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8-Nitro-cGMP attenuates context-dependent fear memory in mice
Biochemical and Biophysical Research Communications 511 (2019) 141e147
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8-Nitro-cGMP attenuates context-dependent fear memory in mice Yusuke Kishimoto a, Shingo Kasamatsu b, c, Shuichi Yanai d, Shogo Endo d, Takaaki Akaike c, Hideshi Ihara a, * a
Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA c Department of Environmental Medicine and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan d Aging Neuroscience Research Team, Tokyo Metropolitan Institute of Gerontology, Itabashi, Tokyo 173-0015, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 January 2019 Accepted 31 January 2019 Available online 14 February 2019
We previously reported that 8-nitroguanosine 30 ,50 -cyclic monophosphate (8-nitro-cGMP) is endogenously produced via nitric oxide/reactive oxygen species signaling pathways and it reacts with protein thiol residues to add cGMP structure to proteins through S-guanylation. S-Guanylation occurs on synaptosomal-associated protein 25 (SNAP-25), which is a part of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that regulates exocytosis. However, the biological relevance of 8-nitro-cGMP in the nervous system remains unclear. Here, we investigated the effects of intracerebroventricular (icv) infusion of 8-nitro-cGMP on mouse brain functions. The results of an open-field test and fear-conditioning task revealed that icv infusion of 8-nitro-cGMP decreased the vertical activity and context-dependent fear memory of mice, which are both associated with the hippocampus. Immunohistochemical analysis revealed increased c-Fos-positive cells in the dentate gyrus in 8-nitro-cGMP-infused mice. Further, biochemical analyses showed that icv infusion of 8-nitro-cGMP increased S-guanylated proteins including SNAP-25 and SNARE complex formation as well as decreased complexes containing complexin, which regulates exocytosis by binding to the SNARE complex, in the hippocampus. These findings suggest that accumulation of 8-nitro-cGMP in the hippocampus affects its functions, including memory, via S-guanylation of hippocampal proteins such as SNAP-25. © 2019 Elsevier Inc. All rights reserved.
Keywords: 8-Nitroguanosine 30 ,50 -cyclic monophosphate Fear-conditioning task Synaptosomal-associated protein 25 S-Guanylation Nitric oxide Reactive oxygen species
1. Introduction Nitric oxide (NO), a well-known gasotransmitter, is endogenously produced by NO synthases and is involved in numerous physiological and pathological functions in various organisms [1]. These functions are mediated through a cyclic guanosine 30 ,50 monophosphate (cGMP)-dependent pathway via activation of cGMP-dependent protein kinase [2,3]. Other pathways may be driven by nitrosylation and nitration of biomolecules such as amino acids, proteins, lipids, and nucleotides through processes induced by reactive nitrogen oxide species, which are derived from NO and reactive oxygen species (ROS), such as peroxynitrite and nitrogen dioxide [4e6]. We have previously reported on the endogenous production of 8-nitroguanosine cGMP (8-nitro-cGMP) as a secondary messenger
* Corresponding author. 608 Bldg. C10, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka, 599-8531, Japan. E-mail address: [email protected] (H. Ihara). https://doi.org/10.1016/j.bbrc.2019.01.138 0006-291X/© 2019 Elsevier Inc. All rights reserved.
of NO/ROS redox signaling [6]. This nitrated nucleotide can react with reactive thiol groups on cysteine residues to add a cGMP structure to the protein, which is a post-translational modification known as protein S-guanylation [6]. Because of its unique chemical properties, 8-nitro-cGMP is related to various physiological and pathophysiological functions, including oxidative stress response [6,7], neurotoxicity [8,9], cell senescence [10], and autophagy [11]. Several target proteins of S-guanylation have been identified and their biological roles have been reported [6,7,10]. We recently reported the endogenous production of 8-nitro-cGMP in the rodent brain and S-guanylation of proteins in the brain including synaptosomal-associated protein 25 (SNAP-25), which is one of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins [12]. SNAP-25 plays a critical role in cooperating with other SNARE proteins and SNARE modulatory proteins such as complexin (cplx), which binds to the SNARE complex, during exocytosis by regulating synaptic vesicle fusion [13]. Post-translational modifications of SNAP-25, such as phosphorylation [14], palmitoylation [15], and
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nitration [16], were reported to be associated with neuronal functions. Additionally, our recent studies revealed that S-guanylation of SNAP-25 enhances SNARE complex formation [12] but attenuates the interaction between the SNARE complex and cplx [17], suggesting that S-guanylation of SNAP-25 is involved in regulating exocytosis. However, the biological implications of 8-nitro-cGMP and S-guanylation of proteins including SNAP-25 in the nervous system are not fully understood. The roles of NO and ROS in the mechanisms underlying memory have been widely examined [18,19]. Additionally, numerous studies have suggested that dysregulation of NO/ROS, which is induced under certain pathological conditions such as Alzheimer's disease (AD), is associated with memory dysfunction [18,20,21]. Thus, NO/ ROS homeostasis is thought to be important for maintaining memory functions. However, the biological importance of 8-nitrocGMP in memory remains unclear. In the present study, to investigate the effects of 8-nitro-cGMP on mouse brain functions, we intracerebroventricularly (icv) infused 8-nitro-cGMP in mice and analyzed brain functions by conducting behavioral tests and evaluated cellular mechanisms regarding synaptic exocytosis through biochemical experiments. Our results indicated that icv administration of 8-nitro-cGMP decreased vertical activity and context-dependent fear memory, likely via S-guanylation of hippocampal SNAP-25 to attenuate the binding affinity of cplx to the SNARE complex. 2. Materials and methods 2.1. Intracerebroventricular infusion of 8-nitro-cGMP into mice Male C57BL/6J mice were obtained from CLEA Japan (Tokyo, Japan). All experiments were approved by the animal experiment committee of the Tokyo Metropolitan Institute of Gerontology and carried out according to its guidelines. Mice (11e12 weeks) were utilized for this study. The detailed experimental design is described in Supplementary information. 8-Nitro-cGMP administration to mice was carried out with a guide cannula and an internal canula (C315GS-5/SPC and C315IS-5/ SPC, Plastics One, Roanoke, VA), that were inserted into the right lateral ventricle (coordinates: anterior-posterior, 0.5 mm; mediallateral, þ1.0 mm; dorsal-ventral, þ1.6 mm), at a rate of 1.0 ml/min of phosphate-buffered saline with or without 5, 10 mM 8-nitro-cGMP, synthesized as described previously [6]. 2.2. Open-field test The open-field test was performed as described previously [22] with modifications. The apparatus for the open-field test was obtained from O'Hara & Co. (Tokyo, Japan). In brief, a mouse was placed in the center of the arena and allowed to explore it for 15 min. On next day of the first 8-nitro-cGMP infusion, the openfield test was carried out under a dark-lit condition (10 lux), then it was performed again under a light-lit condition (300 lux) on the following day as described previously. After recording, the total distance moved, the number and duration of rearing behaviors, the total time spent immobile, and the percentage of time spent in the center region were automatically calculated with Image OFCR (O'Hara & Co.).
