Impaired glutathione synthesis in schizophrenia: convergent genetic and functional evidence

Abstract

Schizophrenia is a complex multifactorial brain disorder with a genetic component. Convergent evidence has implicated oxidative stress and glutathione (GSH) deficits in the pathogenesis of this disease. The aim of the present study was to test whether schizophrenia is associated with a deficit of GSH synthesis. Cultured skin fibroblasts from schizophrenia patients and control subjects were challenged with oxidative stress, and parameters of the rate-limiting enzyme for the GSH synthesis, the glutamate cysteine ligase (GCL), were measured. Stressed cells of patients had a 26% (P = 0.002) decreased GCL activity as compared with controls. This reduction correlated with a 29% (P < 0.001) decreased protein expression of the catalytic GCL subunit (GCLC). Genetic analysis of a trinucleotide repeat (TNR) polymorphism in the GCLC gene showed a significant association with schizophrenia in two independent case-control studies. The most common TNR genotype 7/7 was more frequent in controls [odds ratio (OR) = 0.6, P = 0.003], whereas the rarest TNR genotype 8/8 was three times more frequent in patients (OR = 3.0, P = 0.007). Moreover, subjects with disease-associated genotypes had lower GCLC protein expression (P = 0.017), GCL activity (P = 0.037), and GSH contents (P = 0.004) than subjects with genotypes that were more frequent in controls. Taken together, the study provides genetic and functional evidence that an impaired capacity to synthesize GSH under conditions of oxidative stress is a vulnerability factor for schizophrenia.

Fig. 1.

GCL activity and GCLC protein expression in patients and controls under untreated and t-BHQ-treated conditions. (A) GCL activity of 25 patients and 25 controls, expressed as nanomoles of GSH per minute per milligram of protein. Each box describes 25%, 75%, and median values. The whisker bars show values in the 1.5 box lengths range. The open circle depicts an outlier value. (B) The plot shows GCLC protein levels of 26 patients and 26 controls, determined as α-tubulin corrected arbitrary units. **, P < 0.01; ***, P < 0.001 vs. the respective controls were calculated by using ANOVA test (two-tailed). (C) Representative Western blots of GCLC, GCLM, and α-tubulin of two controls and two patients under untreated and t-BHQ-treated conditions.

Fig. 2.

Different regulation of GSH synthesis in patients and controls. The plots show GCLM and GCLC protein expression under untreated (gray diamonds) and t-BHQ-treated (black triangles) conditions of 26 controls (A) and 26 patients (B). Note that the correlation between GCLM and GCLC is lost in patients. GCLC protein expressions compared with GCL activities under untreated (gray diamonds) and t-BHQ-treated (black triangles) conditions of 24 controls (C) and 24 patients (D). Note that, in patients under oxidative stress, the GCL activity depends on GCLC protein levels. Shown are ratios of GCLM vs. GCLC protein amounts under untreated conditions (gray squares) and after t-BHQ treatment (black triangles) compared with GCL activities of 24 controls (E) and 24 patients (F). Note that, in patients, GCL activity is inversely correlated with the GCLM/GCLC ratio. R is the Spearman correlation; P is the probability of type I error.

Fig. 3.

Functional relevance of the GCLC GAG TNR polymorphism in the Swiss sample. Because genotypes 7/7 and 7/9 were present more often in controls, whereas genotypes 7/8, 8/8, 8/9, and 9/9 were present more often in patients, data of GCL activity, GCLC protein expression, and GSH content were regrouped according their genotypes (7/7 and 7/9 vs. 7/8, 8/8, 8/9, and 9/9). The plots show GCL activity (t-BHQ) (A) and GCLC protein expression (t-BHQ) (B) of 38 subjects with genotypes 7/7 and 7/9 and 11 subjects with genotypes 7/8, 8/8, 8/9, and 9/9. (C) The plot shows GSH content (baseline) of 56 subjects with genotypes 7/7 and 7/9 and 16 subjects with genotypes 7/8, 8/8, 8/9, and 9/9. Each box describes 25%, 75%, and median values. The whisker bars show values in the 1.5 box lengths range. *, P < 0.05; **, P < 0.01 vs. the respective controls was calculated by using Mann–Whitney U test (two-tailed).