Mapping the expression of an ANK3 isoform associated with bipolar disorder in the human brain.

The gene ankyrin-3 (ANK3) has been consistently associated with bipolar disorder (BD) in several genome-wide association studies (GWAS). The exact molecular mechanisms underlying this genetic association remain unknown. The discovery of a loss-of-function variant (rs41283526*G) in an alternatively spliced exon (ENSE00001786716) with a protective effect, suggested that elevated expression of this particular isoform could be a risk factor for developing the disorder. We developed a novel approach for measuring the expression level of all splice forms at a challenging genetic locus using a combination of droplet digital PCR and high-throughput sequencing of indexed PCR amplicons. The combined method was performed on a large collection of 568 postmortem brain samples of BD and SCZ cases and controls. We also studied the expression of the splice forms in a child-development cohort of 41 healthy males. We found that our approach can quantify the splice forms in brain samples, although with less precision than ddPCR. We detected highly significant differences in expression of splice forms and transcription start sites between brain regions, notably with higher expression of the BD-associated isoform in the corpus callosum compared to frontal tissue (mean fold change = 1.80, p < 1e-4). Although the patients in our sample expressed the BD-associated splice form at a similar level to controls, adolescents in our child-development cohort had a clearly higher expression level than younger children (mean fold change = 1.97, p = 5e-3). These results suggest that this ANK3 splice form may play a role in the myelin maturation of the human brain.

Dopamine D3 receptor-dependent changes in alpha6 GABAA subunit expression in striatum modulate anxiety-like behaviour: Responsiveness and tolerance to diazepam.

Increasing evidence indicates that central dopamine (DA) neurotransmission is involved in pathophysiology of anxiety, in particular the DA receptor subtype 3 (D3R). We previously reported that D3R null mice (D3R(-/-)) exhibit low baseline anxiety levels and that acutely administrated diazepam is more effective in D3R(-/-) than in wild type (WT) when tested in the elevated plus maze test (EPM). Here we tested the hypothesis that genetic deletion or pharmacological blockade of D3R affect GABAA subunit expression, which in turn modulates anxiety-like behaviour as well as responsiveness and tolerance to diazepam. D3R(-/-) mice exhibited tolerance to diazepam (0.5mg/kg, i.p.), assessed by EPM, as fast as after 3 day-treatment, performing similarly to untreated D3R(-/-) mice; conversely, WT exhibited tolerance to diazepam after a 14-21 day-treatment. Analysis of GABAA α6 subunit mRNA expression by qPCR in striatum showed that it was about 15-fold higher in D3R(-/-) than in WT. Diazepam treatment did not modify α6 expression in D3R(-/-), but progressively increased α6 expression in WT, to the level of untreated D3R(-/-) after 14-21 day-treatment. BDNF mRNA expression in striatum was remarkably (>10-fold) increased after 3 days of diazepam-treatment in both WT and D3R(-/-); such expression level, however, slowly declined below control levels, by 14-21 days. Following a 7 day-treatment with the selective D3R antagonist SB277011A, WT exhibited a fast tolerance to diazepam accompanied by a robust increase in α6 subunit expression. In conclusion, genetic deletion or pharmacological blockade of D3R accelerate the development of tolerance to repeated administrations of diazepam and increase α6 subunit expression, a GABAA subunit that has been linked to diazepam insensitivity. Modulation of GABAA receptor by DA transmission may be involved in the mechanisms of anxiety and, if occurring in humans, may have therapeutic relevance following repeated use of drugs targeting D3R.