Expression of GABA signaling molecules KCC2, NKCC1, and GAD1 in cortical development and schizophrenia

Abstract

GABA signaling molecules are critical for both human brain development and the pathophysiology of schizophrenia. We examined the expression of transcripts derived from three genes related to GABA signaling [GAD1 (GAD67 and GAD25), SLC12A2 (NKCC1), and SLC12A5 (KCC2)] in the prefrontal cortex (PFC) and hippocampal formation of a large cohort of nonpsychiatric control human brains (n = 240) across the lifespan (from fetal week 14 to 80 years) and in patients with schizophrenia (n = 30-31), using quantitative RT-PCR. We also examined whether a schizophrenia risk-associated promoter SNP in GAD1 (rs3749034) is related to expression of these transcripts. Our studies revealed that development and maturation of both the PFC and hippocampal formation are characterized by progressive switches in expression from GAD25 to GAD67 and from NKCC1 to KCC2. Previous studies have demonstrated that the former leads to GABA synthesis, and the latter leads to switching from excitatory to inhibitory neurotransmission. In the hippocampal formation, GAD25/GAD67 and NKCC1/KCC2 ratios are increased in patients with schizophrenia, reflecting a potentially immature GABA physiology. Remarkably, GAD25/GAD67 and NKCC1/KCC2 expression ratios are associated with rs3749034 genotype, with risk alleles again predicting a relatively less mature pattern. These findings suggest that abnormalities in GABA signaling critical to brain development contribute to genetic risk for schizophrenia.

Figure 1.

Fetal expression patterns of KCC2 and NKCC1 in prefrontal cortex (qRT-PCR). A, KCC2 expression (y-axis) shows a positive correlation with fetal gestational age expressed in weeks during the second trimester (x-axis). Each small dot represents an expression value for a single brain. B, NKCC1 expression also increases with fetal gestational age during the second trimester, with and without inclusion of the 39 week fetal sample. C, The ratio of NKCC1/KCC2 mRNA transcript levels decreases with increasing fetal gestational age. A decreasing ratio indicates that the rate of increase of the “mature” cotransporter, KCC2, is greater than that of the “immature” cotransporter, NKCC1.

Figure 2.

Cotransporter expression patterns in DLPFC and hippocampal formation across the lifespan (qRT-PCR). A, KCC2 expression in the DLPFC increases throughout the lifespan from birth to approximately 20 years of age and then levels off. B, In the DLPFC, NKCC1 also increases after birth and levels off at approximately 20 years of age. C, The DLPFC NKCC1/KCC2 ratio steadily declines during fetal development, reaching a plateau around 3 years of age that is maintained throughout the lifespan. D, KCC2 expression in the hippocampal formation increases from birth to approximately 20 years of age and then levels off, before declining around 60 years of age. E, NKCC1 also increases after birth in the hippocampal formation and levels off around 23 years of age. F, The hippocampal NKCC1/KCC2 ratio steadily declines during fetal development, reaching a plateau around 14 years of age that is maintained until approximately 53 years of age, before starting to rise.

Figure 3.

Expression patterns of GAD67, GAD25, and the GAD25/GAD67 ratio in prefrontal cortex and hippocampal formation across the lifespan (qRT-PCR). A, In the DLPFC, GAD67 expression increases from 14 weeks of gestational age until approximately 10 years of age and then levels off throughout the rest of the lifespan. B, DLPFC GAD25 expression is highest around 14 weeks of gestational age and steadily declines thereafter, reaching a stable level of expression at approximately 10 years of age. C, The DLPFC GAD25/GAD67 ratio rapidly declines during fetal development through the first decade of life after birth and then is stable across the lifespan. D, In marked contrast to the DLPFC expression pattern, in the hippocampus, GAD67 expression is relatively stable from 14 weeks of gestational age across the lifespan. E, As in the DLPFC, hippocampal GAD25 expression is highest around 14 weeks of gestational age and steadily declines thereafter, reaching a stable level of expression around 10 years of age that is maintained for the rest of the lifespan. F, The hippocampus pattern of the GAD25/GAD67 expression ratio is similar to that of the DLPFC, rapidly falling until 15 years of age and then is stable in adulthood.

Figure 4.

KCC2 expression and the NKCC1/KCC2 expression ratio in the hippocampal formation in schizophrenia. A, KCC2 mRNA expression is significantly lower in the hippocampus of adult schizophrenic subjects compared with normal controls. *p = 0.04. B, The NKCC1/KCC2 ratio shows trend towards an increase in the hippocampus of adult schizophrenic subjects compared with normal controls. ^†p = 0.08. Error bars indicate SD.

Figure 5.

GAD1 genotypic variation and KCC2 and the NKCC1/KCC2 ratio. A, KCC2 mRNA expression levels are significantly and selectively lower in the hippocampus of schizophrenic subjects who are homozygous (1/1 = G/G) for the major allele of the GAD1 schizophrenia-associated risk SNP rs3749034. *p < 0.004. There was no association between homozygosity for this SNP and KCC2 expression levels in normal controls in hippocampus or in normal controls or schizophrenic subjects in the DLPFC. B, The NKCC1/KCC2 ratio also was significantly higher in the hippocampus of G/G schizophrenic subjects. *p = 0.05. There was no association between the 1/1 (G/G) genotype for rs3749034 and the cotransporter ratio in normal controls in the hippocampus or in normal controls or schizophrenic subjects in the DLPFC. Error bars indicate SD.

Figure 6.

GAD1 genotypic variation and GAD25 expression and the GAD25/GAD67 expression ratio. A, In a combined cohort of schizophrenic and control subjects, those with a 1/1 (G/G) genotype for the schizophrenia-associated GAD1 risk allele rs3749034 had significantly higher expression of GAD25 compared with carriers of the 2 (A) allele in the hippocampus. *p = 0.03. B, In a cohort of schizophrenic subjects, those with a G/G genotype for the schizophrenia-associated GAD1 risk allele rs3749034 had a significantly higher GAD25/GAD67 expression ratio compared with carriers of the A allele in the hippocampus *p = 0.04. Error bars indicate SD.