Antipsychotic drugs elevate mRNA levels of presynaptic proteins in the frontal cortex of the rat

Abstract

Background: Molecular adaptations are believed to contribute to the mechanism of action of antipsychotic drugs (APDs). We attempted to establish common gene regulation patterns induced by chronic treatment with APDs.

Methods: Gene expression analysis was performed with the Affymetrix U34A array in the frontal cortex (FC) and the striatum of rats chronically treated with two concentrations of either clozapine or haloperidol. Key data were verified with real-time quantitative polymerase chain reaction.

Results: Many genes in the FC affected by APD-treatment contribute to similar functions. mRNAs coding for synaptic vesicle docking- and microtubule-associated proteins were upregulated; mRNAs for serine-threonine protein phosphatases were downregulated, whereas the serine-threonine kinases protein kinase A, protein kinase C, and calcium/calmodulin kinase II alpha and IV were upregulated, indicating increased potential for protein phosphorylation. In the striatum, altered gene expression was less focused on genes of particular function or location, and the high concentration of haloperidol had a different gene expression profile than any of the other APD treatments.

Conclusion: We found an increase in the transcription of genes coding for proteins involved in synaptic plasticity and synaptic activity in the FC. We furthermore found that the gene expression profile of APDs is different between FC and striatum.

Figure 1. Unsupervised, hierarchical clustering of samples in the FC and the striatum

Clustering analysis was performed in the FC (A) and the striatum (B), using 120 genes with the highest variation across treatment groups in each brain area, which were expressed in at least 60% of all samples. In the FC, vehicle samples clustered together (p=0.03), whereas haloperidol-H samples clustered in the striatum (p=0.02). No other significant sample clustering was observed.

Figure 2. Upregulation of mRNAs for presynaptic proteins in the FC

The graphs depict the effect of APDs in the FC, comparing vehicle-treated rats to all APD treatments combined. The percentage induction or reduction of mRNAs is shown. Multiple probe sets of the same gene are joined by brackets. Names of regulated genes, Genbank accession numbers and p-values are shown in the tables below the graphs. The number sign (#) denotes genes that were only regulated in dChip 1.3 but not MAS 5.0. The section sign (§) denotes genes that were also significantly regulated in the striatum. The graph shows the percentage induction or reduction of genes associated with synaptic vesicle function and microtubules. Thirteen genes involved in synaptic vesicle fusion and recycling, or associated with microtubules, were upregulated in the FC. Of four genes that were downregulated with the dChip 1.3 program, three were unchanged in MAS 5.0. Upregulations were concordant between both programs. None of the genes shown were altered in the striatum.

Figure 3. Verification of upregulation of synaptic proteins in the FC with Q-PCR

Upregulation of SNAP 25 (A), VAMP1 (B) and 2 (C), and syntaxin 1b2 (D) by APDs in the FC was verified with Q-PCR. Data were normalized to β-actin, which was not regulated in the gene arrays. N=12 vehicle-treated controls, 23 APDs (6 clozapine-L, 7 clozapine-H, 5 haloperidol-L, 5 haloperidol-H). ** p<=0.01, * p<=0.05.

Figure 4. Downregulation of mRNAs for protein phosphatases in the FC

The graphs depict the effect of APDs in the FC, comparing vehicle-treated rats to all APD treatments combined. The percentage induction or reduction of mRNAs is shown. Multiple probe sets of the same gene are joined by brackets. Names of regulated genes, Genbank accession numbers and p-values are shown in the tables below the graphs. The number sign (#) denotes genes that were only regulated in dChip 1.3 but not MAS 5.0. The section sign (§) denotes genes that were also significantly regulated in the striatum. PP1 and PP2A exist as multimeric holoenzymes with a limited number of catalytic subunits complexed to a variety of regulatory subunits. PP1 is a heterodimeric complex of catalytic and regulatory subunits, while PP2A forms a heterotrimeric complex between the catalytic subunit and the regulatory A and B subunits (Sim et al 2003). PP2C is a monomeric enzyme (Price and Mumby 1999). A downregulation in mRNA levels for protein phosphatases was observed, except for the regulatory A alpha subunit for PP2A, which was upregulated, in essence causing an inhibition of PP2A. Genes that did not reach significant with MAS 5.0 (denoted with #), still showed a pronounced downregulation in that program. None of the genes presented were similarly regulated in the striatum, although three had a significant opposite regulation (denoted with §).

