A simple microfluidic device for live-imaging of the vertical section of epithelial cells.

We investigated the capability of simple microfluidic devices with trenches having vertical sidewalls for live-cell fluorescence imaging of adherent cells. An epithelial cell line that forms a two-dimensional (2D) sheet was cultured to adhere to the vertical sidewall so that its vertical section can be imaged directly using ordinal inverted-type laser-scanning microscopy. The material and the structure of the device were characterized. We show that the detailed distribution of intracellular organelles, such as microtubules and mitochondria, and of intercellular apparatus, such as claudin and zonula occludens, can be imaged with high spatio-temporal resolution with a single scan.

DNA Methylation Profiling in a Neuroblastoma Cell Line Exposed to the Antipsychotic Perospirone.

INTRODUCTION: Accumulating evidence suggests the importance of epigenetic changes in the brain induced by antipsychotic drugs. However, due to the lack of systematic investigation, their effects on epigenetic status remain largely unclear. During the course of examining the epigenetic effects of antipsychotics, we here focused on perospirone, an atypical antipsychotic drug mainly used in Japan. METHODS: Genomic DNA was obtained from human neuroblastoma cells exposed to 2 different doses of perospirone. Comprehensive DNA methylation analysis was performed using the Infinium HumanMethylation450 BeadChip. RESULTS: Of about 470,000 probes, perospirone exposure changed DNA methylation at 4098 probes. These probes were enriched to genes for neural development. Probes showing hypermethylation were mainly found at gene body and intergenic regions, whereas those that showed hypomethylation were located near promoter regions. Additionally, DNA methylation changes were found in the probes for dopamine receptor 2 and serotonin receptor (HTR) 2A and HTR1A, which are the pharmacological targets of atypical antipsychotics. DISCUSSION: Our comprehensive DNA methylation analyses will contribute to a better understanding of detailed pharmacological actions of perospirone.

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.

Comprehensive DNA methylation analysis of human neuroblastoma cells treated with blonanserin.

Blonanserin is a second-generation antipsychotic drug for schizophrenia. The pharmacological actions of blonanserin are shown to be the antagonism of dopamine receptor 2 and serotonin receptors. However, its molecular mechanisms in brain cells have not been fully characterized. Accumulating evidence suggests that antipsychotic drugs and mood stabilizers show epigenetic effects on a wide range of genes in animal and cellular models. We performed genome-wide DNA methylation analysis targeting 479,814 CpG sites of cultured human neuroblastoma cells administered with blonanserin. We found that 3,057 CpG sites showed statistically significant changes in DNA methylation at two different doses of blonanserin (1.36 nM and 13.6 nM). These included hypermethylated CpG sites that were enriched in genes related to axonogenesis and cell morphogenesis involved in neuron differentiation. We also showed that the global effect on DNA methylome depends on the concentration of the drug. With a high dose of blonanserin, the overall methylation levels across all CpG sites significantly increased. These increases in DNA methylation were prominent in the CpG sites distant from promoter regions. We further examined DNA methylation changes in specific genes implicated for the actions of antipsychotic drugs, such as the dopamine receptor 2 (DRD2) gene and the serotonin receptor 2A (HTR2A) gene. We observed that CpG sites that were located within DRD2 and HTR2A genes were significantly hypermethylated by blonanserin. The DNA methylation changes induced by the treatment with blonanserin will be useful for understanding its pharmacological actions at the cellular level.

Increased l1 retrotransposition in the neuronal genome in schizophrenia.

Recent studies indicate that long interspersed nuclear element-1 (L1) are mobilized in the genome of human neural progenitor cells and enhanced in Rett syndrome and ataxia telangiectasia. However, whether aberrant L1 retrotransposition occurs in mental disorders is unknown. Here, we report high L1 copy number in schizophrenia. Increased L1 was demonstrated in neurons from prefrontal cortex of patients and in induced pluripotent stem (iPS) cell-derived neurons containing 22q11 deletions. Whole-genome sequencing revealed brain-specific L1 insertion in patients localized preferentially to synapse- and schizophrenia-related genes. To study the mechanism of L1 transposition, we examined perinatal environmental risk factors for schizophrenia in animal models and observed an increased L1 copy number after immune activation by poly-I:C or epidermal growth factor. These findings suggest that hyperactive retrotransposition of L1 in neurons triggered by environmental and/or genetic risk factors may contribute to the susceptibility and pathophysiology of schizophrenia.

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.

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.

Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice.

