Sleep rebound leads to marked recovery of prolonged sleep deprivation-induced adversities in the stress response and hippocampal neuroplasticity of male rats.

BACKGROUND: Sleep disturbances are not only frequent symptoms, but also risk factors for major depressive disorder. We previously reported that depressed patients who experienced "Hypersomnia" showed a higher and more rapid response rate under paroxetine treatment, but the underlying mechanism remains unclear. The present study was conducted to clarify the beneficial effects of sleep rebound through an experimental "Hypersomnia" rat model on glucocorticoid and hippocampal neuroplasticity associated with antidepressive potency. METHODS: Thirty-four male Sprague-Dawley rats were subjected to sham treatment, 72-h sleep deprivation, or sleep deprivation and subsequent follow-up for one week. Approximately half of the animals were sacrificed to evaluate adrenal weight, plasma corticosterone level, hippocampal content of mRNA isoforms, and protein of the brain-derived neurotrophic factor (Bdnf) gene. In the other half of the rats, Ki-67- and doublecortin (DCX)-positive cells in the hippocampus were counted via immunostaining to quantify adult neurogenesis. RESULTS: Prolonged sleep deprivation led to adrenal hypertrophy and an increase in the plasma corticosterone level, which had returned to normal after one week follow-up. Of note, sleep deprivation-induced decreases in hippocampal Bdnf transcripts containing exons II, IV, VI, and IX and BDNF protein levels, Ki-67-(+)-proliferating cells, and DCX-(+)-newly-born neurons were not merely reversed, but overshot their normal levels with sleep rebound. LIMITATIONS: The present study did not record electroencephalogram or assess behavioral changes of the sleep-deprived rats. CONCLUSIONS: The present study demonstrated that prolonged sleep deprivation-induced adversities are reversed or recovered by sleep rebound, which supports "Hypersomnia" in depressed patients as having a beneficial pharmacological effect.

Modified activity-based anorexia paradigm dampens chronic food restriction-induced hyperadiponectinemia in adolescent female mice.

Anorexia nervosa (AN) is a chronic, life-threatening disease with mental and physical components that include excessive weight loss, persistent food restriction, and altered body image. It is sometimes accompanied by hyperactivity, day-night reversal, and amenorrhea. No medications have been approved specific to the treatment of AN, partially due to its unclear etiopathogenesis. Because adiponectin is an appetite-regulating cytokine released by adipose tissue, we hypothesized that it could be useful as a specific biomarker that reflects the disease state of AN, so we developed a modified AN mouse model to test this hypothesis. Twenty-eight 3-week-old female C57BL/6J mice were randomly assigned to the following groups: 1) no intervention; 2) running wheel access; 3) food restriction (FR); and 4) activity-based anorexia (ABA) that included running wheel access plus FR. After a 10-day cage adaptation period, the mice of the FR and ABA groups were given 40% of their baseline food intake until 30% weight reduction (acute FR), then the body weight was maintained for 2.5 weeks (chronic FR). Running wheel activity and the incidence of the estrous cycle were assessed. Spontaneous food restriction and the plasma adiponectin level were evaluated at the end of the acute and chronic FR phases. An increase in running wheel activity was found in the light phase, and amenorrhea was found solely in the ABA group, which indicates that this is a good model of AN. This group showed a slight decrease in spontaneous food intake accompanied with an attenuated level of normally induced plasma adiponectin at the end of the chronic FR phase. These results indicate that the plasma adiponectin level may be a useful candidate biomarker for the status or stage of AN.

Recovery Sleep Immediately after Prolonged Sleep Deprivation Stimulates the Transcription of Integrated Stress Response-Related Genes in the Liver of Male Rats.

Sleep loss induces performance impairment and fatigue. The reactivation of human herpesvirus-6, which is related to the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), is one candidate for use as an objective biomarker of fatigue. Phosphorylated eIF2α is a key regulator in integrated stress response (ISR), an intracellular stress response system. However, the relation between sleep/sleep loss and ISR is unclear. The purpose of the current study was to evaluate the effect of prolonged sleep deprivation and recovery sleep on ISR-related gene expression in rat liver. Eight-week-old male Sprague-Dawley rats were subjected to a 96-hour sleep deprivation using a flowerpot technique. The rats were sacrificed, and the liver was collected immediately or 6 or 72 h after the end of the sleep deprivation. RT-qPCR was used to analyze the expression levels of ISR-related gene transcripts in the rat liver. The transcript levels of the Atf3, Ddit3, Hmox-1, and Ppp15a1r genes were markedly increased early in the recovery sleep period after the termination of sleep deprivation. These results indicate that both activation and inactivation of ISRs in the rat liver occur simultaneously in the early phase of recovery sleep.