subsequent unconditioned stimulus (US; 0.5 s of electric footshock, 0.12 mA, delivered from 2.5 s after CS starts). One and 24 h after the conditioning, cue-dependent fear memory was analyzed. Forty-eight hours after the conditioning, context-dependent fear memory was assessed. The freezing behavior of the mice was recorded with a camera placed above the chamber and the data were analyzed using automated software (Time FZ1, O'Hara & Co.). Further details are provided in Supplementary information. 2.4. Immunohistochemistry After the context-dependent fear memory test, immunohistochemical analysis of mouse brain sections was performed as previously described [12], with antibodies specific to S-guanylated proteins [6] and c-Fos (Santa Cruz Biotechnology, Dallas, TX), followed by peroxidase-conjugated secondary antibodies (Nichirei Biosciences, Tokyo, Japan). Reactions were visualized with a diaminobenzidine system (Nacalai Tesque, Kyoto, Japan). Immunostained images in several regions in mouse brains were observed by using IX53 inverted microscope (Olympus, Tokyo, Japan). Nissl staining of mouse brain sections was also performed by soaking sections into 0.2% cresyl violet 0.3% acetic acid solution for 15 min. 2.5. Immunoprecipitation and Western blotting Mouse brain samples were harvested, then immunoprecipitation and Western blotting were performed as described previously [12] with slight modifications. Antibodies specific to SNAP-25, syntaxin 1A and vesicle-associated membrane protein 2 (VAMP2) were kindly supplied by Dr. Masami Takahashi (Kitasato University). In brief, hippocampal lysates (50 mg) were incubated with anti-SNAP-25 antibody or anti-S-guanylated protein antibody [6] bound to Protein G magnetic beads (Merck Millipore, Darmstadt, Germany). Immunoprecipitates were collected by 5 min boiling and analyzed by Western blotting with specific antibodies. 2.6. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) Native SNARE complexes with cplx were analyzed using BNPAGE followed by Western blotting as described previously [17,23] with minor modifications. In brief, 20 mg hippocampal lysates, prepared with 1 NativePAGE sample buffer (Thermo Fisher Scientific, Waltham, MA) containing 0.5% Triton X-100 and 0.125% Coomassie Brilliant Blue-G250 (Thermo Fisher Scientific) were separated by BN-PAGE using 4e16% Bis-Tris polyacrylamide gradient gels, followed by Western blotting with anti-SNAP-25 and anti-cplx 1/2 antibodies (R&D systems, Minneapolis, MN). 2.7. Statistical analysis Data are the mean ± standard error of the mean (SEM) of at least three independent experiments unless otherwise specified. For statistical comparisons, we utilized unpaired one-way or two-way analysis of variance (ANOVA) followed by the Tukey's post-hoc test via GraphPad Prism software (GraphPad Software, La Jolla, CA). We considered P < 0.05 as statistically significant. 3. Results
2.3. Fear-conditioning task
3.1. Open-field test
The fear-conditioning task was carried out as described previously [22] with modifications. In brief, fear-conditioning was performed by presenting a conditioned stimulus (CS; 3 s of pure tone, 10 kHz, 70 dB, delivered from 60 s after session starts) and
First, we analyzed the effects of icv infusion of 8-nitro-cGMP on mouse behaviors in open-field tests under both dark-lit and light-lit conditions. As shown in Fig. 1-A, there were no significant differences in the total distance traveled by the mice within 15 min in the
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Fig. 1. Open-field test. The open-field test was carried out under dark-lit and the light-lit conditions to measure activity and anxiety levels in mice. (A) Total distance moved. (B) Rearing frequency and duration. (C) Immobility time. (D) Percentage of time spent in the center region. Data are the mean ± SEM (n ¼ 22 or 23). *P < 0.05, **P < 0.01 vs. control.