BACKGROUND: There is considerable interest in inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although short interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge, especially by systemic administration. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) by using short hairpin RNA-expressing single-stranded adeno-associated virus 9 (ssAAV9-shRNA). RESULTS: Intraperitoneal administration of ssAAV9-shRNA to neonatal mice resulted in highly effective and specific silencing of a target gene in DRG. We observed an approximately 80% reduction in target mRNA in the DRG, and 74.7% suppression of the protein was confirmed by Western blot analysis. There were no major side effects, and the suppression effect lasted for more than three months after the injection of ssAAV9-shRNA. CONCLUSIONS: Although we previously showed substantial inhibition of target gene expression in DRG via intrathecal ssAAV9-shRNA administration, here we succeeded in inhibiting target gene expression in DRG neurons via intraperitoneal injection of ssAAV9-shRNA. AAV9-mediated delivery of shRNA will pave the way for creating animal models for investigating the molecular biology of the mechanisms of pain and sensory ganglionopathies.

A systematic evaluation of whole genome amplification of bisulfite-modified DNA.

BACKGROUND: Studying DNA methylation profiles in detail should be the first step in epigenetic research. Although sodium bisulfite modification of genomic DNA is the gold standard method for DNA methylation analysis, this method results in the loss of the majority of the DNA material. Whole genome amplification (WGA) of bisulfite-modified DNA is expected to provide a rich source of materials, but its validity has not been thoroughly evaluated. In this study, we evaluated the extent of biased amplification in the WGA of bisulfite-modified DNA and the reproducibility of independent WGA reactions. We performed the multiple displacement amplification-based WGA separately three times. Each experiment included two reactions using 10 or 50 ng of bisulfite-modified DNA as template. DNA methylation levels were compared between WGA products and original bisulfite-modified DNA at about 450,000 CpG sites. RESULTS: Using a sufficient amount of bisulfite-modified DNA for WGA was critical for downstream application. The considerable deviations from original bisulfite-modified DNA were found in the middle range of DNA methylation levels. Distribution of hyper- and hypomethylation were equal, which suggested that the deviation at each CpG site occurred randomly. Averaging the data from independently amplified WGA products dramatically improved the overall quality. CONCLUSIONS: WGA of bisulfite-modified DNA could be a valuable tool for epigenetic research, but careful experimental design and data interpretation are required.

Intraperitoneal AAV9-shRNA inhibits target expression in neonatal skeletal and cardiac muscles.

Systemic injections of AAV vectors generally transduce to the liver more effectively than to cardiac and skeletal muscles. The short hairpin RNA (shRNA)-expressing AAV9 (shRNA-AAV9) can also reduce target gene expression in the liver, but not enough in cardiac or skeletal muscles. Higher doses of shRNA-AAV9 required for inhibiting target genes in cardiac and skeletal muscles often results in shRNA-related toxicity including microRNA oversaturation that can induce fetal liver failure. In this study, we injected high-dose shRNA-AAV9 to neonates and efficiently silenced genes in cardiac and skeletal muscles without inducing liver toxicity. This is because AAV is most likely diluted or degraded in the liver than in cardiac or skeletal muscle during cell division after birth. We report that this systemically injected shRNA-AAV method does not induce any major side effects, such as liver dysfunction, and the dose of shRNA-AAV is sufficient for gene silencing in skeletal and cardiac muscle tissues. This novel method may be useful for generating gene knockdown in skeletal and cardiac mouse tissues, thus providing mouse models useful for analyzing diseases caused by loss-of-function of target genes.

Identification of novel HCV subgenome replicating persistently in chronic active hepatitis C patients.

In an effort to clarify the life cycle of HCV, the HCV genome in liver biopsies taken from chronic active hepatitis C patients undergoing interferon treatment was investigated. Molecular cloning by long distance reverse-transcription polymerase chain reaction (RT-PCR) revealed that the HCV genome in two patients with high viral loads in the liver had in-frame deletions of approximately 2 kb between E1 and NS2, which encode the E1-NS2 fusion protein and six other HCV proteins: core, NS3, NS4A, NS4B, NS5A, and NS5B. Among the remaining 21 chronic active hepatitis C patients, these types of deletion were found in another two patients and in two hepatocellular carcinoma patients. Out-of-frame deletions in the structural region were isolated from the other five patients, but the dominant RT-PCR products were non-truncated genomes. Retrospective analysis of a series of serum samples taken from a patient carrying the subgenome with the in-frame deletion revealed that both the subgenome and the full genome persisted through the 2-year period of investigation, with the subgenome being predominant during this period. Sequence analysis of the isolated cDNA suggested that both the subgenome and the full genome evolved independently. Western blotting analysis of HCV proteins from the HCV subgenome indicated that they were processed in the same way as those from the full genome. HCV subgenomes thus appear to be involved in the HCV life cycle.