Memantine improves cognitive deficits via KATP channel inhibition in olfactory bulbectomized mice.

Patients with Alzheimer's disease (AD) demonstrate severely impaired olfactory systems, which occur in the early stages of the disease. Olfactory bulbectomy (OBX) in mice elicits cognitive deficits, and reduces cholinergic activity in the hippocampus. Here, we confirmed that the novel AD drug memantine rescues cognitive deficits via ATP-sensitive potassium (KATP) channel inhibition in OBX mice. Repeated memantine administration at 1-3 mg/kg p.o. for 14 days starting at 10 days after OBX surgery significantly rescued cognitive deficits in OBX mice, as assessed using Y-maze, novel object recognition, and passive avoidance tasks. Consistent with the rescued cognitive deficits in OBX mice, long-term potentiation (LTP) in the hippocampal cornu ammonis (CA) 1 region was markedly restored with memantine administration. As demonstrated by immunoblotting, the reductions of calcium/calmodulin-dependent protein kinase II (CaMKII) α (Thr-286) autophosphorylation and calcium/calmodulin-dependent protein kinase IV (CaMKIV; Thr-196) phosphorylation in the CA1 region of OBX mice were significantly restored with memantine. Conversely, pre-treatment with pinacidil, a KATP channel opener, failed to reinstate hippocampal LTP and CaMKII/CaMKIV activities in the CA1 region. Finally, improvement of cognitive deficits by memantine treatments was observed in OBX-operated Kir6.1 heterozygous (+/-) mice but not in OBX-operated Kir6.2 heterozygous (+/-) mice. Overall, our study demonstrates that memantine rescues OBX-induced cognitive deficits via Kir6.2 channel inhibition in the CA1 region.

Combined Memantine and Donepezil Treatment Improves Behavioral and Psychological Symptoms of Dementia-Like Behaviors in Olfactory Bulbectomized Mice.

Memantine, an uncompetitive N-methyl-D-aspartate receptor antagonist, and the cholinesterase inhibitor, donepezil, are approved in most countries for treating moderate-to-severe Alzheimer's disease (AD). These drugs have different molecular targets; thus, it is expected that the effects of combined treatment would be synergistic. Some reports do show memantine/donepezil synergy in ameliorating cognition in AD model animals, but their combined effects on behavioral and psychological symptoms of dementia (BPSD)-like behaviors have not been addressed. Here, we investigate combined memantine/donepezil effects on cognitive impairment and BPSD-like behaviors in olfactory bulbectomized (OBX) mice. Interestingly, combined administration synergistically improved both depressive-like behaviors and impaired social interaction in OBX mice, whereas only weak synergistic effects on cognitive performance were seen. To address mechanisms underlying these effects, we used in vivo microdialysis study and observed impaired nicotine-induced serotonin (5-HT) release in OBX mouse hippocampus. Combined memantine/donepezil administration, but not single administration of either, significantly antagonized the decrease in nicotine-induced 5-HT release seen in OBX mouse hippocampus. Furthermore, decreased autophosphorylation of calcium/calmodulin dependent protein kinase II (CaMKII) was rescued in hippocampal CA1 and dentate gyrus of OBX mice by combined memantine/donepezil administration. These results suggest that improvement of BPSD-like behaviors by the co-administration of both drugs is in part mediated by enhanced 5-HT release and CaMKII activity in OBX mouse hippocampus.

Stimulation of the Sigma-1 Receptor and the Effects on Neurogenesis and Depressive Behaviors in Mice.

Sigma-1 receptor (Sig-1R) is molecular chaperone regulating calcium efflux from the neuronal endoplasmic reticulum to mitochondria. Recent studies show that Sig-1R stimulation antagonizes depressive-like behaviors in animal models, but molecular mechanisms underlying this effect remain unclear. Here, we focus on the effects of Sig-1R ligands on hippocampal neurogenesis and depressive-like behaviors. Sig-1R stimulation also enhances CaMKII /CaMKIV and protein kinase B (Akt) activities in hippocampus. Therefore, we discuss the fundamental roles of Sig-1R, CaMKII /CaMKIV and protein kinase B (Akt) signaling in amelioration of depressive-like behaviors following Sig-1R stimulation.

The extracellular fragment of GPNMB (Glycoprotein nonmelanosoma protein B, osteoactivin) improves memory and increases hippocampal GluA1 levels in mice.