open-field test among the control and 8-nitro-cGMP-infused groups both under dark-lit and light-lit conditions. In the dark-lit test, 8-nitro-cGMP-administrated mice showed significant decreases in both the frequency and duration of rearing, whereas a significant difference was observed only in rearing duration in 8nitro-cGMP-infused mice but not in the frequency of rearing in the light-lit test, compared to the observations in control mice (Fig. 1-B). As shown in Fig. 1-C and 1-D, there were no significant differences in the total immobility time and percentage of the time mice spent in the center region between control and 8-nitro-cGMPinfused mice. 3.2. Fear-conditioning task We analyzed effects of icv infusion of 8-nitro-cGMP into mice on memory by fear-conditioning task. As shown in Fig. 2-A and 2-B, there were no differences in the ratio of freezing during the cuedependent fear memory test between the control and 8-nitrocGMP-administrated mice at 1 and 24 h after fear conditioning. However, the context-dependent fear memory test performed at 48 h after conditioning indicated that the ratio of freezing decreased in an 8-nitro-cGMP-infused dose-dependent manner and that mice administered 100 nmol 8-nitro-cGMP showed a significantly decreased freezing ratio compared to that of control mice (Fig. 2-C). 3.3. Histochemical analyses of 8-nitro-cGMP-administrated mouse brains To investigate the histological changes in 8-nitro-cGMP-infused mouse brains, we performed Nissl staining and immunohistochemical analysis using antibodies for S-guanylated proteins and cFos. Immunohistochemical analysis indicated that S-guanylated proteins were strongly increased in the hippocampal regions of
cornu ammonis 1 (CA1), CA3, and dentate gyrus, but weakly increased in the cerebral cortex and amygdala (Fig. 3-A, Supplementary Fig. S2-A and S2-B). As shown in Fig. 3-B, neurons showed no morphological changes in the hippocampi of 8-nitrocGMP-infused mouse brains by Nissl staining. To analyze neuronal activity in the hippocampus, we performed immunohistochemical analysis with an antibody specific to c-Fos, which is widely used as a neuronal activity marker [24]. The results showed that c-Fospositive cells in the dentate gyrus were significantly increased in 8nitro-cGMP-administrated animals (Fig. 3-C, 3-D). 3.4. Effects of 8-nitro-cGMP infusion on protein S-guanylation and SNARE complex formation in mouse brains To examine effects of icv administration of 8-nitro-cGMP to mice on the functions of proteins related to memory, we analyzed the Sguanylation of proteins in the hippocampus and cerebral cortex. Western blotting revealed that various S-guanylated protein levels were high in the hippocampus but low in the cerebral cortex collected from 8-nitro-cGMP-infused mice (Fig. 4-A and 4-B). Because SNAP-25 has been reported to be S-guanylated [12,17], we confirmed S-guanylation of SNAP-25 by immunoprecipitation with an anti-SNAP-25 antibody followed by Western blotting using an anti-S-guanylated protein antibody. As shown in Fig. 4-C, increased S-guanylated SNAP-25 was detected in the hippocampi of 8-nitrocGMP-infused mice. Additionally, S-guanylation of SNAP-25 was also confirmed in the sample immunoprecipitated with the anti-Sguanylated protein antibody (Fig. 4-C). Because it has been reported that S-guanylation of SNAP-25 affects SNARE complex formation [12,17], we examined the effects of 8-nitro-cGMP administration on SNARE complex formation in the hippocampus. Although icv infusion of 8-nitro-cGMP did not affect the expression levels of SNARE proteins (i.e., SNAP-25, syntaxin 1A, and VAMP2) and cplx 1/2 (Supplementary Fig. S3-A and
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Fig. 2. Fear-conditioning task. Cue- and context-dependent fear memory tests were carried out to measure short- and long-term fear memory in control and 8-nitro-cGMPadministrated mice. (A) Freezing rate of the cue-dependent fear memory test performed 1 h after conditioning. (B) Freezing rate of the cue-dependent fear memory test performed 24 h after conditioning. (C) Results of the context-dependent fear memory test performed 48 h after conditioning. Data are the mean ± SEM (n ¼ 22 or 23). *P < 0.05 vs. control.
Fig. 3. Histochemical analyses of 8-nitro-cGMP-administrated mouse brains. Immunohistochemistry to detect S-guanylated proteins and c-Fos (A and C) and Nissl staining (B) were carried out using mouse brain tissues. (D) Mean ± SEM of c-Fos-positive cells in the dentate gyrus region marked by dashed lines in C (n ¼ 3 or 4). *P < 0.05 vs. control. Scale bars show 100 mm. CA, cornu ammonis.
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Fig. 4. Effects of 8-nitro-cGMP infusion on protein S-guanylation and SNARE complex formation in mouse brains. Western blotting (WB) with anti-S-guanylated protein or antiactin antibodies (A), immunoprecipitation (IP) followed by WB with antibodies for SNAP-25 or S-guanylated proteins (C) and BN-PAGE followed by WB with antibodies for SNAP-25 or cplx 1/2 (D) were carried out using lysates obtained from the hippocampus or cerebral cortex. (B and E) Densitometric analysis of the immunoblot of A and D, respectively. Data are the mean ± SEM (n ¼ 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control hippocampus; xP < 0.01 vs. control cerebral cortex; #P < 0.01, ##P < 0.001 vs. hippocampus of 8-nitro-cGMP-infused mice with the same dose. C, cerebral cortex; H, hippocampus.
