Eri Hashimoto

Neurons show distinctive DNA methylation profile and higher interindividual variations compared with non-neurons

Epigenome information in mammalian brain cells reflects their developmental history, neuronal activity, and environmental exposures. Studying the epigenetic modifications present in neuronal cells is critical to a more complete understanding of the role of the genome in brain functions. We performed comprehensive DNA methylation analysis in neuronal and non-neuronal nuclei obtained from the human prefrontal cortex. Neuronal nuclei manifest qualitatively and quantitatively distinctive DNA methylation patterns, including relative global hypomethylation, differential enrichment of transcription-factor binding sites, and higher methylation of genes expressed in astrocytes. Non-neuronal nuclei showed indistinguishable DNA methylation patterns from bulk cortex and higher methylation of synaptic transmission-related genes compared with neuronal nuclei. We also found higher variation in DNA methylation in neuronal nuclei, suggesting that neuronal cells have more potential ability to change their epigenetic status in response to developmental and environmental conditions compared with non-neuronal cells in the central nervous system.

Neurotoxic potential of haloperidol in comparison with risperidone: implication of Akt-mediated signal changes by haloperidol

Summary.The neurotoxicity of conventional antipsychotic drugs has emerged as a potential pathogenic event in extrapyramidal side effects (EPS) and in their limited efficacy for negative-cognitive symptoms in schizophrenic patients. The atypical antipsychotics, recently developed, have superior therapeutic efficacy to treat not only positive symptoms but negative symptoms and cognitive dysfunctions with much lower potentials of side effects, although the influence of atypical antipsychotics on the regulation of neuronal survival has been less investigated. It is important to clarify the effects of typical and atypical antipsychotics on neuronal survival and their contributions to the therapeutic development and understanding of the pathophysiology of schizophrenia. We measured the neurotoxicity of two antipsychotic drug treatments, haloperidol and risperidone, in primary cultured rat cortical neurons. Immunoblotting and pharmacological agent analyses were used to determine the signal transduction changes implicated in the mechanisms of the neurotoxicity. Haloperidol induced apoptotic injury in cultured cortical neurons, but risperidone showed weak potential to injure the neurons. Treatment with haloperidol also led the reduction of phosphorylation levels of Akt, and activated caspase-3. The D2 agonist bromocriptine and 5-HT2A antagonist, ketanserin attenuated the haloperidol-induced neuronal toxicity. Moreover, brain-derived neurotrophic factor (BDNF) reduced the caspase-3 activity and protected neurons from haloperidol-induced apoptosis. BDNF also reversed the reduced levels of phosphorylation of Akt caused by treatment with haloperidol. Haloperidol but not risperidone induces caspase-dependent apoptosis by reducing cellular survival signaling, which possibly contributes to the differential clinical therapeutic efficacy and expression of side effects in schizophrenia.

Low serum BDNF and food intake regulation: a possible new explanation of the pathophysiology of eating disorders.

BACKGROUND Several lines of evidence suggest that brain-derived neurotrophic factor (BDNF) plays an important role in weight regulation and eating behavior, and poorly balanced diets lead to a decrease in blood BDNF levels. However, studies regarding BDNF blood levels in eating disorders (ED) have yielded inconsistent results. We measured serum concentrations of BDNF and assessed behavior and cognition related to eating in ED patients and control subjects. METHODS Forty female drug-free patients [19 with anorexia nervosa (AN), 21 with bulimia nervosa (BN)], who did not meet the diagnostic criteria for depressive disorder, and 24 age-matched normal control subjects were enrolled in the current study. We evaluated eating-related psychopathology and depressive symptoms using the Eating Disorder Inventory-2 (EDI-2), Eating Attitude Test-26 (EAT-26) and the Hamilton Depression Rating Scale (HDRS), and measured serum BDNF levels by an enzyme-linked immunosorbent assay. RESULTS Compared to normal controls, serum levels of BDNF were significantly reduced in AN, but not in BN. There was a significant positive correlation between serum BDNF levels and BMI in both AN patients (r=.649, p=.003) and BN patients (r=.626, p=.002). However, no correlation between serum BDNF levels and BMI was detected in the controls. Furthermore, there was a significant negative correlation between serum BDNF levels and the oral control subscale scores of EAT in both AN patients (r=-.506, p=.027) and BN patients (r=-.511, p=.018); whereas, no correlation was detected in normal controls. CONCLUSION Our study demonstrated that individuals showing more extreme food intake regulation were those with lower serum BDNF levels. This finding is contrary to that in mice where mice with reduced BDNF levels showed aberrant eating behavior. This result suggests that BDNF is no longer functioning appropriately in ED patients, which could be an important factor in the pathophysiological of ED.

