Schizophrenia (SZ) is a severe psychiatric disorder and the median incidence of schizophrenia was 15.2 per 100,000 persons.1 Due to its chronic relapsing course, SZ poses considerable burden on caregivers and society.2 Disturbances of the dopaminergic system have been considered as one of the hypotheses for schizophrenia pathogenesis.3 Elevated presynaptic striatal dopaminergic function is most highly correlated with positive symptoms (psychosis), while low prefrontal cortex dopaminergic function is associated with negative symptoms (blunted affect, anhedonia, and avolition).3 Another hypothesis proposes that schizophrenia is a neurodevelopmental disorder involving disturbances in neuronal migration, connections, and neuroplasticity.4,5
Nerve growth factor (NGF) is a neurotrophic factor and neuropeptide essential for the regulation of differentiation, maintenance, and survival of innervating neurons.6 Previous studies have indicated that lower serum levels of NGF such as brain-derived neurotrophic factor (BDNF),6–11 glial cell-derived neurotrophic factor (GDNF),8,9,12 and beta nerve growth factor (β-NGF)13–15 were found in patients with SZ. However, other studies showed no significant differences between patients with SZ and healthy controls in GDNF,16 BDNF13,17,18 and NGF-beta.18–20 Nevertheless, two recent meta-analysis studies discussed these inconsistent findings and concluded that serum levels of BDNF and NGF were moderately lower in patients with SZ than in controls.21,22 These studies support the possible role of neurotrophic factors in the development of SZ.
Serum cytokine alteration correlates with the severity of psychopathology in schizophrenia. Some studies have reported that BDNF serum levels are positively correlated with positive symptoms, as measured with the Positive and Negative Syndrome Scale (PANSS),4 while others have shown negative correlations.7,23 So far, few studies have investigated the association between GDNF and psychopathology.8,16 One study16 reported that higher GDNF serum levels were associated with greater severity of attention deficits on the scale for the assessment of negative symptoms (SANS) in SZ but another more recent study reported contrary results.8 The contradictory results of these studies may be further explained by characteristics of disease severity and length of untreated psychosis, and diverse usage of antipsychotic agents among studies.8,16 Therefore, the association between GDNF and psychopathology remains unknown.
No previous study has simultaneously examined the association between serum BDNF, GDNF and NGF-beta levels and psychopathology in patients with SZ. Moreover, psychotropic drugs may alter BDNF, GDNF, and NGF-beta serum levels.12,16 To further contribute to this issue, the aim of this study was to examine the association between serum BDNF, GDNF, and NGF-beta levels and psychopathology in unmedicated patients with SZ.
Thirty patients from a medical center located in Southern Taiwan admitted from August 2014 to August 2015 were enrolled in the present study. All patients met the diagnostic criteria for schizophrenia according to the Diagnostic and Statistical Manual of Mental Disorder, 5th edition (DSM-5).24 Patients were unmedicated for a minimum of three months before enrollment. We excluded those (1) with mental retardation, neurocognitive disorders, major depressive disorder, bipolar I or II disorder, and substance dependence; (2) who could not provide informed consent. This study was approved by the Ethics Committee of Kaohsiung Veterans General Hospital (VGHKS13-CT5–08).
The Brief Psychiatric Rating Scale (BPRS) is one of the most frequently used screening tools allowing clinicians to quickly assess psychopathology in various psychiatric disorders. The original BPRS consists of 16-items and was developed in 1962,25 it was later extended to 18 items.26 More recently, the expanded version of 24-items BPRS (BPRS-E) was developed to increase the instrument's sensitivity to psychotic and affective disorders.27 The BPRS-E was administered to 30 patients by a psychiatrist (CLL).27 Four conceptual domains (manic excitement/disorganization, depression/anxiety, negative symptoms, and positive symptoms) were considered for sub-analysis in the present study.28 The presence and severity of psychiatric symptoms were rated from 1 (not present) to 7 (extremely severe). The total scores varied from 24 to 168, with higher scores denoting more severe psychopathology.
Venous blood (5 mL) was collected between 7:00 and 8:00 AM to avoid circadian fluctuation of the measured cytokines. After centrifugation at 3000 rpm for 10 min, the serum was separated from the blood and was stored at −70 °C prior to assay.
