In addition to the cyclic

In addition to the cyclic guanosine monophosphate-dependent protein kinase pathway, NO is known to regulate the function of target proteins through S-nitrosylation of cysteine residues (Hess et al., 2005; Jaffrey et al., 2001). One such target is the type 1 ryanodine receptor (RyR1), which is the Ca-induced Ca release (CICR) channel in the endoplasmic reticulum (ER). NO induces the opening of the RyR1 channel through S-nitrosylation (Aghdasi et al., 1997; Eu et al., 2000; Sun et al., 2001). The activity of RyR1 due to this activation mechanism has been implicated in Ca leakage from skeletal muscle Ca stores that has been attributed to certain pathological conditions (Bellinger et al., 2009; Durham et al., 2008). A single cysteine residue at 3635 (C3635) in rabbit RyR1 is responsible for sensitizing the skeletal muscle Ca release channel to NO (Sun et al., 2001). An alanine substitution for C3635 (C3635A) of RyR1 expressed in human embryonic kidney (HEK) 293 neomycin sulfate has been shown to reduce S-nitrosylation levels and abolish the regulation of the skeletal muscle Ca-release channel by physiological concentrations of NO (Sun et al., 2001). In the brain, NO induces Ca release from the ER through S-nitrosylation of RyR1, which results in an increased concentration of intracellular Ca in neurons (Kakizawa et al., 2012). Ca release via RyR1 has also been implicated in NO-induced neuronal cell death, as shown by studies in which cell death is significantly milder in cultured neurons taken from RyR1-deficient mice than in controls (Kakizawa et al., 2012). These studies raise the possibility that NO-induced Ca release (NICR) is involved in certain pathological states in the brain; however, the pathophysiological role of NICR remains to be established. Furthermore, pathophysiological significance of NICR in vivo has not been examined. In this study, we examined whether NICR via S-nitrosylated RyR1 is involved in SE-induced neurodegeneration. In order to study the role of NICR in vivo, we generated a knock-in mouse line, in which the essential cysteine residue at 3636 of mouse RyR1 (corresponding to cysteine 3635 in humans and rabbits) was replaced by alanine (Ryr1C3636A) to prevent its S-nitrosylation. We show that NICR was indeed silenced in neurons from Ryr1C3636A knock-in mice, which allowed us to examine the role of NICR in a kainic acid (KA)-model of temporal lobe epilepsy. Here we provide evidence that NICR exacerbates neurodegeneration in the hippocampus following KA-induced seizures, suggesting that RyR1 is a promising therapeutic target candidate to ameliorate the neurodegenerative effect of SE.
Materials and Methods
Results
Discussion These results indicate the involvement of NICR in neurodegeneration following SE. Moreover, this data set is consistent with the finding that epileptic seizure-induced neurotoxicity is reduced in nNOS-deficient mice (Parathath et al., 2007). Although the targets of NO have been elusive, the present results highlight the importance of the S-nitrosylation of RyR1 at Cys3636 (corresponding to Cys3635 in rabbits and humans). This cysteine residue has been shown to undergo various redox modifications such as oxidation by H2O2 and S-glutathionylation in addition to S-nitrosylation (Aracena-Parks et al., 2006). However, H2O2 has been shown to activate both rabbit RyR1WT and RyR1C3635A (Aracena-Parks et al., 2006). Furthermore, while it was reported that S-nitrosoglutathione (GSNO) preferentially S-glutathionylated Cys3635 (Aracena-Parks et al., 2006), another study found that GSNO activated RyR1C3635A similarly to RyR1WT (Sun et al., 2003). Thus, neither oxidation nor S-glutathionylation of Cys3636 is likely to be involved in the activation of RyR1. The RyR1 antagonist dantrolene is an approved drug for the treatment of malignant hyperthermia, which is a pharmacogenetic disorder in patients characterized by an increased susceptibility of muscular RyR1 to general anesthetics (Hirshey Dirksen et al., 2011). Several studies have raised the possibility of dantrolene as a treatment for neurodegeneration (Mody and MacDonald, 1995; Muehlschlegel and Sims, 2009). However, the precise mechanism for its therapeutic effect has remained elusive. The present results shed light on this important question, and pave the way for exploring RyR1 antagonists as therapeutics for brain damage associated with epileptic seizures.