Glycoprotein nonmelanoma protein B (GPNMB, alias osteoactivin), a type I transmembrane glycoprotein, is cleaved by extracellular proteases, resulting in release of an extracellular fragment (ECF). GPNMB is widely expressed by neurons within the CNS, including the hippocampus; however, its function in the brain remains unknown. Here, we investigated the role of GPNMB in memory and learning by using transgenic (Tg) mice over-expressing GPNMB (Tg mice on a BDF-1 background) and ECF-treated mice. In the hippocampus of both wild-type and Tg mice, GPNMB was highly expressed in neurons and astrocytes. Tg mice exhibited memory improvements in two types of learning tasks but were impaired in a passive-avoidance test. In Tg mice, the hippocampus displayed increased levels of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor subunit GluA1. Intracerebroventricular administration of ECF (50 ng) to Institute of Cancer Research (ICR) mice also improved memory in a passive-avoidance test and increased hippocampal GluA1 levels 24 h after treatment. In Tg mice and ECF (0.25 µg/mL)-treated hippocampal slices, long-term potentiation was promoted. These findings suggest that GPNMB may be a novel target for research on higher order brain functions.

CaMKII activity is essential for improvement of memory-related behaviors by chronic rivastigmine treatment.

Because the cholinergic system is down-regulated in the brain of Alzheimer's disease patients, cognitive deficits in Alzheimer's disease patients are significantly improved by rivastigmine treatment. To address the mechanism underlying rivastigmine-induced memory improvements, we chronically treated olfactory bulbectomized (OBX) mice with rivastigmine. The chronic rivastigmine treatments for 12-13 days starting at 10 days after OBX operation significantly improved memory-related behaviors assessed by Y-maze task, novel object recognition task, passive avoidance task, and Barnes maze task, whereas the single rivastigmine treatment failed to improve the memory. Consistent with the improved memory-related behaviors, long-term potentiation in the hippocampal CA1 region was markedly restored by rivastigmine treatments. In immunoblotting analyses, the reductions of calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and calcium/calmodulin-dependent protein kinase IV (CaMKIV) phosphorylation in the CA1 region in OBX mice were significantly restored by rivastigmine treatments. In addition, phosphorylation of AMPAR subunit glutamate receptor 1 (GluA1) (Ser-831) and cAMP-responsive element-binding protein (Ser-133) as downstream targets of CaMKII and CaMKIV, respectively, in the CA1 region was also significantly restored by chronic rivastigmine treatments. Finally, we confirmed that rivastigmine-induced improvements of memory-related behaviors and long-term potentiation were not obtained in CaMKIIα(+/-) mice. On the other hand, CaMKIV(-/-) mice did not exhibit the cognitive impairments. Taken together, the stimulation of CaMKII activity in the hippocampus is essential for rivastigmine-induced memory improvement in OBX mice.

Melatonin reverses the decreases in hippocampal protein serine/threonine kinases observed in an animal model of autism.

Lower global cognitive function scores are a common symptom of autism spectrum disorders (ASDs). This study investigates the effects of melatonin on hippocampal serine/threonine kinase signaling in an experimental ASD model. We found that chronic melatonin (1.0 or 5.0 mg/kg/day, 28 days) treatment significantly rescued valproic acid (VPA, 600 mg/kg)-induced decreases in CaMKII (Thr286), NMDAR1 (Ser896), and PKA (Thr197) phosphorylation in the hippocampus without affecting total protein levels. Compared with control rats, the immunostaining of pyramidal neurons in the hippocampus revealed a decrease in immunolabeling intensity for phospho-CaMKII (Thr286) in the hippocampus of VPA-treated rats, which was ameliorated by chronic melatonin treatment. Consistent with the elevation of CaMKII/PKA/PKC phosphorylation observed in melatonin-treated rat, long-term potentiation (LTP) was enhanced after chronic melatonin (5.0 mg/kg) treatment, as reflected by extracellular field potential slopes that increased from 56 to 60 min (133.4 ± 3.9% of the baseline, P < 0.01 versus VPA-treated rats) following high-frequency stimulation (HFS) in hippocampal slices. Accordingly, melatonin treatment also significantly improved social behavioral deficits at postnatal day 50 in VPA-treated rats. Taken together, the increased phosphorylation of CaMKII/PKA/PKC signaling might contribute to the beneficial effects of melatonin on autism symptoms.

Preventive effect of propolis on cognitive decline in Alzheimer's disease model mice.

