Elsevier

Neuroscience

Volume 231, 12 February 2013, Pages 206-215
Neuroscience

Nitric oxide/soluble guanylyl cyclase signaling mediates depolarization-induced protection of rat mesencephalic dopaminergic neurons from MPP+ cytotoxicity

https://doi.org/10.1016/j.neuroscience.2012.11.044Get rights and content

Abstract

Neuronal electrical activity has been known to affect the viability of neurons in the central nervous system. Here we show that long-lasting membrane depolarization induced by elevated extracellular K+ recruits nitric oxide (NO)/soluble guanylyl cyclase/protein kinase G signaling pathway, induces 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP)-mediated protein S-guanylation, and confers dopaminergic neuroprotection. Treatment of primary mesencephalic cell cultures with 1-methyl-4-phenylpyridinium (MPP+) for 72 h decreased the number of dopaminergic neurons, whereas the cell loss was markedly inhibited by elevated extracellular concentration of K+ (+40 mM). The neuroprotective effect of elevated extracellular K+ was significantly attenuated by tetrodotoxin (a Na+ channel blocker), amlodipine (a voltage-dependent Ca2+ channel blocker), Nω-nitro-l-arginine methyl ester (l-NAME) (a nitric oxide synthase inhibitor), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (a soluble guanylyl cyclase inhibitor), and KT5823 or Rp-8-bromo-β-phenyl-1,N2-ethenoguanosine 3′,5′-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPS) (protein kinase G inhibitors). Elevated extracellular K+ increased 8-nitro-cGMP production resulting in the induction of protein S-guanylation in cells in mesencephalic cultures including dopaminergic neurons. In addition, exogenous application of 8-nitro-cGMP protected dopaminergic neurons from MPP+ cytotoxicity, which was prevented by zinc protoporphyrin IX, an inhibitor of heme oxygenase-1 (HO-1). Zinc protoporphyrin IX also inhibited the neuroprotective effect of elevated extracellular K+. On the other hand, KT5823 or Rp-8-Br-PET-cGMPS did not inhibit the induction of HO-1 protein expression by 8-nitro-cGMP, although these protein kinase G inhibitors abrogated the neuroprotective effect of 8-nitro-cGMP. These results suggest that protein S-guanylation (leading to HO-1 induction) as well as canonical protein kinase G signaling pathway plays an important role in NO-mediated, activity-dependent dopaminergic neuroprotection.

Highlights

► High K+ depolarization protects dopaminergic neurons from MPP+ cytotoxicity. ► The protective effect is dependent on nitric oxide and protein kinase G (PKG). ► 8-Nitro-cGMP is produced in response to high K+ depolarization. ► 8-Nitro-cGMP confers neuroprotection in a PKG-dependent manner. ► PKG-independent expression of heme oxygenase-1 is also involved in neuroprotection.

Introduction

Parkinson’s disease, characterized by selective loss of dopaminergic neurons in the substantia nigra, is one of the most common neurodegenerative disorders (Dauer and Przedborski, 2003, Fahn and Sulzer, 2004). Various cellular events including mitochondrial dysfunction and oxidative stress have been reported to participate in the pathogenesis of this disorder. On the other hand, several lines of evidence suggest that excitatory neuronal activity plays an important role in the maintenance of the survival of dopaminergic neurons (Michel et al., 2007). For example, sustained depolarizing conditions produced by elevated extracellular K+ concentrations support the survival of dissociated mesencephalic dopaminergic neurons (Michel and Agid, 1996, Murer et al., 1999). In these settings, Ca2+ influx through voltage-dependent Ca2+ channels is implicated in the survival-promoting effect of elevated extracellular K+ (Michel et al., 2007). Conversely, long-term application of tetrodotoxin (TTX), a blocker of voltage-dependent Na+ channels, decreases the number of dopaminergic neurons in organotypic midbrain slice cultures (Katsuki et al., 2001). However, detailed signaling mechanisms involved in activity-dependent survival of dopaminergic neurons, particularly under pathological conditions, remain unclear.

Excitatory neuronal activity associated with membrane depolarization triggers Ca2+ entry into neurons that recruits several Ca2+-dependent signaling pathways. Of these, nitric oxide (NO) production by Ca2+/calmoduin-dependent neuronal NO synthase (nNOS) provides a means to transduce signals both intracellularly and intercellularly because of the gaseous properties of NO (Calabrese et al., 2007). NO is known to activate soluble guanylyl cyclase (sGC) and promotes the formation of cGMP, which activates protein kinase G (PKG). Besides this canonical PKG signaling pathway, NO-induced post-translational protein modifications such as S-nitrosation, which are independent of cGMP formation and PKG activation, may also be involved in NO-mediated regulation of cellular functions (Martínez-Ruiz and Lamas, 2009).

