The mitochondria are the most important cytoplasmic organelles in determining cell survival and death. Mitochondrial dysfunction leads to a wide range of disorders, including neurodegenerative diseases. The central events in the mitochondrial-dependent cell death pathway are the activation of the mitochodrial permeability transition pore (mPTP) and the disruption of mitochondrial membrane potential, which cause the release of apoptogenic molecules and finally lead to cell death. This is thought to be at least partly responsible for the loss of dopaminergic neurons in Parkinson’s disease (PD); thus, the attenuation of mitochondrial dysfunction may contribute to alleviating the severity and progression of this disease. Guanosine is a pleiotropic molecule affecting multiple cellular processes, including cellular growth, differentiation and survival. Its protective effects on the central nervous system and and on several cell types by inhibiting apoptosis have been shown in a number of pathological conditions. This study aimed to analyze the ability of guanosine to protect neuronal PC12 cells from the toxicity induced by 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which mediates selective damage to dopaminergic neurons and causes irreversible Parkinson-like symptoms in humans and primates. Our results demonstrated that the apoptosis of PC12 cells induced by MPP+ was significantly prevented by pre-treatment for 3 h with guanosine. In addition, guanosine attenuated the MPP+-induced collapse of mitochondrial transmembrane potential and prevented the sebsequent activation of caspase-3, thereby protecting dopaminergic neurons against mitochondrial stress-induced damage.
Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the gradually progressive and selective loss of dopaminergic neurons in the substantia nigra (
PC12 cells treated with 1-methyl-4-phenylpyridinium (MPP+) provide a reliable
All reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise.
The PC12 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 4.00 mM L-glutamine, 100 U/ml of penicillin and 100 μg/ml of streptomycin (Gibco, Grand Island, NY, USA). The cultures were maintained in a humidified 5% CO2 atmosphere at 37°C. The culture medium was changed every 3–4 days and the cells were seeded at a density of 30,000 cells/cm2.
Cell viability was measured using the modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a mitochondrial dye which is converted into a blue formazan product by mitochondrial dehydrogenases in metabolically active cells. The PC12 cells were plated at a density of 30,000 cells/cm2 in 96-well plates and incubated for 24 h. To assess the neuroprotective effects of guanosine on MPP+-induced toxicity in PC12 cells, the cells were pre-treated with various concentrations of guanosine (0.01–1,000 μM) for 3 h and were then exposed to 500 μM MPP+ for 72 h, under optimal conditions for the assessment of the neuroprotective effects as previously described (
The morphological signs of apoptosis induced by MPP+ were detected using acridine orange (AO)/ethidium bromide (EB) staining of the PC12 cells. The cells were plated in 6-well plates at a density of 30,000 cells/cm2 and were incubated in DMEM medium at 37°C. After 3 h of pre-treatment with guanosine (10 μM), MPP+ (500 μM) was added to the medium for 72 h. The cells were washed and resuspended in phosphate-buffered saline (PBS) and AO/EB was added at a final concentration of 1 μg/ml. Subsequently, the number of apoptotic cells was randomly counted under a fluorescence microscope (IX71; Olympus, Tokyo, Japan). Viable cells with intact structures stained with AO only showed bright green nuclear staining; the early apoptotic cells were bright green and later apoptotic cells were red-orange with condensed chromatin. The number of apoptotic cells is expressed as a percentage of the total cells counted.
Apoptosis was assessed by measuring DNA fragmentation with single-stranded DNA (ssDNA) apoptosis enzyme-linked immunosorbent assay (ELISA) kits (Chemicon International, Temecula, CA, USA) according to the manufacture’s instructions. The cells plated at a concentration of 30,000 cells/cm2 were cultured for 24 h, followed by treatment with 10 μM guanosine prior to the addition of 500 μM MPP+ for 3 h. The cells were washed 3 times with PBS and formamide was then added which selectively denaturates DNA in apoptotic cells. Anti-ssDNA monoclonal antibody and peroxidase-conjugated secondary antibody were then added to the cells; the ssDNA was then measured at 450 nm using a microplate reader (Epoch; BioTek).
