Risk factors for severe RSV-induced lower respiratory tract infection over four consecutive epidemics

Variability in severity among different respiratory syncytial virus (RSV) seasons may influence hospital admission rates for RSV-induced lower respiratory tract infection (LRTI) in young children. The aim of the present study was to identify through logistic regression analysis, risk factors associated with higher likelihood to acquire RSV-induced LRTI, in children with symptoms severe enough to lead to hospital admission. Over four consecutive RSV seasons (2000–2004), records from children <4 years of age admitted for RSV-induced LRTI (“cases”) were compared with those from children with LRTI not due to RSV and not requiring hospitalization (“controls”). 145 “case-patients” and 295 “control-patients” were evaluated. Independent from the severity of the four epidemic seasons, seven predictors for hospitalization for RSV infection were found in the bivariate analysis: number of children in the family, chronological age at the onset of RSV season, birth weight and gestational age, birth order, daycare attendance, previous RSV infections. In the logistic regression analysis, only three predictors were detected: chronological age at the beginning of RSV season [aOR = 8.46; 95% CI:3.09–23.18]; birth weight category [aOR = 7.70; 95% CI:1.29–45.91]; birth order (aOR = 1.92; 95% CI:1.21–3.06). Conclusions: Independent from the RSV seasonality, specific host/environmental factors can be used to identify children at greatest risk for hospitalization for RSV infection.
Introduction
Lower respiratory tract infection (LRTI) due to respiratory syncytial virus (RSV) is a major cause of morbidity in infants and young children [21]. Although certain risk factors have been traditionally associated with a higher likelihood to develop severe RSV-induced LRTI, the vast majority of children hospitalized for this condition are previously normal babies [3, 21]. Solid epidemiologic data indicate that prematurity is a consistently proven risk factor for severe LTRI induced by RSV [3, 21]. However, there are other independent risk factors, which show a significant variability among studies performed in different countries and even among different RSV seasons within the same country with alternation of minor and major epidemics [1, 3, 4, 21]. This was recently demonstrated in the “Osservatorio study”, which was designed to collect epidemiological data on RSV infection in two consecutive winter epidemics (2000–2002) in Italy [16]. This study showed that, because of differences in severity of the two RSV seasons, some of the risk factors were significant in one but not in the subsequent epidemics [15]. To further expand the RSV surveillance, the “Osservatorio study” was extended for two additional winter epidemics (2002–2004). The aim of the present study was to use the data collected to identify, by means of logistic regression analysis, risk factors associated with higher likelihood to acquire RSV-induced LRTI in children with symptoms severe enough to lead to hospital admission.
Materials and methods
Study design
A case-control study was used to evaluate the risk factors associated with higher likelihood to acquire RSV-induced LRTI, severe enough to lead to hospital admission, over four consecutive winter epidemics in Italy (2000–2004). The pediatric centres involved (scattered over the Italian national territory), the study population, the surveillance, and the laboratory methods have been described elsewhere [13]. Briefly, all consecutive children, aged ≤4 years, referred to emergency departments for acute respiratory infection on one day of the week (Tuesday), between 8 a.m. and 2 p.m., during the “expected” epidemiological seasons (October – April) were enrolled and tested for RSV determination. Children who had received immunoprophylaxis with palivizumab (≤7, each year) were excluded. We expanded the age limit of the children to be enrolled over the “classic” 24 months, to also evaluate possible predictors of RSV-induced LRTI in an older age group. Each study site determined the date of onset and the end of the local RSV season according to a definition previously developed by the Pediatric Investigators Collaborative Network on Infections in Canada (PICNIC) group [20]. The study protocol was approved by the local ethics board of each hospital and written informed consent was sought from parents or guardians of all children evaluated. Medical history and physical examination were recorded and evaluated and clinical assessment, including nasal secretion specimens for RSV detection, were obtained according to the usual protocol at each hospital. Nasal secretion samples were sent to the microbiology laboratory of each hospital to check for RSV by an immunoenzymatic test (TestPack RSV, Abbott, Italy) within 24 hours. Other respiratory viruses were not tested. All children were treated according to standardized protocols and the decision to admit a child to hospital for a respiratory illness was made by local non-study physicians responsible for each child’s care and was not influenced by the child’s participation in the study.
Case definition and control selection
During a given study year, children were considered eligible for the case-control study on the base of his/her first LRTI during that year. “Case of disease” was defined as LRTI due to RSV, occurring in a child ≤4 years of age and requiring hospital admission. From the cases we excluded patients with LRTI induced by agents other than RSV or RSV-positive but not severe enough to require hospitalization. “Controls” were selected from children who had LRTI which was with certainty not due to RSV (negativity on immunoenzymatic test performed on nasal secretions) and who did not require hospitalization. This control population was selected to determine both predictors of acquiring RSV infection and developing severe disease. All controls should have come from the same residence areas as case-patients. Since case patients ranged from 0 to 30 months of age, control patients aged ≥31 months were excluded from the total control population (N = 1064). Within the remaining group of controls (N = 729), a sample (N = 295) was randomly selected (simple random selection procedure via SPSS software) and included in the study to obtain a case-control ratio of 1:2.
For both case patients and controls evaluated in the four epidemic seasons, data related to the characteristics of the family (e.g. mother’s age, number of children in the family, father’s and mother’s educational level and social condition score) and of the children (e.g. chronological age of the child at the beginning of RSV season, birth weight, birth order, gestational age, length of breast feeding) were categorized according to cut-offs partially derived from literature [5, 10, 12] and partially imposed from the original categorization recorded in the “Osservatorio” database. In addition, children were considered to be small for gestational age (SGA) if their weight at birth fell below the 10th percentile based on gestational age and sex according to Oken’s method [15] and using the United States of America national reference.
Statistical analysis and data definitions
Descriptive statistics of the characteristics of the patients were performed and reported in terms of mean and standard deviation (SD) for the quantitative variables (chronological age, birth weight, gestational age, etc.) and in terms of absolute frequencies and percentages for the qualitative variables. Firstly, a bivariate analysis was performed and the comparison of quantitative variables between two groups of subjects (i.e. cases vs controls) was made calculating the likelihood ratio test (LR test), and reporting the bivariate odds ratio (OR) and 95% confidence interval (CI). Comparison of frequency data was performed by the chi-square test or by the Fisher’s exact test in case of expected frequencies less than five.
In order to evaluate the role of different predictors in the association with the outcome (hospitalization for RSV infection), multiple logistic regression analysis (step-down strategy) was performed. Variables that were statistically significant in the bivariate analysis or that were considered a priori important for the outcome were entered in the model. The following predictors were considered: epidemic season, father’s educational level, chronological age category at the beginning of RSV season, birth order, birth weight category, small for gestational age, daycare attendance, and previous RSV infections. The effect was expressed in terms of adjusted (a)OR and 95% CI and the LR test was performed. The models’ predictive ability has been investigated by calculating the area under the ROC curve of the model (AUC). All tests were two-sided and a P value less than 0.05 was considered statistically significant. The statistical packages used were the “Statistica release 6” (StatSoft Corp., Tulsa, OK, USA) for bivariate analyses and the “Stata release 7” (Stata Corporation, College Station, TX, USA) for multivariate analyses.
Results
Patient populations
There were 145 case-patients included in the study (plus three cases not evaluated because of missing values). There were 61 males (42.1%) and 84 females (57.9%), with a median age of 3.5 months and a median birth weight of 3,050 grams. Seventeen of the 145 (11.7%) were born preterm (at less than 36 weeks of gestational age), two had congenital heart conditions and five had a previous history of chronic lung disease.