S3-B), BN-PAGE followed by Western blotting with an anti-SNAP-25 antibody revealed that immunoreactive bands at higher than 480 kDa (indicated as “I” in Fig. 4-D, left) were significantly increased in the hippocampi of 8-nitro-cGMP-infused mice (Fig. 4D and 4-E). Additionally, immunoreactive bands against an antibody specific for cplx 1/2, an exocytosis regulatory protein that binds to the SNARE complex, detected at approximately 200 kDa (indicated in “II” in Fig. 4-D, right) and the band at lower than approximately 150 kDa (indicated in “III” in Fig. 4-D, right) in the hippocampi of 8-nitro-cGMP-administrated animals were significantly decreased and increased, respectively (Fig. 4-D and 4-E). 4. Discussion In the present study, we evaluated the effects of icv administration of 8-nitro-cGMP on mouse brain functions by conducting behavioral tests and biochemical analyses. This is the first study to reveal an association between 8-nitro-cGMP and mouse brain functions. The open-field test revealed that 8-nitro-cGMP did not affect the total distance moved (horizontal activity), total immobility time, and percentage of time spent in the center region (both are indices of anxiety [25]). Additionally, in the dark-lit test, both the frequency and duration of rearing (vertical activity) in 8-nitro-cGMP-infused animals were decreased, while only rearing duration was decreased in the light-lit test (Fig. 1). Some mouse behaviors including rearing have been reported to decrease under light conditions [25]. Therefore, slight differences in rearing frequency may not have been detected in the light-lit test. Rearing is controlled by the
hippocampus [26], and reduced rearing behavior has been reported in mice with hippocampal lesions, whereas horizontal activity is unaffected in these animals [27]. Taken together, our results suggest that 8-nitro-cGMP infusion affects vertical activity, which is controlled by the hippocampus, but does not affect horizontal activity or anxiety. The fear-conditioning task is one of the most well-established methods for measuring fear memory. Cue-dependent and contextdependent fear memory are both associated with the amygdala, while the latter is also associated with the hippocampus [28,29]. 8Nitro-cGMP administration did not alter short- or long-term fear memory associated with the amygdala but reduced long-term fear memory, which is associated with the amygdala and hippocampus, in a dose-dependent manner (Fig. 2). These data suggest that icv infusion of 8-nitro-cGMP attenuates hippocampal functions. We confirmed the elevation of S-guanylated proteins in the brain tissues, particularly in the hippocampi of 8-nitro-cGMPinfused animals (Figs. 3 and 4). However, there were no morphological changes in the hippocampal neurons of 8-nitro-cGMPinfused mice (Fig. 3-B), which is consistent with our previous study showing that 8-nitro-cGMP does not possess cytotoxic effects in rat cerebellar granule neurons [30]. In contrast, c-Fos-positive cells were significantly increased in the dentate gyrus of 8-nitro-cGMPinfused mice (Fig. 3-C and 3-D), indicating increased neuronal activity in the hippocampus. Because increased c-Fos levels were observed in the hippocampus of patients with AD and in a mouse model [31,32] and hippocampal hyperactivation has been reported in patients in the early stages of AD [33], elevated c-Fos levels may be involved in neuronal dysfunction. However, the precise
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mechanisms underlying memory dysfunctions in AD remain unclear. In the present study, we confirmed S-guanylation of SNAP-25 by immunoprecipitation followed by Western blotting (Fig. 4-C), suggesting that icv infusion of 8-nitro-cGMP strongly affects hippocampal proteins including SNAP-25 in mice. In fact, although 8nitro-cGMP infusion did not alter the expression levels of SNARE proteins and cplx (Supplementary Fig. S3), BN-PAGE followed by Western blotting revealed an increase in immunoreactivity against SNAP-25 at higher than 480 kDa in the hippocampi of 8-nitrocGMP-infused mice, which were predicted to be oligomerized SNARE complexes [23,34] that are essential for synaptic vesicle exocytosis [35]. Moreover, BN-PAGE followed by Western blotting detected an 8-nitro-cGMP administration-dependent decrease in immunoreactivity against cplx 1/2 at approximately 200 kDa. Previous studies have shown that SNARE complexes and cplx-attached SNARE complexes were detected at greater than 150 and 200 kDa, respectively, by BN-PAGE [23,34]. Taken together with our previous findings that S-guanylation of SNAP-25 increases SNARE complex formation but decreases the binding affinity of SNARE complex to cplx [12,17], our results strongly suggest that 8-nitro-cGMP in the mouse brain increases SNARE complex formation via S-guanylation of SNAP-25 but decreases cplx-attached SNARE complexes. These alternations may affect neurotransmitter release, neuronal activity, and hippocampal functions including memory; however, further experiments are required to confirm the precise biological roles of 8-nitro-cGMP in brain functions. In conclusion, our behavioral tests indicated that icv administration of 8-nitro-cGMP to mice affected hippocampus-dependent fear memory, and we proposed one possible mechanism underlying this phenomenon involving S-guanylation of SNAP-25 to increase SNARE complex formation and decrease the affinity of the SNARE complex for cplx, potentially leading to impaired exocytosis regulation. Conflicts of interest None declared. Acknowledgements The authors thank Ms. Tomoko Arasaki (Tokyo Metropolitan Institute of Gerontology) and Dr. Masami Takahashi (Kitasato University) for technical helps and the kind gift of the antibodies. This work was supported in part by a Grant-in-Aid for Scientific Research (B), Challenging Exploratory Research, and Innovative Areas "Oxygen Biology: a new criterion for integrated understanding of life" from the Ministry of Education, Sciences, Sports, Technology (MEXT), Japan to H.I. (16H04674, 16K13089, 26111011); the Smoking Research Foundation to H.I.; Graduate Course for System-inspired Leaders in Material Science (SiMS) of Osaka Prefecture University by MEXT, Japan to Y.K. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.01.138. References [1] J.V. Esplugues, NO as a signalling molecule in the nervous system, Br. J. Pharmacol. 135 (2002) 1079e1095. https://doi.org/10.1038/sj.bjp.0704569. [2] S. Endo, G-substrate: the cerebellum and beyond, Prog. Mol. Biol. Transl. Sci. 106 (2012) 381e416. https://doi.org/10.1016/b978-0-12-396456-4.00004-3. [3] J. Garthwaite, Concepts of neural nitric oxide-mediated transmission, Eur. J. Neurosci. 27 (2008) 2783e2802. https://doi.org/10.1111/j.1460-9568.2008. 06285.x.