Brazilian green propolis (propolis) is a chemically complex resinous substance that is a potentially viable therapeutic agent for Alzheimer's disease. Herein, propolis induced a transient increase in intracellular Ca2+ concentration ([Ca2+]i) in Neuro-2A cells; moreover, propolis-induced [Ca2+]i elevations were suppressed prior to 24-h pretreatment with amyloid-ß. To reveal the effect of [Ca2+]i elevation on impaired cognition, we performed memory-related behavioral tasks in APP-KI mice relative to WT mice at 4 and 12 months of age. Propolis, at 300-1000 mg/kg/d for 8 wk, significantly ameliorated cognitive deficits in APP-KI mice at 4 months, but not at 12 months of age. Consistent with behavioral observations, injured hippocampal long-term potentiation was markedly ameliorated in APP-KI mice at 4 months of age following repeated propolis administration. In addition, repeated administration of propolis significantly activated intracellular calcium signaling pathway in the CA1 region of APP-KI mice. These results suggest a preventive effect of propolis on cognitive decline through the activation of intracellular calcium signaling pathways in CA1 region of AD mice model.

Novel nootropic drug sunifiram enhances hippocampal synaptic efficacy via glycine-binding site of N-methyl-D-aspartate receptor.

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR-dependent long-term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10-100 nM significantly enhanced LTP in a bell-shaped dose-response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7-chloro-kynurenic acid (7-ClKN), an antagonist for glycine-binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α-amino-3-hydroxy-5-methylisozazole-4-propionate receptor (AMPAR) through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1-1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose-dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser-657) and Src family (Tyr-416) activities with the same bell-shaped dose-response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser-657) and Src (Tyr-416) induced by sunifiram was inhibited by 7-ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 µM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine-binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation.

Novel cognitive enhancer ST101 enhances acetylcholine release in mouse dorsal hippocampus through T-type voltage-gated calcium channel stimulation.

We recently developed a novel cognitive enhancer, ST101 (spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one), that activates T-type voltage-gated calcium channels (VGCCs). Here, we address whether T-type VGCC activation with ST101 mediates its cognitive effects in vivo and the relevance of T-type VGCC activation to acetylcholine (ACh) release in the hippocampus. Acute intraperitoneal administration of ST101 (1 mg/kg, i.p.) improved memory-related behaviors in both olfactory bulbectomized (OBX) and scopolamine-treated mice. Effects of ST101 administration were abolished by both intraperitoneal and intracerebroventricular pre-administration of the T-type VGCC inhibitor mibefradil. Acute administration of ST101 enhanced basal and nicotine-induced ACh release in the dorsal hippocampus in both OBX and sham-treated mice. Enhanced ACh release was abolished by infusion with mibefradil (10 µM) but not with the L-type VGCC inhibitor nifedipine (10 µM). As expected, significantly reduced CaMKIIα, PKCα, and ERK phosphorylation was restored by acute ST101 administration in the OBX mouse hippocampal CA1 region. Enhancement of CaMKIIα and PKCα but not ERK phosphorylation was inhibited by mibefradil (20 mg/kg, i.p.) preadministration. Increased CaMKIIα and PKCα phosphorylation was confirmed by increased phosphorylation of GluR1, synapsin I, and NR1. Taken together, stimulation of T-type VGCCs is critical for the enhanced hippocampal ACh release and improved cognitive function seen following ST101 administration.

Reduced calcium/calmodulin-dependent protein kinase II activity in the hippocampus is associated with impaired cognitive function in MPTP-treated mice.

Parkinson's disease (PD) patients frequently reveal deficit in cognitive functions during the early stage in PD. The dopaminergic neurotoxin, MPTP-induced neurodegeneration causes an injury of the basal ganglia and is associated with PD-like behaviors. In this study, we demonstrated that deficits in cognitive functions in MPTP-treated mice were associated with reduced calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and impaired long-term potentiation (LTP) induction in the hippocampal CA1 region. Mice were injected once a day for 5days with MPTP (25mg/kg i.p.). The impaired motor coordination was observed 1 or 2week after MPTP treatment as assessed by rota-rod and beam-walking tasks. In immunoblotting analyses, the levels of tyrosine hydroxylase protein and CaMKII autophosphorylation in the striatum were significantly decreased 1week after MPTP treatment. By contrast, deficits of cognitive functions were observed 3-4weeks after MPTP treatment as assessed by novel object recognition and passive avoidance tasks but not Y-maze task. Impaired LTP in the hippocampal CA1 region was also observed in MPTP-treated mice. Concomitant with impaired LTP induction, CaMKII autophosphorylation was significantly decreased 3weeks after MPTP treatment in the hippocampal CA1 region. Finally, the reduced CaMKII autophosphorylation was closely associated with reduced AMPA-type glutamate receptor subunit 1 (GluR1; Ser-831) phosphorylation in the hippocampal CA1 region of MPTP-treated mice. Taken together, decreased CaMKII activity with concomitant impaired LTP induction in the hippocampus likely account for the learning disability observed in MPTP-treated mice.

