Testosterone Depletion Induces Demethylation of Murine Reelin Promoter CpG Dinucleotides: A Preliminary Study

Abstract

Schizophrenia (SZ) is a debilitating mental disorder characterized by psychotic events, abnormal social behavior, false beliefs, and auditory hallucinations. Hypermethylation of the promoter region of reelin (RELN), a gene involved in regulation of neuronal positioning during telencephalic development, is strongly associated with low protein expression in several cortical structures and promoter hypermethylation in brain from postmortem SZ subjects. Recent experimental data suggests that testosterone is able to promote RELN demethylation, although no direct evidence of hormonal influence on reelin promoter methylation was obtained. We investigated if reduced levels of plasma testosterone in adult male mice lead to Reln promoter demethylation. Animals were administered with flutamide, an antiandrogenic compound, and reelin promoter methylation was assessed using methylationspecific PCR using bisulfite DNA from cerebellum. We found that flutamide was able to significantly lower plasma testosterone when compared to control mice, and treatment did not influence animal survival and body weight. We also show that low plasma testosterone was associated with demethylation of a cytosine residue located at -860 in reelin promoter region. These preliminary data suggest that androgenic hormones can influence cerebral reelin demethylation. To our knowledge, this is the first experimental approach directly linking testosterone depletion and RELN promoter methylation.

Figure 1

(a) Murine Reln CpG island prediction and MSP primer design. Blue regions delimited on mouse reelin gene promoter sequence (first 1200 bases) indicate CpG islands predicted by MethPrimer software (http://www.urogene.org/methprimer/). General parameters for CG-rich regions are depicted in Section 2.4. The small red lines indicate C residues from CpG dinucleotides alongside Reln promoter region. Green boxes indicate annealing regions for MSP forward (UF3) and reverse (UR3) primers. (b) Alignment of segments of DNA sequences from reelin promoter regions of different species showing the conserved cytosine at position −860.

Figure 2

(a) Kaplan-Meier survival curves from control (full line) and flutamide-treated mice (dashed line). The x-axis shows days after treatment; the y-axis shows proportion of mice that survived. Animals were euthanized at day 30. Differences in survival were measured by log-rank (Mantel-Cox) test (p = 0.453). (b) Mean body weight of flutamide-treated mice. Measurements were done every three days until day 30. Values represent median ± SEM (n = 5). Data is representative of three independent experiments.

Figure 3

Total plasma testosterone of vehicle and flutamide-treated mice. Values represent median ± SEM (n = 5 per group). Difference was considered significant (p = 0.035, Mann Whitney exact test). Data is representative of three independent experiments.

Figure 4

Methylation specific PCR evaluation of reelin promoter after flutamide administration. (a) MSP analysis of CpG cytosines located at position −860 of mouse reelin promoter. Gel images show results obtained from control mice (lanes 1–3 and 9–11) and flutamide-treated animals (lanes 4–6 and 12–14) using specific primers designed to detect unmethylated or methylated cytosines. In vitro methylated mouse DNA (lanes 7 and 15) serves as positive and negative controls for each MSP primer, respectively. Genomic double-stranded DNA (lanes 8 and 16) was used as additional control to show that MSP primers do not amplify unmodified DNA. L, 100 bp DNA ladder. (b) Semiquantitative end-point comparison of band intensity was carried out using Fiji Software; n = 5 mice per group. Values represent median ± SEM; ^∗ p < 0.05.