Epigenetic Histone Methylation of PPARγ and CPT1A Signaling Contributes to Betahistine Preventing Olanzapine-Induced Dyslipidemia.

As a partial histamine H1 receptor agonist and H3 antagonist, betahistine has been reported to partially prevent olanzapine-induced dyslipidemia and obesity through a combination therapy, although the underlying epigenetic mechanisms are still not known. Recent studies have revealed that histone regulation of key genes for lipogenesis and adipogenesis in the liver is one of the crucial mechanisms for olanzapine-induced metabolic disorders. This study investigated the role of epigenetic histone regulation in betahistine co-treatment preventing dyslipidemia and fatty liver caused by chronic olanzapine treatment in a rat model. In addition to abnormal lipid metabolism, the upregulation of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein (C/EBPα), as well as the downregulation of carnitine palmitoyltransferase 1A (CPT1A) in the liver induced by olanzapine, were significantly attenuated by betahistine co-treatment. In addition, betahistine co-treatment significantly enhanced the global expression of H3K4me and the enrichment of H3K4me binding on the promoter of Cpt1a gene as revealed by ChIP-qPCR, but inhibited the expression of one of its site-specific demethylases, lysine (K)-specific demethylase 1A (KDM1A). Betahistine co-treatment also significantly enhanced the global expression of H3K9me and the enrichment of H3K9me binding on the promoter of the Pparg gene, but inhibited the expression of two of its site-specific demethylases, lysine demethylase 4B (KDM4B) and PHD finger protein 2 (PHF2). These results suggest that betahistine attenuates abnormal adipogenesis and lipogenesis triggered by olanzapine through modulating hepatic histone methylation, and thus inhibiting the PPARγ pathway-mediated lipid storage, while at the same time promoting CP1A-mediated fatty acid oxidation.

Epigenetic histone acetylation modulating prenatal Poly I:C induced neuroinflammation in the prefrontal cortex of rats: a study in a maternal immune activation model.

Introduction: Neuroinflammation in the central nervous system, particularly the prefrontal cortex (PFC), plays a role in the pathogenesis of schizophrenia, which has been found to be associated with maternal immune activation (MIA). Recent evidence suggests that epigenetic regulation involves in the MIA-induced neurodevelopmental disturbance. However, it is not well-understood how epigenetic modulation is involved in the neuroinflammation and pathogenesis of schizophrenia. Methods: This study explored the modulation of histone acetylation in both neuroinflammation and neurotransmission using an MIA rat model induced by prenatal polyriboinosinic-polyribocytidylic acid (Poly I:C) exposure, specifically examining those genes that were previously observed to be impacted by the exposure, including a subunit of nuclear factor kappa-B (Rela), Nod-Like-Receptor family Pyrin domain containing 3 (Nlrp3), NMDA receptor subunit 2A (Grin2a), 5-HT2A (Htr2a), and GABAA subunit ß3 (Gabrb3). Results: Our results revealed global changes of histone acetylation on H3 (H3ace) and H4 (H4ace) in the PFC of offspring rats with prenatal Poly I:C exposure. In addition, it revealed enhancement of both H3ace and H4ace binding on the promoter region of Rela, as well as positive correlations between Rela and genes encoding histone acetyltransferases (HATs) including CREB-binding protein (CBP) and E1A-associated protein p300 (EP300). Although there was no change in H3ace or H4ace enrichment on the promoter region of Nlrp3, a significant enhancement of histone deacetylase 6 (HDAC6) binding on the promoter region of Nlrp3 and a positive correlation between Nlrp3 and Hdac6 were also observed. However, prenatal Poly I:C treatment did not lead to any specific changes of H3ace and H4ace on the promoter region of the target genes encoding neurotransmitter receptors in this study. Discussion: These findings demonstrated that epigenetic modulation contributes to NF-κB/NLRP3 mediated neuroinflammation induced by prenatal Poly I:C exposure via enhancement of histone acetylation of H3ace and H4ace on Rela and HDAC6-mediated NLRP3 transcriptional activation. This may further lead to deficits in neurotransmissions and schizophrenia-like behaviors observed in offspring.