The T-type voltage-gated calcium channel as a molecular target of the novel cognitive enhancer ST101: enhancement of long-term potentiation and CaMKII autophosphorylation in rat cortical slices.

In this study, we report that spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ST101; previously coded as ZSET1446) targets T-type voltage-gated calcium channels in mediating improved cognition in the CNS. We prepared rat somatosensory cortical and hippocampal slices, treated them with 0.01 to 100 nM ST101, and performed immunoblotting and electrophysiological analyses using various voltage-gated calcium channel (VGCC) inhibitors. Treatment of rat cortical slices with a range of ST101 concentrations significantly increased calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation following a bell-shaped dose-response curve, with 0.1 nM ST101 representing the maximally effective concentration. protein kinase Cα autophosphorylation was also significantly increased by 0.1 nM ST101 treatment. ST101 treatment had a moderate effect on CaMKII autophosphorylation but no effect on hippocampal protein kinase Cα autophosphorylation in slice preparations. Consistent with increased cortical CaMKII autophosphorylation, AMPA-type glutamate receptor subunit 1 (Ser-831) phosphorylation as a CaMKII post-synaptic substrate was significantly increased by treatment with 0.1-1 nM ST101, whereas phosphorylation of the pre-synaptic substrate synapsin I (Ser-603) remained unchanged. Notably, enhanced CaMKII autophosphorylation seen following 0.1 nM ST101 treatment was significantly inhibited by pre-treatment with 1 µM mibefradil, a T-type VGCC inhibitor, but not with N-type (ω-conotoxin), P/Q-type (ω-agatoxin) or L-type (nifedipine) VGCC inhibitors. Similarly, 0.1 nM ST101 significantly potentiated long-term potentiation in cortical but not hippocampal slices. Enhanced long-term potentiation in cortical slices was totally inhibited by 1 µM mibefadil treatment. Finally, whole-cell patch-clamp analysis of Neuro2A cells over-expressing recombinant human Ca(V) 3.1 (α1G) T-channels and treated with 0.1 nM ST101 showed significant increases in T-type VGCC currents. These results indicate that T-type VGCCs are direct molecular targets for the novel cognitive enhancer ST101, a potential Alzheimer disease therapeutic.

Aberrant hippocampal spine morphology and impaired memory formation in neuronal platelet-derived growth factor ß-receptor lacking mice.

The physiological role of platelet-derived growth factor (PDGF) in the central nervous system (CNS) synaptic function remains uncharacterized. Here we identify physiological roles of PDGF receptor-ß (PDGFR-ß) in the CNS by conditional knockout of the gene encoding it. In the hippocampus, PDGFR-ß colocalized immunohistochemically with both presynaptic synaptophysin and postsynaptic density-95 (PSD-95). In the hippocampal CA1 region, expression levels of postsynaptic proteins, including spinophilin, drebrin, and PSD-95, were significantly decreased in PDGFR-ß knockout mice, although presynaptic synaptophysin levels remained comparable to controls. Interestingly, in hippocampal CA1 pyramidal neurons, dendritic spine density in PDGFR-ß knockout mice was significantly decreased compared with that seen in wild-type mice, although spine length and number of dendritic branches remained unchanged. Consistent with these findings, impairment in hippocampal long-term potentiation (LTP) and in hippocampus-dependent memory formation were seen in PDGFR-ß knockout mice. These results suggest PDGFR-ß plays critical roles in spine morphology and memory formation in mouse brain.

Ca2+/calmodulin-dependent protein kinase II and protein kinase C activities mediate extracellular glucose-regulated hippocampal synaptic efficacy.