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8-Nitro-cGMP attenuates context-dependent fear memory in mice Yusuke Kishimoto a, Shingo Kasamatsu b, c, Shuichi Yanai d, Shogo Endo d, Takaaki Akaike c, Hideshi Ihara a, * a
Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA c Department of Environmental Medicine and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan d Aging Neuroscience Research Team, Tokyo Metropolitan Institute of Gerontology, Itabashi, Tokyo 173-0015, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 January 2019 Accepted 31 January 2019 Available online 14 February 2019
We previously reported that 8-nitroguanosine 30 ,50 -cyclic monophosphate (8-nitro-cGMP) is endogenously produced via nitric oxide/reactive oxygen species signaling pathways and it reacts with protein thiol residues to add cGMP structure to proteins through S-guanylation. S-Guanylation occurs on synaptosomal-associated protein 25 (SNAP-25), which is a part of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that regulates exocytosis. However, the biological relevance of 8-nitro-cGMP in the nervous system remains unclear. Here, we investigated the effects of intracerebroventricular (icv) infusion of 8-nitro-cGMP on mouse brain functions. The results of an open-field test and fear-conditioning task revealed that icv infusion of 8-nitro-cGMP decreased the vertical activity and context-dependent fear memory of mice, which are both associated with the hippocampus. Immunohistochemical analysis revealed increased c-Fos-positive cells in the dentate gyrus in 8-nitro-cGMP-infused mice. Further, biochemical analyses showed that icv infusion of 8-nitro-cGMP increased S-guanylated proteins including SNAP-25 and SNARE complex formation as well as decreased complexes containing complexin, which regulates exocytosis by binding to the SNARE complex, in the hippocampus. These findings suggest that accumulation of 8-nitro-cGMP in the hippocampus affects its functions, including memory, via S-guanylation of hippocampal proteins such as SNAP-25. © 2019 Elsevier Inc. All rights reserved.
Keywords: 8-Nitroguanosine 30 ,50 -cyclic monophosphate Fear-conditioning task Synaptosomal-associated protein 25 S-Guanylation Nitric oxide Reactive oxygen species
1. Introduction Nitric oxide (NO), a well-known gasotransmitter, is endogenously produced by NO synthases and is involved in numerous physiological and pathological functions in various organisms [1]. These functions are mediated through a cyclic guanosine 30 ,50 monophosphate (cGMP)-dependent pathway via activation of cGMP-dependent protein kinase [2,3]. Other pathways may be driven by nitrosylation and nitration of biomolecules such as amino acids, proteins, lipids, and nucleotides through processes induced by reactive nitrogen oxide species, which are derived from NO and reactive oxygen species (ROS), such as peroxynitrite and nitrogen dioxide [4e6]. We have previously reported on the endogenous production of 8-nitroguanosine cGMP (8-nitro-cGMP) as a secondary messenger
* Corresponding author. 608 Bldg. C10, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka, 599-8531, Japan. E-mail address: [email protected] (H. Ihara). https://doi.org/10.1016/j.bbrc.2019.01.138 0006-291X/© 2019 Elsevier Inc. All rights reserved.
of NO/ROS redox signaling [6]. This nitrated nucleotide can react with reactive thiol groups on cysteine residues to add a cGMP structure to the protein, which is a post-translational modification known as protein S-guanylation [6]. Because of its unique chemical properties, 8-nitro-cGMP is related to various physiological and pathophysiological functions, including oxidative stress response [6,7], neurotoxicity [8,9], cell senescence [10], and autophagy [11]. Several target proteins of S-guanylation have been identified and their biological roles have been reported [6,7,10]. We recently reported the endogenous production of 8-nitro-cGMP in the rodent brain and S-guanylation of proteins in the brain including synaptosomal-associated protein 25 (SNAP-25), which is one of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins [12]. SNAP-25 plays a critical role in cooperating with other SNARE proteins and SNARE modulatory proteins such as complexin (cplx), which binds to the SNARE complex, during exocytosis by regulating synaptic vesicle fusion [13]. Post-translational modifications of SNAP-25, such as phosphorylation [14], palmitoylation [15], and
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nitration [16], were reported to be associated with neuronal functions. Additionally, our recent studies revealed that S-guanylation of SNAP-25 enhances SNARE complex formation [12] but attenuates the interaction between the SNARE complex and cplx [17], suggesting that S-guanylation of SNAP-25 is involved in regulating exocytosis. However, the biological implications of 8-nitro-cGMP and S-guanylation of proteins including SNAP-25 in the nervous system are not fully understood. The roles of NO and ROS in the mechanisms underlying memory have been widely examined [18,19]. Additionally, numerous studies have suggested that dysregulation of NO/ROS, which is induced under certain pathological conditions such as Alzheimer's disease (AD), is associated with memory dysfunction [18,20,21]. Thus, NO/ ROS homeostasis is thought to be important for maintaining memory functions. However, the biological importance of 8-nitrocGMP in memory remains unclear. In the present study, to investigate the effects of 8-nitro-cGMP on mouse brain functions, we intracerebroventricularly (icv) infused 8-nitro-cGMP in mice and analyzed brain functions by conducting behavioral tests and evaluated cellular mechanisms regarding synaptic exocytosis through biochemical experiments. Our results indicated that icv administration of 8-nitro-cGMP decreased vertical activity and context-dependent fear memory, likely via S-guanylation of hippocampal SNAP-25 to attenuate the binding affinity of cplx to the SNARE complex. 2. Materials and methods 2.1. Intracerebroventricular infusion of 8-nitro-cGMP into mice Male C57BL/6J mice were obtained from CLEA Japan (Tokyo, Japan). All experiments were approved by the animal experiment committee of the Tokyo Metropolitan Institute of Gerontology and carried out according to its guidelines. Mice (11e12 weeks) were utilized for this study. The detailed experimental design is described in Supplementary information. 8-Nitro-cGMP administration to mice was carried out with a guide cannula and an internal canula (C315GS-5/SPC and C315IS-5/ SPC, Plastics One, Roanoke, VA), that were inserted into the right lateral ventricle (coordinates: anterior-posterior, 0.5 mm; mediallateral, þ1.0 mm; dorsal-ventral, þ1.6 mm), at a rate of 1.0 ml/min of phosphate-buffered saline with or without 5, 10 mM 8-nitro-cGMP, synthesized as described previously [6]. 2.2. Open-field test The open-field test was performed as described previously [22] with modifications. The apparatus for the open-field test was obtained from O'Hara & Co. (Tokyo, Japan). In brief, a mouse was placed in the center of the arena and allowed to explore it for 15 min. On next day of the first 8-nitro-cGMP infusion, the openfield test was carried out under a dark-lit condition (10 lux), then it was performed again under a light-lit condition (300 lux) on the following day as described previously. After recording, the total distance moved, the number and duration of rearing behaviors, the total time spent immobile, and the percentage of time spent in the center region were automatically calculated with Image OFCR (O'Hara & Co.).