Continuous psychosocial stress stimulates BMP signaling in dorsal hippocampus concomitant with anxiety-like behavior associated with differential modulation of cell proliferation and neurogenesis.

Bone morphogenetic protein (BMP) signaling in the hippocampus regulates psychiatric behaviors and hippocampal neurogenesis in non-stress conditions; however, stress-induced changes in hippocampal BMP signaling have not yet been reported. Therefore, we sought to examine whether psychosocial stress, which induces psychiatric symptoms, affects hippocampal BMP signaling. A total of 32 male Sprague-Dawley rats were exposed to a psychosocial stress using a Resident/Intruder paradigm for ten consecutive days. Subsequently, rats were subjected to a battery of behavioral tests (novelty-suppressed feeding test, sucrose preference test, and forced swimming test) for the evaluation of adult neurogenesis and activity of BMP signaling in the dorsal and ventral hippocampus. Repeated social defeat promoted anxiety-like behaviors, but neither anhedonia nor behavioral despair. Socially defeated rats exhibited an increase in the number of Ki-67-positive cells, decrease in the number of doublecortin (DCX)-positive cells, and decrease only in the dorsal hippocampus of the ratio of DCX-positive to Ki-67-positive cells, a proxy for newly-born cell maturation speed and survival. In contrast, no differences were observed in the number of 5-Bromo-2'-deoxyuridine (BrdU)-positive cells, indicating survival of newly-born cells both in the dorsal and ventral hippocampus. Furthermore, psychosocial stress significantly increased the BMP-4 and phosphorylated Smad1/5/9 expression levels specifically in the dorsal hippocampus. Our findings suggest that repeated psychosocial stress activates BMP signaling and differently affects cell proliferation and neurogenesis exclusively in the dorsal hippocampus, potentially exacerbating anxiety-related symptoms. Targeting BMP signaling is a potential therapeutic strategy for psychiatric disorders.

Chronic administration of quetiapine stimulates dorsal hippocampal proliferation and immature neurons of male rats, but does not reverse psychosocial stress-induced hyponeophagic behavior.

Quetiapine, an atypical antipsychotic, has been used for the treatment of several neuropsychiatric disorders. However, the underlying mechanism of the broad therapeutic range of quetiapine remains unknown. We previously reported that several aversive conditions affect dorsal/ventral hippocampal neurogenesis differentially. This study was aimed to elucidate the positive effects of chronic treatment with quetiapine on regional differences in hippocampal proliferation and immature neurons and behavioral changes under psychosocial stress using the Resident-Intruder paradigm. Twenty-three male Sprague-Dawley rats were intraperitoneally administered a vehicle or quetiapine (10 mg/kg) once daily for 28 days. Two weeks after starting the injections, animals were exposed to intermittent social defeat (four times over two weeks). The behavioral effects of stress and quetiapine were evaluated by the Novelty-Suppressed Feeding (NSF) test. The stereological quantification of hippocampal neurogenesis was estimated using immunostaining with Ki-67 and doublecortin (DCX). Chronic quetiapine treatment stimulated the Ki-67- and DCX-positive cells in the dorsal hippocampus, but not in the ventral subregion. The stress-induced changes in neurogenesis and hyponeophagic behavior were not reversed by repeated administration of quetiapine. Future study with additional behavioral tests is needed to elucidate the functional significance of the quetiapine-induced increase in dorsal hippocampal neurogenesis.

Prolonged sleep deprivation decreases cell proliferation and immature newborn neurons in both dorsal and ventral hippocampus of male rats.