To define how extracellular glucose levels affect synaptic efficacy and long-term potentiation (LTP), we evaluated electrophysiological and neurochemical properties in hippocampal CA1 regions following alterations in glucose levels in the ACSF. In rat hippocampal slices prepared in ACSF with 3.5mM glucose, fEPSPs generated by Schaffer collateral/commissural stimulation markedly increased when ACSF glucose levels were increased from 3.5 to 7.0mM. The paired-pulse facilitation reflecting presynaptic transmitter release efficacy was significantly suppressed by elevation to 7.0mM glucose because of potentiation of the input-output relationship (I/O relationship) of fEPSPs by single pulse stimulation. Prolonged potentiation of fEPSPs by elevation to 7.0mM glucose coincided with increased autophosphorylation both of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and protein kinase Cα (PKCα). The increased I/O relationship of fEPSPs was also associated with markedly increased synapsin I phosphorylation by CaMKII. Transmitter-evoked postsynaptic currents were also measured in CA1 neurons by electrophoretical application of NMDA and AMPA to the apical dendrites of pyramidal neurons. NMDA- and AMPA-evoked currents were significantly augmented by elevation to 7.0mM. Notably, high frequency stimulation of the Schaffer collateral/commissural pathway failed to induce LTP in the CA1 region at 3.5mM glucose but LTP was restored dose-dependently by increasing glucose levels to 7.0 and 10.0mM. LTP induction in the presence of 7.0mM glucose was closely associated with further increases in CaMKII autophosphorylation without changes in PKCα autophosphorylation. Taken together, CaMKII and PKC activation likely mediate potentiation of fEPSPs by elevated glucose levels, and CaMKII activity is also associated with LTP induction in the hippocampal CA1 region.

Propolis Promotes Memantine-Dependent Rescue of Cognitive Deficits in APP-KI Mice.

Propolis is a complex resinous substance that is relevant as a therapeutic target for Alzheimer's disease (AD) and other neurodegenerative diseases. In this study, we confirmed that propolis (Brazilian green propolis) further enhances the rescue of cognitive deficits by the novel AD drug memantine in APP-KI mice. In memory-related behavior tasks, administration of a single dose of propolis at 1-100 mg/kg p.o. significantly enhanced the rescue of cognitive deficits by memantine at 1 mg/kg p.o. in APP-KI mice. In in vitro studies, propolis significantly increased intracellular Ca2+ concentration and calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation in Kir6.2-overexpressed N2A cells treated with memantine. Propolis also significantly increased adenosine 5'-triphosphate (ATP) contents and CaMKII autophosphorylation, which was impaired in Aß-treated Kir6.2-overexpressed N2A cells. Similarly, repeated administration of propolis at 100 mg/kg p.o. for 8 weeks further enhanced the rescue of cognitive deficits by memantine in APP-KI mice. Consistent with the rescued cognitive deficits in APP-KI mice, repeated administration of propolis markedly ameliorated memantine-dependent rescue of injured long-term potentiation (LTP) in APP-KI mice, concomitant with increased CaMKII autophosphorylation and calcium/calmodulin-dependent protein kinase IV (CaMKIV) phosphorylation in the hippocampal CA1 region. Furthermore, repeated administration of both memantine and propolis significantly restored the decreased ATP contents in the CA1 region of APP-KI mice. Finally, we confirmed that repeated administration of memantine at 1 mg/kg p.o. and propolis at 100 mg/kg p.o. for 8 weeks failed to restore the cognitive deficits in Kir6.2-/- mice. Our study demonstrates that propolis increases ATP contents and promotes the amelioration of cognitive deficits rescued by memantine via Kir6.2 channel inhibition in the CA1 region.

Sigma-1 receptor stimulation by dehydroepiandrosterone ameliorates cognitive impairment through activation of CaM kinase II, protein kinase C and extracellular signal-regulated kinase in olfactory bulbectomized mice.