subsequent unconditioned stimulus (US; 0.5 s of electric footshock, 0.12 mA, delivered from 2.5 s after CS starts). One and 24 h after the conditioning, cue-dependent fear memory was analyzed. Forty-eight hours after the conditioning, context-dependent fear memory was assessed. The freezing behavior of the mice was recorded with a camera placed above the chamber and the data were analyzed using automated software (Time FZ1, O'Hara & Co.). Further details are provided in Supplementary information. 2.4. Immunohistochemistry After the context-dependent fear memory test, immunohistochemical analysis of mouse brain sections was performed as previously described [12], with antibodies specific to S-guanylated proteins [6] and c-Fos (Santa Cruz Biotechnology, Dallas, TX), followed by peroxidase-conjugated secondary antibodies (Nichirei Biosciences, Tokyo, Japan). Reactions were visualized with a diaminobenzidine system (Nacalai Tesque, Kyoto, Japan). Immunostained images in several regions in mouse brains were observed by using IX53 inverted microscope (Olympus, Tokyo, Japan). Nissl staining of mouse brain sections was also performed by soaking sections into 0.2% cresyl violet 0.3% acetic acid solution for 15 min. 2.5. Immunoprecipitation and Western blotting Mouse brain samples were harvested, then immunoprecipitation and Western blotting were performed as described previously [12] with slight modifications. Antibodies specific to SNAP-25, syntaxin 1A and vesicle-associated membrane protein 2 (VAMP2) were kindly supplied by Dr. Masami Takahashi (Kitasato University). In brief, hippocampal lysates (50 mg) were incubated with anti-SNAP-25 antibody or anti-S-guanylated protein antibody [6] bound to Protein G magnetic beads (Merck Millipore, Darmstadt, Germany). Immunoprecipitates were collected by 5 min boiling and analyzed by Western blotting with specific antibodies. 2.6. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) Native SNARE complexes with cplx were analyzed using BNPAGE followed by Western blotting as described previously [17,23] with minor modifications. In brief, 20 mg hippocampal lysates, prepared with 1 NativePAGE sample buffer (Thermo Fisher Scientific, Waltham, MA) containing 0.5% Triton X-100 and 0.125% Coomassie Brilliant Blue-G250 (Thermo Fisher Scientific) were separated by BN-PAGE using 4e16% Bis-Tris polyacrylamide gradient gels, followed by Western blotting with anti-SNAP-25 and anti-cplx 1/2 antibodies (R&D systems, Minneapolis, MN). 2.7. Statistical analysis Data are the mean ± standard error of the mean (SEM) of at least three independent experiments unless otherwise specified. For statistical comparisons, we utilized unpaired one-way or two-way analysis of variance (ANOVA) followed by the Tukey's post-hoc test via GraphPad Prism software (GraphPad Software, La Jolla, CA). We considered P < 0.05 as statistically significant. 3. Results
2.3. Fear-conditioning task
3.1. Open-field test
The fear-conditioning task was carried out as described previously [22] with modifications. In brief, fear-conditioning was performed by presenting a conditioned stimulus (CS; 3 s of pure tone, 10 kHz, 70 dB, delivered from 60 s after session starts) and
First, we analyzed the effects of icv infusion of 8-nitro-cGMP on mouse behaviors in open-field tests under both dark-lit and light-lit conditions. As shown in Fig. 1-A, there were no significant differences in the total distance traveled by the mice within 15 min in the
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Fig. 1. Open-field test. The open-field test was carried out under dark-lit and the light-lit conditions to measure activity and anxiety levels in mice. (A) Total distance moved. (B) Rearing frequency and duration. (C) Immobility time. (D) Percentage of time spent in the center region. Data are the mean ± SEM (n ¼ 22 or 23). *P < 0.05, **P < 0.01 vs. control.