There were 292 control-patients (plus three cases not evaluated because of missing values). There were 124 males (42.5%) and 168 females (57.5%), with a median age of 5 months. Eighteen were preterm infants (6.2%) and their birth weight was greater but not significantly different (mean = 2,310; median = 2,175 grams) from the preterm case-patients’ birth weight (mean = 1,993; median = 1,817.5) (P = 0.21).
Risk factors associated with a higher likelihood to acquire RSV-induced LRTI, severe enough to lead to hospital admission
In the bivariate analysis we considered several risk factors that could be associated with a higher likelihood to acquire RSV-induced LRTI, severe enough to lead to hospital admission.
In Table 1 only the statistically significant risk factors associated with a higher likelihood to acquire RSV-induced LRTI, severe enough to lead to hospital admission, are reported. The bivariate analysis showed that, although the severity of RSV-induced LRTI with hospital admission was different during the four epidemics, a variety of other risk factors could be detected. The only demographic-social characteristic associated with severe RSV-induced LRTI requiring hospitalization was the number of children in the family ≥2. Geographical area, social condition level and mother’s age category were not significant risk factors (not shown). The major predictors were the characteristics of the child itself as follows: low chronological age at the beginning of RSV season, being at least the second baby in the family, having a low birth weight (1,500–2,499 g), and a low gestational age (<33 weeks). In addition, the absence of daycare attendance was also a risk factor and higher risks could be seen in children without previous exposure to RSV infections. Having a birth weight small for the gestational age was also a risk factor but the role of the birth weight uncorrected for gestational age was a stronger predictor. Breast feeding had no significant role due to the small number of data available (n = 138). Mother’s educational level was available only in a very small subset of patients (n = 80) (not shown) and because of the strong association with father’s educational level (not shown) (P < 0.0001) only this latter variable was considered in subsequent analysis.
Table 1Distribution of risk factors associated with a higher likelihood to acquire RSV-induced LRTI, severe enough to lead to hospital admission Cases (N = 145) N (%)Controls (N = 292) N (%)P valueOR (95%CI)Epidemic season 2003–200429 (20.0%)56 (19.2%)Reference 2002–200350 (34.5%)68 (23.3%)1.42 (0.80–2.53) 2001–200241 (28.3%)122 (41.8%)0.65 (0.37–1–15) 2000–200125 (17.2%)46 (15.7%)P = 0.0241.05 (0.54–2.03)Epidemic period (October to April): Yes137 (94.6%)247 (84.6%)P = 0.0033.12 (1.43–6.81)Sex: Female84 (57.9%)168 (57.5%)P = 0.941.02 (0.68–1.52)Exposure to cigarette smoking: Yes (N = 432)74 (51.7%)165 (57.1%)P = 0.290.81 (0.54–1.21)Family history of allergy: Yes (N = 333)50 (44.6%)75 (33.9%)P = 0.061.57 (0.99–2.50)No. of children in the family: ≥2 (N = 393)97 (72.9%)155 (59.6%)P = 0.0091.83 (1.16–2.88)Chronological age at the beginning of RSV season ≥12 months6 (4.1%)48 (16.4%)P < 0.0001Reference 6–11 months31 (21.4%)98 (33.6%)2.53 (0.99–6.48) 3–5 months48 (33.1%)85 (29.1%)4.52 (1.80–11.33) <3 months60 (41.4%)61 (20.9%)7.87 (3.13–19.75) Birth order: ≥ 2nd (N = 420)95 (69.3%)151 (53.4%)P = 0.0021.98 (1.28–3.05)Birth weight category (N = 430) ≥2500 grams113 (80.1%)259 (92.2%)P= 0.013aReference 1500–2499 grams24 (17.0%)28 (7.2%)1.96 (1.09–3.54) <1500 grams4 (2.8%)2 (0.6%)4.58 (0.83–25.39)Gestational age category (N = 430) ≥36 weeks126 (88.1%)269 (93.7%)P = 0.023aReference 33–35 weeks8 (5.6%)14 (4.9%)1.22 (0.50–2.98) <33 weeks9 (6.3%)4 (1.4%)5.35 (1.45–15.89)SGA: Yes (N = 426)36 (25.7%)63 (22.0%)P = 0.401.23 (0.76–1.96)Breast feeding (N = 138) None or <3 months25 (71.4%)68 (66.0%)P = 0.14aReference 3–6 months9 (25.7%)20 (19.4%)1.22 (0.49–3.04) ≥7 months1 (2.9%)15 (14.6%)0.18 (0.02–1.44)Daycare attendance: No (N = 430)138 (97.9%)265 (78.0%)P = 0.0134.17 (1.23–14.08)Previous RSV infections: No (N = 426)126 (88.7%)230 (81.0%)P = 0.0421.85 (1.02–3.36)P values in the table refer to chi-square test, unless otherwise specifieda Fisher’s exact test
In the logistic regression analysis variables that were statistically significant in the bivariate analysis or that were considered a priori important for the outcome were entered in the model (see Table 1). As reported in Table 2 only three independent predictors were associated with severe RSV infection with hospitalization: a lower age category of the child, a lower birth weight category (<1500 grams), and birth order (being at least the second child in the family). The number of children in the family and gestational age were not entered in the logistic model because of the strong autocorrelation, respectively, with the variables birth order and birth weight. Indeed, the vast majority of children (84.6%) belonging to the lowest gestational age category (<33 weeks) and more than 60% of those belonging to the intermediate gestational age category (33–35 weeks) had a birth weight <2,500 grams.
Table 2Best fitting logistic regression model for independent predictors of severe RSV infection with hospitalization (N = 419)Explanatory variables aOR95% CIP valueaChronological age at the beginning of RSV season  (reference category: ≥12 months)6–11 months2.4670.879–6.925P< 0.00013–5 months4.1531.506–11.451<3 months8.4623.088–23.185Birth weight category (reference category: ≥2,500 grams)1,500–2,499 grams2.4331.289–4.595P = 0.0023<1,500 grams7.7011.292–45.907Birth order (reference category: first)≥ second1.9241.210–3.058P = 0.0049a Likelihood ratio testThe area under the ROC curve of the model was 0.712
Discussion
Evaluating data collected over four consecutive years in Italy, we found that, in addition to the severity of the RSV epidemics in a specific year, a variety of risk factors were associated with a higher likelihood to acquire RSV-induced LRTI, severe enough to lead to hospital admission. The only condition related to demographic and social characteristics of the family was the number of children in the family (being at least the second baby was a “risk-factor”). Among factors related to the characteristics of the children, major predictors were chronological age of the child at the onset of RSV season, birth weight and gestational age category, birth order, daycare attendance and previous episodes of RSV infections. However, in the logistic regression analysis, only three predictors turned out to be significant: chronological age at the beginning of RSV season, birth weight category, and birth order.
These results only partially confirm previous observations regarding risk factors for severe RSV infection [1, 2, 8, 11, 14, 17].
Chronological age of the child at the beginning of RSV season is a known factor for severe RSV-induced LRTI [1, 10, 17–19]. Indeed, in our study population only six children (4.1%) with a chronological age at the beginning of the season >12 months (and none between 2 to 4 years) had RSV-induced LRTI severe enough to lead to hospital admission. Aside from the incomplete development of the lung structure and the small size of the conducting airways related to the young age, data also suggest that such infants are less likely to have RSV-neutralizing maternal antibody as compared with infants born after the peak of the RSV season [6].