Previous studies have indicated that sleep deprivation negatively affects hippocampal neurogenesis, which may explain the reason for the relation between sleep loss and depression. Increasing evidence indicates that the hippocampus is anatomically and functionally segregated along a dorsolateral (cognitive function)/ventromedial (control for mood and stress response) axis. Thus, the present study was conducted to elucidate regional differences in the adverse effects of sleep deprivation on hippocampal neurogenesis. Male Sprague-Dawley rats were subjected to sleep deprivation using the "platform on the water" method for 24- or 72-h. Quantification of hippocampal cell proliferation and immature newborn neurons was stereologically estimated using immunostaining with Ki-67 and doublecortin (DCX), respectively, by optical fractionator method. A consecutive three days of sleep deprivation significantly reduced the density of Ki-67- and DCX-immunopositive cells both in the dorsal and ventral hippocampal subgranular zone and the decrease in DCX-labeled cells was more pronounced in the ventral hippocampus than in dorsal region. Our results indicate that prolonged sleep deprivation decreases hippocampal cell proliferation and neurogenesis in both the dorsal and ventral dentate gyrus. Future studies will be needed to clarify the impact of sleep deprivation-induced decreases in hippocampal neurogenesis on the development of depression.

A high fat diet-induced decrease in hippocampal newly-born neurons of male mice is exacerbated by mild psychological stress using a Communication Box.

BACKGROUND: Obese persons have a higher incidence of depression than healthy-weight persons. Several studies indicated that the exposure to a high fat diet (HFD) results in a decrease in hippocampal neurogenesis, which leads to higher stress response and stress-induced depression. Although stress is a risk factor for obesity and depression, no studies to date have investigated the effect of stress on the hippocampal neurogenesis of HFD-induced obese animals. The aim of this study was to elucidate whether or not obese HFD-fed mice are vulnerable to stress-induced depression by investigating hippocampal neurogenesis. METHODS: Sixty-four male ICR mice (four weeks of age) were fed a control (N=24) or 45%HFD (N=40) for seven weeks. Of the HFD-fed group, twenty-four mice met the criteria for "diet-induced obesity". The animals were then exposed to three consecutive days of psychological stress using a Communication Box. Half were sacrificed to evaluate the physiological changes, and the other half were perfused to quantify hippocampal neuroblasts/immature neurons by the estimation of doublecortin-immunopositive cells. RESULTS: In the HFD-fed mice, psychological stress resulted in increases in caloric intake and visceral adipose tissue and a significant decrease in doublecortin-positive cells in the dentate gyrus; however, no such differences were found in the control diet-fed group. Limitations Further study using other neurogenic markers to assess the stage-specific changes in hippocampal neurogenesis will be required CONCLUSIONS: Our findings suggest that an HFD-induced decrease in hippocampal newly-born neurons leads to stress vulnerability, which may contribute to a high risk of stress-induced depression for obese persons.

Chronic treatment with tandospirone, a serotonin 1A receptor partial agonist, inhibits psychosocial stress-induced changes in hippocampal neurogenesis and behavior.

BACKGROUND: The 5-hydroxytryptamine (5-HT) 1A receptors are considered a potential target for the treatment of mental and neuropsychiatric disorders. Several studies have indicated that 5-HT1A receptor agonists increase hippocampal neurogenesis, which is implicated in the action mechanism of antidepressants. However, these agents have not been applied to humans due to intolerable side effects. We recently showed that chronic administration of tandospirone, a clinically available 5-HT1A receptor partial agonist, increased hippocampal neurogenesis dose-dependently. The present study was done to determine if chronic tandospirone treatment has antidepressant potential from the standpoint of hippocampal neurogenesis and behavior. METHODS: Male Sprague-Dawley rats were intraperitoneally administered a vehicle or tandospirone (10mg/kg) once daily for 28 days. Two weeks after starting the injections, animals were exposed to intermittent social defeat (four times over two weeks). The effects of stress and tandospirone on the rodents׳ behavior were evaluated by the Novelty-Suppressed Feeding (NSF) test. The quantification of hippocampal neurogenesis was estimated using immunostaining with Ki-67 and doublecortin (DCX). RESULTS: Chronic tandospirone treatment reversed the psychosocial stress-induced increase in the latency in the NSF test and decrease in the density of DCX-positive cells in the dentate gyrus of the dorsal and ventral hippocampus. However, no difference in the density of Ki-67-positive cells was observed between the vehicle- and tandospirone-administered groups. LIMITATIONS: To clarify the antidepressant potential of TDS, the other behavioral tests for depression will be required. CONCLUSIONS: Our findings suggest that tandospirone has antidepressant potential through an inhibiting effect on stress-induced changes in hippocampal neurogenesis.

