| Background and ObjectivesSchizophrenia is a disease that can affect all aspects of mental function including thinking, feeling, mood and sociability, with an incidence rate of almost 1% all over the world. Schizophrenia is often characterized by both positive symptoms such as hallucinations, delusions and psychosis, as well as negative symptoms including emotional withdrawal, anhedonia, stereotyped thinking and social withdrawal; and some patients could present cognitive symptoms. GABAergic neurotransmitter system may contribute to negative and cognitive symptoms for the fact that lower Gamma-aminobutyric acid (GABA) concentration exist in such schizophrenia patients.GABA is the major inhibitory neurotransmitter in vertebrates’ central nervous system (CNS), synthesized from L-glutamate via the enzyme called glutamate decarboxylase (GAD), working with GABA receptors located in the postsynaptic membrane, and being removed by GABA transporters (GAT) promptly to maintaining a low extracellular level. This process is also called GABAergic transmission. GABA exerts its effects through two classes of receptors designated as GABA (A) receptors (ionotropic receptors) and GABA (B) receptors (metabotropic receptors). GABA (A) receptors are pentamers consisted of five subunits (al-α6,β1-β3, γ1-γ3, ρ1-ρ3,θ, ε,δ and π subunits), and mediate fast synaptic transmission. Although GABA (A) receptors have many isoforms, the most common type in the brain consists of two a’s, two β’s, and a γ (α1β2γ2). GABA (B) receptors are heterodimer of GABBR1 and GABBR2, and mediate slow synaptic transmission.In this GABAergic transmission pathway, the most interesting gene we focused on is GABRB2 which encoding β2 subunit of GABA (A) receptor. We have proved its contribution to schizophrenia through various experiments including clinical case-control analyzing, cell functional analyzing and mice functional analyzing, revealed that possible pathogenesis factors might be specific single nucleotide polymorphism (SNP), abnormal mRNA expression, protein dysfunction or abnormal epigenetic modification. What’s more, other genes in this pathway, GAD1 and GAD2, are also candidate genes for schizophrenia. The three genes are registered in SZgene database. Whereas GAT1, the gene which encoding GAT in brain is still under determined.Schizophrenia is caused by both genetic and environmental factors, which mediated by epigenetic regulation. GAD genes are well known in GABAergic system for schizophrenia, and various studies including epigenetics on GAD1 have been reported. Dong and Tremolizzo implemented L-methionine (MET) treatment of mice to generate some schizophrenia-like behavioral changes, with promoter hypermethylation as well as mRNA expression downregulation of RELN and GAD1 in GABAergic neurons. What’s more, when intervened with Valproate acid (VPA), those abnormal behaviors were gone and promoter methylation level of RELN decreased.Our previous work on GABRB2 also revealed an imprinting phenomenon that allele specific methylation at the disease-associated SNPs in introns 8 and 9, which indicates methylation alteration as a plausible mechanism underlying schizophrenia. In addition, we have found a hypermethylation at GABRB2 specific promoter regions in schizophrenia patients than normal people.Zebrafish is a booming model animal which is highly homologous to human, easy to be maintained, observed and controlled over. What’s more, despite of similar brain structure, zebrafish also share a conserved regulatory processes underlie behaviors with human, which makes zebrafish ideal for neuropsychiatric disease study. Speaking of GABAergic system, it is also the major inhibitory neurotransmitter in zebrafish brain with a similar distribution of both GAD protein and GABA receptors; and GABA (A) receptor could mediate fast synaptic transmission as well.The most successful utilization of zebrafish for neuropsychiatric disease studying is depression. First, different methods could be used for disease modeling, such as chronic stress method, drug implement method and gene edition method. Second, a rounded behavioral testing system was established, including novel tank test, light/ dark box test and other tests. Third, a chemical mensuration of cortisol and 5-HT could reflect the pathophysiological state of the zebrafish depression model. More meaningfully, the further drug screening test is available now. To our knowledge, few zebrafish schizophrenia modeling studies have been reported via+MK-801 to antagonize NMD A.Considering all above, we intend to focus on gabrb2 (GABRB2 in human) for complementing the promoter methylation and mRNA expression dynamics data during zebrafish development first; then treat adult zebrafish with MET for schizophrenia modeling and comprehensive analysis of any triggered schizophrenia-like behaviors with promoter methylation changes of GABAergic system genes (gabrb2; gad1b, GAD1 in human; gad2, GAD2 in human; scl6a1b, GAT1 in human); in order to better understand the plausible epigenetic mechanism of GABAergic system underlying schizophrenia pathogenesis.Materials and methods1. Animals:Wildtype AB strain zebrafish were obtained from and maintained in the department of developmental biology of Southern Medical University. For general reproduction,6-18 months old fish used, and for MET treatment,6-11 months old fish used with approximate body length.2. Methods:(1) Both the embryo and adult fish were taken care of as Westerfield suggested. Important parameters as followed:maintaining fish room of light:dark= 14:10; maintaining system (or tank) water of pH 7.2-7.6,28.5-29.5 ℃, conductivity 450-550 μs/cm.