Birth weight was also an important risk factor in our study population. Low birth weight is often linked to low gestational age and there is no question that premature birth is, in general, associated with acute and chronic pulmonary morbidity [3, 5, 7–10, 14, 22]. Prematurity, in RSV-induced LRTI, can lead to increased need for and lengthened duration of hospital admission, intensive care, mechanical ventilation and supplemental oxygen, as well as increased mortality [1, 21]. Increased risk to develop severe RSV-induced LRTI may also be observed in the “less premature” infants, i.e. those with a gestational age of 32 weeks or greater, in the presence of other concomitant host/environmental unfavourable conditions, such as day-care attendance, low chronological age at the onset of RSV season, presence of preschool age sibling(s), and low birth weight [3, 5, 10, 21].
In the present study, gestational age, a strong risk factor in the bivariate analysis, was not included in the logistic model because of the strong autocorrelation with birth weight. Indeed, a great proportion of children belonging to two lower gestational age categories (<33 weeks and 33–35 weeks) had a birth weight <2,500 grams.
SGA has been only recently examined as an independent risk factor for hospitalization for RSV infection and, in agreement with the result of the present study, was found to be significant in a population of infants born at 33 through 35 weeks of gestation [10]. An interesting finding of the present report is that a low birth weight, corrected or uncorrected for gestational age, was a strong predictor for severe RSV-induced LRTI.
The third important predictor in our study population was birth order, i.e. being at least the second child in the family was found to be a risk factor for severe RSV-induced LRTI. A variety of studies demonstrated a trend toward higher numbers of RSV hospitalization rates in infants with any older siblings, including preschool age siblings (younger than 6 years old) who attend daycare and school age siblings (older than 3 years) [1, 3, 5–7, 10, 18, 19, 21].
In agreement with the majority of reports, other conditions related to demographic and social characteristics of the family (the number of children in the family) or of the environment (daycare attendance) were found in the bivariate analysis to be able to increase the likelihood to be hospitalized for RSV-induced LRTI [1, 3, 5–7, 10, 18, 19, 21]. While the number of children in the family was not entered in the logistic model because of the strong autocorrelation with the variable birth order, daycare attendance did not reach a significant value in the logistic regression analysis, probably because of the small number of children attending daycare in our study population [3 (2.1%) in the “case” group and 24 (22.0%) in the “control” group]. Despite other conditions, such as father’s and mother’s educational level, mother’s age category, social conditions, breast feeding, family history of allergy and gender and exposure to cigarette smoking, that were not statistically relevant in our study population, we cannot exclude that other socio-economical and environmental factors might be important [1, 3, 5–7, 10, 18, 19, 21]. As shown for other Mediterranean countries [5], exposure to tobacco smoke at home has decreased during recent years in Italy as a result of anti-smoking campaigns and better health education, and this may explain our surprising results.
Due to the low number of children with specific characteristics, other risk factors such as presence of chronic lung diseases or of congenital heart diseases, immunodeficiency, haematological malignancies, bone-marrow or organ transplants, and cystic fibrosis were not analysed. Moreover, since only in the second, third and fourth RSV season a limited number of children had received immunoprophylaxis with palivizumab, its protective effect could not be evaluated.
In summary, evaluating four RSV seasons, characterized by inter-annual alternation of minor and major epidemics, we found, in addition to prematurity, a limited number of other conditions which appear to influence the severity of RSV-induced LTRI in infants and young children. However, before translating to clinical practice these indications, other similar studies performed in different countries are required, including a broader number of children and covering other RSV epidemic seasons. This information should be valuable to better determine hospital resource management and individualize RSV prophylaxis programs in preterm and term infants.

infants
respiratory syncytial virus infections
emergency rooms


MPP+-induced cytotoxicity in neuroblastoma cells: Antagonism and reversal by guanosine

Guanosine exerts neuroprotective effects in the central nervous system. Apoptosis, a morphological form of programmed cell death, is implicated in the pathophysiology of Parkinson’s disease (PD). MPP+, a dopaminergic neurotoxin, produces in vivo and in vitro cellular changes characteristic of PD, such as cytotoxicity, resulting in apoptosis. Undifferentiated human SH-SY5Y neuroblastoma cells had been used as an in vitro model of Parkinson’s disease. We investigated if extracellular guanosine affected MPP+-induced cytotoxicity and examined the molecular mechanisms mediating its effects. Exposure of neuroblastoma cells to MPP+ (10 μM–5 mM for 24–72 h) induced DNA fragmentation in a time-dependent manner (p < 0.05). Administration of guanosine (100 μM) before, concomitantly with or, importantly, after the addition of MPP+ abolished MPP+-induced DNA fragmentation. Addition of MPP+ (500 μM) to cells increased caspase-3 activity over 72 h (p < 0.05), and this was abolished by pre- or co-treatment with guanosine. Exposure of cells to pertussis toxin prior to MPP+ eliminated the anti-apoptotic effect of guanosine, indicating that this effect is dependent on a Gi protein-coupled receptor, most likely the putative guanosine receptor. The protection by guanosine was also abolished by the selective inhibitor of the enzyme PI-3-K/Akt/PKB (LY294002), confirming that this pathway plays a decisive role in this effect of guanosine. Neither MPP+ nor guanosine had any significant effect on α-synuclein expression. Thus, guanosine antagonizes and reverses MPP+-induced cytotoxicity of neuroblastoma cells via activation of the cell survival pathway, PI-3-K/Akt/PKB. Our results suggest that guanosine may be an effective pharmacological intervention in PD.
Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative disorder caused primarily by selective degeneration of dopaminergic neurons in the substantia nigra pars compacta [1, 2]. Apoptosis, a morphological form of programmed cell death [3], has been implicated in the pathophysiology of PD [4–6]. The biochemical pathways that mediate apoptosis may be initiated by extrinsic (e.g. activation of the death receptors) or intrinsic factors (e.g. mitochondrial insult) [7] and result in the activation of a set of cysteine-dependent aspartyl proteases (caspases) [8]. Both apoptotic pathways stimulate the effector enzyme, caspase-3 [7, 9, 10], which in turn activates a specific nuclear DNAse that degrades DNA into 200 base pair oligonucleosomal fragments, a hallmark of apoptosis [11, 12]. Apoptosis-related alterations have been reported in the dopaminergic brain regions in post-mortem tissues of PD patients, including increased caspase-3 activity in the substantia nigra [13, 14], and elevated immunoreactivity of the pro-apoptotic protein, Bax [15], indicating that apoptosis may play a role in the pathophysiology of PD.
The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been used extensively to study PD [16, 17]. MPTP selectively targets and damages the dopaminergic neurons causing parkinsonism in humans and other primates [18, 19]. MPTP readily crosses the blood-brain barrier and is converted into the active metabolite, 1-methyl-4-phenyl pyridinium (MPP+) [17]. MPP+ is taken up by the dopaminergic neurons and accumulates in the mitochondria, where it inhibits complex I of the electron transport chain and ultimately causes neuronal cell death [5]. The mechanisms of MPP+-induced cytotoxicity most likely involve oxidative stress [20]. Several groups reported that treatment of mice with MPTP induces a Parkinson-like syndrome [16], accompanied by apoptosis of the dopaminergic neurons in the substantia nigra [21] and up-regulation of α-synuclein, a pre-synaptic protein, which plays an important role in PD pathogenesis [22]. In vitro studies using human SH-SY5Y neuroblastoma cells have produced comparable results [23, 24]. Exposure of these cells to MPP+ led to fragmentation of nuclear DNA [25], activation of caspase-3 [23, 26] and to up-regulation of α-synuclein [27–29]. In cultured SH-SY5Y cells dopamine-dependent cytotoxicity was enhanced by the accumulation of α-synuclein [30]. Therefore, targeting the molecular pathways activated during apoptosis may lead to novel treatment strategies for PD [5, 31].