Mitochondrial membrane potential is a key indicator of mitochondrial function and cell death or injury, which can be detected using the mitochondrial dye, 3,3-dihexyloxacarbocyanine iodide [DiOC6(3)]. This dye is a lipophilic fluorescent stain and becomes highly fluorescent when incorporated into membranes. The cells at a concentration of 30,000 cells/cm2 were cultured in 24-well plates for 24 h, followed by treatment with 10 μM guanosine prior to the addition of 500 μM MPP+ for 3 h. Following 72 h of incubation, 1 ml of serum-free culture medium containing DiOC6(3) was added to each well with the final concentration of 1 μM, and the cells were cultured in a humidified incubator for 15 min. The cells were collected and centrifuged at 1,000 × g for 5 min, and the cell pellets were resuspended in PBS containing 0.5 mM EDTA. The intensity of DiOC6(3) fluorescence was recorded using a flow cytometer (Becton-Dickinson, San Diego, CA, USA).
Following treatment, the PC12 cells were collected and lysed with cell lysis solution containing 4% sodium dodecyl sulfate (SDS), 2 mM EDTA and 50 mM Tris-HCl, pH 6.8. Equal amounts of protein were loaded onto a 12% SDS-polyacrylamide gel. Following electrophoretic separation, the polyacrylamide gels were transferred onto PVDF transfer membranes (Amersham Biosciences, Uppsala, Sweden). The membranes were incubated in Tris-buffered saline/Tween-20 (TBST) supplemented with 5% fat-free milk for 1 h to block non-specific binding. The blots were incubated using rabbit anti-Bax, anti-B-cell lymphoma 2 (Bcl-2) antibodies. Horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies were used as the secondary antidodies.
Intracellular ROS produced during the inhibition of mitochondrial complex I was detected using 2′–7′-dichlorofluorescein diacetate (DCFH-DA). This is a non-fluorescent cell-permeating compound that can easily diffuse into cells and be converted into dichlorofluorescin (DCFH) by intracellular esterase. DCFH is then trapped within the cell and oxidized into fluorescent dichlorofluorescein (DCF) by intracellular ROS. Following treatment, the cells were incubated in BSA-free DMEM with DCFH-DA at a final concentration of 20 μM for 30 min at 37°C. Thereafter, 10,000 cells of each group were analyzed by flow cytometry using the FL1 flow cytometer detection channels. The excitation wavelength was 485 nm and the reading was performed at 530 nm.
GSH levels were measured using GSH reductase, as previously described (
Caspase-3 activity was measured using an ApoAlert caspase-3 assay kit according to the manufacturer’s instructions. Briefly, the cells were lysed and centrifuged at 1,000 × g for 10 min, then the supernatant was added to the reaction mixture containing dithiothreitol and caspase-3 substrate (N-acetyl-Asp-Glu-Val-Asp p-nitroanilide). The cells were incubated for 1 h at 37°C, and the absorbance of the chromophore p-nitroanilide produced was measured at 450 nm. The standard curves were obtained from the absorbance of p-nitroanilide standard reagent diluted with cell lysis buffer. One unit of the enzyme was defined as the activity producing 1 nmol of p-nitroanilide.
Data are expressed as the means ± standard error of the mean (SEM). Statistical analysis was performed by one-way analysis of variance, followed by Dunnett’s multiple-comparisons test. Differences between mean values were considered statistically different at p<0.05.
