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The present study investigated the therapeutic effects of the curcumin derivative 3-[(1E)-2-(1H-indol-6-yl)ethenyl]-5-[(1E)-2-[2-methoxy-4-(2-pyridylmethoxy)phenyl]ethenyl]-1H-pyrazole (GT863) in amyotrophic lateral sclerosis (ALS). The inhibitory effect of GT863 on superoxide dismutase 1 (SOD1) aggregation was evaluated in cell-free assays. GT863 interfered with the conformational changes of the SOD1 protein and later, oligomeric aggregation. Furthermore, its antioxidant, anti-inflammatory, and neuroprotective effects were evaluated in cell-free and cultured cell assays. GT863 inhibited H 2 O 2 - and glutamate-induced cytotoxicity and activated an antioxidant responsive element pathway. Additionally, in vivo effects of GT863 in the ALS mice model were evaluated by its oral administration to H46R mutant SOD1 transgenic mice. Rotarod test showed that GT863 administration significantly slowed the progression of motor dysfunction in the mice. In addition, GT863 substantially reduced highly-aggregated SOD1, further preserving large neurons in the spinal cord of GT863-treated mice. Collectively, these results indicated that GT863 could be a viable therapeutic agent with multiple vital actions for the treatment of ALS.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and is known to be the most common cause of dementia. We previously described the benefits of aromatherapy on the cognitive function of patients with AD utilizing various aromatic essential oils; however, its mechanism of action remains poorly understood. Consequently, in the present study, this mechanism was thoroughly evaluated employing a dementia mice model, specifically the senescence-accelerated mouse prone 8. The mice were exposed to a mixture of lemon and rosemary oil at nighttime as well as to a mixture of lavender and orange oil in the daytime for 2 months. The cognitive function of the mice was assessed before and after treatment with the aromatic essential oils using the Y-maze test. Moreover, the brain levels of amyloid beta (Aβ), abnormally phosphorylated tau, and brain-derived neurotrophic factor (BDNF) were measured following treatment. The benefits of aromatherapy on the cognitive function in mice were confirmed. It was also established that the brain levels of Aβ and abnormally phosphorylated tau were considerably lower in the aromatherapy group, while the levels of BDNF were marginally higher. These results suggest that aromatherapy employing these aromatic essential oils is beneficial for the prevention and treatment of AD., Competing Interests: The authors have declared that no competing interests exist.

Alzheimer's disease (AD) is proposed to be induced by abnormal aggregation of amyloidβ in the brain. Here, we designed a brain-permeable peptide nanofiber drug from a fragment of heat shock protein to suppress aggregation of the pathogenic proteins. To facilitate delivery of the nanofiber into the brain, a protein transduction domain from Drosophila Antennapedia was incorporated into the peptide sequence. The resulting nanofiber efficiently suppressed the cytotoxicity of amyloid βby trapping amyloid β onto its hydrophobic nanofiber surface. Moreover, the intravenously or intranasally injected nanofiber was delivered into the mouse brain, and improved the cognitive function of an Alzheimer transgenic mouse model. These results demonstrate the potential therapeutic utility of nanofibers for the treatment of AD., Competing Interests: The authors have declared that no competing interests exist.

Amyloid-β (Aβ) peptides play a crucial role in the pathogenesis of Alzheimer's disease (AD). Aβ production, aggregation, and clearance are thought to be important therapeutic targets for AD. Curcumin has been known to have an anti-amyloidogenic effect on AD. In the present study, we performed screening analysis using a curcumin derivative library with the aim of finding derivatives effective in suppressing Aβ production with improved bioavailability of curcumin using CHO cells that stably express human amyloid-β precursor protein and using human neuroblastoma SH-SY5Y cells. We found that the curcumin derivative GT863/PE859, which has been shown to have an inhibitory effect on Aβ and tau aggregation in vivo, was more effective than curcumin itself in reducing Aβ secretion. We further found that GT863 inhibited neither β- nor γ-secretase activity, but did suppress γ-secretase-mediated cleavage in a substrate-dependent manner. We further found that GT863 suppressed N -linked glycosylation, including that of the γ-secretase subunit nicastrin. We also found that mannosidase inhibitors that block the mannose trimming step of N -glycosylation suppressed Aβ production in a similar fashion, as was observed as a result of treatment with GT863. Collectively, these results suggest that GT863 downregulates N -glycosylation, resulting in suppression of Aβ production without affecting secretase activity., Competing Interests: H.S. is President of Green Tech Co., Ltd. Other authors declare no conflict of interest.