Guanosine and other non-adenine-based purines exert neuroprotective effects in the central nervous system [32]. In vitro guanosine also protects rat astrocytes from staurosporine-induced apoptosis [33] and SH-SY5Y cells from β-amyloid-induced apoptosis [34]. In both cases, the anti-apoptotic effect of guanosine was mediated by stimulation of the phosphatidylinositol-3-kinase (PI-3-K)/Akt/protein kinase B (PKB) and mitogen-activated protein kinase (MAPK) cell survival pathways [33, 34]. These pathways were activated via the pertussis toxin-sensitive Gi protein-coupled putative guanosine receptor that we have recently characterized pharmacologically [35–37].
In PD, symptoms appear only after the majority of the dopaminergic neurons in the substantia nigra pars compacta have degenerated, so putative neuroprotective drugs for PD should be tested in models in which the neurodegenerative process is already underway [38]. Therefore, we examined the effects of guanosine on MPP+-induced caspase-3 activation and DNA fragmentation in SH-SY5Y human neuroblastoma cells not only when it was added prior to, or at the same time as MPP+, but also when it was added to the cultures up to 48 h after MPP+ treatment, when the pro-apoptotic pathways were already activated. We also studied the molecular mechanisms that mediate the neuroprotective effects of guanosine.
Materials and methods
Tissue culture and treatment of human SH-SY5Y neuroblastoma cells
Human SH-SY5Y neuroblastoma cells (ATCC, Manassas, VA, USA) were cultured using 45% minimum essential medium, 45% F12 Hamilton’s medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 U/ml of streptomycin at a pH of 7.4 (all from Invitrogen, Burlington, ON, Canada). Culture medium was changed every 3–4 days, and cells were maintained in a humidified 5% CO2 atmosphere at 37°C and subcultured at a ratio of 1:20 every 7–10 days. Culture medium was changed to 1% FBS for 24 h before the start of each experiments. All experiments were performed using 70–80% confluent cultures.
Apoptosis was induced by adding MPP+ (Sigma-Aldrich) to SH-SY5Y neuroblastoma cells, using a fresh stock solution of MPP+ (10 mM in reverse osmosis purified H2O). In all experiments, guanosine (Sigma-Aldrich) was dissolved in 0.1 M NaOH and added to the culture medium at a final concentration of 0.001 M NaOH. Some cells (control) were exposed to 0.001 M NaOH. In experiments in which the protective effect of guanosine was tested, guanosine (100 μM) was added to SH-SY5Y neuroblastoma cells either: 1 h prior to the administration of MPP+ (pre-treatment), or at the same time as MPP+ (co-treatment) or 24 or 48 h after the addition of MPP+(post-treatment at 24 h, or post-treatment at 48 h, respectively; Fig. 1). Guanosine and MPP+ remained in the culture medium for the duration of the experiment (24, 48 or 72 h). In the first post-treatment experimental group at 24 h, cells were exposed to MPP+ for 24 h, followed by MPP+ plus guanosine for an additional 24 h (Fig. 1). In the second post-treatment experimental group at 48 h, cells were treated with MPP+ for 48 h, followed by MPP+ plus guanosine for an additional 24 h (Fig. 1).
Fig. 1Experimental design for guanosine post-treatment of SH-SY5Y neuroblastoma cells added at 24 or 48 h after MPP+. SH-SY5Y neuroblastoma cells were exposed to either vehicle, or guanosine (100 μM) or MPP+ (500 μM) for various times. For post-treatment experiments with guanosine at 24 h, cells were exposed to MPP+ for 24 h, followed by MPP+ plus guanosine for an additional 24 h (total MPP+ exposure = 48 h). For post-treatment experiments with guanosine at 48 h, cells were exposed to MPP+ for 48 h followed by MPP+ plus guanosine for a further 24 h (total MPP+ exposure = 72 h). Once added, guanosine and MPP+ remained in the culture medium for the duration of the experiment. DNA fragmentation, or caspase-3 activity, was determined at 24, 48 or 72 h as described in the “Materials and methods” section
In experiments in which enzyme inhibitors were tested, SH-SY5Y cells were pre-treated with various agents for 20 min prior to the addition of guanosine. These treatments included: the potent and selective inhibitor of PI-3-K, [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-1-hydrochloride] (LY294002; IC50 = 1.4 μM; Sigma), or the selective inhibitor of MAP kinase kinase (MEK), [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-1] (PD98059; IC50 = 2 μM; Sigma). In experiments using the ADP-ribosylating factor of the inhibitory guanosine nucleotide binding protein (Gi), pertussis toxin [PTX, 200 ng/ml; in 50% glycerol, 0.5 M NaCl, and 0.05 M Tris-glycine (pH 7.5); Sigma] was added overnight (16 h). LY294002 and PD98059 were dissolved in and added to the culture medium at a final concentration of 0.01% dimethyl sulfoxide (DMSO).
Evaluation of cell death and DNA fragmentation
In initial experiments, cell death was determined using acridine orange-ethidium bromide (AO-EB, Sigma-Aldrich) staining. Cells were seeded onto 6-well plates (diameter 10 mm; 2.5 × 104 cells/well) and exposed to MPP+. After 24, 48 or 72 h, cells were washed three times with phosphate-buffered saline (PBS) and AO-EB solution (6 μM/ml) was added to the wells. Cells were examined using a fluorescence microscope (Nikon, Tokyo, Japan). The number of fragmented nuclei and/or condensed chromatin was determined by counting >200 cells. The percentage of apoptotic cells is defined as: (total number of cells with apoptotic nuclei/total number of cells) × 100.
In subsequent experiments, DNA fragmentation was evaluated by the oligonucleosomal ELISA (Cell Death Detection ELISAPLUS; Roche Diagnostics, Laval, QC, Canada) and carried out according to the manufacturer’s instructions. This assay measures the amount of oligonucleosomal fragments and is a marker of apoptosis [39]. Cells were exposed to MPP+ alone, or in combination with guanosine or pre-treated with the various inhibitors as described above. Following various treatments, cells were isolated for analysis of DNA fragmentation as described by Pettifer et al. [34]. For the oligonucleosomal ELISA, 10,000 viable cells/treatment were lysed and centrifuged (200 g for 10 min) to isolate fragmented oligonucleosomal DNA. The cytosolic fractions of cell lysates were transferred into streptavidin-coated microplate wells, and a mixture of biotin-linked anti-histone antibody and peroxidase-linked anti-DNA antibody was added and incubated for 2 h at room temperature. Plates were washed with incubation buffer to remove the unfixed anti-DNA antibody and the peroxidase activity was determined spectrophotometrically with 2,2′-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) as the substrate (absorbance of 405 nm). The amount of DNA fragmentation is expressed as a percentage of the positive control provided with the kit.