The long-lasting effects of early antipsychotic exposure during juvenile period on adult behaviours - A study in a poly I:C rat model.

Second generation antipsychotic drugs including aripiprazole, olanzapine and risperidone are prescribed increasingly (mostly off-label) to treat various mental disorders in children and adolescents. Early treatment with antipsychotics during this period may have long-lasting behavioural impacts, but to date there have been only limited investigations. Maternal infection could be implicated in the aetiology of various mental disorders including schizophrenia. Exposure of pregnant rodents to polyriboinosinic-polyribocytidylic acid (Poly I:C) causes schizophrenia-like behavioural abnormalities and neurodevelopmental conditions such as autism spectrum disorders in offspring. This study, using a Poly I:C rat model, investigated the long-lasting effects of early aripiprazole, olanzapine and risperidone treatment in the childhood/adolescent period (postnatal day 22-50) on adult behaviours of male rats. The study showed that early treatment with three antipsychotics had different effects on long-term behavioural changes in adults. Prenatal Poly I:C exposure (5 mg/kg) at gestation day 15 caused deficits in pre-pulse inhibition and social interaction, as well as cognitive impairments, that could be partially improved by early antipsychotic treatment in the juvenile period. Early antipsychotic treatment during the childhood-adolescent period resulted in similar long-lasting effects on pre-pulse inhibition, anxiety- and depressive-related behaviours in both Poly I:C and healthy (control) male rats. Overall, these results suggest that both prenatal Poly I:C exposure and early antipsychotic treatment in the childhood/adolescent period had long-lasting effects on adult behaviours of male rats, while early antipsychotic treatment could partly prevent the onset of behavioural abnormalities resulting from prenatal insult.

Effects of Risperidone and Prenatal Poly I:C Exposure on GABAA Receptors and AKT-GSK3ß Pathway in the Ventral Tegmental Area of Female Juvenile Rats.

The ventral tegmental area (VTA) in the ventral midbrain is the origin of the dopaminergic neurotransmission pathways. Although GABAA receptors and AKT-GSK3ß signaling are involved in the pathophysiology of mental disorders and are modulated by antipsychotics, an unmet task is to reveal the pathological changes in these biomarkers and antipsychotic modulations in the VTA. Using a juvenile polyriboinosinic-polyribocytidylic acid (Poly I:C) psychiatric rat model, this study investigated the effects of adolescent risperidone treatment on GABAA receptors and AKT/GSK3ß in the VTA. Pregnant female Sprague-Dawley rats were administered Poly I:C (5mg/kg; i.p) or saline at gestational day 15. Juvenile female offspring received risperidone (0.9 mg/kg, twice per day) or a vehicle from postnatal day 35 for 25 days. Poly I:C offspring had significantly decreased mRNA expression of GABAA receptor ß3 subunits and glutamic acid decarboxylase (GAD2) in the VTA, while risperidone partially reversed the decreased GAD2 expression. Prenatal Poly I:C exposure led to increased expression of AKT2 and GSK3ß. Risperidone decreased GABAA receptor ß2/3, but increased AKT2 mRNA expression in the VTA of healthy rats. This study suggests that Poly I:C-elicited maternal immune activation and risperidone differentially modulate GABAergic neurotransmission and AKT-GSK3ß signaling in the VTA of adolescent rats.

Prenatal Poly I:C Challenge Affects Behaviors and Neurotransmission via Elevated Neuroinflammation Responses in Female Juvenile Rats.