Serum levels of NGF-beta (Cat. No. DY256), GDNF (Cat. No. DY212), and BDNF (Cat. No. DBD00) were measured using a sandwich enzyme-linked-immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA), according to the protocols provided by the manufacturer. Serum samples were diluted 2-, 2-, and 10-fold with PBS for detection of NGF-beta, GDNF, and BDNF, respectively. All the experiments were performed in duplicate. The intra- and inter-assay levels of variation were less than 10%. The detection limits for NGF-beta, GDNF, and BDNF were 31.3, 31.3, and 62.5 pg/mL, respectively.
2.4. Statistical analysis
Patient demographics and clinical characteristics are presented as mean ± standard deviation (SD) for continuous variables and as percentages for categorical variables. Kolmogorov-Smirnov one sample test (K-S test) was performed to test non-parametric variables. We used the median of the BPRS total and sub-domain scores to separate patients into high and low scoring groups, as like in previous studies.29,30 Mann-Whitney U test was used to examine group differences between the high and low groups in NGF-beta, GDNF, and BDNF. In addition, Spearman's correlation was performed to examine the correlations among NGF-beta, GDNF, and BDNF and BPRS and sub-domain scores, as cytokines of interest (NGF-beta, GDNF, BDNF) were non-normally distributed (K-S test; all p < 0.05). Given that we conducted multiple correlations across items, it is vital to account for the risk of Type I errors. Therefore, we applied the Benjamini-Hochberg procedure because it has been shown to demonstrate greater power than Bonferroni correction.31,32 All statistical analyses were conducted using SPSS 19.0 and differences were considered statistically significant when p values were less than 0.05.
3.1. Basic demographic and clinical characteristics of patients
In this study, we recruited 30 patients with schizophrenia, including 17 men (56.7%) and 13 women (43.3%). Table 1 presents the data on age, gender, education, body mass index (BMI), age at illness onset, disease duration, and psychopathology (total and sub-domains). The mean age, age at illness onset, and disease duration of recruited patients were 47.3 ± 8.7, 25.9 ± 8.9, and 21.5 ± 9.6 years old, respectively. In addition, the mean total score of BPRS was 66.0 ± 11.2.
3.2. Differences between the high BPRS total and sub-domains scoring group and low scoring group in serum NGF-beta, BDNF, and GDNF
Table 2 presents the significantly higher serum levels of BDNF that were detected in the high than in the low BPRS positive symptoms group (corrected p = 0.013). Besides, significantly higher serum levels of GDNF that were detected in the low than in the high BPRS manic symptoms and total symptoms group (both raw p = 0.041), but the significance disappeared after the Benjamini-Hochberg procedure (both corrected p = 0.082).
3.3. Serum NGF-beta, GDNF, BDNF, and confounders
Serum NGF-beta, GDNF, and BDNF did not significantly correlate with age, sex, education, BMI, age at illness onset, and disease duration (all p > 0.05). In addition, BPRS total and subsets scores did not correlate with age, sex, education, BMI, age at illness onset, and disease duration (all p > 0.05). Moreover, Table 3 presents the Spearman's correlations to examine the association among the cytokines of interest and the BPRS subset and total scores. A significant correlation was found between the BPRS total scores and, GDNF (r = −0.533, corrected p = 0.002), and BDNF (r = 0.480, corrected p = 0.007). The correlation between the BPRS total scores and NGF-beta was no longer significant after multiple correction (raw p = 0.033, corrected p = 0.165).
In addition, significant correlation was found between the BPRS manic score and GDNF (r = −0.456, corrected p = 0.033). After correction for multiple testing, several correlations did not reach significance with regard to the association between the BPRS manic and anxiety score and BDNF (raw p = 0.032, corrected p = 0.128 for manic; raw p = 0.048, corrected p = 0.336 for anxiety) and the association between the BPRS positive score and GDNF (raw p = 0.037, corrected p = 0.222). No correlations were found between BPRS negative symptom subscales and the cytokines of interest.
To the best of our knowledge, this is the first study to explore the association between serum levels of BDNF, GDNF, and NGF-beta and the psychopathology of unmedicated patients with schizophrenia. We found that: (1) patients with higher BPRS-positive subtest scores had significant higher serum levels of BDNF compared with those with lower BPRS-positive subtest scores; (2) a negative correlation between GDNF levels and BPRS-total and BPRS-manic subtest scores; (3) BDNF serum levels showed a positive correlation with BPRS-total, subscale scores.
