Hiroki Ozawa

Impaired phosphorylation of cyclic AMP response element binding protein in the hippocampus of dementia of the Alzheimer type.

Cyclic AMP (cAMP) is disrupted in the brain in dementia of the Alzheimer type (DAT). We investigated whether the cAMP reduction is accompanied by an alteration in the cAMP response element binding protein (CREB) downstream in DAT and control hippocampi. Immunoreactivity of pCREB was significantly decreased in DAT, while total CREB level was unchanged. These findings indicate that impaired cAMP signaling may contribute to the pathophysiology of the disease.

Reduced phosphorylation of cyclic AMP-responsive element binding protein in the postmortem orbitofrontal cortex of patients with major depressive disorder

Summary. In this study, we examined the amounts of cAMP-responsive element binding protein (CREB) and its phosphorylated form in homogenate preparations from postmortem orbitofrontal cortices of antidepressant drug-free patients with major depressive disorder and age-matched controls by immunoblotting. Immunoreactivies of both CREB and phosphorylated CREB were significantly decreased in depressive subjects compared to controls. The immunoreactivity of phosphorylated CREB was diminished to a greater extent than that of CREB in depressive patients. It has been indicated from animal studies that a transcription factor likely mediates neural plasticity in the mammalian brain and neural tissues. Our results suggest that alterations in the cAMP signaling system, especially in CREB, may be involved in the pathophysiology of depression and be potential targets for antidepressant treatment.

Decreased expression of NEFH and PCP4/PEP19 in the prefrontal cortex of alcoholics

Patients with alcoholism exhibit behavioral adaptations to ethanol such as tolerance, dependence, and addiction. Molecular mechanisms that underlie these altered behavioral responses to ethanol are largely unclear. We have performed oligonucleotide microarray analysis in postmortem prefrontal cortices of alcoholics. Among about 12,000 genes represented on microarray, a total of 79 genes showed differential expression changes in alcoholics compared with control subjects, consisting of 54 up- and 25 down-regulated genes. Altered expressions in alcoholics were observed in genes having a wide range of biological functions. The remarkable findings were up-regulation of myelin-related genes and molecular chaperones in alcoholics. Among the genes identified, decreased expressions of NEFH and PCP4/PEP19 were further examined. NEFH encodes a component of neurofilament protein in neurons. PCP4/PEP19 encodes protein involved in calcium signaling and neuronal apoptosis. Observation of their down-regulations in alcoholics in microarray analysis was confirmed by real-time quantitative RT-PCR, and was also confirmed in the independent set of postmortem brains of alcoholics. The present results may provide some insights into understanding the mechanism of ethanol-induced altered behavioral responses at the molecular level.

Role of central ATP-sensitive potassium channels in the analgesic effect and spinal noradrenaline turnover-enhancing effect of intracerebroventricularly injected morphine in mice

Glibenclamide is one of the most potent sulfonylurea-derived antidiabetic drugs which block the adenosine triphosphate-sensitive potassium (KATP) channels. In the present study, we found that none of morphine, U-50,488H (a selective kappa agonist) and baclofen (a selective GABAB agonist) added to the incubation medium at concentrations up to 10(-4) M had appreciable effect on the specific binding of [cyclohexyl-2,3-3H(N)]glibenclamide ([3H]glibenclamide) to the isolated mouse brain microsomes. The analgesic activity induced by intracerebroventricular injection (i.c.v.) of morphine but not U-50,488H was antagonized by pretreatment with either i.c.v. glibenclamide or beta-funaltrexamine (beta-FNA; a selective mu antagonist) in mice. Furthermore, the increasing effect of i.c.v. morphine on the spinal noradrenaline (NA) turnover was greatly antagonized by i.c.v. pretreatment with either beta-FNA or glibenclamide. From these results, we demonstrated that KATP channels play an important role as indirect modulators of the supraspinal analgesia induced by mu agonist but not kappa agonist in mice, and the activation of descending noradrenergic system induced by i.c.v. morphine appears to be suppressed by the blockade of KATP channels.

Hippocampal level of neural specific adenylyl cyclase type I is decreased in Alzheimer's disease.