Chronic Treatment with the 5-HT1A Receptor Partial Agonist Tandospirone Increases Hippocampal Neurogenesis.

INTRODUCTION: A large-scale clinical trial, the Sequence Trial Alternatives to Relieve Depression (STAR*D) study, concluded that about one-third of the studied patients with major depressive disorder remitted during the initial treatment with selective serotonin reuptake inhibitors and that approximately half of the remitted subjects relapsed over a 1-year follow-up. The development of new therapeutic approaches with potent efficacy and good tolerability for the treatment of depressive disorders is of great importance. Adult hippocampal neurogenesis has been proposed to be important for understanding and treating depression and anxiety. The present study aimed to elucidate whether or not 5-hydroxytryptamine 1A (5-HT1A) receptor partial agonists have a potential therapeutic effect for the treatment of depressive and anxiety disorders, from the standpoint of neurogenesis. METHODS: Male Sprague-Dawley rats were subcutaneously administered a vehicle or tandospirone (TDS) (1 or 10 mg/kg) once daily for 14 days. The effects of chronic TDS treatment on neurogenesis were evaluated on the day after the last injection. The quantification of hippocampal neurogenesis was estimated using immunostaining with doublecortin (DCX), a marker protein of newborn neurons. RESULTS: Chronic TDS treatment resulted in a significant increase in the number of DCX-positive cells per volume of dentate gyrus in a dose-dependent manner. CONCLUSION: The results strongly suggest that 5-HT1A receptor partial agonists would be useful and beneficial in the treatment of depressive and anxiety disorders through increased hippocampal neurogenesis.

Severe sleepiness and excess sleep duration induced by paroxetine treatment is a beneficial pharmacological effect, not an adverse reaction.

BACKGROUND: Severe sleepiness and excess sleep duration, "Hypersomnia", induced by paroxetine treatment are generally considered adverse drug reactions, however, our experience indicates that patients with depressive disorder who experience "Hypersomnia" during paroxetine treatment have good clinical response. The aim of this study was to determine if "Hypersomnia" during paroxetine treatment is a beneficial pharmacological effect or an adverse drug reaction, and to investigate the impact of genetic polymorphisms on individual differences in the occurrence of "Hypersomnia" induced by paroxetine. METHODS: A consecutive series of 46 Japanese patients with depressive disorder were treated with paroxetine. Patients who complained of great drowsiness or who slept for more than 12-h per day over seven days were identified as having experienced "Hypersomnia". For the clinical improvement rates and genotype distribution of the circadian locomotor output cycles kaput (CLOCK), serotonin transporter and cytochrome P450 2D6 (CYP2D6), the group that showed "Hypersomnia" induced by paroxetine treatment and the group that did not show "Hypersomnia" were compared statistically. RESULTS: Patients who experienced "Hypersomnia" (17.4%) showed a significantly higher response rate at two weeks than did patients who did not experience "Hypersomnia" (p=0.0127). No significant association between the occurrence of "Hypersomnia" and genetic polymorphisms was found. LIMITATIONS: We cannot exclude the risk of false positive errors due to the relatively small sample sizes. CONCLUSIONS: "Hypersomnia" during paroxetine treatment for depression is a beneficial pharmacological effect, not an adverse drug reaction.

Reduction of group II metabotropic glutamate receptors during development of benzodiazepine dependence.

