DNA methyltransferase 1 regulates reelin mRNA expression in mouse primary cortical cultures

Abstract

The polygenic nature of complex psychiatric disorders suggests a common pathway that may be involved in the down-regulation of multiple genes through an epigenetic mechanism. To investigate the role of methylation in down-regulating the expression of mRNAs that may be associated with the schizophrenia phenotype, we have adopted a cell-culture model amenable to this line of investigation. We have administered methionine (2 mM) to primary cultures of cortical neurons prepared from embryonic day 16 mice and show that this treatment down-regulated reelin and glutamic acid decarboxylase 67 (GAD67) mRNA expression but not that corresponding to neuron-specific enolase mRNA. Moreover, methionine increased methylation of the reelin promoter, suggesting a possible mechanism for the observed change. These cultures contain a mixed population of neurons and glia. Approximately 83% of the neurons are GABAergic based on GAD immunoreactivity, and these neurons coexpress high levels of reelin and DNA methyltransferase (Dnmt) 1 immunoreactivity. To examine whether Dnmt1 regulates reelin gene expression, we used an antisense approach to reduce (knock down) Dnmt1 expression. The reduced Dnmt1 mRNA and protein were accompanied by increased reelin mRNA expression. More importantly, the Dnmt1 knockdown blocked the methionine-induced reelin and GAD67 mRNA down-regulation. These data support the hypothesis that the reduced amounts of reelin and GAD67 mRNAs documented in postmortem schizophrenia brain may be the consequence of a Dnmt1-mediated hypermethylation of the corresponding promoters.

Fig. 1.

Immunohistochemistry of the mouse cortical cultures. The photomicrographs show the colocalization of reelin with additional histochemical markers in the primary cortical neuronal cultures. (A) Reelin IR, color-coded green (Left); GAD IR, color-coded red (Center); overlay of reelin and GAD (Right). (Scale bar: 10 mm.) (B) Reelin IR, color-coded green (Left); DNMT1 IR, color-coded red (Center); overlay of Reelin and DNMT1 (Right). (Scale bar: 10 mm.) The figure shows representative images taken from random fields. A more complete statistical analysis of these data are presented in Table 1.

Fig. 2.

Treatment of mouse cortical cultures with methionine down-regulates mRNA expression. Mouse primary cortical cultures were prepared and treated with 2 mM methionine as outlined in Methods. After 72 h, RNA was harvested, and the amounts of specific transcripts were quantified by using competitive RT-PCR with internal standards. These included reelin (A), GAD67 (B), Dnmt1 (C), and NSE (D) mRNAs. mRNA units are pg/μg total RNA. The data represent mean ± SE of three independent RNA measurements from three separate treatments. The vehicle-treated group was time-matched and performed in parallel. *, significance at P < 0.001.

Fig. 3.

Methylation patterns of genomic DNA isolated from saline- and methionine-treated primary mouse cortical cultures. After the saline/methionine treatment, genomic DNA was isolated and treated with sodium bisulfite. PCR primers were used to amplify the reelin promoter region. The amplified material was subcloned, and individual clones were sequenced to determine the pattern and total number of methylated bases. A minimum of 11 clones from each treatment set were sequenced. The saline-treated controls were time-matched for each group and performed in parallel. (A) Twenty-four-hour treatment of saline (S24) and methionine (M24). The total number of methylated cytosines for S24 is 27, and for M24 is 24. (B) Thirty-six-hour treatment of saline (S36) and methionine (M36). Total number of methylated cytosines for S36 is 22, and for M36 is 41. (C) Seventy-two-hour treatment of saline (S72) and methionine (M72). The total number of methylated cytosines for S72 is 25, and for M72 is 37.

Fig. 4.

Time dependence of the methionine-induced reelin mRNA down-regulation. Data presented in Fig. 2 show the effects after a 72-h methionine treatment on reelin mRNA levels. Fig. 3 shows that changes in reelin promoter methylation begin to be significant somewhat earlier. In this experiment, we treated mouse primary cortical cultures for 24 and 36 h with 2 mM methionine and harvested RNA for analysis. The data show that whereas there appears to be a trend toward down-regulation at 24 h (P < 0.08), this becomes significant after 36 h of treatment (P < 0.001). RNA levels were quantified by using competitive RT-PCR with internal standards.

Fig. 5.

Dnmt1 antisense oligonucleotide increases reelin mRNA expression. The Dnmt1 antisense oligonucleotide was incubated with the primary cortical cultures to down-regulate the corresponding mRNA and protein (see Methods). A shows a representative Western blot (Upper) demonstrating a down-regulation of Dnmt1 protein (Oligo, antisense treated; Control, scrambled oligo). The amount of residual Dnmt1 after antisense treatment was measured to be ≈18%; t test, P < 0.02 (Lower). B shows the results of a competitive RT-PCR assay showing that the Dnmt1 mRNA is also reduced by the antisense treatment. C demonstrates that the Dnmt1 antisense increases reelin mRNA levels (P < 0.001) but does not affect the levels of the NSE mRNA (D).

Fig. 6.

Dnmt1 antisense blocks the methionine-mediated reelin and GAD67 mRNA down-regulation. Primary cortical cultures were treated with the Dnmt1 antisense oligonucleotide before the methionine treatment to test whether Dnmt1 is a component of the pathway from methionine to the reelin promoter. See Methods for specific details of the treatment paradigm. In brief, cultures were transfected with the Dnmt1 antisense (Oligo, hatched bars) and in parallel the control oligo (open bars) and were subsequently treated with methionine (Meth) for 72 h. RNA was harvested, and the levels of reelin (A), NSE (B), and GAD67 (C) mRNAs were quantified by using competitive RT-PCR with internal standards (28). The data show that whereas the treatment did not affect NSE mRNA expression, the methionine-mediated down-regulation was attenuated in the presence of the Dnmt1 antisense knockdown. *, P < 0.001.