open-field test among the control and 8-nitro-cGMP-infused groups both under dark-lit and light-lit conditions. In the dark-lit test, 8-nitro-cGMP-administrated mice showed significant decreases in both the frequency and duration of rearing, whereas a significant difference was observed only in rearing duration in 8nitro-cGMP-infused mice but not in the frequency of rearing in the light-lit test, compared to the observations in control mice (Fig. 1-B). As shown in Fig. 1-C and 1-D, there were no significant differences in the total immobility time and percentage of the time mice spent in the center region between control and 8-nitro-cGMPinfused mice. 3.2. Fear-conditioning task We analyzed effects of icv infusion of 8-nitro-cGMP into mice on memory by fear-conditioning task. As shown in Fig. 2-A and 2-B, there were no differences in the ratio of freezing during the cuedependent fear memory test between the control and 8-nitrocGMP-administrated mice at 1 and 24 h after fear conditioning. However, the context-dependent fear memory test performed at 48 h after conditioning indicated that the ratio of freezing decreased in an 8-nitro-cGMP-infused dose-dependent manner and that mice administered 100 nmol 8-nitro-cGMP showed a significantly decreased freezing ratio compared to that of control mice (Fig. 2-C). 3.3. Histochemical analyses of 8-nitro-cGMP-administrated mouse brains To investigate the histological changes in 8-nitro-cGMP-infused mouse brains, we performed Nissl staining and immunohistochemical analysis using antibodies for S-guanylated proteins and cFos. Immunohistochemical analysis indicated that S-guanylated proteins were strongly increased in the hippocampal regions of
cornu ammonis 1 (CA1), CA3, and dentate gyrus, but weakly increased in the cerebral cortex and amygdala (Fig. 3-A, Supplementary Fig. S2-A and S2-B). As shown in Fig. 3-B, neurons showed no morphological changes in the hippocampi of 8-nitrocGMP-infused mouse brains by Nissl staining. To analyze neuronal activity in the hippocampus, we performed immunohistochemical analysis with an antibody specific to c-Fos, which is widely used as a neuronal activity marker [24]. The results showed that c-Fospositive cells in the dentate gyrus were significantly increased in 8nitro-cGMP-administrated animals (Fig. 3-C, 3-D). 3.4. Effects of 8-nitro-cGMP infusion on protein S-guanylation and SNARE complex formation in mouse brains To examine effects of icv administration of 8-nitro-cGMP to mice on the functions of proteins related to memory, we analyzed the Sguanylation of proteins in the hippocampus and cerebral cortex. Western blotting revealed that various S-guanylated protein levels were high in the hippocampus but low in the cerebral cortex collected from 8-nitro-cGMP-infused mice (Fig. 4-A and 4-B). Because SNAP-25 has been reported to be S-guanylated [12,17], we confirmed S-guanylation of SNAP-25 by immunoprecipitation with an anti-SNAP-25 antibody followed by Western blotting using an anti-S-guanylated protein antibody. As shown in Fig. 4-C, increased S-guanylated SNAP-25 was detected in the hippocampi of 8-nitrocGMP-infused mice. Additionally, S-guanylation of SNAP-25 was also confirmed in the sample immunoprecipitated with the anti-Sguanylated protein antibody (Fig. 4-C). Because it has been reported that S-guanylation of SNAP-25 affects SNARE complex formation [12,17], we examined the effects of 8-nitro-cGMP administration on SNARE complex formation in the hippocampus. Although icv infusion of 8-nitro-cGMP did not affect the expression levels of SNARE proteins (i.e., SNAP-25, syntaxin 1A, and VAMP2) and cplx 1/2 (Supplementary Fig. S3-A and
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Fig. 2. Fear-conditioning task. Cue- and context-dependent fear memory tests were carried out to measure short- and long-term fear memory in control and 8-nitro-cGMPadministrated mice. (A) Freezing rate of the cue-dependent fear memory test performed 1 h after conditioning. (B) Freezing rate of the cue-dependent fear memory test performed 24 h after conditioning. (C) Results of the context-dependent fear memory test performed 48 h after conditioning. Data are the mean ± SEM (n ¼ 22 or 23). *P < 0.05 vs. control.
Fig. 3. Histochemical analyses of 8-nitro-cGMP-administrated mouse brains. Immunohistochemistry to detect S-guanylated proteins and c-Fos (A and C) and Nissl staining (B) were carried out using mouse brain tissues. (D) Mean ± SEM of c-Fos-positive cells in the dentate gyrus region marked by dashed lines in C (n ¼ 3 or 4). *P < 0.05 vs. control. Scale bars show 100 mm. CA, cornu ammonis.
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Fig. 4. Effects of 8-nitro-cGMP infusion on protein S-guanylation and SNARE complex formation in mouse brains. Western blotting (WB) with anti-S-guanylated protein or antiactin antibodies (A), immunoprecipitation (IP) followed by WB with antibodies for SNAP-25 or S-guanylated proteins (C) and BN-PAGE followed by WB with antibodies for SNAP-25 or cplx 1/2 (D) were carried out using lysates obtained from the hippocampus or cerebral cortex. (B and E) Densitometric analysis of the immunoblot of A and D, respectively. Data are the mean ± SEM (n ¼ 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control hippocampus; xP < 0.01 vs. control cerebral cortex; #P < 0.01, ##P < 0.001 vs. hippocampus of 8-nitro-cGMP-infused mice with the same dose. C, cerebral cortex; H, hippocampus.