Prolonged use of benzodiazepines often leads to dependence and withdrawal syndrome. However, the cellular mechanisms underlying benzodiazepine dependence have not been fully clarified. Several investigators have shown an involvement of metabotropic glutamate receptors (mGluRs) in the pathophysiology of dependence or withdrawal. This study was performed to elucidate the role of mGluRs in benzodiazepine dependence. Withdrawal signs were precipitated in mice by flumazenil injection (25 mg/kg) after continuous subcutaneous infusion of benzodiazepines for 7 days, and the effects of several Gi-coupled receptor ligands on forskolin-stimulated cyclic AMP accumulation were examined in the cerebral cortex of mice. The mRNA expression for mGluRs was determined by RT-PCR. A single injection of flumazenil precipitated typical withdrawal signs such as tail elevation and tremor in mice treated with diazepam or alprazolam, but not quazepam. The inhibitory effect of nonselective mGluR ligands on adenylate cyclase activity was diminished in mice that showed signs of benzodiazepine withdrawal. The mRNA expression levels of mGluR2 and mGluR3 were lowered in the cerebral cortex of mice pretreated with diazepam or alprazolam. Our findings suggest that the reduction in the expression of group II mGluRs subunits may be involved in the development of benzodiazepine dependence.

Effects of the serotonin 1A, 2A, 2C, 3A, and 3B and serotonin transporter gene polymorphisms on the occurrence of paroxetine discontinuation syndrome.

Paroxetine discontinuation symptoms can at times be severe enough to reduce the quality of life. However, it is currently not possible to predict the occurrence of discontinuation syndrome before the initiation or discontinuation of paroxetine treatment. In this study, we investigated the effects of genetic polymorphisms in the serotonin 1A, 2A, 2C, 3A, and 3B receptor, the serotonin transporter, and the cytochrome P450 2D6 (CYP2D6) genes on the occurrence of paroxetine discontinuation syndrome. A consecutive series of 56 Japanese patients who had a diagnosis of major depressive or anxiety disorder according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, were treated with paroxetine. Paroxetine discontinuation syndrome was found in 35.7% of the patients by direct interview. Patients who stopped taking paroxetine abruptly experienced paroxetine discontinuation syndrome significantly more often than patients who had a tapering off of the dosage of medication. Patients who had the -1019C allele experienced paroxetine discontinuation syndrome more frequently than patients who had the -1019G homozygote (nominal P = 0.0423) of the serotonin 1A receptor gene. However, this result did not remain significant after the Bonferroni correction for multiple comparisons. The findings suggest that the abrupt stoppage of medication is a major risk factor for the occurrence of paroxetine discontinuation syndrome and that C(-1019)G polymorphism of the serotonin 1A receptor gene may be related to the occurrence of the syndrome.

Effect of smoking and CYP2D6 polymorphisms on the extent of fluvoxamine-alprazolam interaction in patients with psychosomatic disease.

PURPOSE: Fluvoxamine (FVX) is metabolized by cytochrome P450 (CYP) 2D6 and CYP1A2 and inhibits CYP3A4. The aim of this study was to investigate the factors responsible for interindividual variability in the extent of interaction between FVX and alprazolam (ALP). METHODS: Blood samples were taken from 49 depressive patients to determine plasma concentration of FVX, ALP or both. Twenty-four samples were taken during the FVX-alone period, 21 samples during the ALP-alone period and 30 samples during the FVX-ALP period. Subjects were also genotyped for CYP2D6. RESULTS: The concentration-to-dose (C/D) ratio of ALP during the FVX-treatment period was significantly higher than that during the ALP-alone period. The CYP2D6 genotype affected neither the C/D ratios of FVX nor the extent of interaction. The mean C/D ratio of FVX in smokers was reduced by more than 30% in comparison with that in non-smokers. The mean C/D ratio of ALP in non-smokers was increased by FVX, while that in smokers was unchanged. CONCLUSIONS: The extent of interaction between FVX and ALP may be affected by smoking, which alters the C/D ratio of FVX. Therefore, when FVX and ALP are concomitantly administered, it should be noted that non-smokers may exhibit greater drug interaction than smokers.

Genetic polymorphisms in the 5-hydroxytryptamine type 3B receptor gene and paroxetine-induced nausea.