The ability of guanosine to reverse the cytotoxicity to PC12 cells induced by MPP+ was investigated using MTT, which is a mitochondrial dye and can be converted into a blue formazan product by mitochondrial dehydrogenases; therefore, it can partially detect the levels of metabolically active cells. The measurements revealed a significant decrease in the viability of the PC12 cells following exposure to 500 μM MPP+ for 72 h; however, the cells treated with guanosine alone did not show a decrease in cell viability. Pre-treatment with 10 μM guanosine significantly decreased the MPP+-induced cytotoxicity (
To determine whether guanosine prevents MPP+-induced apoptosis in PC12 cells, AO/EB and DNA fragmentation assays were performed. Apoptosis is a process of programmed cell death characterized by a series of distinct nuclear morphological changes. These changes can be detected by AO/EB staining. This assay identified 3 types of cells under a fluorescence microscope: live cells (green), early apoptotic cells (bright green with condensed chromation) and later apoptotic cells (red-orange with condensed chromation). The administration of guanosine alone did not induce changes in the number of apoptotic cells, while the administration of MPP+ significantly increased the number of apoptotic cells compared to the control group (p<0.01). Pre-treatment with 10 μM guanosine significantly decreased the number of apoptotic cells induced by exposure to MPP+ (p<0.01;
Bax and Bcl-2 are key members of the Bcl-2 family of proteins that contribute to the opening of mPTP, leading to the induction of apoptosis. To investigate the changes in Bax and Bcl-2 protein expression levels, western blot analysis was performed on the untreated cells and the cells treated with 500 μM MPP+ alone or 500 μM MPP+ in the presence of 10 μM of guanosine. The administration of MPP+ significantly increased the levels of Bax expression and decreased Bcl-2 expression. These changes were be markedly reversed by pre-treatment with guanosine. Treatment with guanosine alone did not induce changes in the expression levels of these proteins (
Mitochondrial membrane potential maintenance is essential for living cells, and its collapse is a key event in the activation of the mitochondrial-dependent pathway. The collapse of mitochondrial transmembrane potential was assessed by measuring the response to the mitochondrial dye, DiOC6(3), which is converted into a highly green fluorescent dye following incorporation into mitochondrial membranes, thereby allowing the qualitative assessment of mitochondrial membrane potential. The administration of MPP+ in comparison with the control cells induced a significant decrease in fluorescence intensity, indicating the increasing percentage of the cells with collapse of mitochondrial membrane potential. The results also revealed a marked reduction in the number of cells with the collapse of mitochondrial membrane potential, when guanosine was administered prior to exposure to MPP+; no significant change was observed following treatment with guanosine alone (
The levels of ROS production were evaluated by flow cytometry with DCFH-DA. DCFH-DA is a stable compound that can easily diffuse into cells, where it is converted into DCFH by intracellular esterase. DCFH is then trapped within cells and oxidized to highly fluorescent DCF by intracellular ROS; thus, the intensity of fluorescence produced by DCF may reflect an intracellular oxidative state.
The administration of guanosine alone, compared with the control group, did not elicit changes in the levels of DCFH oxidation. The administration of MPP+ induced a significant increase in DCFH oxidation in the PC12 cells, which was markedly reversed by pre-treatment with guanosine (
GSH protein is a major non-enzymatic antioxidant that plays a crucial role in protecting neurons from oxidative damage in the central nervous system (
Caspase-3 is an effector caspase that cleaves a wide range of signal transduction proteins in the apoptotic process (
The non-adenine-based purine, guanosine, is a multifaceted intercellular signaling molecule affecting multiple cellular processes, including cellular growth, differentiation and survival (
PD is a common neurodegenerative disease clinically characterized by rigidity, resting tremor, bradykinesia and postural instability caused by the degeneration and death of dopaminergic neurons in the pars compacta of the substantia nigra (
Our results revealed that treatment with MPP+ induced the adverse expression levels of two Bcl-2 proteins and the disruption of the mitochondrial membrane potential, supporting the involvement of mitochondrial dysfunction in dopaminergic neuronal degeneration. These changes were reversed by the administration of guanosine prior to exposure to MPP+, demonstrating the protective role of guanosine in mitochondrial-stress induced cell damage, which was partly mediated through the regulation of the expression of proteins involved in the mitochondrial stage of the apoptotic cascade. However, the underlying mechanism responsible for this effect of guanosine is unclear.
A number of studies have indicated that the anti-apoptotic effects of guanosine are mediated by modulating the phosphatidylinositol 3-kinase (PI3K)/Akt/protein kinase B (PKB) and the mitogen-activated protein kinase (MAPK) cell survival pathways (
Oxidative stress is another pathological event associated with cell death mechanisms in PD. Studies using postmortem samples of PD have demonstrated that oxidative markers, including soluble protein carbonyl modifications, lipid peroxidation and DNA oxidative damage are selectively observed in the dopaminergic neurons in the pars compacta of the substantia nigra, indicating the correlation of oxidative damage with striatal dopaminergic neurodegeneration (
In conclusion, this study clearly demonstrates that the neuroprotective effects of guanosine promote dopaminergic neuronal survival by alleviating mitochontrial dysfunction in a cellular model of PD. These neuroprotective effects are partly mediated through the stabilization of mitochondrial membrane potential via the modulation of the expression levels of intrinsic apoptotic proteins involved in the mitochondrial apoptotic pathway. Further studies are required to fully elucidate the mechanisms responsible for the protective effects of guanosine in neurodegenerative diseases, which may promote the development of potentially effective treatments for neurodegenerative diseases by targeting mitochondrias-mediated neuronal damage.