BACKGROUND: Exposure to polyriboinosinic-polyribocytidylic acid (Poly I:C) in pregnant rats has been reported to cause schizophrenia-like behaviors and abnormal neurotransmissions in adult, particularly male, offspring. However, what is less well understood are the effects of maternal Poly I:C exposure on adolescent behaviors and neurotransmission in female juvenile rats. METHODS: Female adolescent Poly I:C offspring were constructed by treating with 5 mg/kg Poly I:C on timed pregnant rats (gestation day 15). A battery of behavioral tests was conducted during postnatal day 35-60. Neurotransmitter receptors and inflammation markers in brain regions were evaluated by RT-qPCR on postnatal day 60. RESULTS: Open field, elevated plus maze, and forced swimming tests revealed that prenatal Poly I:C exposure led to elevated anxiety-like and depression-like behaviors in female adolescent offspring. Deficits in pre-pulse inhibition and social interaction were also observed. However, the Poly I:C rats had better performance than the controls in the novel object recognition memory test, which demonstrated a behavioral phenotype with improved cognitive function. Prenatal Poly I:C exposure caused brain region-specific elevation of the P2X7 receptor- and NF-κB-NLRP3-IL-1ß inflammatory signaling in female juvenile rats. Prenatal Poly I:C exposure decreased expression of GABAA receptor subunits Gabrb3 in the prefrontal cortex and Gabrb1 and dopamine D2 receptor in the hippocampus, but increased NMDA receptor subunit Grin2a in the prefrontal cortex, 5-HT2A in the hippocampus, and Gabrb3 and D2 receptor in the nucleus accumben. CONCLUSIONS: Prenatal Poly I:C challenge causes behavioral deficits and brain-specific neurotransmission changes via elevated neuroinflammation responses in female adolescent offspring rats.

Cevimeline co-treatment attenuates olanzapine-induced metabolic disorders via modulating hepatic M3 muscarinic receptor: AMPKα signalling pathway in female rats.

BACKGROUND: Olanzapine is one of the most commonly used antipsychotic drugs; however, its metabolic disorders are the main obstacle in the clinic. Olanzapine is a potent antagonist of the M3 acetylcholine muscarinic receptor (M3R), while the downregulated hepatic M3R-AMPKα signalling pathway is involved in metabolic disorders. AIM: This study investigated the effects of chronic co-treatment with cevimeline (an agonist of M3Rs) in attenuating olanzapine-induced metabolic disorders and the underlying mechanisms. METHODS: Forty-eight adult female Sprague-Dawley rats were treated orally with olanzapine (2 mg/kg, 3 times/day (t.i.d.)) and/or cevimeline (9 mg/kg, t.i.d.), or control (vehicle) for 9 weeks. RESULTS: Cevimeline co-treatment significantly attenuated olanzapine-induced body weight gain and glucolipid metabolic disorders. Importantly, cevimeline co-treatment attenuated olanzapine-induced upregulation of M3Rs, while the co-treatment improved olanzapine-induced downregulation of AMPKα in the liver. Cevimeline co-treatment attenuated olanzapine-induced dyslipidaemia by modulating the hepatic M3R-AMPKα downstream pathways. Cevimeline co-treatment also improved lower activated AKT-GSK3ß signalling to reverse impairment of glucose metabolism and insulin resistance caused by chronic olanzapine treatment. CONCLUSION: These results not only support the important role of M3R antagonism and its related AMPKα and downstream pathways in antipsychotic-induced metabolic disorders but also indicate that these pathways might be promising targets for pharmacological intervention to control these side effects caused by antipsychotic therapy.

Epigenetic histone modulations of PPARγ and related pathways contribute to olanzapine-induced metabolic disorders.

The antipsychotic drug olanzapine is widely used in the treatment of schizophrenia, bipolar and other mental disorders; however, it causes serious metabolic disorders, including dyslipidemia. Our previous studies have identified that olanzapine activated expression of the sterol regulatory element binding transcription factor 1 (SREBP-1) gene, a key transcriptional factor for lipogenesis in the liver and adipocytes. SREBP-1 has been reported to positively regulate the peroxisome proliferator-activated receptor gamma (PPARγ), a master regulator in the process of adipogenesis. This study aimed to investigate epigenetic modulations of the hepatic PPARγ pathway in olanzapine-induced lipid dysfunctions. Olanzapine led to significant increases of body weight gain, white adipose tissue, fasting triglyceride, and fat accumulation in the liver. A significant upregulation of PPARγ was observed in olanzapine-treated rats. ChIP-deep sequencing showed the increase of H3K4me2 binding on the whole gene loci of key regulators of adipogenesis and lipogenesis, the Pparg, Srebp-1, Cebps families (Cebpa, Cebpb and Cebpd), the Signal transducer and activator of transcription 5 families (Stat5a and Stat5b) and Klfs families (Klf9 and Klf15), as well as muscarinic M3 receptor (Chrm3). ChIP-qPCR revealed that H3K9me3 binding on the promoter of Pparg2 was significantly decreased. Consistently, KDM4B, KDM1A and PHF2, the three histone demethylases responsible for site-specific erasure of H3K9me, was increased in olanzapine-treated rats. These results suggested that olanzapine acted as stimuli to trigger the cascade of adipogenesis and lipogenesis through modulating hepatic histone modifications and subsequently upregulating key transcriptional factors. These findings provided new insight into effective strategies for the prevention and treatment of metabolic side-effects induced by antipsychotic medication.