Determination of caspase-3 activity
Caspase-3 activity was determined using the caspase-3 colourimetric assay (Sigma-Aldrich). Cells were exposed to different treatments as described above; then cells were isolated, re-suspended in 100 μl/107 cells of lysis buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 5 mM 3[(3-cholamidopropyl) dimethylammonio]-propanesulfonic acid (CHAPS), 5 mM dithiothreitol (DTT)) and incubated on ice for 20 min. Cell lysates were centrifuged (18,000 g for 10 min at 4°C) and the supernatants (5 μl/well) were transferred to 96-well plates. A selective caspase-3 inhibitor (N-acetyl-Asp-Glu-Val-Asp-CHO; 10 μl/well at 200 μM) was added to supernatants to control for the nonspecific hydrolysis of the substrate. Purified caspase-3 human recombinant protein, provided with the assay, was used (5 μl/well at 5 μg/ml) as a positive control for caspase-3 activity. Cell lysates were incubated in an assay buffer [40 mM HEPES, pH 7.4, 1% CHAPS, 10 mM DTT, 4 mM ethylenediaminetetraacetic acid (EDTA)], containing N-acetyl-Asp-Glu-Val-Asp p-nitroaniline (10 μl/well at 2 mM) overnight at 37°C and the formation of p-nitroaniline was measured at 405 nm, using a microtiter plate reader. The activity of caspase-3 was calculated from the absorbance values using a calibration curve, and it is expressed as μg of substrate cleaved/min per ml. As the number of cells in 1 ml of cell lysate is adjusted to 1 × 108 cells, this caspase-3 activity is generated by 1 × 108 cells.
Measurement of α-synuclein protein concentration by ELISA
The concentration of α-synuclein protein in SH-SY5Y neuroblastoma cells was determined by an ELISA (BioSource, San Diego, CA, USA). Cells were exposed to various treatments as described above and subsequently lysed using a cell extraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulfate, 0.5% deoxycholate; BioSource, San Diego, CA, USA), containing protease inhibitors [1 mM phenylmethanesulfonyl fluoride, 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 130 μM bestatin, 14 μM E-64, 1 μM leupeptin, 0.3 μM aprotinin; Sigma]. Cells were incubated on ice in cell extraction buffer for 30 min and centrifuged (14,000 g for 10 min at 4°C). Aliquots of supernatants (25 μl) were removed for the determination of the total protein concentration using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). We measured α-synuclein protein concentration using an ELISA kit according to the manufacturer’s instructions. Briefly, cell lysates (diluted 1:10) were added to α-synuclein antibody-coated wells in duplicate, allowed to bind and then were treated with the anti-α-synuclein antibody. This was followed by the addition of an anti-rabbit IgG-horse radish peroxidase-linked antibody, and by the addition of a stabilized chromogen (tetramethylbenzidine). The reaction was terminated by adding 1 M HCl (stop solution) provided with the kit, and the absorbance of each well was read at 450 nm. Standard curves were prepared using purified recombinant α-synuclein protein (100 ng/ml) provided with the kit. The concentration of the α-synuclein protein in each sample was determined from a nonlinear regression, and it is expressed as a ratio of α-synuclein protein (in ng/ml) to total protein concentration (in ng/ml).
Statistical analysis
Data obtained in experiments testing the concentration dependence of the protective effect of guanosine on MPP+-induced DNA fragmentation (Fig. 3) and the effect of PTX, and inhibitors of the PI-3-K, and the MAPK pathways on DNA fragmentation (Fig. 6 and Table 1) were analyzed by a one-way analysis of variance (ANOVA) followed by a Student-Newman-Keul post-hoc test. Two-way ANOVA, followed by a Bonferroni post-hoc test, was used to analyze the data obtained from experiments evaluating the effects of guanosine, administered at different time points on MPP+-induced DNA fragmentation (Fig. 4) and on MPP+-induced caspase-3 activation (Fig. 5). A p value of 0.05 or less was considered statistically significant. Results represent the mean ± SEM of a minimum of three independent experiments.
Table 1Effect of selected inhibitors of cell survival pathways on the anti-apoptotic effect of guanosine in SH-SY5Y cellsTreatmentDNA fragmentation (% of positive control)No inhibitorLY294002 10 μMPD98059 10 μMControl10.1 ± 1.316.3 ± 3.124.0 ± 4.7MPP+ (500 μM)34.5 ± 4.3a––Guanosine pre-treatment15.9 ± 4.838.6 ± 10.3b38.3 ± 9.1bGuanosine co-treatment11.0 ± 0.732.9 ± 5.7b28.7 ± 10.4n.s.Guanosine post-treatment12.4 ± 1.848.2 ± 4.3c28.4 ± 4.8n.s.SH-SY5Y cells were exposed to MPP+ (500 μM) for 48 h to induce DNA fragmentation. Some cells were treated with guanosine (100 μM) either for 1 h prior to MPP+ (pre-treatment), or at the same time as MPP+ (co-treatment) or 24 h after MPP+ (post-treatment, see Fig. 1). Some cultures were exposed to LY294002 (inhibitor of PI-3-K) or to PD98059 (inhibitor of MAP kinase kinase, MEK) for 20 min prior to the addition of guanosine. DNA fragmentation was determined at 48 h (n = 3) and is expressed as a percentage of the positive control. LY294002 abolished the protective effect of guanosine in cells pre-treated, co-treated or post-treated with this nucleoside. PD98059 also abolished the protective effect of guanosine in cells pre-treated with this nucleoside, but had no significant effect in cells co-treated or post-treated with guanosineap < 0.001 compared to controlbp < 0.01 compared to same treatment without inhibitorcp < 0.05 compared to same treatment without inhibitorn.s. not significant
Results
MPP+ induces DNA fragmentation in SH-SY5Y neuroblastoma cells
SH-SY5Y neuroblastoma cells were exposed to different concentrations of MPP+ (10–500 μM) for various times (0–72 h) to optimize the experimental conditions and to confirm that MPP+-induced DNA fragmentation was concentration and time dependent (Fig. 2). DNA fragmentation in untreated control cells was around 10% and remained unchanged for up to 72 h (Fig. 2). Treatment of cells with 500 μM MPP+ increased DNA fragmentation significantly after 48 h (p < 0.01) and 72 h (p < 0.001; Fig. 2) compared to untreated controls. Since exposure of cells to 500 μM MPP+ for 48 h induced robust DNA fragmentation, these conditions were used in the following studies.
Fig. 2MPP+ induces DNA fragmentation in SH-SY5Y cells. MPP+ (10, 100 or 500 μM) was added to SH-SY5Y cells and DNA fragmentation determined after 24, 48 or 72 h by oligonucleosomal ELISA as described in the “Materials and methods” section. Data are presented as the percentage of DNA fragmentation relative to a positive control (n > 3). MPP+ (500 μM) increased DNA fragmentation significantly at 48 h (*p < 0.01) and at 72 h (**p < 0.001) compared to that of untreated cells at zero time
Pre-treatment of SH-SY5Y neuroblastoma cells with guanosine attenuates MPP+-induced DNA fragmentation
SH-SY5Y cells were exposed to various concentrations of guanosine (1–300 μM) for 1 h, followed by the addition of 500 μM MPP+ for a further 48 h, and DNA fragmentation was determined at 49 h by oligonucleosomal ELISA. As we observed in the previous experiment the addition of MPP+ (500 μM) led to significantly increased DNA fragmentation compared to controls (p < 0.001; Fig. 3). Pre-treatment of cells for 1 h with various concentrations of guanosine (10–300 μM) significantly reduced MPP+-induced DNA fragmentation (p < 0.05) for all four concentrations of guanosine compared to cells treated with MPP+ alone (Fig. 3). Based on these results, we used 100 μM guanosine in all subsequent experiments.