Detection of Chromosomal Structural Alterations in Single Cells by SNP Arrays: A Systematic Survey of Amplification Bias and Optimized Workflow

Background In single-cell human genome analysis using whole-genome amplified product, a strong amplification bias involving allele dropout and preferential amplification hampers the quality of results. Using an oligonucleotide single nucleotide polymorphism (SNP) array, we systematically examined the nature of this amplification bias, including frequency, degree, and preference for genomic location, and we assessed the effects of this amplification bias on subsequent genotype and chromosomal copy number analyses. Methodology/Principal Findings We found a large variability in amplification bias among the amplified products obtained by multiple displacement amplification (MDA), and this bias had a severe effect on the genotype and chromosomal copy number analyses. We established optimal experimental conditions for pre-screening for high-quality amplified products, processing array data, and analyzing chromosomal structural alterations. Using this optimized protocol, we successfully detected previously unidentified chromosomal structural alterations in single cells from a lymphoblastoid cell line. These alterations were subsequently confirmed by karyotype analysis. In addition, we successfully obtained reproducible chromosomal copy number profiles of single cells from the cell line with a complex karyotype, indicating the applicability and potential of our optimized workflow. Conclusions/Significance Our results suggest that the quality of amplification products should be critically assessed before using them for genomic analyses. The method of MDA-based whole-genome amplification followed by SNP array analysis described here will be useful for exploring chromosomal alterations in single cells.

Attenuation of brain derived neurotrophic factor (BDNF) by ethanol and cytoprotective effect of exogenous BDNF against ethanol damage in neuronal cells

Summary.Ethanol-induced cell damage was investigated using human neuroblastomas SH-SY5Y cells, which can be differentiated by retinoic acid. With 100 mM or more of ethanol, cytotoxicity was significantly higher in undifferentiated cells than in differentiated cells. Thus, a severer effect of ethanol was observed in undifferentiated cells. In differentiated cells it was shown that the secreted amount of brain derived neurotrophic factor (BDNF) and the cyclic AMP responsive element binding protein (CREB) activity were significantly reduced by ethanol. These effects may be involved in ethanol-induced cell damage in differentiated cells. It was reported that neurotrophic factors have protective effects and that the hippocampus exclusively was damaged by ethanol. Since SH-SY5Y cell is a cell line (a neuronal cell model) and similar cytotoxic effect of ethanol was observed in both SH-SY5Y and primary culture neuronal cells, it will be favorable to use primary culture cells to test a protective effect of BDNF. Exogenous BDNF was shown to have a protective effect against ethanol-induced damage in primary culture neurons from rat hippocampi.

Neuroprotective effects of Yi-Gan San against beta amyloid-induced cytotoxicity on rat cortical neurons.

INTRODUCTION Recent clinical studies have demonstrated that Yi-Gan San (YGS, Yokukan-San in Japanese), a Chinese herbal medicine, alleviates various dementia-related symptoms. However, Chinese herbal medicines have rarely been investigated scientifically and the underlying mechanism of YGS remains uncertain. In this study, we investigated the effect of YGS on beta amyloid protein (Abeta)-induced cytotoxicity in a primary culture of rat cortical neurons. METHODS Cortical neurons prepared from rat embryos were exposed to Abeta in the presence or absence of YGS. The protective effect of YGS was measured as the % of control (unexposed neurons) by using MTT assay and LDH assay. RESULTS Abeta significantly decreased the number of surviving cortical neurons at a dose of 20 microM and higher. In the presence of 20 microM Abeta, YGS concentrations of 10(-5) g/L (W/V) and higher significantly increased the number of viable neurons. CONCLUSION Our study demonstrated a neuroprotective effect of Yi-Gan San against Abeta-induced cytotoxicity. Since according to traditional herbal medicine beliefs, YGS most likely exerts its clinical effects not through a single constituent but as a mixture of several herbal ingredients, the true mechanism of this neuroprotective action remains unclear. However, our results suggest that this Chinese herbal medicine might be a valuable treatment for clinical symptoms associated with dementia having fewer side effects and possible additional neuroprotective effects in the elderly.

Effect of antidepressants on brain-derived neurotrophic factor (BDNF) release from platelets in the rats.