Moreover, recent studies identified 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) as a novel signaling molecule (Sawa et al., 2007, Fujii et al., 2010). That is, NO can react with GTP in the cytoplasm to form 8-nitro-GTP, and then 8-nitro-cGMP is produced by sGC that can utilize 8-nitro-GTP as a substrate (Fujii et al., 2010). 8-Nitro-cGMP can act as an endogenous, phosphodiesterase-resistant cGMP analog to activate PKG (Sawa et al., 2007). Another interesting feature of 8-nitro-cGMP is that this nitrated cyclic nucleotide can produce protein S-guanylation, namely, formation of protein-8-RS-cGMP adducts onto the sulfhydryl group of cysteine residues (Sawa et al., 2007, Fujii et al., 2010). In rat C6 glioma cells (Fujii et al., 2010) and mouse RAW 264.7 macrophages (Sawa et al., 2007) stimulated by lipopolysaccharide and cytokines, one of the targets of S-guanylation has been identified as kelch-like ECH-associated protein 1 (Keap1) (Itoh et al., 1999). S-Guanylation of Keap1 results in liberation of its associated transcription factor, nuclear factor-erythroid 2-related factor 2 (Nrf2), and induces the expression of heme oxygenase-1 (HO-1) (Sawa et al., 2007, Fujii et al., 2010). Whether these signaling mechanisms play a neuroprotective role in the central nervous system remains largely unclear, although our recent study demonstrated 8-nitro-cGMP production in midbrain slice cultures in response to a neuroprotective retinoic acid receptor agonist (Kurauchi et al., 2011).

In this context, we have previously reported that NO/cGMP-dependent upregulation of HO-1 expression acts as an endogenous neuroprotective system to limit inflammatory degeneration of dopaminergic neurons in rat organotypic midbrain slice cultures (Kurauchi et al., 2009, Kurauchi et al., 2012). Accordingly, in the present study we set out for the experiments to verify whether NO/cGMP-related molecular events including 8-nitro-cGMP formation, PKG activation and HO-1 upregulation are involved in activity-dependent neuroprotection of midbrain dopaminergic neurons.

Section snippets

Drugs

Drugs used in the present study were 1-methyl-4-phenylpyridinium (MPP+; Sigma, St. Louis, MO, USA), TTX (Nacalai Tesque, Kyoto, Japan), amlodipine besylate (Wako Chemicals, Osaka, Japan), Nω-nitro-l-arginine methyl ester (l-NAME; Sigma), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Wako, Osaka, Japan), KT5823 (Wako), Rp-8-bromo-β-phenyl-1,N2-ethenoguanosine 3′,5′-cyclic monophosphorothioate sodium salt hydrate (Rp-8-Br-PET-cGMPS; Sigma), 8-bromoguanosine-3′,5′-cyclic monophosphate sodium

Elevated extracellular K+ protects dopaminergic neurons from MPP+ cytotoxicity

Elevation of extracellular K+ concentration is a conventional method to cause membrane depolarization and neuronal excitation (Gallo et al., 1987, Katsuki et al., 2001). We examined the effect of elevated extracellular K+ against the cytotoxicity of a dopaminergic neurotoxin MPP+, in rat primary mesencephalic cell cultures. Treatment of mesencephalic cultures with MPP+ (5 μM) for 72 h significantly decreased the number of TH-positive dopaminergic neurons, and the majority of remaining TH-positive

Discussion

Neuronal activity plays a critical role in maintaining the survival of dopaminergic neurons (Michel et al., 2007) and the phenotype of dopaminergic neurons with regard to TH expression (Aumann and Horne, 2012). However, the role of neuronal activity under neuropathological conditions has been poorly understood. Here we found that NO-mediated signaling pathway induced by neuronal activity protects dopaminergic neurons from MPP+ cytotoxicity. We also demonstrated the neuroprotective effect of

Conclusion

Prolonged depolarization protected dopaminergic neurons from MPP+ cytotoxicity. This effect was mediated by NO/sGC, which caused PKG activation along with production of cGMP and 8-nitro-cGMP. NO/sGC also caused PKG-independent upregulation of HO-1, where protein S-guanylation may be involved. Interestingly, the protective effect of prolonged depolarization was attenuated by the inhibition of either PKG or HO-1, suggesting that both PKG-dependent and PKG-independent (but HO-1-dependent) pathways

Acknowledgments

This work was supported by Takeda Science Foundation, MEXT KAKENHI Grant No. 23117714, and JSPS KAKENHI Grant No. 24659118.

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    Present address: Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-Ku, Tokyo 108-8641, Japan.

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