Nitric oxide promotes survival of cerebellar granule neurons cultured in vitro through the Akt pathway＊＊＊☆

Lin Wang, Mei Li, Lihua Zhou

1Department of Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China

2Department of Neurology, Second Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China

lNTRODUCTlON

Nitric oxide (NO) plays a critical role in cell proliferation, apoptosis, immune regulation,and neurotransmission[1-6]. In cultured cerebellar granule neurons (CGNs), NO is primarily generated by the N-methyl-D-aspartic acid (NMDA) receptor,which is activated by excitatory amino acids[6-7]. The physiological actions of NO are largely mediated via stimulation of soluble guanylate cyclase, which results in accumulation of cyclic guanosine monophosphate (cGMP) and subsequent activation of protein kinase G (PKG)[8].

NO-induced cGMP signaling can prevent apoptosis via activation of the phosphoinositide-3-kinase (PI3-K)/protein kinase B/Akt pathway[9].

Blockade of NO production induces apoptotic death in differentiating CGNs through activation of caspase-3. Furthermore, NO is an effector of the Akt/GSK-3 survival pathway in a cGMP dependent manner,and neuronal death can be prevented by agents known to transduce survival signals, such as insulin, forskolin, and lithium[10]. NO donors can function as endogenous regulators of apoptosis in primary cultured neurons[11-13]. We hypothesized that inhibition of NO production should induce CGN apoptosis. Therefore,we investigated the pathways by which NO sustains CGN survival in vitro.

RESULTS

lnfluence of NO on cultured CGN viability in vitro

Five groups were established in this study:control, MK-801, 1H-[1, 2, 4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), MK-801 + sodium nitroprusside (SNP), and ODQ + SNP groups. After 7 days in culture, CGNs in these groups were treated, respectively, with either no additive (control), MK-801 (an antagonist of the NMDA receptor, 30 μmol/L),ODQ (an antagonist of sGC, 50 μmol/L),MK-801 + SNP (an exogenous NO donor,10 μmol/L) or ODQ + SNP. After 24 hours,cells were examined using an inverted phase contrast microscope. Mature neurons displayed shrinkage of the cell body and damaged neurites after MK-801 (Figure 1B)and ODQ (Figure 1C) treatments, compared with the control group (Figure 1A). The neurons returned to a normal appearance after SNP treatment (Figures 1D, E). Cell viability was assessed using the MTT assay(Figure 1F). MK-801 and ODQ decreased CGN viability compared with the control group (P ＜ 0.05). The antagonist-induced decrease in cell viability could be reversed by SNP (P ＜ 0.05).

Effect of MK-801, ODQ and SNP on caspase-3 expression in CGNs

Immunofluorescence staining showed that MK-801 (Figure 2B) and ODQ (Figure 2C) induced caspase-3 expression compared with the control group (Figure 2A).

This increase in caspase-3 was diminished by SNP treatment (Figures 2D, E). Western blot analysis showed enhanced caspase-3 expression in the MK-801 and ODQ groups compared with the control group (P ＜ 0.05),which was significantly reduced by SNP treatment (P ＜0.05). These results were consistent with the immunofluorescence data (Figure 2F).

Figure 1 MK-801 and ODQ decreased the viability of cultured cerebellar granule neurons (CGNs; arrows). The CGNs were integrated in a dense network and formed numerous synapses in the control group (A). The MK801(B) and ODQ (C) groups exhibited cell clumping with shrunken cell bodies and damaged neurites. Sodium nitroprusside (SNP) treatment prevented these degenerative changes, resulting in a phenotype similar to that of cells in the control group (D, E). CGN viability was assessed using the MTT assay (by a person blinded to the different treatments) after incubating the cells with 10 μmol/L SNP, 30 μmol/L MK-801, and 50 μmol/L ODQ,respectively, for 24 hours (F). Each antagonist, including MK-801 and ODQ, reduced cell viability. The MK-801 and ODQ-induced decrease in cell viability was reversed by SNP. aP ＜ 0.05, vs. control group; bP ＜ 0.05, vs. MK-801 group; cP ＜ 0.05, vs. ODQ group. Scale bars: 20 μm.Survival rate (%)=Aexperimental/Acontrol×100%. Statistical calculations and data handling were performed by one-way analysis of variance as well as Bonferroni post hoc multiple comparison test. Data were expressed as mean ± SD. Each experiment was repeated six times.Three sets of results were obtained for each group after one experiment, and each group comprised 18 wells.ODQ: 1H-[1, 2, 4]oxadiazolo-[4, 3-a]quinoxalin-1-one.

lnfluence of MK-801, ODQ and SNP on phosphorylated-Akt (P-Akt) expression in CGNs

Recent evidence demonstrated that Akt/PKB kinase promotes survival in many systems. Therefore, we hypothesized that NO acts as a survival factor for CGNs through Akt activation. Indeed, Akt activity is high in CGNs under normal conditions, most likely due to the presence of growth factors in the serum-containing medium used for culture[14]. Incubating CGNs with either the NMDA receptor antagonist (MK801) or the sGC antagonist (ODQ) resulted in a progressive decrease of P-Akt,as determined by western blot analysis. However, P-Akt was significantly upregulated by SNP treatment (P ＜0.05; Figure 3).