Selective serotonin reuptake inhibitor (SSRI)-induced nausea can be severe enough to lead to early treatment discontinuation. However, it is currently not possible to predict the occurrence of nausea before the initiation of SSRI treatment. In this study, we investigated the effect of genetic polymorphisms in the 5-hydroxytryptamine type 2A, 3A, and 3B (5-HT3B) receptors, 5-HT transporter, and CYP2D6 genes on the incidence of paroxetine-induced nausea. A consecutive series of 72 Japanese patients with depressive or anxiety disorders were treated with paroxetine. Paroxetine-induced nausea was assessed by a pharmacist and was observed in 29.2% of the patients. A significant (nominal p=0.00286) association was found between the incidence of nausea and the -100_-102AAG insertion/deletion polymorphism of the 5-HT3B receptor gene. No significant associations were observed between the other genetic polymorphisms and the incidence of nausea. The -100_-102AAG deletion variant of the 5-HT3B receptor gene may affect paroxetine-induced nausea.

Edrophonium provocative testing for the evaluation of upper gastrointestinal hypersensitivity in patients with nonulcer dyspepsia.

The aim of this study was to examine if edrophonium provocative testing is useful for evaluating upper gastrointestinal hypersensitivity in patients with nonulcer dyspepsia (NUD). A questionnaire rating dyspeptic symptoms was done for 58 patients with NUD. The patients were then given an intravenous infusion of saline followed by 5 mg of edrophonium. Baseline esophageal manometry was also done. Patients whose usual symptoms were reproduced (48.3%) had significantly higher symptom scores (13.0 [8.5, 17.0] vs. 8.5 [6.0, 11.0]; P = 0.015) and a significantly higher number of symptoms (4.0 [2.5, 6.0] vs. 3.0 [1.0, 4.0]; P = 0.010) than patients whose usual symptoms were not reproduced. The presence of an esophageal motility disorder was not significantly different between the two groups. These findings suggest upper gastrointestinal hypersensitivity in the patients whose symptoms were reproduced. Edrophonium provocative testing might be useful for evaluating upper gastrointestinal hypersensitivity in patients with NUD.

The pharmacokinetics and pharmacodynamics of tandospirone in rats exposed to conditioned fear stress.

The 5-HT1, agonist tandospirone is generally thought to have a weak anxiolytic effect with a slow onset of action. Our recent clinical study suggested that a comparatively high dose of tandospirone has excellent anxiolytic efficacy and is without significant adverse effects. The present study was designed to clarify the relationship between the anxiolytic effect of tandospirone and its plasma and brain concentrations. The anxiolytic effect was estimated by determining the conditioned fear stress-induced freezing behavior in rats after tandospirone administration. Obvious correlations between anxiolytic effect and brain concentration of tandospirone were observed 0.5 and 4 h after tandospirone administration, while the anxiolytic effect was dependent on the plasma concentration of at 0.5 h but not 4 h after tandospirone administration. The plasma concentration was significantly correlated with the brain concentration. These findings suggest that the potency of the anxiolytic effect is dependent on both the plasma and brain concentration.

[Enlightenment activities about home palliative care for a terminal stage of cancer].

In Palliative and End-of-Life Care, it is very important that we support cancer patients being at home as well as for the removal of physical and psychological symptoms. In doing so, Hyogo Prefectural Awaji Hospital and Awaji Medical Association founded the "Awaji Palliative Care Team" in April, 2004. We have conducted a monthly study, for a total of 34 times to date. And we have an open citizen lecture 3 times a year. A doctor from Awaji Medical Association or a nurse from a home nursing station are the main lecturers on "home medical care". A doctor from the Hyogo Prefectural Awaji Hospital gave a special lecture "the latest cancer treatment". We also invited experts of palliative care from outside of the island and carried out a citizen open lecture. We held a total of 6 special lectures so far.

Proteolytic degradation of glutamate decarboxylase mediates disinhibition of hippocampal CA3 pyramidal cells in cathepsin D-deficient mice.