Dehydroepiandrosterone (DHEA) is one of the most abundant neurosteroids synthesized de novo in the CNS. We here found that sigma-1 receptor stimulation by DHEA improves cognitive function through phosphorylation of synaptic proteins in olfactory bulbectomized (OBX) mouse hippocampus. We have previously reported that calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) were impaired in OBX mouse hippocampus. OBX mice were administered once a day for 7-8 days with DHEA (30 or 60 mg/kg p.o.) 10 days after operation. The spatial, cognitive and conditioned fear memories in OBX mice were significantly improved as assessed by Y-maze, novel object recognition and passive avoidance task, respectively. DHEA also improved impaired hippocampal long-term potentiation in OBX mice. Notably, DHEA treatment restored PKCα (Ser-657) autophosphorylation and NR1 (Ser-896) and myristoylated alanine-rich protein kinase C substrate (Ser-152/156) phosphorylation to the control levels in the hippocampal CA1 region. Likewise, DHEA treatment improved CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation to the control levels in the CA1 region. Furthermore, DHEA treatment improved ERK and cAMP-responsive element-binding protein (Ser-133) phosphorylation to the control levels. Finally, NE-100, sigma-1 receptor antagonist, significantly inhibited the DHEA-induced improvement of memory-related behaviors and CaMKII, PKC and ERK phosphorylation in CA1 region. Taken together, sigma-1 receptor stimulation by DHEA ameliorates OBX-induced impairment in memory-related behaviors and long-term potentiation in the hippocampal CA1 region through activation of CaMKII, PKC and ERK.

Reduced expression of the ATRX gene, a chromatin-remodeling factor, causes hippocampal dysfunction in mice.

Mutations of the ATRX gene, which encodes an ATP-dependent chromatin-remodeling factor, were identified in patients with α-thalassemia X-linked mental retardation (ATR-X) syndrome. There is a milder variant of ATR-X syndrome caused by mutations in the Exon 2 of the gene. To examine the impact of the Exon 2 mutation on neuronal development, we generated ATRX mutant (ATRX(ΔE2)) mice. Truncated ATRX protein was produced from the ATRX(ΔE2) mutant allele with reduced expression level. The ATRX(ΔE2) mice survived and reproduced normally. There was no significant difference in Morris water maze test between wild-type and ATRX(ΔE2) mice. In a contextual fear conditioning test, however, total freezing time was decreased in ATRX(ΔE2) mice compared to wild-type mice, suggesting that ATRX(ΔE2) mice have impaired contextual fear memory. ATRX(ΔE2) mice showed significantly reduced long-term potentiation in the hippocampal CA1 region evoked by high-frequency stimulation. Moreover, autophosphorylation of calcium-calmodulin-dependent kinase II (αCaMKII) and phosphorylation of glutamate receptor, ionotropic, AMPA 1 (GluR1) were decreased in the hippocampi of the ATRX(ΔE2) mice compared to wild-type mice. These findings suggest that ATRX(ΔE2) mice may have fear-associated learning impairment with the dysfunction of αCaMKII and GluR1. The ATRX(ΔE2) mice would be useful tools to investigate the role of the chromatin-remodeling factor in the pathogenesis of abnormal behaviors and learning impairment.

Pharmacological study on Alzheimer's drugs targeting calcium/calmodulin-dependent protein kinase II.

In the brain of Alzheimer's disease patients, down-regulation of both cholinergic and glutamatergic systems have been found and is thought to play an important role in impairment of cognition, learning, and memory. Nefiracetam is a pyrrolidine-related nootropic drug exhibiting various pharmacological actions such as a cognitive-enhancing effect. The present study was undertaken to elucidate mechanisms underlying the action of nefiracetam on glutamatergic receptors and intracellular protein kinases. N-Methyl-D-aspartate (NMDA)-evoked currents were recorded from rat cortical neurons in long-term cultured primary neurons using the whole-cell patch-clamp technique. NMDA-evoked currents were greatly and reversibly potentiated by bath application of nefiracetam, resulting in a bell-shaped dose-response curve. The maximum potentiation of 170% relative to the control was produced at 10 nM. Treatment with an inhibitor of the glycine binding site of the NMDA receptor, 7-chlorokynurenic acid, at 1 µM prevented augmentation of NMDA-evoked currents by nefiracetam. In rat hippocampal CA1 slices, field excitatory postsynaptic potentials were recorded by stimulation of Schaffer collateral/commissural pathways. Nefiracetam treatment significantly enhanced long-term potentiation (LTP) with the same bell-shaped dose-response curve. Furthermore, nefiracetam-induced LTP enhancement was closely associated with calcium/calmodulin-dependent protein kinase II (CaMKII) activation with concomitant increase in phosphorylation of AMPA-type glutamate receptor subunit 1 (GluA1) (Ser-831) as a postsynaptic CaMKII substrate. In conclusion, nefiracetam enhances NMDA-receptor function through stimulation of its glycine binding site and nefiracetam-induced CaMKII activation likely contributes to improvement of cognition, learning, and memory.

