Effect of mood stabilizers on DNA methylation in human neuroblastoma cells.

Unraveling the epigenetic status of neuronal cells in the brain is critical to our understanding of the pathophysiology of psychiatric disorders, which may reflect a complex interaction between genetic and environmental factors. Several epigenetic studies of mood disorders have been conducted with postmortem brains. However, proper interpretation of the results is hampered by our scant understanding of the effects of mood stabilizers on the epigenetic status of neuronal cells. We performed both comprehensive and gene-specific analyses to examine DNA methylation in human neuroblastoma SK-N-SH cells treated with three mood stabilizers: lithium, valproate and carbamazepine. Measurement of the level of DNA methylation of about 27 000 CpG sites revealed a profound epigenetic effect of lithium, compared with the two other mood stabilizers. In addition, we found that the mood stabilizers have common epigenetic targets and a propensity to increase DNA methylation. Gene-specific analysis involved detailed analysis of the methylation of promoter regions of SLC6A4 and BDNF, both of which have been reported to show altered DNA methylation in bipolar disorder patients or suicide victims, by extensive bisulfite sequencing. We did not observe significant changes in DNA methylation at BDNF promoter IV. However, we found that CpG sites of SLC6A4, which were hypermethylated in patients with bipolar disorder, were hypomethylated in the neuroblastoma cells treated with mood stabilizers. Our results will contribute to a better understanding of the epigenetic changes associated with mood disorders, and they also provide new insight into the mechanisms of action of mood stabilizers.

Identification and functional characterization of the extremely long allele of the serotonin transporter-linked polymorphic region.

SLC6A4, which encodes the serotonin transporter, has a functional polymorphism called the serotonin transporter-linked polymorphic region (5-HTTLPR). The 5-HTTLPR consists of short (S) and long (L) alleles, each of which has 14 or 16 tandem repeats. In addition, the extralong (XL) and other rare alleles have been reported in 5-HTTLPR. Although they are more frequent in Asian and African than in other populations, the extent of variations and allele frequencies (AFs) were not addressed in a large population. Here, we report the AFs of the rare alleles in a large number of Japanese subjects (N = 2894) consisting of two cohorts. The first cohort (case-control study set, CCSS) consisted of 1366 subjects, including 485 controls and 881 patients with psychosis (bipolar disorder or schizophrenia). The second cohort (the Arao cohort study set, ACSS) consisted of 1528 elderly subjects. During genotyping, we identified 11 novel 5-HTTLPR alleles, including 3 XL alleles. One novel allele had the longest subunit ever reported, consisting of 28 tandem repeats. We named this XL28-A. An in vitro luciferase assay revealed that XL28-A has no transcriptional activity. XL28-A was found in two unrelated patients with bipolar disorder in the CCSS and one healthy subject in the ACSS who did not show depressive symptoms or a decline in cognitive function. Therefore, it is unlikely that XL28-A is associated with psychiatric disorders, despite its apparent functional deficit. Our results suggest that unraveling the complex genetic variations of 5-HTTLPR will be important for further understanding its role in psychiatric disorders.

Promoter Activity-Based Case-Control Association Study on SLC6A4 Highlighting Hypermethylation and Altered Amygdala Volume in Male Patients With Schizophrenia.

Associations between altered DNA methylation of the serotonin transporter (5-HTT)-encoding gene SLC6A4 and early life adversity, mood and anxiety disorders, and amygdala reactivity have been reported. However, few studies have examined epigenetic alterations of SLC6A4 in schizophrenia (SZ). We examined CpG sites of SLC6A4, whose DNA methylation levels have been reported to be altered in bipolar disorder, using 3 independent cohorts of patients with SZ and age-matched controls. We found significant hypermethylation of a CpG site in SLC6A4 in male patients with SZ in all 3 cohorts. We showed that chronic administration of risperidone did not affect the DNA methylation status at this CpG site using common marmosets, and that in vitro DNA methylation at this CpG site diminished the promoter activity of SLC6A4. We then genotyped the 5-HTT-linked polymorphic region (5-HTTLPR) and investigated the relationship among 5-HTTLPR, DNA methylation, and amygdala volume using brain imaging data. We found that patients harboring low-activity 5-HTTLPR alleles showed hypermethylation and they showed a negative correlation between DNA methylation levels and left amygdala volumes. These results suggest that hypermethylation of the CpG site in SLC6A4 is involved in the pathophysiology of SZ, especially in male patients harboring low-activity 5-HTTLPR alleles.

Effects of quetiapine on DNA methylation in neuroblastoma cells.

Epigenetic regulation may be involved in the pathophysiology of mental disorders, such as schizophrenia and bipolar disorder, and in the pharmacological action of drugs. Characterizing the epigenetic effects of drugs is an important step to optimal treatment. We performed comprehensive and gene-specific DNA methylation analyses of quetiapine using human neuroblastoma cells. Human neuroblastoma cells were cultured with quetiapine for 8 days, and DNA methylation analysis was performed using Infinium HumanMethylation27 BeadChip. A total of 1173 genes showed altered DNA methylation. Altered DNA methylation predominantly occurred as hypomethylation within the CpG island compared to DNA isolated from non-treated cells. Gene ontology analysis revealed that these genes were related to the cellular process of intracellular protein binding. There was no common effect of quetiapine with three mood stabilizers (lithium, valproate, and carbamazepine). However, common DNA methylation changes in eight genes, including ADRA1A, which encodes adrenoceptor alpha 1A, were found with quetiapine and lithium treatments. Finally, bisulfite-sequencing analysis revealed that quetiapine decreased the DNA methylation level of the promoter region of SLC6A4, where hypermethylation with bipolar disorder and hypomethylation with mood stabilizers have been reported.

DNA methylation analysis of BDNF gene promoters in peripheral blood cells of schizophrenia patients.

Accumulating evidence suggests that epigenetic alterations in brain-derived neurotrophic factor (BDNF) promoters are associated with the pathophysiology of psychiatric disorders. Epigenetic changes in BDNF were reported not only in brain tissues but also in other tissues, including peripheral blood cells (PBC) and saliva. We examined DNA methylation levels of BDNF promoters I and IV using genomic DNA derived from PBC of healthy controls (n=100), and patients with schizophrenia (n=100), all from the Japanese population, by pyrosequencing. The examined CpG sites were chosen based on previous epigenetic studies that reported altered DNA methylation. We found a significantly higher level of methylation at BDNF promoter I in patients with schizophrenia compared to controls, although the difference was small. Subsequent analysis revealed that in controls, the methylation level of BDNF promoters was associated with sex, and the methylation difference observed in promoter I was more prominent in male patients with schizophrenia. Epigenetic alteration of BDNF in the PBC might reflect the pathophysiology of schizophrenia, and could be a potential biomarker.

Practical synthesis of photochromic diarylethenes in integrated flow microreactor systems.

An effective method for the synthesis of photochromic diarylethenes through the generation of heteroaryllithiums and subsequent reaction with octafluorocyclopentene has been developed by using integrated flow microreactor systems. Reactions can be conducted without using cryogenic conditions by virtue of effective temperature and residence time control, although much lower temperatures (<-78 °C) are needed for batch macroreactions. Moreover, the synthesis of unsymmetrical diarylethenes, which is difficult to achieve when using conventional batch macrosystems, has been accomplished based on the selective introduction of one aryl group to give arylheptafluorocyclopentene followed by the introduction of another aryl group. The productivity of the laboratory-scale system is approximately 0.5 mmol min(-1). Therefore, the present integrated flow microreactor method serves as a practical way of synthesizing various photochromic diarylethene derivatives.