Role of glutamic acid decarboxylase 67 in regulating cortical parvalbumin and GABA membrane transporter 1 expression: implications for schizophrenia

Abstract

Markers of GABA neurotransmission are altered in multiple regions of the neocortex in individuals with schizophrenia. Lower levels of glutamic acid decarboxylase 67 (GAD67) mRNA and protein, which is responsible for most cortical GABA synthesis, are accompanied by lower levels of GABA membrane transporter 1 (GAT1) mRNA. These alterations are thought to be most prominent in the parvalbumin (PV)-containing subclass of interneurons, which also contain lower levels of PV mRNA. Since GAT1 and PV each reduce the availability of GABA at postsynaptic receptors, lower levels of GAT1 and PV mRNAs have been hypothesized to represent compensatory responses to an upstream reduction in cortical GABA synthesis in schizophrenia. However, such cause-and-effect hypotheses cannot be directly tested in a human illness. Consequently, we used two mouse models with reduced GAD67 expression specifically in PV neurons (PV(GAD67+/-)) or in all interneurons (GABA(GAD67+/-)) and quantified GAD67, GAT1 and PV mRNA levels using methods identical to those employed in studies of schizophrenia. Cortical levels of PV or GAT1 mRNAs were not altered in PV(GAD67+/-) mice during postnatal development or in adulthood. Furthermore, cellular analyses confirmed the predicted reduction in GAD67 mRNA, but failed to show a deficit in PV mRNA in these animals. Levels of PV and GAT1 mRNAs were also unaltered in GABA(GAD67+/-) mice. Thus, mouse lines with cortical reductions in GAD67 mRNA that match or exceed those present in schizophrenia, and that differ in the developmental timing and cell type-specificity of the GAD67 deficit, failed to provide proof-of-concept evidence that lower PV and GAT1 expression in schizophrenia are a consequence of lower GAD67 expression. Together, these findings suggest that the correlated decrements in cortical GAD67, PV and GAT1 mRNAs in schizophrenia may be a common consequence of some other upstream factor.

Figure 1. GAD67, but not PV or GAT1, mRNA levels are reduced in PV^GAD67+/− mice

A: representative film autoradiograms illustrating the expression of GAD67, PV, and GAT1 mRNA in the frontal lobe of P50 PV^GAD67+/+ mice. The density of hybridization signal is presented in pseudocolor according to the calibration bars. The white contours denote the quantified regions in M1/M2 and PFC. Note that PV mRNA expression is much lower in PFC than in M1/M2. B: comparison of M1/M2 GAD67, PV, and GAT1 mRNA levels by film OD in P50 PV^GAD67+/+ and PV^GAD67+/− mice (GAD67: t₅=2.23, p=0.038; all others, p≥0.350). C: comparison of PFC GAD67, PV, and GAT1 mRNA levels by film OD in P50 PV^GAD67+/+ and PV^GAD67+/− mice (all transcripts, p≥0.146). Mean values for each group are indicated by hash marks.

Figure 2. GAD67, but not PV or GAT1, mRNA levels are reduced in PV^GAD67+/− mice across development

Comparison of GAD67 (A), PV (B), and GAT1 (C) mRNA levels by film OD in PV^GAD67+/+ and PV^GAD67+/− mice (GAD67 P30: t₅=6.33, p=0.001; GAD67 P50: t₅=2.23, p=0.038; all others, p≥0.051). Mean values for each condition are indicated by hash marks.

Figure 3. Cellular levels of GAD67, but not PV, mRNA are reduced in PV^GAD67+/− mice

A: representative micrographs of GAD67 grains in P50 PV^GAD67+/+ (left) and PV^GAD67+/− (right) mice. B: comparison of GAD67 (left) and PV (right) mRNA levels in M1 by grain counting in litter-matched pairs of P50 PV^GAD67+/+ and PV^GAD67+/− mice (GAD67: t₅=9.55, p<0.001; PV: p≥0.483). Mean values for each condition are indicated by hash marks.

Figure 4. GAD67, but not PV or GAT1, mRNA levels are reduced in GABA^GAD67+/− mice

A: representative film autoradiograms illustrating the expression of GAD67, PV, and GAT1 mRNA in the frontal lobe of P56 GABA^GAD67+/+ mice. The density of hybridization signal is presented in pseudocolor according to the calibration bars. The white contours denote the quantified regions in M1/M2 and PFC. B: comparison of GAD67, PV, and GAT1 mRNA levels in M1/M2 by film OD in P56 GABA^GAD67+/+ and GABA^GAD67+/− mice (GAD67: t₆=7.89, p<0.001; all others: p≥0.344). C: comparison of GAD67, PV, and GAT1 mRNA levels in PFC by film OD of P56 GABA^GAD67+/+ and GABA^GAD67+/− mice [GAD67: t₆=7.67, p<0.001; all others: p≥0.235]. Mean values for each group are indicated by hash marks. PFC GAD67 OD levels are from Eggan et al., 2012.

Figure 5. GAD67 protein levels are reduced in GABA^GAD67+/− mice

A: lanes from a representative Western blot loaded with brain tissue homogenate (spanning Bregma +1.10 to +0.50) from a GABA^GAD67+/+ and GABA^GAD67+/− litter-matched pair. B: comparison of tubulin-normalized GAD67 protein levels in pairs of P56 GABA^GAD67+/+ and GABA^GAD67+/− mice (t₅=7.22, p<0.001). Mean values for each condition are indicated by hash marks.