Methylation pattern and mRNA expression of synapse-relevant genes in the MAM model of schizophrenia in the time-course of adolescence

Abstract

Schizophrenia is highly heritable and aggregating in families, but genetics alone does not exclusively explain the pathogenesis. Many risk factors, including childhood trauma, viral infections, migration, and the use of cannabis, are associated with schizophrenia. Adolescence seems to be the critical period where symptoms of the disease manifest. This work focuses on studying an epigenetic regulatory mechanism (the role of DNA methylation) and its interaction with mRNA expression during development, with a particular emphasis on adolescence. The presumptions regarding the role of aberrant neurodevelopment in schizophrenia were tested in the Methyl-Azoxy-Methanol (MAM) animal model. MAM treatment induces neurodevelopmental disruptions and behavioral deficits in off-springs of the treated animals reminiscent of those observed in schizophrenia and is thus considered a promising model for studying this pathology. On a gestational day-17, adult pregnant rats were treated with the antimitotic agent MAM. Experimental animals were divided into groups and subgroups according to substance treatment (MAM and vehicle agent [Sham]) and age of analysis (pre-adolescent and post-adolescent). Methylation and mRNA expression analysis of four candidate genes, which are often implicated in schizophrenia, with special emphasis on the Dopamine hypothesis i.e., Dopamine receptor D₂ (Drd2), and the "co-factors" Disrupted in schizophrenia 1 (DISC1), Synaptophysin (Syp), and Dystrobrevin-binding protein 1 (Dtnbp1), was performed in the Gyrus cingulum (CING) and prefrontal cortex (PFC). Data were analyzed to observe the effect of substance treatment between groups and the impact of adolescence within-group. We found reduced pre-adolescent expression levels of Drd2 in both brain areas under the application of MAM. The "co-factor genes" did not show high deviations in mRNA expression levels but high alterations of methylation rates under the application of MAM (up to ~20%), which diminished in the further time course, reaching a comparable level like in Sham control animals after adolescence. The pre-adolescent reduction in DRD2 expression might be interpreted as downregulation of the receptor due to hyperdopaminergic signaling from the ventral tegmental area (VTA), eventually even to both investigated brain regions. The notable alterations of methylation rates in the three analyzed co-factor genes might be interpreted as attempt to compensate for the altered dopaminergic neurotransmission.

Fig. 1. Mean methylation rates and expression levels (CNRQs, calibrated normalized relative quantities) of MAM and Sham pre-adolescent groups for the four investigated genes in the PFC.

A Mean methylation rates and expression levels of Drd2, B mean methylation rates and expression levels of DISC 1, C mean methylation rates and expression levels of Syp and D mean methylation rates and expression levels of Dtnp1. Important significant differences between two groups are indicated by a line. Less important significant results (as the groups are not directly related) are indicated by dashed lines. The number of asterisks indicates the level of significance: *p < 0.05, **p < 0.01; ***p < 0.001. MAM animals were treated with Methylazoxymethanol-Acetate, Sham animals were only treated with vehicle. Drd2 dopamine receptor 2, DISC1 disrupted in schizophrenia 1, Syp synaptophysin, Dtnbp1 dysbindin. For the analysis 7 pre-adolescent Sham, 6 pre-adolescent MAM, 7 post-adolescent Sham and 6 post-adolescent MAM animals have been included.