Fig. 3Pre-treatment of SH-SY5Y cells with guanosine decreases MPP+-induced DNA fragmentation. SH-SY5Y cells were exposed to MPP+ (500 μM) for 48 h to induce DNA fragmentation. Some cells were pre-treated with guanosine (1–300 μM) for 1 h prior to MPP+. Guanosine and MPP+ remained in the cultures for the duration of the experiment. DNA fragmentation was determined after 48 h by oligonucleosomal ELISA as described in the “Materials and methods” section and is expressed as the percentage of the positive control (n > 3). MPP+ increased DNA fragmentation significantly at 48 h compared to that of untreated controls (*p < 0.001). Pre-treatment with guanosine (10–300 μM) reduced significantly the MPP+-induced DNA fragmentation compared to that in cells treated with MPP+ for the same time (#p < 0.05, at all four concentrations)
Post-treatment with guanosine protects SH-SY5Y neuroblastoma cells against MPP+-induced DNA fragmentation
We next examined whether guanosine exerted any effect on MPP+-induced DNA fragmentation when it was added to neuroblastoma cells after apoptosis was already in progress. Cells were pre-treated with 100 μM guanosine prior to MPP+ or co-treated with guanosine plus MPP+ or, as described in Fig. 1, guanosine was added as post-treatment. DNA fragmentation was determined in cells exposed to various treatments after 24, 48 and 72 h of MPP+ administration (Fig. 4). The addition of MPP+ led to a significant increase in DNA fragmentation compared to controls at 48 h (p < 0.01) and 72 h (p < 0.001) (Fig. 4). Guanosine alone had no significant effect on DNA fragmentation (Fig. 4). Pre-treatment of cells with guanosine significantly reduced DNA fragmentation after 48 and 72 h compared to MPP+ treatment alone, at the respective time points (p < 0.05, at both time points; Fig. 4). Similarly, co-treatment of cells with guanosine plus MPP+ significantly reduced DNA fragmentation after 48 and 72 h compared to cells treated with MPP+ alone at these times (p < 0.05, at both time points; Fig. 4). Addition of guanosine 24 h after MPP+ (post-treatment at 24 h) or 48 h after MPP+ (post-treatment at 48 h) reduced DNA fragmentation significantly compared to cells treated with MPP+ alone for 48 or 72 h, respectively (p < 0.05, for both treatments; Fig. 4). These results demonstrate that pre-treatment or co-treatment of cells with guanosine and MPP+ attenuates MPP+-induced DNA fragmentation, and guanosine added 24 or 48 h after the start of the DNA fragmentation (post-treatment) can actually reverse this process.
Fig. 4Guanosine antagonizes and reverses MPP+-induced DNA fragmentation in SH-SY5Y cells. SH-SY5Y cells were exposed to MPP+ (500 μM) for 48 h to induce DNA fragmentation. Some cells were treated with guanosine (100 μM) either for 1 h prior to MPP+ (pre-treatment), or at the same time as MPP+ (co-treatment) or 24 or 48 h after MPP+ (post-treatment, see Fig. 1). DNA fragmentation was determined after 24, 48 or 72 h of MPP+ addition by oligonucleosomal ELISA as described in the “Materials and methods” section and it is expressed as a percentage of the positive control (n > 3). MPP+ induced a significant increase in DNA fragmentation at 48 (*p < 0.01) and at 72 h (**p < 0.001) compared to that of untreated control cells at the same time. Pre-treatment or co-treatment of cells with guanosine significantly reduced MPP+-induced DNA fragmentation at 48 and 72 h compared to that in cells treated with MPP+ alone at the same time (#p < 0.05 at both time points, for both treatments). Post-treatment with guanosine at 24 or 48 h also reduced MPP+-induced DNA fragmentation at 48 and 72 h compared to that in cells treated with MPP+ alone at the same time (#p < 0.05, at both time points)
Guanosine inhibits MPP+-induced caspase-3 activity
Exposure of SH-SY5Y cells to MPP+ leads to apoptosis via caspase-3 activation [23, 26]. We therefore examined if under our experimental conditions treatment of neuroblastoma cells with MPP+ activated caspase-3 and whether the addition of guanosine to MPP+-treated cells at various times (pre-treatment, co-treatment or post-treatment) had any effect on the activity of this enzyme. In untreated control cells caspase-3 activity was low and remained unchanged for up to 72 h (Fig. 5). Addition of guanosine to cells had no significant effect on caspase-3 activity (Fig. 5). In MPP+-treated cells caspase-3 activity increased significantly from 24 to 72 h (p < 0.001, at all time points; Fig. 5). Pre-treatment of cells with guanosine prior to MPP+ addition significantly reduced caspase-3 activity after 48 or 72 h compared to that in cells treated with MPP+ alone for the same time (p < 0.01, at both time points; Fig. 5). Similar results were also obtained when cells were co-treated with guanosine and MPP+ for 48 and 72 h (p < 0.01, at both time points; Fig. 5). Post-treatment with guanosine at 24 h (see Fig. 1) also reduced caspase-3 activity significantly at 48 h compared to that in cells treated with MPP+ alone at this time point (p < 0.01; Fig. 5). When guanosine was added to cells 48 h after the addition of MPP+ (post-treatment at 48 h), caspase-3 activity determined after 72 h was unaffected compared to MPP+ treatment alone at this time point (Fig. 5). These results thus demonstrate that pre-treatment, co-treatment or post-treatment at 24 h of neuroblastoma cells with guanosine prevents MPP+-induced activation of caspase-3. Administration of guanosine to cells 48 h after MPP+ however has no effect on caspase-3 activity.
Fig. 5Guanosine inhibits MPP+-induced caspase-3 activation in SH-SY5Y cells. SH-SY5Y cells were exposed to MPP+ (500 μM) for 48 h to induce to induce caspase-3 activation. Some cells were treated with guanosine (100 μM) either for 1 h prior to MPP+ (pre-treatment), or at the same time as MPP+ (co-treatment) or 24 or 48 h after MPP+ (post-treatment, see Fig. 1). Caspase-3 activity was determined as described in the “Materials and methods” section and is expressed as the amount of N-acetyl-Asp-Glu-Val-Asp p-nitroaniline cleaved by 1 × 108 cells, in μg/min per ml (n > 3). MPP+ significantly increased caspase-3 activity from 24 to 72 h (*p < 0.001, at all three time points) compared to that of control cells at the same time. Pre-treatment or co-treatment of cells with guanosine significantly reduced MPP+-induced caspase-3 activity, at 48 and 72 h, compared to that in cells treated with MPP+ alone at the same time (#p < 0.01, at both time points, for both treatments). Post-treatment with guanosine at 24 h reduced MPP+-induced caspase-3 activity at 48 h compared to that in cells treated with MPP+ alone at the same time (#p < 0.01). Guanosine added to cultures 48 h after MPP+ had no effect on caspase-3 activity
Guanosine has no effect on α-synuclein protein expression in MPP+-treated neuroblastoma cells
Several authors reported that addition of MPP+ to SH-SY5Y cells increased α-synuclein protein expression [27, 29]. We therefore determined α-synuclein protein concentration in neuroblastoma cells under our experimental conditions (500 μM MPP+ for 48 h) and evaluated the effects of guanosine on the expression of this protein in MPP+-treated cells. The ratio of α-synuclein protein/total protein in untreated control cells after 48 h was very low and exposure of neuroblastoma cells to MPP+ for 48 h had no significant effect on the expression of this protein (data not shown). Treatment of cells with guanosine prior to the addition of MPP+, or at the same time as MPP+ or 24 h after MPP+ had no significant effect on of α-synuclein protein expression (data not shown). These results indicate that under the present experimental conditions neither MPP+ nor a combination of guanosine plus MPP+ has any effect on α-synuclein protein expression.