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family, and enhances the growth and maintenance of several neuronal systems. In addition, BDNF may promote neurogenesis and protect against hippocampal volume loss in depressive disorders. Although first detected in brain, BDNF also exists in peripheral tissues and is mainly stored in platelets and circulates in blood. Recent reports indicate that serum BDNF levels in depressive patients are lower than in control subjects, and antidepressant treatment increases serum BDNF levels in responders. A single report suggests that decreased serum BDNF in major depression is related to mechanisms of platelet BDNF release; however, the mechanisms of changes in BDNF blood levels are still poorly understood. In the present study, we investigated the direct influence of antidepressants on BDNF release from platelets and their effects on serum levels. We used samples of washed platelets prepared from rat blood, and investigated the platelet BDNF release and serum BDNF concentration changes in response to adding antidepressants. We found that BDNF was dose-dependently released from platelets by direct treatment with various kinds of antidepressants in vitro, and serum BDNF concentration was also increased by intravenous antidepressant treatment. These results confirm that BDNF release from platelets is affected by antidepressants, which may relate to the circulating BDNF level change in peripheral blood. The response of BDNF release differs depending on the type and amount of antidepressants, making BDNF a serious candidate as a predictor of antidepressant treatment response.

Quantitative reduction of type I adenylyl cyclase in human alcoholics

The amounts of adenylyl cyclase type I (AC I) were examined in various parts of the postmortem brains from alcoholics who prior to death had been abstinent from alcohol for at least 6 months and compared with controls using immunoblot analysis with anti-AC I specific antibody. It was revealed that a significant reduction of AC I was observed in both frontal and temporal cortices. On the other hand, in other areas (occipital cortex, caudate nucleus, putamen, and hippocampus) the amounts were comparable between alcoholics and controls. In the next step, we examined two subtypes of human AC mRNA levels (AC I and AC VIII) in blood cells by quantitative RT-PCR using [alpha-32P]dCTP with two sets of the synthetic oligonucleotide primers based on the DNA sequences reported elsewhere (Villacres, E.C. et al., Genomics 16 (1993) 473-478; J. Parma et al., Biochem. Biophys. Res. Commun. 179 (1991) 455-462). The amounts of amplified DNAs of both AC I and AC VIII were significantly smaller in alcoholics than in controls. On the other hand, the amounts of amplified DNA of beta-actin DNA were almost equal between alcoholics and controls. It appears from these results that a reduction in the amount of AC subtypes may be a biological marker for alcoholics.

Changes in the cAMP-related signal transduction mechanism in postmortem human brains of heroin addicts.

Summary. Immunoreactivities of adenylyl cyclase (AC) type I (AC-I), and basal, forskolin- and Mn2+-stimulated AC activities with or without calcium and calmodulin (Ca2+/CaM) were estimated in temporal cortex (TC)-and nucleus accumbens (NAc) membranes from brains of heroin addicts and controls. Immunoreactivity of AC-I was significantly decreased in TC from brains of heroin addicts, but that did not change in NAc. Ca2+/CaM-sensitive AC activity was significantly lower in TC from brains of heroin addicts, but that activity in NAc did not show significant difference compared with the control. Some previous reports demonstrated that Ca2+/CaM-sensitive AC activity in membranes from postmortem human brain reflected the function of AC-I. Therefore, the downregulation of AC-I in TC plays an important role in the molecular mechanism of chronic opiate addiction in human brain.

Epidemiological studies of tobacco smoking and dependence in Japan

In this study, we attempted to determine the prevalence of tobacco or nicotine dependence in current smokers in Japan and to assess the relationship between alcoholism and tobacco or nicotine dependence. The subjects consisted of 246 alcohol-dependent and 1,111 non-alcohol-dependent individuals. We used a questionnaire, consisting of items obtained from the World Health Organizations The ICD-10 Classification of Mental and Behavioural Disorders: Clinical Descriptions and Diagnostic Guidelines (ICD-10) and the American Psychiatric Associations Diagnostic and Statistical Manual of Mental Disorders (4th ed.; DSM-IV) criteria for tobacco or nicotine dependence. The prevalence of tobacco dependence diagnosed according to the ICD-10 criteria was 23.9% among all subjects. The prevalence of tobacco dependence diagnosed according to the ICD-10 criteria was higher in alcohol-dependent individuals (58.1%) than in nondrinkers or social drinkers (12.8%). Alcohol-dependent subjects consumed significantly more nicotine per day than did nondrinkers or social drinkers. The prevalence of nicotine physical dependence diagnosed by using DSM-IV criteria for nicotine withdrawal was 2.4% in alcohol-dependent individuals, whereas only 0.3% of nondrinkers or social drinkers exhibited nicotine physical dependence. These results indicate to us that the potential for nicotine physical dependence is not much stronger than that reported among current smokers.