Reductions in midbrain GABAergic and dopamine neuron markers are linked in schizophrenia

Abstract

Reductions in the GABAergic neurotransmitter system exist across multiple brain regions in schizophrenia and encompass both pre- and postsynaptic components. While reduced midbrain GABAergic inhibitory neurotransmission may contribute to the hyperdopaminergia thought to underpin psychosis in schizophrenia, molecular changes consistent with this have not been reported. We hypothesised that reduced GABA-related molecular markers would be found in the midbrain of people with schizophrenia and that these would correlate with dopaminergic molecular changes. We hypothesised that downregulation of inhibitory neuron markers would be exacerbated in schizophrenia cases with high levels of neuroinflammation. Eight GABAergic-related transcripts were measured with quantitative PCR, and glutamate decarboxylase (GAD) 65/67 and GABA_A alpha 3 (α3) (GABRA3) protein were measured with immunoblotting, in post-mortem midbrain (28/28 and 28/26 control/schizophrenia cases for mRNA and protein, respectively), and analysed by both diagnosis and inflammatory subgroups (as previously defined by higher levels of four pro-inflammatory cytokine transcripts). We found reductions (21 - 44%) in mRNA encoding both presynaptic and postsynaptic proteins, vesicular GABA transporter (VGAT), GAD1, and parvalbumin (PV) mRNAs and four alpha subunits (α1, α2, α3, α5) of the GABA_A receptor in people with schizophrenia compared to controls (p < 0.05). Gene expression of somatostatin (SST) was unchanged (p = 0.485). We confirmed the reduction in GAD at the protein level (34%, p < 0.05). When stratifying by inflammation, only GABRA3 mRNA exhibited more pronounced changes in high compared to low inflammatory subgroups in schizophrenia. GABRA3 protein was expressed by 98% of tyrosine hydroxylase-positive neurons and was 23% lower in schizophrenia, though this did not reach statistical significance (p > 0.05). Expression of transcripts for GABA_A receptor alpha subunits 2 and 3 (GABRA2, GABRA3) were positively correlated with tyrosine hydroxylase (TH) and dopamine transporter (DAT) transcripts in schizophrenia cases (GABRA2; r > 0.630, GABRA3; r > 0.762, all p < 0.001) but not controls (GABRA2; r < - 0.200, GABRA3; r < 0.310, all p > 0.05). Taken together, our results support a profound disruption to inhibitory neurotransmission in the substantia nigra regardless of inflammatory status, which provides a potential mechanism for disinhibition of nigrostriatal dopamine neurotransmission.

Keywords: GABA; GABRA; GAD1; GAD65/67; Neuroinflammation; Parvalbumin; Schizophrenia; Somatostatin; Substantia nigra; Tyrosine hydroxylase.

Fig. 1

Gene (GAD1) and protein (GAD65/67) expression levels of glutamate decarboxylase (GAD) in the midbrain in schizophrenia. a GAD1 mRNA was 30.96% lower in schizophrenia cases relative to controls, and this was not exacerbated by inflammatory status. b GAD65/67 protein was 34.14% lower in schizophrenia cases compared to controls, and this was also independent of inflammatory status. Pink dots are low inflammatory/schizophrenia cases, dark red dots are high inflammatory/schizophrenia cases. c A GAD65/67 protein band (~ 66 kDa) was detected in all control and schizophrenia cases. β-actin (42 kDa) was used as a loading control. IC internal control, C control, SH schizophrenia high inflammatory, SL schizophrenia low inflammatory. ^& inflammatory status unknown. Statistical results for analysis by inflammatory subgroup are in Table 3. Bars indicate mean ± SEM. *p < 0.05, ****p < 0.0001