To our knowledge, the present study is the first to show a significant negative correlation between GDNF levels and psychopathology, as measured by BPRS-total and BPRS-manic subtest scores. Lower GDNF serum levels are related to more severe psychiatric symptoms in patients with schizophrenia. These findings are consistent with most previous studies that focused on cognition and showed that lower GDNF serum levels are related to more profound attention impairment and verbal fluency deficits.8 In addition, the usage of antipsychotic agents might explain the inconsistent results between these studies and the one by Niitsu et al.,16 which reported higher GDNF serum levels linking to poor attention ability. Similarly, Xiao et al.12 enrolled first-episode drug-naïve patients with schizophrenia, our patients were unmedicated for a minimum of 3 months prior to enrollment, and therefore, the effect induced by antipsychotic treatment was not considered.
Another finding of this study was that the BDNF serum levels showed a significant positive correlation with BPRS-total scores and higher BDNF serum levels were present in the higher positive subscale scoring group. These findings are in line with a previous report with drug-naïve patients with first episode schizophrenia from China by Chen et al.,4 which showed a positive correlation between BDNF levels and PANS positive sub-scores. However, other reports from Eastern-Asian counties have showed that BDNF levels are negatively associated with negative symptoms.10,11 Similarly, Buckley et al.23 and Pillai A et al.7 reported contrary results in western populations, showing negative correlations between BDNF and psychopathology in first-episode never-medicated patients with schizophrenia. The association between BDNF and psychopathology is more consistent in Caucasian7,23 but not in East Asian population.4,10,11 The heterogeneity of schizophrenic symptoms, effects of antipsychotic agents, and disease duration should be considered in future studies to elucidate the exact mechanism of neurotrophic factors on the pathogenesis of schizophrenia.
In this study, we found serum levels of NGF-beta were positively associated with BPRS total scores initially, but significance disappeared after multiple corrections. Another study found that NGF-beta was negatively associated with PANSS in first episode unmedicated chronic schizophrenia, and tended to correlate with BPRS.14 In addition, several studies reported that plasma NGF-beta levels did not correlate with PANSS total or subscales scores.33,34 The exact reason for this discrepancy remains unknown. As these studies enrolled drug-naïve or unmedicated patients with schizophrenia, effects other than those of antipsychotic agents, such as differences in the clinical status of recruited patients, ethnic differences, and different scales used among studies should be considered. The contribution of these effects should be further investigated in future studies.
Although the precise mechanisms underpinning the association between neurotropic factors and psychopathology in SZ are still unknown, several possible reasons may explain the pathological mechanisms involved in the development of SZ. For example, neurotropic factors included BDNF, GDNF, and NGF play a vital role in the development, regeneration, survival, and maintenance of neuronal function.35 They could modulate neurotransmitter synthesis, metabolism and release that influence synaptic plasticity.12,36 Specific, GDNF possesses neuroprotective features in neuropsychiatric disorders via the regulation of dopaminergic and serotonergic neurons12 and it promotes neuronal survival and signals via GFR-alpha.6 Additionally, recombinant adeno-mediated GDNF combined with early rehabilitation enhanced neuroprotection and improved motor function in the spinal cords of traumatized rats.37 With regard to NGF, neuronal cell death associated with injury could be prevented following 2 weeks of NGF treatment in an animal study of rat.38 Taken together, neurotrophic factors show neuroprotective properties in neurobasic and clinical aspects.
Some limitations should be noted. First, this study had a small sample size. Only 30 patients with schizophrenia were enrolled, limiting the statistical power and generalization. Second, this study lacked a healthy control group. However, previous meta-analysis studies have confirmed that patients with schizophrenia have lower serum levels of BDNF, GDNF, and NGF-beta among than healthy controls. Third, schizophrenia as a syndrome with “genetic heterogeneity” may contribute to the inconsistencies observed among studies.39 Accounting for the subtypes of schizophrenia, it would be reasonable to examine the role of the cytokines of interest in the psychopathology. However, we did not perform this analysis due to the small sample size. Finally, smoking should be considered as a confounding factors, as it may affect serum NGF-beta, GDNF and BDNF levels. But we did not consider smoking as a confounder in the present study.
In conclusion, this study suggests that BDNF and GDNF may be playing an important role in the psychopathology of patients with schizophrenia. A larger sample controlled study would be required to further elucidate this association.
We express our gratitude to proteomics core laboratory, molecular medicine research center, Chang Gung University to data and lab analysis. We are grateful to our patients and their parents who voluntarily participated in our study and gave valuable information. This work was supported by grants VGHKS13-CT5–08 from Kaohsiung Veterans General Hospital, Taiwan.
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