Pertussis toxin abolishes the anti-apoptotic effect of guanosine
We reported recently that specific binding sites exist for guanosine in rat brain membranes [35], in cultured rat astrocytes [36] and in SH-SY5Y neuroblastoma cells (Traversa and Caciagli, 2005, personal communications). We have further shown that guanosine binding in these cells is sensitive to treatment with PTX [37, 40], indicating that the putative guanosine receptor is coupled to Gi protein. We therefore tested whether the anti-apoptotic effect of guanosine in the MPP+-treated cells is mediated via this putative guanosine receptor. Cells were exposed to PTX (200 ng/ml for 16 h) prior to the various treatments with guanosine and MPP+ (pre-treatment, co-treatment or post-treatment at 24 h) and DNA fragmentation was determined at 48 h. As in previous experiments, MPP+ induced a significant increase in DNA fragmentation and the addition of guanosine alone had no significant effect on this process (Fig. 6). Exposure of neuroblastoma cells to PTX alone increased DNA fragmentation slightly under basal conditions, but this was not statistically significant. Pre-treatment, co-treatment or post-treatment at 24 h of cells with guanosine and MPP+ significantly reduced DNA fragmentation compared to cells treated with MPP+ only (p < 0.01, for all three treatments; Fig. 6). Addition of PTX to neuroblastoma cells prior to any of the three guanosine plus MPP+ treatments reversed the anti-apoptotic effect of guanosine (Fig. 6). These results demonstrate that regardless of the time of its addition to MPP+-treated cells, the protective effect of guanosine is abolished by PTX. These findings are consistent with the interpretation that these effects are mediated by a Gi protein-coupled receptor, most likely the putative guanosine receptor.
Fig. 6Pertussis toxin abolishes the anti-apoptotic effect of guanosine in SH-SY5Y cells. SH-SY5Y cells were exposed to MPP+ (500 μM) for 48 h to induce DNA fragmentation. Some cells were treated with guanosine (100 μM) either for 1 h prior to MPP+ (pre-treatment), or at the same time as MPP+ (co-treatment) or 24 h after MPP+ (post-treatment, see Fig. 1). Pertussis toxin (PTX, 200 ng/ml for 16 h) was added to some cultures prior to pre-treatment or co-treatment or post-treatment with guanosine at 24 h. DNA fragmentation was determined at 48 h and is expressed as a percentage of the positive control (n > 3). MPP+ increased DNA fragmentation significantly at 48 h (*p < 0.001) compared to that of untreated control cells at the same time. Pre-treatment, or co-treatment or post-treatment of cells with guanosine at 24 h reduced significantly MPP+-induced DNA fragmentation compared to that in cells treated with MPP+ alone at the same time (§p < 0.01). PTX reversed the protective effect of guanosine. DNA fragmentation in cells exposed to PTX plus pre-treatment, or co-treatment or post-treatment with guanosine was significantly higher compared to that of cells without PTX (#p < 0.05, for all three treatments). These values were comparable to that obtained in cells treated with MPP+ alone
Guanosine protects against MPP+-induced apoptosis by activating the cell survival pathways
We have shown earlier that guanosine activates the PI-3-K/Akt/PKB and MAPK pathways in neuroblastoma cells [34]. We therefore evaluated if the protective effects of guanosine against MPP+-induced DNA fragmentation were also mediated via these signaling pathways. Cells were exposed to either LY294002, or to PD98059, selective inhibitors of PI-3-K or MEK, respectively, for 20 min prior to various treatments with guanosine and MPP+ (pre-treatment, co-treatment or post-treatment at 24 h). Exposure of neuroblastoma cells to LY294002 alone had no significant effect on DNA fragmentation under basal conditions. Although in cells exposed to PD98059 alone DNA fragmentation increased slightly, this was not statistically significant (Table 1). As in previous experiments, the addition of MPP+ induced significant DNA fragmentation and treatment of cells with guanosine under various conditions significantly reduced DNA fragmentation compared to cells treated with guanosine (Table 1).
Exposure of cells to LY294002 prior to pre-treatment, or co-treatment or post-treatment at 24 h with guanosine and MPP+ reversed the protective effect of guanosine. DNA fragmentation in cells exposed to LY294002 plus guanosine plus MPP+ was significantly reduced compared to cells treated with guanosine plus MPP+ without the inhibitor (p < 0.05, for pre-treatment and co-treatment; p < 0.01, for post-treatment at 24 h; Table 1). Addition of PD98059 to cells before pre-treatment with guanosine and MPP+ also abolished the protective effect of guanosine (p < 0.05; Table 1). But this inhibitor had no significant effect on MPP+-induced DNA fragmentation in cells co-treated or post-treated at 24 with guanosine and MPP+ (Table 1). These results demonstrate that the protective effects of guanosine are abolished by LY294002 in MPP+-treated cells regardless of the time of its administration and are consistent with a role of the PI-3-K/Akt/PKB cell survival pathway in guanosine-mediated reduction of DNA fragmentation. In contrast, the effect of PD98059 is limited to pre-treatment of cells with guanosine prior to MPP+, thus indicating that the MAPK pathway contributes to the protective effects of guanosine only when it is added before MPP+.
Discussion
We have shown earlier that pre-treatment with guanosine protects different cell types against apoptosis [33, 34]. Here, we report that guanosine not only protects human SH-SY5Y neuroblastoma cells against MPP+-induced apoptosis, but more importantly, it can reverse MPP+-induced cytotoxicity after the pro-apoptotic pathways have already been activated. Thus, these findings are novel and demonstrate for the first time that guanosine can in fact ‘rescue’ neuroblastoma cells from apoptosis when it is added to cells under experimental conditions that are ‘clinically relevant’ to the treatment of PD.
Undifferentiated dopaminergic human SH-SY5Y neuroblastoma cells have been used widely to study the molecular pathways that mediate MPP+-induced apoptosis [23–29]. MPTP, after conversion to MPP+, activates the same intracellular pathways in vivo and in vitro as in idiopathic PD [2]. Thus, MPP+ is taken up by dopaminergic neurons, where it inhibits complex I of the mitochondrial electron transport chain. This leads to enhanced production of reactive oxygen species (ROS) and decreased synthesis of ATP [2]. Prolonged exposure to MPP+ results in the up-regulation of the pro-apoptotic protein Bax, and following its translocation to the mitochondria it promotes the release of mitochondrial cytochrome c. This may be initiated by the DNA damage caused by MPP+. Cytochrome c, together with pro-caspase-9, is incorporated into the complex, the apoptosome, which activates caspase-9, and this enzyme in turn activates caspase-3 [2, 41]. At the same time, the anti-apoptotic protein, Bcl-2, is down-regulated [42]. Taken together these data demonstrate that exposure of dopaminergic cells to MPP+ leads to cytotoxicity and cell death by apoptosis [2, 17]. Our findings are consistent with previous reports that MPP+ induces DNA fragmentation and activation of caspase-3 in SH-SY5Y cells [23, 25]. Interestingly, we detected no significant change in α-synuclein protein expression in MPP+-treated cells (data not shown). This may be the result of the relatively low MPP+ concentration (500 μM) and short duration of our experiments (48 h). Others reported that high concentrations of MPP+ (5 mM for 12 h [29]) or prolonged exposure of neuroblastoma cells to lower concentrations of MPP+ (5 μM for 4 days [28] or 1 mM for 3 days [27]) actually increased α-synuclein protein concentration. Mutations of this protein have been associated with a familial form of PD [43] and the wild-type protein is a major constituent of Lewy bodies [44]. In MPTP-treated mice α-synuclein expression is up-regulated [45], and in SH-SY5Y cells accumulation of this protein contributes to dopamine-dependent apoptosis [30]. Under our experimental conditions that were optimized to detect DNA fragmentation, we observed no change in α-synuclein protein expression.