Previous studies reported disruption of adenylyl cyclase (AC)-cyclic AMP (cAMP) signal transduction in brain of Alzheimers disease (AD). We also demonstrated that basal and stimulated AC activities in the presence of calcium and calmodulin (Ca(2+)/CaM) were significantly decreased in AD parietal cortex. In the present study, we examined the amounts of Ca(2+)/CaM-sensitive types I and VIII AC, and Ca(2+)/CaM-insensitive type VII AC in the postmortem hippocampi from AD patients and age-matched controls using immunoblotting. The specificities of the anti-type VII and VIII AC antibodies were confirmed by preabsorption with their specific blocking peptides. We observed a significant decrease in the level of type I AC and a tendency to decrease in the level of type VIII AC in AD hippocampus. On the other hand, the level of type VII AC showed no alteration between AD and controls. A body of evidence from the studies with invertebrates and vertebrates suggests that types I and VIII AC may play an essential role in learning and memory. Our finding thus firstly demonstrated that a specific disruption of the Ca(2+)/CaM-sensitive AC isoforms is likely involved in the pathophysiology in AD hippocampus.

Imbalance of the Gs and Gi/o function in post-mortem human brain of depressed patients

The amounts of various G protein subunits in postmortem brain samples from the parietal and temporal cortices were the same in controls and depressive patients as demonstrated by immunoblotting. However, photoaffinity GTP labeling (AAGTP) of Gi/oα, but not Gsα, was significantly increased in depressives in both cortex regions. Furthermore, the ratio of Gs/Gi/o AAGTP incorporation revealed a significant reduction in depressives in these regions. The present findings suggest that an imbalance of second messengers via G protein function may be involved in the pathophysiology of depression.

Reduced immunoreactivity of adenylyl cyclase in dementia of the Alzheimer type.

THE amounts of three adenylyl cyclase (AC) subtypes In membranes from brain obtained post-mortem from patients with dementia of Alzheimer type (DAT) and controls were studied by Western blotting using poly-clonal antibodies. AC subtypes were characterized by preabsorption by complementary peptides, species difference, tissue distribution and by using AC-VI-transfected cells. Immunoreactivities of AC-I and AC-II subtypes, which are predominant in brain tissue were decreased 40.5% (p < 0.02) and 52.0% (p < 0.02), respectively in DAT compared with controls. No significant difference in immunoreactivity of AC-V/VI subtypes distributed in various other tissues was observed. Our results are the first to indicate that reduced levels of brain-specific AC subtypes occur in DAT.

Ca2+/CaM-sensitive adenylyl cyclase activity is decreased in the Alzheimer's brain: possible relation to type I adenylyl cyclase.

SummaryImmunoreactivities of four subtypes of adenylyl cyclase (AC) (types I, II, IV and V/VI), and basal, forskolin- and Mn2+-stimulated AC activities with or without calcium and calmodulin (Ca2+/CaM) were estimated in parietal cortex membranes from cases with dementia of the Alzheimer type (DAT) and age-matched controls. Immunoreactivities of AC-I and AC-II were significantly decreased, but those of AC-IV and AC-V/VI did not change in DAT brains. There was a significant correlation of AC-I immunoreactivity with Ca2+/CaM-sensitive AC activity, but not with the Ca2+/CaM-insensitive activity. Ca2+/CaM-sensitive AC activity was significantly lower in DAT than in the control, indicating that impairment of Ca2+/CaM-sensitive AC-I is clearly involved in the pathophysiology of DAT.

Alterations of guanine nucleotide-binding proteins in post-mortem human brain in alcoholics.

Qualitative and quantitative alterations of G proteins in membrane preparations from parietal and temporal cortex regions in post-mortem brains obtained from alcoholics and controls matched with respect to age and post-mortem delay were investigated by Western-blotting with polyclonal antibodies against specific G protein subunits and functional photoaffinity GTP binding. Quantitative immunoblotting showed that only Gs alpha (52 kDa species) in temporal cortex was significantly decreased (30%, P < 0.05) in alcoholics compared with controls. Moreover, ethanol-stimulated photoaffinity GTP labeling of Gs alpha and Gi/o alpha was decreased in alcoholics in both cortex regions. These results suggest that disturbances of G protein-mediated signal transduction may be involved in the pathophysiology of alcoholics.

Induction of cysteine string protein after chronic antidepressant treatment in rat frontal cortex

We have previously identified 204 partial cDNA fragments (ADRG1-204) as antidepressant related genes/expressed sequence tags. Then, we developed our original cDNA microarrays, on which the 194 clones out of ADRG1-204 were spotted. With this ADRG microarray, we found that the expression of a spot, ADRG55, which representing cysteine string protein (CSP), was significantly increased in rat brain after chronic treatment with a selective serotonin reuptake inhibitor, sertraline. In the present study, reverse transcription-polymerase chain reaction analysis confirmed the induction of CSP at mRNA levels in rat frontal cortex after chronic treatment with two different classes of antidepressants, imipramine or sertraline. Western blot analysis also revealed that CSP-immunoreactivity was increased after antidepressant treatment. In conclusion, our data suggest that CSP is one of the common functional molecules induced after chronic antidepressant treatment.