Different expression profile of mRNA and long noncoding RNA in autoimmune thyroid diseases patients.

Increasing evidence has found that long noncoding RNAs (lncRNAs) and message RNAs (mRNAs) play an important role in the progress of autoimmune thyroid diseases (AITD). So, in this study, the different expressed of lncRNA and mRNA was screened by microarray analysis and quantitative real-time polymerase chain reaction (PCR). To further investigate the relationship among the differentially expressed genes, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene ontology (GO) were used for biofunctions and signaling pathways analysis, respectively. Finally, the interaction relationship between lncRNA and mRNAs was analysis with lncRNA-mRNA coexpression network. The result found that the abnormal expression of lncRNAs and mRNAs were 1615 and 1913, respectively. The altered genes included CD40LG, IFNG, CTLA4, FAS, STAT1, STAT3, and STAT4. These were enriched in presentation and antigen processing, Th1 and Th2 cell differentiation, natural killer cell-mediated cytotoxicity, B cell receptor signaling pathway, Th17 cell differentiation, and cell adhesion molecules (CAMs), all of which had been suggested to be associated with immunopathogenic mechanisms and AITD-induced pathophysiologic changes. A coexpression network profile was contained with 126 network nodes and 477 connections which were based on seven mRNAs and 119 interacted lncRNAs. The outcomes of differentially expressed lncRNAs and their coreralated mRNAs in our study revealed that lncRNAs involved in immunopathogenic mechanisms may play a crital role in the pathogenesis of AITD.

[Analysis of GCDH gene mutations in 3 patients from Fujian area with glutaric academia type I].

OBJECTIVE: To explore clinical features and mutation types in patients from Fujian area with glutaric academia type I(GA I). METHODS: Serum acylcarnitine and urine organic acid of 3 patients were determined with tandem mass spectrometry and gas chromatographic mass spectrometry. The patients also underwent magnetic resonance imaging analysis for the cranial region. Genomic DNA was extracted from peripheral blood samples, and the 12 exons and flanking regions of the GCDH gene were amplified with PCR and subjected to direct DNA sequencing. One hundred healthy newborns were used as controls. RESULTS: Mutations of the GCDH gene were identified in all of the 3 patients. Two patients have carried compound heterozygous mutations including c.1244-2A>C and c.1147C>T(p.R383C), c.406G>T(p.G136C) and c.1169G>A(p.G390E), respectively. One has carried homozygous c.1244-2A>C mutation. The same mutations were not detected among the 100 healthy newborns. Only one patient received early intervention and did not develop the disease. The other two had irreversible damagesto their intelligence. CONCLUSION: c.1169G>A(p.G390E) is likely pathogenic mutations for GA I patients from Fujianarea. Early screening of neonatal metabolic diseases is crucial for such patients.

Mutation of glucose-6-phosphate dehydrogenase deficiency in Chinese Han children in eastern Fujian.

