Dataset Information

Expression of D-Amino Acid Oxidase (DAO/DAAO) and D-Amino Acid Oxidase Activator (DAOA/G72) during Development and Aging in the Human Post-mortem Brain.

ABSTRACT: In the brain, D-amino acid oxidase (DAO/DAAO) mainly oxidizes D-serine, a co-agonist of the N-methyl-D-aspartate (NMDA) receptors. Thus, DAO can regulate the function of NMDA receptors via D-serine breakdown. Furthermore, DAO activator (DAOA)/G72 has been reported as both DAOA and repressor. The co-expression of DAO and DAOA genes and proteins in the human brain is not yet elucidated. The aim of this study was to understand the regional and age span distribution of DAO and DAOA (mRNA and protein) in a concomitant manner. We determined DAO and DAOA mRNA and protein expression across six brain regions in normal human post-mortem brain samples (16 weeks of gestation to 91 years) using quantitative real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. We found higher expression of DAO mRNA in the cerebellum, whereas lower expression of DAO protein in the cerebellum compared to the other brain regions studied, which suggests post-transcriptional regulation. We detected DAOA protein but not DAOA mRNA in all brain regions studied, suggesting a tightly regulated expression. To understand this regulation at the transcriptional level, we analyzed DNA methylation levels at DAO and DAOA CpG sites in the cerebellum and frontal cortex of control human post-mortem brain obtained from Gene Expression Omnibus datasets. Indeed, DAO and DAOA CpG sites in the cerebellum were significantly more methylated than those in the frontal cortex. While investigating lifespan effects, we found that DAO mRNA levels were positively correlated with age <2 years in the cerebellum and amygdala. We also detected a significant positive correlation (controlled for age) between DAO and DAOA protein in all of the brain regions studied except for the frontal cortex. In summary, DAO and DAOA expression in the human brain are both age and brain region dependent.

SUBMITTER: Jagannath V

PROVIDER: S-EPMC5382383 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Expression of D-Amino Acid Oxidase (DAO/DAAO) and D-Amino Acid Oxidase Activator (DAOA/G72) during Development and Aging in the Human Post-mortem Brain.

Jagannath Vinita V Marinova Zoya Z Monoranu Camelia-Maria CM Walitza Susanne S Grünblatt Edna E

Frontiers in neuroanatomy 20170406

In the brain, D-amino acid oxidase (DAO/DAAO) mainly oxidizes D-serine, a co-agonist of the N-methyl-D-aspartate (NMDA) receptors. Thus, DAO can regulate the function of NMDA receptors via D-serine breakdown. Furthermore, DAO activator (DAOA)/G72 has been reported as both DAOA and repressor. The co-expression of DAO and DAOA genes and proteins in the human brain is not yet elucidated. The aim of this study was to understand the regional and age span distribution of DAO and DAOA (mRNA and ...[more]