A novel cognitive enhancer, ZSET1446/ST101, promotes hippocampal neurogenesis and ameliorates depressive behavior in olfactory bulbectomized mice.

In the adult brain, neurogenesis persistently occurs in the subgranular zone of the hippocampal dentate gyrus (DG), and impaired neurogenesis is implicated in depressive behaviors and poor learning memory. Here, we investigated the effects of oral administration of spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ZSET1446/ST101), a novel cognitive enhancer stimulating acetylcholine release, on adult neurogenesis in olfactory bulbectomized (OBX) mice. OBX mice showed significant decreases in the number of newborn cells in the DG by immunohistochemical analysis of 5-bromo-2-deoxyuridine incorporation. Impaired neurogenesis observed in OBX mice was significantly improved by chronic administration with ZSET1446. We confirmed that administration with mecamylamine, a nicotinic acetylcholine receptor antagonist, inhibits ZSET1446-enhanced neurogenesis in the DG. ZSET1446 administration also restored decreased phosphorylation of Akt and extracellular signal-regulated kinase in the DG of OBX mice. Consistent with restored neurogenesis, chronic but not single ZSET1446 administration promoted significant decreases in immobility in tail suspension tests and improved cognitive behaviors in OBX mice. Taken together, chronic ZSET1446 administration antagonized impaired neurogenesis seen in OBX mice, an effect closely associated with improvement of depressive behavior.

The induction of reactive oxygen species and loss of mitochondrial Omi/HtrA2 is associated with S-nitrosoglutathione-induced apoptosis in human endothelial cells.

The pathophysiological relevance of S-nitrosoglutathione (GSNO)-induced endothelial cell injury remains unclear. The main objective of this study was to elucidate the molecular mechanisms of GSNO-induced oxidative stress in endothelial cells. Morphological evaluation through DAPI staining and propidium iodide (PI) flow cytometry was used to detect apoptosis. In cultured EA.hy926 endothelial cells, exposure to GSNO led to a time- and dose-dependent apoptotic cascade. When intracellular reactive oxygen species (ROS) production was measured in GSNO-treated cells with the fluorescent probes 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, we observed elevated ROS levels and a concomitant loss in mitochondrial membrane potential, indicating that GSNO-induced death signaling is mediated through a ROS-mitochondrial pathway. Importantly, we found that peroxynitrite formation and Omi/HtrA2 release from mitochondria were involved in this phenomenon, whereas changes of death-receptor dependent signaling were not detected in the same context. The inhibition of NADPH oxidase activation and Omi/HtrA2 by a pharmacological approach provided significant protection against caspase-3 activation and GSNO-induced cell death, confirming that GSNO triggers the death cascade in endothelial cells in a mitochondria-dependent manner. Taken together, our results indicate that ROS overproduction and loss of mitochondrial Omi/HtrA2 play a pivotal role in reactive nitrogen species-induced cell death, and the modulation of these pathways can be of significant therapeutic benefit.

[Donepezil-induced neuroprotection of acetylcholinergic neurons in olfactory bulbectomized mice].

In the brain of Alzheimer's patients, the cholinergic neurons innervated the hippocampus and cerebral cortex degenerates before accumulation of beta-amyloid protein. Donepezil, a potent acetylcholinesterase (AChE) inhibitor is reported to rescue neurons from excitotoxic injury in culture. However, there is no evidence to confirm its neuroprotective effect on ACh neurons in vivo. Using olfactory bulbectomy (OBX) mice, we defined the neuroprotective mechanisms of donepezil on the medial septum cholinergic neurons with concomitant improvement of the impaired cognitive function. Bilateral olfactory bulbs of DDY mouse were removed by surgery. After olfactory bulbectomized (OBX), donepezil (1 or 3 mg/kg/day) was administered for 15 days and mouse brain was fixed with paraformaldehyde perfusion at day 18. Then, the neuroprotective effect of donepezil was evaluated by counting the number of Chdine acetyltrans-ferase (ChAT) immunoreactive neurons in the medial septum. The number of ChAT immunoreactive neurons in the medial septum reduced by 40% of that in sham-operated animals. The reduced ChAT positive neurons were restored by donepezil treatments. Consistent with these observations, the cognitive deficits observed in OBX mice were significantly improved by the donepezil treatment. Taken together, donepezil treatment rescues the cholinergic neurons in the medial septum from the neurodegeneration by OBX. We will also discuss the mechanism underlying the donepezil-induced neuroprotection in the medial septum cholinergic neurons.