Putative presynaptic dopamine dysregulation in schizophrenia is supported by molecular evidence from post-mortem human midbrain

Abstract

The dopamine hypothesis of schizophrenia posits that increased subcortical dopamine underpins psychosis. In vivo imaging studies indicate an increased presynaptic dopamine synthesis capacity in striatal terminals and cell bodies in the midbrain in schizophrenia; however, measures of the dopamine-synthesising enzyme, tyrosine hydroxylase (TH), have not identified consistent changes. We hypothesise that dopamine dysregulation in schizophrenia could result from changes in expression of dopamine synthesis enzymes, receptors, transporters or catabolic enzymes. Gene expression of 12 dopamine-related molecules was examined in post-mortem midbrain (28 antipsychotic-treated schizophrenia cases/29 controls) using quantitative PCR. TH and the synaptic dopamine transporter (DAT) proteins were examined in post-mortem midbrain (26 antipsychotic-treated schizophrenia cases per 27 controls) using immunoblotting. TH and aromatic acid decarboxylase (AADC) mRNA and TH protein were unchanged in the midbrain in schizophrenia compared with controls. Dopamine receptor D2 short, vesicular monoamine transporter (VMAT2) and DAT mRNAs were significantly decreased in schizophrenia, with no change in DRD3 mRNA, DRD3nf mRNA and DAT protein between diagnostic groups. However, DAT protein was significantly increased in putatively treatment-resistant cases of schizophrenia compared to putatively treatment-responsive cases. Midbrain monoamine oxidase A (MAOA) mRNA was increased, whereas MAOB and catechol-O-methyl transferase mRNAs were unchanged in schizophrenia. We conclude that, whereas some mRNA changes are consistent with increased dopamine action (decreased DAT mRNA), others suggest reduced dopamine action (increased MAOA mRNA) in the midbrain in schizophrenia. Here, we identify a molecular signature of dopamine dysregulation in the midbrain in schizophrenia that mainly includes gene expression changes of molecules involved in dopamine synthesis and in regulating the time course of dopamine action.

Figure 1

Dopamine synthesis enzymes, TH mRNA and protein and AADC mRNA, levels in the substantia nigra in control (blue circles) and schizophrenia cases (red circles). (a) TH immunohistochemistry in a human midbrain representative of our cohort. Dark brown staining is TH expression in cell bodies and processes. Dashed lines bound the area of tissue dissected and homogenised to enrich for midbrain dopamine neurons of the substantia nigra (black). Scale bar=1 cm. Inset shows TH-positive neurons with TH expression in the cytoplasm (arrow) and processes, scale bar=100 μm. (b) TH mRNA in the substantia nigra was not significantly different between control and schizophrenia cases (F=0.74; df=54; P=0.395). (c) TH protein in the substantia nigra was not significantly different between control and schizophrenia cases (F=0.304; df=53; P=0.584). (d) A single TH protein band (~59 kDa) and a single β-actin band (~42 kDa) were detected in all samples using immunoblotting. (IC, internal control; C control; S, schizophrenia). (e) AADC mRNA was decreased 22.49% in the substantia nigra in schizophrenia cases when compared with controls; however, this only reached a trend level (F=3.417; df=54; P=0.070). Data are mean±s.e.m., ^&P<0.01. AADC, aromatic acid decarboxylase; RN, red nucleus; SN, substantia nigra; TH, tyrosine hydroxylase; VTA, ventral tegmental area.

Figure 2

Dopamine receptor (DRD2S, DRD2L and DRD2Longer, DRD3, DRD3nf) and dopamine breakdown enzyme (MAOA, MAOB and COMT) gene expression in the substantia nigra in control (blue circles) and schizophrenia cases (red circles). (a) DRD2S mRNA was significantly decreased in substantia nigra from schizophrenia cases compared with control cases (F=3.05, df=55, P=0.018). (b) There was a significant decrease in expression of DRD2L mRNA in schizophrenia (F=3.98, df=56, P=0.051). (c) There was a trend for decreased expression for DRD2Longer mRNA (F=3.49, df=56, P=0.067). (d) DRD3 mRNA in the substantia nigra was not significantly different between control and schizophrenia cases (F=1.471; df=53; P=0.231). (e) DRD3nf mRNA in the substantia nigra was not significantly different between control and schizophrenia cases (F=0.949; df=55; P=0.334). (f) MAOA mRNA in the substantia nigra was significantly increased in schizophrenia cases compared with control cases (F=6.34; df=56; P=0.015). (g) MAOB mRNA in the substantia nigra was not significantly different between control and schizophrenia cases (F=0.81; df=53; P=0.371). (h) COMT mRNA in the substantia nigra was not significantly different between control and schizophrenia cases (U=462, z=0.89, P=0.371). Data are mean±s.e.m., *P⩽0.05. COMT, catechol-O-methyl transferase; DRD, dopamine receptor D; DRD3nf, DRD3 non-functional; MAOA, midbrain monoamine oxidase A.

Figure 3

Gene expression of dopamine transporters, DAT and VMAT2, and DAT protein expression, in the substantia nigra in control (blue circles) and schizophrenia cases (red circles). (a) DAT mRNA levels were significantly decreased in schizophrenia (F=17.73; df=55; P<0.0001). (b) VMAT2 mRNA levels were significantly decreased in schizophrenia (F=6.54; df=54; P=0.014). (c) DAT protein in the substantia nigra was not significantly different between control and schizophrenia cases (t=−1.361; df=52; P=0.179). (d) Single DAT (~75 kDa) and β-actin bands (~42 kDa) were detected in all samples using immunoblotting. Data are mean±s.e.m.. *P⩽0.05, ****P⩽0.0001. C, control; DAT, dopamine transporter; IC, internal control; S, schizophrenia; VMAT2, vesicular monoamine transporter 2.

Figure 4

Proposed model of dopamine dysregulation in the nigrostriatal pathway in schizophrenia. Gene expression of multiple DA-regulating molecules (red box) involved in autoreception (DRD2), transport (DAT and VMAT) and catabolism (MAO) are changed in the substantia nigra in schizophrenia, and together may contribute to DA dysregulation at the level of the DA cell bodies and/or at the DA terminals in the striatum. DA is synthesised from tyrosine (tyr) by TH and AADC, but these do not appear to be changed in level. MAO and COMT breakdown DA to metabolites, DOPAC and HVA and MAO mRNA is increased. DAT removes DA from the synaptic cleft and is reduced at the mRNA level but not at the protein level in the midbrain. VMAT packages DA into vesicles and is reduced at the mRNA level. DRD2 (blue triangle) autoreception inhibits DA firing, DA synthesis and DA release, and we found a significant decrease in DRD2S mRNA. DRD3 mRNA was unchanged. DA activation of DRD2 (blue triangle) on the postsynaptic neuron inhibits AC leading to disinhibition of the inhibitory medium spiny neurons. Increased DRD2 expression in the striatum in schizophrenia would further contribute to DA dysregulation. AADC, aromatic acid decarboxylase; AC, adenylate cyclase; COMT, catechol-O-methyl transferase; DA, dopamine; DRD, dopamine receptor D; MAO, monoamine oxidase; TH, tyrosine hydroxylase; VMAT2, vesicular monoamine transporter 2.