PMID: 28428746

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Project description:BackgroundSchizophrenia is a neurodegenerative disorder that occurs worldwide and can be difficult to diagnose. It is the foremost neurological disorder leading to suicide among patients in both developed and underdeveloped countries. D-amino acid oxidase activator (DAOA), also known as G72, is directly implicated in the glutamateric hypothesis of schizophrenia. It activates D-amino acid oxidase, which oxidizes D-serine, leading to modulation of the N-methyl-D-aspartate receptor.MethodsMODELLER (9v10) was utilized to generate three dimensional structures of the DAOA candidate gene. The HOPE server was used for mutational analysis. The Molecular Evolutionary Genetics Analysis (MEGA5) tool was utilized to reconstruct the evolutionary history of the candidate gene DAOA. AutoDock was used for protein-ligand docking and Gramm-X and PatchDock for protein-protein docking.ResultsA suitable template (1ZCA) was selected by employing BLASTp on the basis of 33% query coverage, 27% identity and E-value 4.9. The Rampage evaluation tool showed 91.1% favored region, 4.9% allowed region and 4.1% outlier region in DAOA. ERRAT demonstrated that the predicted model had a 50.909% quality factor. Mutational analysis of DAOA revealed significant effects on hydrogen bonding and correct folding of the DAOA protein, which in turn affect protein conformation. Ciona was inferred as the outgroup. Tetrapods were in their appropriate clusters with bifurcations. Human amino acid sequences are conserved, with chimpanzee and gorilla showing more than 80% homology and bootstrap value based on 1000 replications. Molecular docking analysis was employed to elucidate the binding mode of the reported ligand complex for DAOA. The docking experiment demonstrated that DAOA is involved in major amino acid interactions: the residues that interact most strongly with the ligand C28H28N3O5PS2 are polar but uncharged (Gln36, Asn38, Thr 122) and non-polar hydrophobic (Ile119, Ser171, Ser21, Ala31). Protein-protein docking simulation demonstrated two ionic bonds and one hydrogen bond involving DAOA. Lys-7 of the receptor protein interacted with Lys-163 and Asp-2037. Tyr-03 interacted with Arg-286 of the ligand protein and formed a hydrogen bond.ConclusionThe predicted interactions might serve to inhibit the disease-related allele. It is assumed that current bioinformatics methods will contribute significantly to identifying, analyzing and curing schizophrenia. There is an urgent need to develop effective drugs for schizophrenia, and tools for examining candidate genes more accurately and efficiently are required.

Project description:BackgroundPolymorphisms at the G72/G30 locus on chromosome 13q have been associated with schizophrenia or bipolar disorder in more than ten independent studies. Even though the genetic findings are very robust, the physiological role of the predicted G72 protein has thus far not been resolved. Initial reports suggested G72 as an activator of D-amino acid oxidase (DAO), supporting the glutamate dysfunction hypothesis of schizophrenia. However, these findings have subsequently not been reproduced and reports of endogenous human G72 mRNA and protein expression are extremely limited. In order to better understand the function of this putative schizophrenia susceptibility gene, we attempted to demonstrate G72 mRNA and protein expression in relevant human brain regions.MethodsThe expression of G72 mRNA was studied by northern blotting and semi-quantitative SYBR-Green and Taqman RT-PCR. Protein expression in human tissue lysates was investigated by western blotting using two custom-made specific anti-G72 peptide antibodies. An in-depth in silico analysis of the G72/G30 locus was performed in order to try and identify motifs or regulatory elements that provide insight to G72 mRNA expression and transcript stability.ResultsDespite using highly sensitive techniques, we failed to identify significant levels of G72 mRNA in a variety of human tissues (e.g. adult brain, amygdala, caudate nucleus, fetal brain, spinal cord and testis) human cell lines or schizophrenia/control post mortem BA10 samples. Furthermore, using western blotting in combination with sensitive detection methods, we were also unable to detect G72 protein in a number of human brain regions (including cerebellum and amygdala), spinal cord or testis. A detailed in silico analysis provides several lines of evidence that support the apparent low or absent expression of G72.ConclusionOur results suggest that native G72 protein is not normally present in the tissues that we analysed in this study. We also conclude that the lack of demonstrable G72 expression in relevant brain regions does not support a role for G72 in modulation of DAO activity and the pathology of schizophrenia via a DAO-mediated mechanism. In silico analysis suggests that G72 is not robustly expressed and that the transcript is potentially labile. Further studies are required to understand the significance of the G72/30 locus to schizophrenia.

Dataset Information

Expression of D-Amino Acid Oxidase (DAO/DAAO) and D-Amino Acid Oxidase Activator (DAOA/G72) during Development and Aging in the Human Post-mortem Brain.

Publications

Expression of D-Amino Acid Oxidase (<i>DAO</i>/<i>DAAO</i>) and D-Amino Acid Oxidase Activator (<i>DAOA/G72</i>) during Development and Aging in the Human Post-mortem Brain.

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