We aim to investigate the mutation types of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Chinese Han children in eastern Fujian Province.A total of 904 Chinese Han neonates (male: 733 with positive G6PD deficiency and 28 with weakly positive deficiency; female: 73 with positive G6PD deficiency and 70 with weakly positive deficiency) received G6PD screening in our center from January 2014 to December 2016 were included in this study. Additionally, 904 age-matched normal Chinese Han individuals (male: 761; female: 143) were selected as control. Neonatal G6PD deficiency screening was performed through blood sample collection from the heels, using the commercial kits. Multicolor melting curve analysis (MMCA) method was used to determine the G6PD mutation type in the 904 cases. If it failed to detect mutations in the cases with abnormal enzyme activity, the polymerase chain reaction (PCR) and gene sequencing were used to determine the mutation sites. PCR and gene sequencing were used to determine the mutation sites in the 904 individuals with normal enzyme activity. Three most common mutation types in Chinese population were compared between Fujian and other provinces.Among the 904 neonates with abnormal G6PD enzyme activity, 17 mutation types were detected including 15 single point mutations and 7 complex mutations. Three most common mutation types were c.1376G > T, c.1388G > A, and c.95A > G accounted for 72.6% of the total mutations in eastern Fujian.The proportion of mutational types in G6PD and the degree of enzyme activity change in various mutational types were found in the neonates of Fujian Province. Our study may enrich the molecular diagnosis of G6PD deficiency meaning Fujian Province.

[Analysis of CYP21A2 gene mutations among patients with classical steroid 21-hydroxylase deficiency].

OBJECTIVE: To assess the frequencies of CYP21A2 gene mutations among patients from Fujian area with classical 21-hydroxylase deficiency. METHODS: For 19 probands from different families affected with classical steroid 21-hydroxylase deficiency and 74 family members, mutations of the CYP21A2 gene were analyzed with combined nested polymerase chain reaction, Sanger sequencing and multiplex ligation-dependent probe amplification. Time resolved fluorescence immunoassay was performed to determine the level of 17-hydroxyprogesterone (17-OHP) in all family members. Clinical data and laboratory results of the probands and their family members were analyzed. RESULTS: Eleven mutations were identified among the 38 alleles from the 19 probands. 92.1% (35/38) of the mutant CYP21A2 alleles were due to recombination between CYP21A2 and CYP21A1P. Gene conversion and deletions were identified in 84.2% (32/38) and 7.9% (3/38) of the alleles, respectively. IVS2-13A/C>G and chimeras were the most common mutations, which respectively accounted for 34.2% (13/38) and 18.4% (7/38) of all mutant alleles. Among these, IVS2+1G>A and Q318X+356W were first reported in China. 74.3% (55/74) of the family members were carriers of heterozygous mutations. However, no significant difference was found in the 17-OHP levels between carriers and non-carriers (P>0.05). CONCLUSION: There seems to be a specific spectrum of CYP21A2 gene mutations in Fujian area, where IVS2-13A/C>G and chimeras are the most common mutations.

[Association of thyroperoxidase gene polymorphisms with dyshormonogenesis in congenital hypothyroidism].

OBJECTIVE: To assess the association of thyroperoxidase (TPO) gene polymorphisms with dyshormonogenesis in congenital hypothyroidism (CH). METHODS: The 17 exons and flanking introns of the TPO gene from 30 randomly selected samples were sequenced for the selection of single nucleotide polymorphisms (SNPs). In 136 patients with dyshormonogenetic CH and 141 healthy controls from the same region, the selected SNPs were genotyped by polymerase chain reaction (PCR) and direct sequencing or PCR-restriction fragment length polymorphism (RFLP). RESULTS: Six SNPs (rs9678281, rs376413622, rs1126797, rs4927611, rs732609 and rs1126799) were selected to determine the genotype for each sample. Among these, rs4927611 and rs732609 showed a significant difference between the two groups in both allelic and genotypic frequencies. With a recessive model of inheritance, rs732609 CC (OR=0.484, 95%CI: 0.253-0.927, P=0.04) and rs4927611 TT (OR=0.32, 95%CI: 0.112-0.915, P=0.047) were greater in the patients. CONCLUSION: rs4927611 and rs732609 may be associated with dyshormonogenetic CH. rs4927611 TT and rs732609 CC are genotypes associated with potential risk for the disease.

[Risk factors of 125 cases of neonatal congenital hypothyroidism during perinatal period].