The molecular mechanisms by which guanosine exerts its protective effects are complex. Results from the present study show that the effects of both pre-treatment and co-treatment with guanosine are pertussis toxin sensitive, and these may depend on the putative plasma membrane localized guanosine receptor [35–37] that is coupled to Gi proteins. There may be other possible explanations, which account for the effects of pertussis toxin on MPP+-induced DNA fragmentation. Since pertussis toxin enters cells and ADP-ribosylates the α-subunit of Gi proteins [46, 47], it may also attenuate signaling by other Gi protein-coupled receptors, resulting in the elevation of intracellular cAMP [48, 49] and this in turn may activate cell survival pathways, such as the MAPK [56]. Guanosine may also enter neuroblastoma cells via the equilibrative or the concentrative nucleoside transporters [50], cause an alteration in intracellular concentrations of guanine nucleotides [40] and this in turn may affect G protein activity.
However, as we have shown before, guanosine binding to its cognate receptor leads to rapid activation of the cell survival pathways, PI-3-K/Akt/PKB and MAPK [33, 34], and these play a critical role in protecting neurons against cell death [51–56]. Although these signaling pathways are most often triggered by growth factor binding to their cognate receptors, G protein-coupled receptors have also been shown to activate them [57–60]. Thus, binding of guanosine to its Gi protein-coupled putative receptor may activate the class IB PI-3-K (PI-3-Kγ), via the dissociated β,γ-subunits of Gi proteins, which promote the synthesis of phosphatidylinositol (3,4,5) trisphosphate at the plasma membrane and recruit the protein kinase, Akt/PKB [60, 61]. Activation of Akt/PKB by phosphorylation at Thr 308 and Ser 473 regulates cell survival and apoptosis by both transcription-dependent and transcription-independent pathways [7, 54, 61]. Thus, Akt/PKB may suppress apoptosis by phosphorylating and inactivating the pro-apoptotic protein Bad [62], inhibiting the release of mitochondrial cytochrome c [63] and inhibiting the activation of caspase-9 and caspase-3 [64]. Expression of cell survival genes may also be triggered by the PI-3-K/Akt/PKB pathway, via activation of the transcription factors CREB and NF-κB, and inhibition of Forkhead transcription factors of the FOXO family [54].
Similarly, activation of the MAPK pathway, specifically the extracellular signal-regulated kinases 1 and 2 (ERK1/2), also prevent apoptosis and promote cell survival by both transcription-dependent and transcription-independent pathways [55, 56, 65, 66]. The specific downstream targets of the ERK1/2 pathway include inhibition of caspase-9 by phosphorylation [67], suppression of the pro-apoptotic protein Bad by phosphorylation and activation of the transcription factor CREB [55]. Guanosine binding to its putative receptor may also stimulate this pathway via the dissociated β,γ-subunits of Gi proteins [59]. Reports suggest that the PI-3-K/Akt/PKB and MAPK pathways do not function independently, but there is significant cross-talk between them [57, 68]. It has been suggested that whereas the PI-3-K/Akt/PKB pathway is the main signaling pathway for maintaining trophic support by pro-survival factors, ERK1/2 mediates protection against damage-induced cell death [56]. Our results are thus consistent with the interpretation that protection of cells from MPP+-induced DNA fragmentation by guanosine pre-treatment is dependent on the activation of both these pathways. Following co-treatment with guanosine and MPP+, however, it is the PI-3-K/Akt/PKB pathway, which plays the essential role.
In the present study, we also examined the effect of guanosine on MPP+-induced DNA fragmentation by exposing cells to guanosine after caspase-3 activity was elevated and DNA fragmentation was already underway (post-treatment). This strategy is novel and is based on a recent proposal by Meissner et al. [38], who advocate that potential neuroprotective agents for the treatment of PD should be tested under conditions that are relevant to the clinical situation. Because clinical symptoms of PD are apparent only after the majority of nigrostriatal neurons have died, patients do not present for treatment until after the process of cell death is well established [38]. Most in vitro studies have been conducted by adding potential neuroprotective agents to cells prior to MPP+-triggered apoptosis [69–71] or at the same time as MPP+ [27, 72, 73], making these results irrelevant to the treatment of PD, where the process of neurodegeneration has already begun.
Addition of guanosine to cells after MPP+-induced DNA fragmentation is already in progress may involve additional protective mechanisms. As DNA fragmentation is a late event in apoptosis [11, 12], some cells with damaged DNA may actually undergo DNA repair and survive [74, 75]. The preferred survival strategy of a cell is to attempt to restore any DNA damage by using a complex set of DNA repair pathways [74]. Only when the damage to the DNA becomes excessive and the repair capacity of the cell is overwhelmed will it undergo apoptosis. Therefore, adding guanosine 24 or 48 h after DNA fragmentation has been initiated may activate some of the DNA repair mechanisms, antagonizing and reversing the MPP+-induced DNA fragmentation. The protective effects of guanosine in these cells thus may include activation of both cell survival and DNA repair pathways.
In contrast, post-treatment with guanosine had no significant effect on MPP+-induced activation of caspase-3. As activation of this enzyme requires a cleavage of the interdomain region of the inactive dimer of caspase-3, once this process is initiated it cannot be reversed [8–10]. So addition of guanosine to cells after proteolytic cleavage of this enzyme will not alter caspase-3 activity.
Recently administration of MPTP to mice has been reported to promote caspase-independent cell death of dopaminergic neurons [76]. This “apoptosis-like” programmed cell death [77] is mediated by the release of the apoptosis-inducing factor (AIF) from the mitochondria and its subsequent translocation to the nucleus, where it induces DNA fragmentation [78–80]. If this process plays a significant role in MPP+-induced cell death of neuroblastoma cells, DNA fragmentation will still be reversed by the protective action of guanosine, but caspase-3 activation will not be affected.
Several authors suggested recently that targeting programmed cell death in neurodegenerative diseases may lead to successful therapy [5, 31]. Drugs that will be effective in PD should protect the dopaminergic cells from apoptotic insult by either interrupting the signaling pathways that mediate cell death or activating the cell survival pathways. Our present results show that the non-adenine-based purine nucleoside guanosine, when added to MPP+-treated neuroblastoma cells, promotes their survival by inhibiting caspase-3 activation and DNA fragmentation via activation of the PI-3-K/Akt/PKB pathway. Furthermore, guanosine exerts these effects not only when it is pre- or co-administered with MPP+, but even when it is added to cells after caspase-3 is activated and DNA fragmentation is already in progress. Thus, these findings reveal a unique neuroprotective effect of guanosine with a potential for effective pharmacological intervention in PD.

cytotoxicity
guanosine
apoptosis
parkinson’s disease
dna fragmentation
caspase-3
cell survival
sh-sy5y human neuroblastoma cells
1-methyl–4-phenyl-pyridinium (mpp+)