Abstract
The spatio-temporal regulation of genes involved in the synthesis and degradation of D-serine and D-aspartate such as serine racemase (SR), D-amino acid oxidase (DAO), G72 and D-aspartate oxidase (DDO), play pivotal roles in determining the correct levels of these D-amino acids in the human brain. Here we provide a comprehensive analysis of mRNA expression and DNA methylation status of these genes in post-mortem samples from hippocampus, dorsolateral prefrontal cortex, and cerebellum from patients with schizophrenia and non-psychiatric controls. DNA methylation analysis was performed at an ultradeep level, measuring individual epialleles frequency by single molecule approach. Differential CpG methylation and expression was detected across different brain regions, although no significant correlations were found with diagnosis. G72 showed the highest CpG and non-CpG methylation degree, which may explain the repression of G72 transcription in the brain regions considered here. Conversely, in line with the sustained SR mRNA expression in the analyzed areas, very low methylation levels were detected at this gene’s regulatory regions. Furthermore, for DAO and DDO, our single-molecule methylation approach demonstrated that analysis of epiallele distribution was able to detect differences in DNA methylation representing area-specific methylation signatures, which are likely not detectable with targeted or genome-wide classic methylation analyses.
Highlights
Free D-serine (D-Ser) and D-aspartate (D-Asp) act as a co-agonist and agonist at N-methyl-D-aspartate receptors (NMDARs), respectively, and influence numerous brain functions dependent by this subclass of glutamate receptors[1,2,3,4]
As temporal and regional D-Asp and D-Ser levels are thought to play a role in NMDAR-related synaptic plasticity, morphology and functioning[11,34], it is likely that the expression program of the genes regulating the metabolism of these D-amino acids is strictly orchestrated in the mammalian brain
Using high-coverage targeted bisulfite sequencing, we investigated the DNA methylation state at 10 CpG sites (−40; −30; −26; +11; +30; +74; +82; +105; +153 and +193) surrounding the D-amino acid oxidase (DAO) transcriptional start site (TSS) (Fig. 1A)
Summary
Free D-serine (D-Ser) and D-aspartate (D-Asp) act as a co-agonist and agonist at N-methyl-D-aspartate receptors (NMDARs), respectively, and influence numerous brain functions dependent by this subclass of glutamate receptors[1,2,3,4] These D-amino acids are present in the mammalian brain with an age- and region-specific distribution pattern[5,6,7]. Add-on therapy with a DAO inhibitor, sodium benzoate, improves schizophrenia symptomatology, even in clozapine-resistant patients[28,29] Another protein, pLG72, called D-amino acid oxidase activator (DAOA), encoded by the G72 gene, has been proposed to function as a DAO modulator. We performed an ultra-deep analysis of DNA methylation, at CpG and non-CpG sites, as well as expression profiles of all known D-Ser and D-Asp modulating genes (SR, DAO, G72 and DDO) in several brain areas of controls and subjects with schizophrenia. In addition to conventional average-based DNA methylation analysis, we applied a single molecule methylation approach (epiallele classes and distribution analyses) in order to finely map cell to cell methylation differences in specific brain areas and to evaluate whether these ultra-deep methylation profiles may distinguish brain areas within each individual and/or be associated to schizophrenia diagnosis
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