OBJECTIVE: To understand the relationship between perinatal factors and congenital hypothyroidism (CH) and provide scientific evidence for the prevention of CH. METHODS: A case-control study was conducted among 125 neonates with CH (case group) and 375 neonates without CH (control group) in Fujian Neonatal Screening Center from January in 2012 to December in 2013. Univariate and multivariate logistic regression analysis were performed to identify the risk factors to CH during perinatal period. RESULTS: Univariate logistic regression analysis indicated that compared with control group, gestational hypertension, gestational diabetes mellitus, gestational thyroid disease and older age of mother were the risk factors to CH, the difference was statistically significant (P < 0.05) and the risk of CH was higher in female babies, preterm babies, post-term babies low birth weight babies, macrosomia, twins, babies with birth defects and infection in cases group than those in control group, the difference was statistically significant (P < 0.05). Multivariate logistic analysis showed that older age of mother (OR = 2.518, 95% CI: 1.186-5.347), gestational diabetes mellitus (OR = 1.904, 95% CI: 1.190-3.045), gestational hypothyroidism or hyperthyroidism (OR = 12.883 and 30.797, 95% CI: 2.055-80.751 and 3.309-286.594), preterm birth (OR = 4.238, 95% CI: 1.269-14.155), and post-term birth (OR = 12.799, 95% CI: 1.257-130.327), low birth weight (OR = 3.505, 95% CI: 1.059-11.601), macrosomia (OR = 3.733, 95% CI: 1.415-9.851), twin or multiparous delivery (OR = 5.493, 95% CI: 1.701-17.735), birth defects (OR = 3.665, 95% CI: 1.604-8.371) and fetal distress (OR = 3.130, 95% CI: 1.317-7.440) were the high risk factors to CH (P < 0.05). CONCLUSION: CH was correlated with mother's age, gestational diabetes, gestational thyroid disease as well as neonate's birth weight and gestational age, foetus number, fetal distress and other complicated birth defects at certain degree. More attention should be paid to perinatal care to reduce risk factors and the incidence of CH.

[Genetic analysis of 36 children affected with phenylalanine hydroxylase deficiency from Fujian].

OBJECTIVE: To study the characteristics of phenylalanine hydroxylase gene (PAH) mutations in patients with PAH deficiency in Fujian population. METHODS: Peripheral blood samples of 36 patients and their parents with classical type phenylketouria (PKU) were collected. Genomic DNA was extracted. Following PCR amplification, DNA sequencing was carried out to identify the origins of mutations. RESULTS: Twenty types mutations were identified in 63 of the 72 alleles. The most common mutations were R241C, R408Q and Ex6-96A>G, which respectively accounted for 15.9%, 12.7% and 11.1% of all mutant alleles. The c.189_190dupTGAC mutation was first reported. R241C was associated with 28% of mild hyperphenylalaninemia and R408Q is associated with 25% of classical PKU. CONCLUSION: There is a specific spectrum of PAH gene mutation in Fujian region. R241C, R408Q and Ex6-96A>G are the most common mutations.

A novel dual oxidase maturation factor 2 gene mutation for congenital hypothyroidism.

Congenital hypothyroidism (CH) is caused by thyroid hormone deficiency present at birth. DUOXA2 (dual oxidase maturation factor 2) is one of the prerequisites for thyroid hormone synthesis. The present study explored the novel mutations of DUOXA2 in CH patients. Genomic DNA was extracted from peripheral blood of 47 unrelated CH patients, their family members and 100 healthy controls. All 6 exons and their flanking sequences of the DUOXA2 gene were PCR amplified and sequenced. Sequencing results were compared with the standard. Compound heterozygosity with DUOXA2 gene mutations at c.413-414insA (Y138X) and c.738C>G (Y246X) was identified in one patient, and absent in 100 healthy controls. Among them, the c.413­414insA (Y138X) mutation was a novel one. The patient with the c.413-414insA (Y138X) mutation had mild CH symptoms. This study is the first to report a novel c.413-414insA (Y138X) mutation for CH, thereby expanding the mutational spectrum of the DUOXA2 gene.