Figure 2 Caspase-3 expression in CGNs after treatment with MK-801, ODQ or SNP. Y-axis: Ratio of absorbance of caspase-3/β-actin. Fluorescence micrographs (A-E)demonstrate that caspase-3 was progressively activated in the MK-801 (B) and ODQ (C) groups compared with the control group (A). After SNP treatment, caspase-3 expression was significantly downregulated (D, E). Scale bars: 20 μm. Results of western blot analysis were consistent with the immunofluorescence results (F).aP ＜ 0.05, vs. control group; bP ＜ 0.05, vs. MK-801 group;cP ＜ 0.05, vs. ODQ group. Statistical calculations and data handling were performed by one-way analysis of variance as well as a Bonferroni post hoc multiple comparison test.Data were expressed as mean ± SD. Three sets of results were obtained for each group for one experiment, and each group comprised 18 wells. ODQ: 1H-[1, 2, 4]oxadiazolo-[4, 3-a]quinoxalin-1-one. SNP: sodium nitroprusside; CGNs: cerebellar granule neurons. SNP:Sodium nitroprusside.

Figure 3 Ratio of phosphorylated-AKT (p-Akt)/Akt in cultured CGNs after treatment with MK-801, ODQ or SNP as determined by western blot analysis. Semiquantitative changes in the levels of p-Akt, representing activated Akt,were determined by absorbance measurements and expressed as fold changes. Statistical analysis showed that the levels of p-Akt in the MK-801 and ODQ groups were significantly lower than in the control group.However, after SNP treatment, p-Akt levels were similar to those in the control group. aP ＜ 0.05, vs. control group;bP ＜ 0.05, vs. MK-801 group; cP ＜ 0.05, vs. ODQ group.Each experiment was repeated six times. Data were expressed as mean ± SD. Three sets of results were obtained for each group after one experiment, and each group comprised 18 wells. Y axis: Ratio of absorbance of p-Akt/Akt. The experiments were performed by a person who was blinded to different treatments. Statistical calculations and data handling were performed by one-way analysis of variance as well as Bonferroni post hoc multiple comparison test. ODQ: 1H-[1, 2, 4]oxadiazolo-[4, 3-a]quinoxalin-1-one; SNP: sodium nitroprusside; CGNs: cerebellar granule neurons.

DlSCUSSlON

This study demonstrates that NO is essential for the survival of differentiating CGNs in vitro. Blockade of NO production results in extensive neuronal death, while a NO donor significantly reverted this effect. We also found that endogenous NO contributes to the survival of differentiating CGNs by a cGMP-dependent mechanism,while exogenous NO does so by a cGMP-independent mechanism. This observation is consistent with previous results[15-17]and demonstrates a functional link between the inhibition of NO production, caspase-3 activation,and apoptotic cell death.

A previous study[18]reported that the NMDA receptor antagonist, MK-801, which reduces the production of endogenous NO[19], and the sGC antagonist, ODQ,which blocks the NO-cGMP pathway, decreased cell viability. This reduction in cell viability was prevented by SNP. Our study demonstrates that the number of caspase-3-labeled cells increased, while p-Akt expression decreased, after treatment with either MK-801 or ODQ,and that this could be reversed by SNP treatment. A biochemical link between cGMP and Akt was previously suggested by experiments on hepatocytes[20]. Our data suggest that the NO-Akt signaling pathway is critical for the survival of CGNs in vitro, and that endogenous NO may act through a cGMP-dependent pathway,while the regulation of p-Akt by exogenous NO appears to be independent of cGMP. Stimulation of the NO-GC pathway by NO results in the production of the second messenger cGMP, which exerts its effects via cGMP-dependent kinases, channels, and phosphodiesterases[8,21-23]. In addition to these cGMP-dependent actions, NO has been proposed to mediate a variety of effects via cGMP-independent mechanisms as well[24].

MATERlALS AND METHODS

Design

A randomized, controlled animal experiment.