Although of clinical importance, little is known about the mechanism of seizure in neuronal ceroid lipofuscinosis (NCL). In the present study, we have attempted to elucidate the mechanism underlying the seizure of cathepsin D-deficient (CD-/-) mice that show a novel type of lysosomal storage disease with a phenotype resembling late infantile NCL. In hippocampal slices prepared from CD-/- mice at post-natal day (P)24, spontaneous burst discharges were recorded from CA3 pyramidal cells. At P24, the mean amplitude of IPSPs after stimulation of the mossy fibres was significantly smaller than that of wild-type mice, which was substantiated by the decreased level of gamma-aminobutyric acid (GABA) contents in the hippocampus measured by high-performance liquid chromatography (HPLC). At this stage, activated microglia were found to accumulate in the pyramidal cell layer of the hippocampal CA3 subfield of CD-/- mice. However, there was no significant change in the numerical density of GABAergic interneurons in the CA3 subfield of CD-/- mice at P24, estimated by counting the number of glutamate decarboxylase (GAD) 67-immunoreactive somata. In the hippocampus and the cortex of CD-/- mice at P24, some GABAergic interneurons displayed extremely high somatic granular immunoreactivites for GAD67, suggesting the lysosomal accumulation of GAD67. GAD67 levels in axon terminals abutting on to perisomatic regions of hippocampal CA3 pyramidal cells was not significantly changed in CD-/- mice even at P24, whereas the total protein levels of GAD67 in both the hippocampus and the cortex of CD-/- mice after P24 were significantly decreased as a result of degradation. Furthermore, the recombinant human GAD65/67 was rapidly digested by the lysosomal fraction prepared from the whole brain of wild-type and CD-/- mice. These observations strongly suggest that the reduction of GABA contents, presumably because of lysosomal degradation of GAD67 and lysosomal accumulation of its degraded forms, are responsible for the dysfunction of GABAergic interneurons in the hippocampal CA3 subfield of CD-/- mice.

Functional assessment of ABCG2 (BCRP) gene polymorphisms to protein expression in human placenta.

The aim of the present study was to assess the contribution of polymorphisms in the breast cancer resistance protein/ATP-binding cassette transporter G2 (BCRP/ABCG2) gene to the placental expression from a new perspective, allelic imbalance. Polymorphisms were screened by polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis followed by sequencing with DNA extracted from 100 placentas. To examine whether polymorphisms of the BCRP gene correlate with the placental BCRP expression, we determined mRNA and protein levels by quantitative real-time PCR and Western blotting, respectively. In placentas, G34A (Val(12)Met) and C421A (Gln(141)Lys) were frequently observed (18-36%), but C376T, which creates a stop codon (Gln(126) stop codon), was found with an allelic frequency of 1%. The mean of the BCRP protein level was significantly lower (p < 0.05) in homozygotes for the A421 allele than in those for the C421 allele, and heterozygotes had an intermediate value. To evaluate whether the C421A polymorphism acts as a cis-element in BCRP transcription, allelic imbalance was determined using informative lymphoblasts and 56 samples of placental cDNA. In most of the placental samples we tested, the difference in expression levels between the two alleles was small, and only two samples indicated a monoallelic expression (i.e., preferential expression of one allele). These results suggest that 1) the predominant allelic expression pattern of BCRP in placental samples is biallelic, and 2) the mutation C421A is not a genetic variant acting in cis, but is considered to influence the translation efficiency.

Functional analysis of SNPs variants of BCRP/ABCG2.

PURPOSE: The aim of the current study was to identify the effect of single nucleotide polymorphisms (SNPs) in breast cancer resistance protein (BCRP/ABCG2) on its localization, expression level, and transport activity. METHODS: The cellular localization was identified using the wild type and seven different SNP variants of BCRP (V12M, Q141K, A149P, R163K, Q166E, P269S, and S441N BCRP) after transfection of their cDNAs in plasmid vector to LLC-PK1 cells. Their expression levels and transport activities were determined using the membrane vesicles from HEK293 cells infected with the recombinant adenoviruses containing these kinds of BCRP cDNAs. RESULTS: Wild type and six different SNP variants of BCRP other than S441N BCRP were expressed on the apical membrane, whereas S441N BCRP showed intracellular localization. The expression levels of Q141K and S441N BCRP proteins were significantly lower compared with the wild type and the other five variants. Furthermore, the transport activity of E1S, DHEAS, MTX, and PAH normalized by the expression level of BCRP protein was almost the same for the wild type, V12M, Q141K, A149P, R163K, Q166E, and P269S BCRP. CONCLUSIONS: These results suggest that Q141K SNPs may associate with a lower expression level, and S441N SNPs may affect both the expression level and cellular localization. It is possible that subjects with these polymorphisms may have lower expression level of BCRP protein and, consequently, a reduced ability to export these substrates.