Figure 5. Altered regulation of protein kinase genes

The graphs depict the effect of APDs in the FC, comparing vehicle-treated rats to all APD treatments combined. The percentage induction or reduction of mRNAs is shown. Multiple probe sets of the same gene are joined by brackets. Names of regulated genes, Genbank accession numbers and p-values are shown in the tables below the graphs. The number sign (#) denotes genes that were only regulated in dChip 1.3 but not MAS 5.0. The section sign (§) denotes genes that were also significantly regulated in the striatum. Many Ca²⁺-activated kinases were upregulated in the FC, while members of the MAP kinase pathways were mostly downregulated. Not all regulations were significant in MAS 5.0.

Figure 6. Upregulation of genes involved in energy metabolism in the FC

The graphs depict the effect of APDs in the FC, comparing vehicle-treated rats to all APD treatments combined. The percentage induction or reduction of mRNAs is shown. Multiple probe sets of the same gene are joined by brackets. Names of regulated genes, Genbank accession numbers and p-values are shown in the tables below the graphs. The number sign (#) denotes genes that were only regulated in dChip 1.3 but not MAS 5.0. The section sign (§) denotes genes that were also significantly regulated in the striatum. A predominant upregulation in mRNA levels for proteins involved in glycolysis was observed with the dChip 1.3 program. None of the genes were changed in the striatum. Note the significant upregulation of GAPDH, a gene frequently used as a normalization control.

Figure 7. Downregulation of G protein alpha subunits in the FC

The graphs depict the effect of APDs in the FC, comparing vehicle-treated rats to all APD treatments combined. The percentage induction or reduction of mRNAs is shown. Multiple probe sets of the same gene are joined by brackets. Names of regulated genes, Genbank accession numbers and p-values are shown in the tables below the graphs. The number sign (#) denotes genes that were only regulated in dChip 1.3 but not MAS 5.0. The section sign (§) denotes genes that were also significantly regulated in the striatum. A downregulation of mRNA levels of G alpha subunits, and of G beta 2, was observed in the FC. RGS4 and RGS8 were upregulated.

Figure 8. Gene families regulated by APDs in the striatum

The four gene families that were most affected by APD treatment in the striatum were (A) cyclins and cyclin-dependent kinases, (B) MAP kinases, (C) antioxidants and (D) ANIAs. Z scores were lower than in the FC, and many of the genes did not reach significance with MAS 5.0 (#).

Figure 9. mRNA regulation of PKC beta 1 and PKC zeta by APDs in the striatum

A, B, PKC beta 1 (K03486), C, D, PKC zeta (M18332). A, C, gene array data, B, D, Q-PCR analysis normalized to β-actin.

Figure 10. Sample clustering demonstrates a unique molecular profile induced by high doses of haloperidol in the striatum

The 100 genes with the biggest difference in expression levels between haloperidol-H and vehicle (p-level of <=0.05; ‘present’ in >=60 % of all samples) within each brain area were used for hierarchical clustering in the striatum (A) and the FC (B). In the striatum, these genes clustered haloperidol-H samples (p<0.001; A). In contrast, in the FC, haloperidol-H samples were spread between the other APD samples (B), while vehicle samples were clustered.

Figure 11. Differential effect of Haloperidol-H on the expression of various gene families in the striatum

Compared to the other APD treatments, haloperidol-H treatment induced a downregulation of presynaptic proteins (A), a dysregulation of glutamate-related proteins (B), GTPases (C), and a downregulation of Na+/K+ ATPases, in the striatum.

Figure 12. Genes affected by clozapine-treatment in the FC are predictive of chronic haloperidol exposure

Onehundred genes with the biggest difference in expression between clozapine (H and L) and vehicle samples in the FC were used for hierarchical clustering of haloperidol samples. A. The ‘clozapine’ genes cluster samples of rats treated with 0.2 mg/kg haloperidol, p = 0.01. B. The ‘clozapine’ genes also cluster samples of rats treated with 0.5 mg/kg haloperidol, p = 0.05. Onehundred genes that were not regulated by clozapine did also not cluster haloperidol 0.2 mg/kg (C) or haloperidol 0.5 mg/kg (D) samples.