Time and setting

The experiments were performed at the Laboratory of Human Anatomy, Zhongshan Medical School of Sun Yat-sen University, China, from 2008 to 2011.

Materials

Two hundred healthy, 7-day-old, Sprague-Dawley rats were purchased from the Animal Experimental Center of Sun Yat-sen University, China. Experimental procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals,formulated by the Ministry of Science and Technology of the People’s Republic of China[25].

Methods

In vitro culture of CGNs

CGNs were cultured as described previously[26]. In brief,the entire cerebellum was removed from each rat and all cerebella from a single litter were pooled for each replicate. The cerebella were dissociated, and cells were plated at a density of 1 × 106per dish precoated with 10 mg/L poly-L-lysine (Sigma, St Louis, MO, USA). Cells were maintained in Basal Eagle’s culture medium containing 10% fetal bovine serum (Globepharm, Delaware,USA), 100 mg/mL gentamicin and 25 mol/L KCl. Cultures were treated with medium containing 10 mol/L cytosine arabinoside (Sigma) after 24 hours to inhibit proliferation of non-neuronal cells. The purity of these cultures exceeded 98%.

Assessment of cell viability following exposure to MK-801, ODQ and SNP

Mitochondrial activity, as a measure of cell death, was assessed by measuring dehydrogenase activity using the MTT assay as previously described[27]. Briefly, MTT(0.5 mg/mL; Sigma) was added to each well of CGNs,cultured at 37 °C and 5% CO2, for 4 hours. Subsequently,medium was removed, and 150 mL of dimethyl sulfoxide was added to each well to solubilize the formazan salt.

The resulting purple azo-dye was detected at 490 nm with the Multiskan Ascent microplate reader. Data were expressed as mitochondrial activity relative to that detected in untreated wells, expressed as a percentage, to normalize for differences in plating density between individual experiments.

Western blot analysis of P-Akt and caspase-3 expression

Proteins were extracted from cell pellets using a RIPA buffer (Santa Cruz Biotechnologies, Santa Cruz, CA,USA). Protein concentration was measured using the BCA method with the Bio-Rad Protein Assay Reagent(Bio-Rad, Hercules, CA, USA). For western blot analysis,samples (20 mg protein/lane) were separated by 12%sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Pall, California North Carolina, USA). To detect p-Akt and caspase-3, the membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 at room temperature for 2 hours, and then incubated with rat anti-p-Akt antibody (1: 1 000;Santa Cruz Biotechnologies) or with rat anti-Akt polyclonal antibody (1: 1 000; Santa Cruz Biotechnologies)and rat anti-caspase-3 antibody (1: 1 000; Cell signaling, USA) overnight at 4°C. After washing and incubating for 2 hours at room temperature with goat anti-rat antibody conjugated to horseradish peroxidase (1:4 000; Amersham Biosciences, Piscataway, NJ, USA),the blots were washed and visualized by chemiluminescence according to the manufacturer’s protocol (Super-Signal West Pico Chemiluminescence Substrate,Pierce, Florida, USA). Blots were exposed to X-ray film(Fujifilm, Shantou, China), and the intensity was quantified for both bands. The relative protein levels in the individual lanes were compared by analyzing scanned images using the NIH Image program. The absorbance ratio of each target band/β-actin band was calculated. All experiments were performed a minimum of three times using independent cultures.

Caspase-3 immunofluorescence labeling

Immunofluorescence labeling was performed according to standard protocols. Briefly, CGNs were fixed with 4%paraformaldehyde and washed in PBS three times for 30 minutes, followed by incubation in 0.1% bovine serum albumin, 0.3% Triton X-100 for 30 minutes. Cells were then incubated overnight with rabbit anti-caspase-3 (1:6 400; Sigma) at 4 °C for 48 hours. After washes in PBS,cells were incubated with anti-rabbit IgG tetra rhodamine isothiocyanate conjugate (1: 800; Sigma) in the dark for 1 hour.

Statistical analysis

Statistical calculations and data handling were performed by one-way analysis of variance as well as Bonferroni post hoc multiple comparison test using SPSS version 11.0 (SPSS, Chicago, IL, USA). Data were expressed as the mean ± SD, and a value of P ＜ 0.05 was considered statistically significant.

Author contributions:Lihua Zhou and Mei Li designed this study. Lin Wang conducted experiments, analyzed data, and wrote the manuscript. Lihua Zhou was in charge of funds.

Funding:The study was supported by the National Natural Science Foundation of China, No. 81070995; Guangdong Provincial Science Foundation, No. 05001726, 1050175.

Conflicts of interest:None declared.

Ethical approval:This study received permission from Guangdong General Hospital and Guangdong Academy of Medical Sciences.

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