it is not directly linked to the signaling of either the PARP-1 dependent or the non-programmed cell death pathways.

Introduction

Understanding the mechanisms involved in mediating neuronal cell death linked to stroke/ischemia has been the focus of numerous laboratories over the past four decades. One of the most widely utilized models for initiating cell death in neurons is through the activation of a subset of ionotropic glutamate receptors by the agonist N-methyl-D-aspartate (NMDA). Studies have shown that exposure to NMDA causes an expression of morphological features that are indicative of both apoptosis and necrosis (Siliprandi et al., 1992, Bonfoco et al., 1995, Lam et al., 1999, Goebel and Winkler, 2006). Furthermore, it has been shown that the preference for a particular mode of cell death is dependent upon the degree of insult, with a low concentration of NMDA favoring energy-dependent pathways linked to programmed cell death, and higher doses favoring non-programmed cell death, e.g., necrosis (Bonfoco et al., 1995).

Blockade of the NMDA receptor with the noncompetitive antagonist MK-801 or memantine has been shown to block NMDA-induced neuronal cell death (Fix et al., 1993, Fix et al., 1995, Levy and Lipton, 1990, Pellegrini and Lipton, 1993), leading to the conclusion that over stimulation of the NMDA receptor is directly responsible for the initiation of both programmed and non-programmed cell death pathways. However, MK-801 treatment alone induces cell death of select populations of cortical neurons in rats (Fix et al., 1993), and in human clinical trials causes serious side effects in healthy patients (Davis et al., 1997, Davis et al., 2000, McBurney, 1997). These results have led to the understanding that irreversible NMDA-receptor blockade alone is not a viable option for therapeutic intervention against NMDA-receptor mediated cell death.

As an alternative to full NMDA-receptor blockade, attention has been directed to regulatory proteins that are integral to the propagation of specific cell death pathways. Of particular interest is CaMKII, as both the alpha and beta isoforms of this enzyme are highly expressed in neuronal tissues (Braun and Schulman, 1995). In particular, CaMKII-α has been shown to directly interact with the NR2A and NR2B NMDA-receptor subunits to regulate their interaction with cytosolic scaffolding proteins such as PSD-95 and Sap102 (Watanabe et al., 2003, Iwamoto et al., 2004) by CaMKII-dependent phosphorylation (Gardoni et al., 2006). It has also been shown in retinal neurons to be upregulated, both at the gene and protein levels following NMDA-receptor stimulation (Laabich et al., 2000, Fan et al., 2005). Other retinal studies have shown that specific blockade of the CaMKII-α autophospholylating site, with the highly specific myristoylated autocamtide-2 related inhibitory peptide (AIP) (Ishida et al., 1995), attenuates NMDA-induced caspase-3 activation (Laabich and Cooper, 2000, Laabich et al., 2000, Laabich et al., 2001, Lin et al., 2004, Ishida et al., 2001); however, the effects of this antagonism on cell death pathways not linked to caspase-3 pathway have not been explored.

Using an in vivo rat retinal toxicity model, the present study confirmed that blockade of CaMKII-α activity prevents NMDA-induced caspase-3 activation and provides significant neuroprotection against NMDA-induced ganglion cell loss. However, antagonizing CaMKII-α had only a limited effect on altering NMDA-induced loss of plasma membrane selectivity (LPMS: e.g. necrosis) in cells residing in the ganglion cell- (GCL) and inner nuclear (INL) layers of the retina. In addition, this blockade had no effect on preventing NMDA-induced elevation of poly(ADP-ribosyl)ation (PAR) through the PARP-1 pathway, and did not block TUNEL labeling following a moderate excitotoxic insult by NMDA.

Section snippets

Caspase-3 activation

To measure the effectiveness of AIP in blocking the caspase-3-dependent pathway, retinas exposed to NMDA/AIP, NMDA-alone and appropriate controls were assessed for caspase-3 activity at 1 h, 2 h, 4 h or 6 h post-treatment (PT) (Fig. 1a). NMDA treatment alone resulted in an increase in caspase-3 activity at 1 h-PT and peaked by 2 h-PT (Fig. 1a: n = 5 for each treatment and time point ,⁎⁎⁎ indicates p < 0.001, ⁎ p < 0.05 significance than NMDA activity at 2 h, ANOVA). A decline in activity was noted at

Discussion

The present data supports the hypothesis that upstream activation of CaMKII-α is required for the signaling of the caspase-3 dependent cell death pathway (Laabach and Cooper, 2000; Fan et al., 2005). Blockade with the CaMKII-α specific antagonist AIP, like the caspase-3 specific antagonist Ac-DEVD-CHO, fully attenuated NMDA-induced caspase-3 activity up to 6 h following NMDA insult (Fig. 1a). However, unlike the caspase-3 inhibitor, which did not prevent the generation of the 19 and 17 kDa

Materials

N-methyl-D-aspartate, alkaline phosphatase-linked goat anti-rabbit-IgG, mouse monoclonal anti-β-actin, PJ-34 (Sigma, St Louis, MO); myristoylated autocamtide-2 related inhibitory peptide, Ac-DEVD-CHO (Calbiochem-Novabiochem Corp., LaJolla, CA); Ac-DEVD-fmc (Bachem Bioscience Inc., Torrance, CA); caspase-3 inhibitor I (Ac-Asp-Glu-Val-Asp-CHO, or Ac-DEVD-CHO) (Calbiochem, San Diego, CA); peroxidase-linked sheep anti-mouse-IgG (Amersham, Piscataway, NJ); In situ cell death detection kit, rabbit

Acknowledgments

I would like to thank Dr. Fei Yu, Dr. Ivanova Elena, and my assistant Eren D. Berberoglu for their advice and technical expertise. Also, I wish to acknowledge the data displayed in Fig. 6 to Ms Tania Borboni and dedicate this work in memory of her energetic smile and enthusiasm she brought to our lab. Her tragic departure from this world at such a young age raises many unanswerable questions and her absence, will be greatly missed by all that had the opportunity to know her. Supported by NIH

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