Lower expression of glutamic acid decarboxylase 67 in the prefrontal cortex in schizophrenia: contribution of altered regulation by Zif268

Abstract

Objective: Cognitive deficits of schizophrenia may be due at least in part to lower expression of the 67-kDa isoform of glutamic acid decarboxylase (GAD67), a key enzyme for GABA synthesis, in the dorsolateral prefrontal cortex of individuals with schizophrenia. However, little is known about the molecular regulation of lower cortical GAD67 levels in schizophrenia. The GAD67 promoter region contains a conserved Zif268 binding site, and Zif268 activation is accompanied by increased GAD67 expression. Thus, altered expression of the immediate early gene Zif268 may contribute to lower levels of GAD67 mRNA in the dorsolateral prefrontal cortex in schizophrenia.

Method: The authors used polymerase chain reaction to quantify GAD67 and Zif268 mRNA levels in dorsolateral prefrontal cortex area 9 from 62 matched pairs of schizophrenia and healthy comparison subjects, and in situ hybridization to assess Zif268 expression at laminar and cellular levels of resolution. The effects of potentially confounding variables were assessed in human subjects, and the effects of antipsychotic treatments were tested in antipsychotic-exposed monkeys. The specificity of the Zif268 findings was assessed by quantifying mRNA levels for other immediate early genes.

Results: GAD67 and Zif268 mRNA levels were significantly lower and were positively correlated in the schizophrenia subjects. Both Zif268 mRNA-positive neuron density and Zif268 mRNA levels per neuron were significantly lower in the schizophrenia subjects. These findings were robust to the effects of the confounding variables examined and differed from other immediate early genes.

Conclusions: Deficient Zif268 mRNA expression may contribute to lower cortical GAD67 levels in schizophrenia, suggesting a potential mechanistic basis for altered cortical GABA synthesis and impaired cognition in schizophrenia.

FIGURE 1. qPCR Determination for Zif268 mRNA in Schizophrenia and Comparison Subjects and Effect of Potential Confounding Variables on Zif268 mRNA Expression Levels^a

^aqPCR=quantitative polymerase chain reaction. In panel A, the scatterplots indicate Zif268 mRNA level for each comparison and schizophrenia subject in a pair. Data points below the diagonal unity line indicate lower levels for Zif268 mRNA in the schizophrenia subject relative to the comparison subject. Mean Zif268 mRNA level was significantly lower in the schizophrenia subjects relative to the comparison subjects (mean=0.041 [SD=0.023] and mean=0.059 [SD=0.020], respectively; −32%; F=21.5, df=1, 53, p<0.001). In panel B, Zif268 mRNA levels in dorsolateral prefrontal cortex were significantly negatively correlated with age in both subject groups (schizophrenia subjects: N=58; r=−0.38, p=0.003; comparison subjects: N=61; r=−0.39, p=0.002). In panel C, in which schizophrenia subjects are grouped by potential confounding factors, the circles represent Zif268 mRNA expression levels for individual subjects and the bars represent mean Zif268 mRNA levels for the indicated group. Schizophrenia subjects taking benzodiazepines or sodium valproate at the time of death had lower Zif268 mRNA levels relative to those not taking these medications (F=7.64, df=1, 50, p=0.008). ^bInformation on nicotine use was not available for all subjects. ^cCause of death was undetermined for one schizophrenia subject.

FIGURE 2. In Situ Hybridization Film Analysis and Cellular Grain Counting Analysis for Zif268 mRNA in Schizophrenia Subjects Relative to Matched Comparison Subjects^a

^aPanel A presents representative pseudocolored film autoradiographs of dorsolateral prefrontal cortex sections illustrating Zif268 mRNA expression levels in a schizophrenia and matched comparison subject pair. The solid and dotted lines indicate the pial surface and the gray/white matter border, respectively. In panel B, the scatterplot indicates average Zif268 mRNA levels across the gray matter for each comparison and schizophrenia subject pair. Data points below the diagonal unity line indicate lower Zif268 mRNA levels in the schizophrenia subject relative to the matched comparison subject. Mean Zif268 mRNA levels were significantly lower in schizophrenia subjects relative to comparison subjects (mean=38.0 [SD=22.1] and mean=57.6 [SD=15.4], respectively; −34.0%; F=15.4, df=1, 34, p<0.001). Panels C and D show laminar expression of mean Zif268 mRNA levels across all cortical layers (panel C) and in each cortical layer (panel D) in schizophrenia and comparison subjects (error bars indicate standard deviation). Laminar analysis revealed that mean Zif268 mRNA levels were lower by 36.8% in layer 2, 37.7% in layer 3, 40.6% in layer 4, 42.8% in layer 5, and 38.7% in layer 6 in schizophrenia subjects relative to comparison subjects. Panels E and F present scatterplots of Zif268-positive neurons per mm² and grains per positive neuron, respectively, in layers deep 3–4 for each comparison and schizophrenia subject pair. Data points below the diagonal unity lines indicate lower Zif268 mRNA levels in the schizophrenia subject relative to the comparison subject. Cellular analysis revealed that the mean number of Zif268-positive neurons/mm² was lower in layers deep 3–4 in schizophrenia subjects relative to comparison subjects (mean=53.8 [SD=18.3] and mean=112.7 [SD=24.4], respectively; −52.3%; F=266.3, df=1, 15, p<0.001). The mean grain density per Zif268-positive neuron was also lower in layers deep 3–4 in schizophrenia subjects relative to comparison subjects (mean=43.8 [SD=10.8] and mean=68.1 [SD=10.5], respectively; −35.6%; F=71.4, df=1, 15, p<0.001). *p<0.001.

FIGURE 3. qPCR Determination for GAD67 mRNA Levels and Correlation With Zif268 mRNA Levels in Schizophrenia Subjects and Matched Comparison Subjects^a

^aqPCR=quantitative polymerase chain reaction. In panel A, the scatterplot indicates GAD67 mRNA level for each schizophrenia and matched comparison subject pair. Data points below the diagonal unity line indicate lower levels for GAD67 mRNA in the schizophrenia subject relative to the matched comparison subject. Mean mRNA levels for GAD67 were significantly lower in schizophrenia subjects relative to comparison subjects (mean=0.044 [SD=0.011] and mean=0.052 [SD=0.008], respectively; −14.3%; F=12.6, df=1, 57, p=0.001). In panel B, Zif268 mRNA levels were positively correlated with GAD67 mRNA levels in schizophrenia subjects (N=58; r=0.29, p=0.027), but not in comparison subjects (N=61; r=−0.15, p=0.25).

FIGURE 4. qPCR Analyses of Other Immediate Early Genes in Schizophrenia Subjects and Matched Comparison Subjects^a

^aqPCR=quantitative polymerase chain reaction. Scatterplots indicate mRNA levels of immediate early genes for each schizophrenia and matched comparison subject pair. Data points below the diagonal unity lines indicate lower mRNA levels in the schizophrenia subject relative to the comparison subject and vice versa. Mean mRNA level for c-fos did not differ between schizophrenia (mean=0.018, SD=0.026) and comparison subjects (mean=0.017, SD=0.012). In contrast, mean mRNA levels for c-jun were statistically higher in schizophrenia subjects relative to comparison subjects (mean=0.031 [SD=0.018] and mean=0.023 [SD=0.007], respectively; +34.2%; F=10.9, df=1, 56, p=0.002), while mean mRNA level for EGR-2 was lower, but fell short of significance, in schizophrenia subjects relative to comparison subjects (mean=0.0042 [SD=0.0038] and mean=0.0056 [SD=0.0024], respectively; −23.8%; F=3.49, df=1, 55, p=0.067).