S3-B), BN-PAGE followed by Western blotting with an anti-SNAP-25 antibody revealed that immunoreactive bands at higher than 480 kDa (indicated as “I” in Fig. 4-D, left) were significantly increased in the hippocampi of 8-nitro-cGMP-infused mice (Fig. 4D and 4-E). Additionally, immunoreactive bands against an antibody specific for cplx 1/2, an exocytosis regulatory protein that binds to the SNARE complex, detected at approximately 200 kDa (indicated in “II” in Fig. 4-D, right) and the band at lower than approximately 150 kDa (indicated in “III” in Fig. 4-D, right) in the hippocampi of 8-nitro-cGMP-administrated animals were significantly decreased and increased, respectively (Fig. 4-D and 4-E). 4. Discussion In the present study, we evaluated the effects of icv administration of 8-nitro-cGMP on mouse brain functions by conducting behavioral tests and biochemical analyses. This is the first study to reveal an association between 8-nitro-cGMP and mouse brain functions. The open-field test revealed that 8-nitro-cGMP did not affect the total distance moved (horizontal activity), total immobility time, and percentage of time spent in the center region (both are indices of anxiety [25]). Additionally, in the dark-lit test, both the frequency and duration of rearing (vertical activity) in 8-nitro-cGMP-infused animals were decreased, while only rearing duration was decreased in the light-lit test (Fig. 1). Some mouse behaviors including rearing have been reported to decrease under light conditions [25]. Therefore, slight differences in rearing frequency may not have been detected in the light-lit test. Rearing is controlled by the
hippocampus [26], and reduced rearing behavior has been reported in mice with hippocampal lesions, whereas horizontal activity is unaffected in these animals [27]. Taken together, our results suggest that 8-nitro-cGMP infusion affects vertical activity, which is controlled by the hippocampus, but does not affect horizontal activity or anxiety. The fear-conditioning task is one of the most well-established methods for measuring fear memory. Cue-dependent and contextdependent fear memory are both associated with the amygdala, while the latter is also associated with the hippocampus [28,29]. 8Nitro-cGMP administration did not alter short- or long-term fear memory associated with the amygdala but reduced long-term fear memory, which is associated with the amygdala and hippocampus, in a dose-dependent manner (Fig. 2). These data suggest that icv infusion of 8-nitro-cGMP attenuates hippocampal functions. We confirmed the elevation of S-guanylated proteins in the brain tissues, particularly in the hippocampi of 8-nitro-cGMPinfused animals (Figs. 3 and 4). However, there were no morphological changes in the hippocampal neurons of 8-nitro-cGMPinfused mice (Fig. 3-B), which is consistent with our previous study showing that 8-nitro-cGMP does not possess cytotoxic effects in rat cerebellar granule neurons [30]. In contrast, c-Fos-positive cells were significantly increased in the dentate gyrus of 8-nitro-cGMPinfused mice (Fig. 3-C and 3-D), indicating increased neuronal activity in the hippocampus. Because increased c-Fos levels were observed in the hippocampus of patients with AD and in a mouse model [31,32] and hippocampal hyperactivation has been reported in patients in the early stages of AD [33], elevated c-Fos levels may be involved in neuronal dysfunction. However, the precise
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mechanisms underlying memory dysfunctions in AD remain unclear. In the present study, we confirmed S-guanylation of SNAP-25 by immunoprecipitation followed by Western blotting (Fig. 4-C), suggesting that icv infusion of 8-nitro-cGMP strongly affects hippocampal proteins including SNAP-25 in mice. In fact, although 8nitro-cGMP infusion did not alter the expression levels of SNARE proteins and cplx (Supplementary Fig. S3), BN-PAGE followed by Western blotting revealed an increase in immunoreactivity against SNAP-25 at higher than 480 kDa in the hippocampi of 8-nitrocGMP-infused mice, which were predicted to be oligomerized SNARE complexes [23,34] that are essential for synaptic vesicle exocytosis [35]. Moreover, BN-PAGE followed by Western blotting detected an 8-nitro-cGMP administration-dependent decrease in immunoreactivity against cplx 1/2 at approximately 200 kDa. Previous studies have shown that SNARE complexes and cplx-attached SNARE complexes were detected at greater than 150 and 200 kDa, respectively, by BN-PAGE [23,34]. Taken together with our previous findings that S-guanylation of SNAP-25 increases SNARE complex formation but decreases the binding affinity of SNARE complex to cplx [12,17], our results strongly suggest that 8-nitro-cGMP in the mouse brain increases SNARE complex formation via S-guanylation of SNAP-25 but decreases cplx-attached SNARE complexes. These alternations may affect neurotransmitter release, neuronal activity, and hippocampal functions including memory; however, further experiments are required to confirm the precise biological roles of 8-nitro-cGMP in brain functions. In conclusion, our behavioral tests indicated that icv administration of 8-nitro-cGMP to mice affected hippocampus-dependent fear memory, and we proposed one possible mechanism underlying this phenomenon involving S-guanylation of SNAP-25 to increase SNARE complex formation and decrease the affinity of the SNARE complex for cplx, potentially leading to impaired exocytosis regulation. Conflicts of interest None declared. Acknowledgements The authors thank Ms. Tomoko Arasaki (Tokyo Metropolitan Institute of Gerontology) and Dr. Masami Takahashi (Kitasato University) for technical helps and the kind gift of the antibodies. This work was supported in part by a Grant-in-Aid for Scientific Research (B), Challenging Exploratory Research, and Innovative Areas "Oxygen Biology: a new criterion for integrated understanding of life" from the Ministry of Education, Sciences, Sports, Technology (MEXT), Japan to H.I. (16H04674, 16K13089, 26111011); the Smoking Research Foundation to H.I.; Graduate Course for System-inspired Leaders in Material Science (SiMS) of Osaka Prefecture University by MEXT, Japan to Y.K. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.01.138. References [1] J.V. Esplugues, NO as a signalling molecule in the nervous system, Br. J. Pharmacol. 135 (2002) 1079e1095. https://doi.org/10.1038/sj.bjp.0704569. [2] S. Endo, G-substrate: the cerebellum and beyond, Prog. Mol. Biol. Transl. Sci. 106 (2012) 381e416. https://doi.org/10.1016/b978-0-12-396456-4.00004-3. [3] J. Garthwaite, Concepts of neural nitric oxide-mediated transmission, Eur. J. Neurosci. 27 (2008) 2783e2802. https://doi.org/10.1111/j.1460-9568.2008. 06285.x.
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