Parkinson’s disease
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
1-methyl-4-phenylpyridinium
mitochodrial permeability transition pore
2′7′-dichlorodihydrofluorescein
B-cell lymphoma 2
reactive oxygen species
Effect of guanosine on 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity in PC12 cells. PC12 cells were pre-treated with guanosine (0.01–1,000 μM) for 3 h, and then exposed to 500 μMMPP+ for 72 h. Cell viability was determined by MTT assay as described in the Materials and methods. Data are presented as the means ± standard error of the mean (SEM) of 3 independent experiments performed in sixplicate. ##p<0.01 vs. control and *p<0.05 or **p<0.01 compared to treatmen with MPP+.
(A) Effects of guanosine on 1-methyl-4-phenylpyridinium (MPP+)-induced neuronal cell apoptosis were analyzed by acridine orange (AO)/ethidium bromide (EB) assay. (a) Control; (b) treatment with 10 μM guanosine; (c) treatment with 500 μM MPP+ ; (d) treatment with 10 μM guanosine plus 500 μM MPP+. (B) Histograms show percentages of values in total cells. Data are presented as the means ± standard error of the mean (SEM). n=3. ##p<0.01 vs. control and **p<0.01 compared to treatment with MPP+.
Effects of guanosine on 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of PC12 cells was determined by single-stranded DNA assay. The histograms show percentages of values in total cells. Data are presented as the means ± standard error of the mean (SEM). n=3. ##p<0.01 vs. control and *p<0.01 compared to treatmen with MPP+.
Effects of guanosine on the levels of Bax and Bcl-2 protein expression in 1-methyl-4-phenylpyridinium (MPP+)-exposed PC12 cells. PC12 cells were either untreated, or treated with 500 μM MPP+ alone or 500 μM MPP+ in the presence of 10 μM guanosine. The protein expression levels of Bax and Bcl-2 were determined by western blot analysis, and actin levels were measured as a loading control.
Mitochondrial transmembrane potential was determined by flow cytometry with the mitochondrial dye, 3,3-dihexyloxacarbocyanine iodide [DiOC6(3)]. The x-axis shows the log scale of fluorescence intensity and the y-axis represents the cell count. Data are presented as the means ± standard error of the mean (SEM) of 3 independent experiments. ##p<0.01 vs. control and **p<0.01 compared to treatment with 1-methyl-4-phenylpyridinium (MPP+).
Effects of guanosine on 1-methyl-4-phenylpyridinium (MPP+)-induced reactive oxygen species (ROS) and glutathione (GSH) production. PC12 cells were pre-treated with guanosine for 3 h and then either exposed or not to MPP+ for 72 h. The production of ROS in the cells was measured by flow cytometry with DCFH-DA, and GSH levels were measured by enzyme-linked immunosorbent assay (ELISA). The graphs display the relative levels of (A) ROS production and (B) GSH production compared to the production in the control. Data are presented as the means ± standard error of the mean (SEM). n=3. ##p<0.01 vs. control and *p<0.05 or **p<0.01 compared to treatmen wiht MPP+.
Effects of guanosine on the 1-methyl-4-phenylpyridinium (MPP+)-induced caspase-3 activity in PC12 cells. The cells were either untreated, or treated with 500 μM MPP+ alone or 500 μM MPP+ in the presence of 10 μM guanosine. Caspase-3 activity was determined by enzyme-linked immunosorbent assay (ELISA). Data are expressed as units for caspase-3 activity, and presented as the means ± standard error of the mean (SEM). n=3. ##p<0.01 vs. control and **p<0.01 compared to treatment with MPP+.