(2) Methylation dynamic pattern during zebrafish development was assayed with Hpa Ⅱ/Msp Ⅰ enzyme digestion followed by Fluorescence polarization based measurement of DNA methylation (FPDM) method for global genome, and quantitative real-time PCR (qPCR) method for gabrb2 specific promoter regions.(3) gabrb2 mRNA expression pattern during zebrafish development was assayed with qPCR method after total RNA reverse transcript into total cDNA.(4) gabrb2 mRNA distribution during zebrafish central nervous system (CNS) development was assayed with Whole-mount in-situ hybridization (WISH). In the present work, we checked eight important time points.(5) Drug treatment was carried out with three concentrations of 1.5 mM,3.0 mM and 6.0 mM for 7 days and MET replacement once each day.(6) Three Behavioral tests performed, all of which were restricted to 4.5-9 hours after light onset (13:00-18:00), with 30 minutes pre-acclimation in the testing room. EthoVision 2.1 software was used to analyze video data for novel tank test and social interaction test, whereas T-maze test was observed by blind trained examiner. Novel tank test was for general mental status and locomotor activity measurement. Social interaction test was for social preference judgment. T-maze tank test was for cognitive ability assay.(7) After MET exposure, methylation changes of global genome was detected by FPDM, and specific promoter regions of gabrb2, gad1b, gad2 and slc6a1b was detected by qPCR. Corresponding mRNA expression changes was assayed with qPCR method after total RNA reverse transcript into total cDNA.3. Statistics:Statistical analyses were carried out using SPSS 20:data of developmental methylation and expression, novel tank test, MET-treated methylation and expression were analyzed by one-way analysis of variance (ANOVA) or Brown-Forsythe analysis, followed by LSD or Dunnett T3 as post hoc test; data of social interaction test, and T-maze test were analyzed by independent-samples t test; Correlation of methylation and expression was analyzed by Spearman’s correlation method;p< 0.05 was considered statistically significant.Results1. We identified a significant increased global methylation pattern during zebrafish development, with a relatively greater variance in the beginning. After pre-GABA neuron period, the global methylation level was stabilized with limited increase.2. For gabrb2 promoter region, two target CCGG sites showed a similar significant decreasing methylation pattern during zebrafish development, while-1336 site seemed steeper. gabrb2 mRNA expression pattern during zebrafish development was decreased in the very beginning with a following increasing later, and the turning point is 16 hpf-24 hpf. What’s more, adult zebrafish expression is much higher than larvae.3. WISH results were in accord with qPCR results. The original signal appeared at 24 hpf, located in the forebrain, midbrain, boundary of midbrain and hindbrain; after 2 dpf, hindbrain also showed WISH signal; during 3 dpf-6 dpf, both the signal and brain structure were stable.4. Behavioral testing revealed that high dose of MET exposure induced a social interaction deficit of zebrafish which is the core negative symptom of schizophrenia, as well as an impaired learning and memory which is the cognitive symptom of schizophrenia, meanwhile, lowerd the anxiety level, but unchanged locomotor activity of zebrafish.5. MET exposure elevated methylation level of global genome as well as promoter regions of four target genes involved in GABAergic transmission pathway, especially at gabrb2-1336 CCGG site and gadlb-1397 CCGG site, however, still failed to change mRNA expression of four target genes involved in GABAergic transmission pathway significantly.6. The significant correlation of promoter methylation (-1336 CCGG site) with mRNA expression of gabrb2 in developmental zebrafish demonstrated that gabrb2 mRNA expression was under regulated by DNA methylation. However, in MET-treated adult zebrafish, no any correlation was observed between promoter methylation and mRNA expression. Methylation at gad1b-1397 CCGG site shared no correlation with mRNA expression either.Conclusion1. The methylation of gabrb2 promoter (especially at-1336 CCGG site) could negatively regulate its mRNA expression, especially after GABAergic neurons are detected.2. High dose of MET exposure was able to mimic some negative symptom and cognitive symptom of schizophrenia in zebrafish, which could be looked as a schizophrenia model.3. Disruptions in the epigenetic regulation of gabrb2 and gad1b genes expression (methylation at gabrb2-1336 and gad1b-1397 CCGG sites in the present work), as well as hypermethylation promoter of GABAergic transsimition genes were part of the mechanism underling this schizophrenia model. |
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