Fig. 2

Gene expression level of interneuron markers and vesicular GABA transporter in control and schizophrenia midbrain. a PV mRNA was lower in the midbrain of schizophrenia cases (by 40.70%) relative to controls and this was not exacerbated by inflammatory status. b SST mRNA did not show significant changes in the midbrain between schizophrenia cases and controls. c VGAT mRNA was 39.69% lower in schizophrenia cases compared with controls and this was also not exacerbated by inflammatory status. Pink dots are low inflammatory/schizophrenia cases, dark red dots are high inflammatory/schizophrenia cases. Statistical results for analysis by inflammatory subgroup are in Table 3. Bars indicate mean ± SEM. *p < 0.05, ***p < 0.001

Fig. 3

GABA_A receptor subunit gene expression in the midbrain in schizophrenia. a GABRA1 mRNA was 43.77% lower in the midbrain from schizophrenia cases compared with controls. b GABRA2 mRNA was 21.53% lower in the midbrain from schizophrenia cases compared with controls. c GABRA5 mRNA showed a decrease of 20.98% in the midbrain of schizophrenia cases compared with controls. Pink dots are low inflammatory/schizophrenia cases, dark red dots are high inflammatory/schizophrenia cases. Reductions in GABRA1, 2 and 5 mRNA levels were not exacerbated by neuroinflammation. Statistical results for analysis by inflammatory subgroup are in Table 3. Bars indicate mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001

Fig. 4

GABA_A alpha 3 subunit gene expression in the midbrain of control and schizophrenia cases. a GABRA3 mRNA was 21.98% lower in patients with schizophrenia relative to controls. b Reductions in GABRA3 mRNA were exacerbated in the high inflammatory/schizophrenia subgroup. GABRA3 mRNA was lower in high inflammatory/schizophrenia cases compared with both low inflammatory/schizophrenia cases and controls. There was no difference in GABRA3 mRNA between low inflammatory/schizophrenia cases compared with controls. c A GABA_A α3 subunit protein band was detected at ~ 55 kDa in human midbrain by an antibody raised against amino acids 6–125. β-actin (42 kDa) was used as a loading control. d GABRA3 protein was 23.24% lower in schizophrenia cases relative to controls, though this change did not reach statistical significance. Pink dots are low inflammatory/schizophrenia cases, dark red dots are high inflammatory/schizophrenia cases. SCZ schizophrenia, IC internal control, C control, SH schizophrenia high inflammatory, SL schizophrenia low inflammatory. Statistical results for analysis by inflammatory subgroup are also in Table 3. Bars indicate mean ± SEM. **p < 0.01., ****p < 0.0001

Fig. 5

GABA_A alpha 3 subunit expression was abundant in dopaminergic neurons in the midbrain. a Double-labelling immunohistochemistry revealed GABRA3⁺/TH⁺ neurons with variable levels of GABRA3 (yellow arrowheads) and GABRA3⁺/TH⁻ cells (white arrowhead) in the substantia nigra. GABRA3 expression was identified along the cell membrane and processes (b) and in the cytoplasm of TH⁺ neurons (c). GABRA3⁺/TH⁻ cells (d) and GABRA3⁻/TH⁺ neurons (e) were occasionally identified in the midbrain in schizophrenia and controls cases. Scale bars = 50 μm (a) and 20 μm (b–e)

Fig. 6

TH and DAT gene expression in the midbrain of controls and schizophrenia cases, and the relationships between TH and GABRA2 and GABRA3 mRNAs in the midbrain in control and schizophrenia cases. a TH mRNA and b DAT mRNA levels in the midbrain were significantly lower in the schizophrenia group relative to the controls. c TH and GABRA2 mRNAs were not correlated in controls, d but were strongly positively correlated in schizophrenia cases. The strength of these correlations was significantly different (z = − 3.13, p = 0.002) . Similarly, TH and GABRA3 mRNA were not significantly correlated in controls, e but were strongly positively correlated in schizophrenia cases (f). The strength of these correlations was significantly different (z = − 3.04, p = 0.002). Pink dots are low inflammatory/schizophrenia cases, dark red dots are high inflammatory/schizophrenia cases. Bars indicate mean ± SEM. *p < 0.05., ***p < 0.001