July 21, 2014. A malfunctioning dopamine system can lower expression of nearly 2000 genes in prefrontal cortex neurons, according to a study published online in Molecular Psychiatry on July 15. Led by Emiliana Borrelli at the University of California, Irvine, the study finds disparate consequences of deleting dopamine D2 autoreceptors in the midbrain, which normally limit dopamine synthesis and release. Mice without D2 autoreceptors show massive epigenetic reprogramming in the prefrontal cortex, but not in the striatum, as well as behaviors related to anxiety and psychosis.
The study suggests that epigenetic reprogramming of cortex may follow the dopamine dysfunction found in schizophrenia (see SRF hypothesis). This might also help explain why antipsychotic drugs, which all block dopamine D2 receptors (D2Rs), are relatively effective against psychosis, but not the negative or cognitive symptoms of the disorder.
In an interesting twist, the researchers also found that the gene suppression in cortex could be alleviated by activating D2Rs—the opposite of what current antipsychotics do. This suggests that dopamine dysfunction can give rise to different outcomes, with too much dopamine signaling in the striatum but too little in the cortex.
Though hyperactive dopamine signaling in the striatum is widely accepted as a driver of psychosis, the dopamine situation in the cortex in schizophrenia remains unclear (see SRF related news story). Previous work by Borrelli’s group has found evidence for site-specific effects of dopamine dysfunction in mice. In 2012, the group published a paper examining the effects of removing D2 autoreceptors, which are found on the presynaptic terminals of midbrain dopamine neurons (Anzalone et al., 2012). When activated by excess dopamine, D2 autoreceptors inhibit dopamine synthesis and release. Removing this negative feedback in mice led to increased dopamine synthesis in striatum and cortex, but had different effects on actual dopamine release, which seemed to depend on the local circuitry in which a dopaminergic terminal found itself.
Reshaping a genomic landscape
First authors Karen Brami-Cherrier, Andrea Anzalone, and Maria Ramos found that DA-D2RKO mice showed behaviors that have been used as proxies for psychosis. When placed in a new environment, they exhibited anxiety-like hyperactivity; when given amphetamine, they exhibited enhanced locomotion; and in the paired pulse inhibition (PPI) test, which measures the ability of a preceding tone to subdue a startle response to a following tone, DA-D2RKO mice showed PPI impairment. The DA-D2RKO mice also trailed behind wild type mice in measures of working memory, which depends on prefrontal cortex.
With a microarray, the researchers next profiled the gene expression patterns of neurons from prefrontal cortex and ventral striatum, both targets of midbrain dopamine neurons. This revealed drastic changes in prefrontal cortex: 1930 genes were downregulated compared to wild type transcripts. In contrast, only 20 genes were downregulated (and 132 total showed differential expression) in ventral striatum, again suggesting site-specific effects of dopamine dysfunction. Gene ontology analysis indicated that the cortex’s downregulated genes were enriched for transcription modulators, including chromatin remodelers.
The gene suppression in prefrontal cortex was accompanied by an increase in histone modifications, specifically methylation of lysine residues (H3K9me2 and H3K9me3); in contrast, such changes were not observed in striatum. These methyl groups interfere with the transcription machinery that might otherwise descend upon the DNA wrapped around the histone. Visualizing the pattern of H3K9me3 in the cortex with immunohistochemistry showed that these modifications were especially prominent in deep layers of the cortex.
The researchers estimated that, of the genes downregulated in prefrontal cortex, 36 had ties to schizophrenia. Of these, the researchers focused on NR4A2 and AKT1, to get a higher resolution view of the histone modifications. Using chromatin immunoprecipitation (ChIP), the researchers found enrichment for H3K9me2 and H3K9me3 marks on the promoters of both genes in tissue from the prefrontal cortex not found in wild type mice. Protein levels of these genes were also diminished.
This gene silencing was not permanent, however. Stimulating D2Rs with the agonist quinpirole for two weeks restored the H3K9me3 marks down to wild type levels, while blocking D2Rs with clozapine did not. Similarly, protein levels of NR4A2 and AKT1 in prefrontal cortex almost doubled with quinpirole treatment relative to saline treated animals. This suggests that, in DA-D2RKO mice, the cortex sees a decrease in dopamine release.
The researchers did not report whether stimulating D2Rs could help working memory or hyperactive phenotypes. But the work emphasizes site-specific outcomes of dopamine dysfunction, and supports the idea that casting schizophrenia as a problem of hyperactive dopamine signaling is too simplistic.—Michele Solis.
K Brami-Cherrier, A Anzalone, M Ramos, I Forne, F Macciardi, A Imhof and E Borrelli. Epigenetic reprogramming of cortical neurons through alteration of dopaminergic circuits. Mol Psychiatry advance online publication, July 15, 2014. Abstract