Bone embrittlement with aging, namely osteoporosis, is characterized by low bone mass and deterioration of bone tissue, and can lead to increased risk of fracture. The development of functional foods that can prevent geriatric diseases is in progress. Our focus was on brown rice because of its properties. An interventional study using of ultra-high hydrostatic pressurized brown rice (UHHPBR) for human has not yet been conducted. In this study, we investigated whether long-term dietary intake of UHHPBR prevents aging-related decline of bone mineral density in elderly Japanese individuals. Elderly participants (n=40; mean 73.1 y) in Iinan-cho, Shimane, Japan, were randomly divided into two groups. The UHHPBR-intake group (n=20) consumed 100 g of UHHPBR and 100 g of white rice (WR) per day for 12 mo, while the WR-intake group (n=20) consumed 200 g of WR per day. Pre- and 12-mo post-intervention, bone mineral density was evaluated by quantitative ultrasound. After 12 mo of intervention, the UHHPBR group's bone mineral density was significantly higher than the WR group's bone mineral density. Moreover, chronic intake of UHHPBR had no adverse side effects on participants. Long-term oral UHHPBR intake may have beneficial effects on bone mineral density decline and may attenuate osteoporosis in the elderly.

Alzheimer's disease (AD) is the most common form of dementia and its prevention and treatment is a worldwide issue. Many natural components considered to be effective against AD have been identified. However, almost all clinical trials of these components for AD reported inconclusive results. We thought that multiple factors such as amyloid β (Aβ) and tau progressed the pathology of AD and that a therapeutic effect would be obtained by using multiple active ingredients with different effects. Thus, in this study, we treated ferulic acid (FA), phosphatidylserine (PS) and curcumin (Cur) in combination or alone to APPswe/PS1dE9 transgenic mice and evaluated cognitive function by Y-maze test. Consequently, only the three-ingredient group exhibited a significant improvement in cognitive function compared to the control group. In addition, we determined the amounts of Aβ, brain-derived neurotrophic factor (BDNF), interleukin (IL)-1β, acetylcholine and phosphorylated tau in the mouse brains after the treatment. In the two-ingredient (FA and PS) group, a significant decrease in IL-1β and an increasing trend in acetylcholine were observed. In the Cur group, significant decreases in Aβ and phosphorylated tau and an increasing trend in BDNF were observed. In the three-ingredient group, all of them were observed. These results indicate that the intake of multiple active ingredients with different mechanisms of action for the prevention and treatment of AD.

Brown rice contains many ingredients that might protect against cognitive decline and Alzheimer's disease. However, brown rice is very hard, difficult to cook, and is poorly digested; thus, it is difficult to eat long-term. To solve these problems, ultra-high hydrostatic pressurizing brown rice (UHHPBR) was prepared. We investigated the effects of dietary UHHPBR administration for 24 mo on cognitive function and mental health in the elderly. Healthy elderly participants (n=52) were randomized into UHHPBR and polished white rice (WR) groups. The UHHPBR group consumed 100 g of UHHPBR per day for 24 mo and the WR group consumed white rice. At baseline, and after 12 and 24 mo, cognitive functions were assessed using the Revised Hasegawa's Dementia Scale, Mini-Mental State Examination, Frontal Assessment Battery (FAB), and the Cognitive Assessment for Dementia, iPad version (CADi). Mental health condition was also assessed using the Apathy Scale and the Zung Self-Rating Depression Scale, and serum biochemical parameters were determined. From baseline to month 24, the mean change in the FAB-sub item 1 scores was higher in the UHHPBR group compared to the WR group. Furthermore, apathy scores decreased, as well as the time required to answer all questions of the CADi, and plasma epinephrine levels increased. These results indicate that a 2-y oral consumption of UHHPBR increases information processing speed (as a measure of cognitive function) and improves apathy in the elderly, suggesting a protective effect of UHHPBR administration against age-related decline in brain cognition and motivation.

Background: Alzheimer's disease (AD) is the most common form of dementia and the number of AD patients continues to increase worldwide. Components of the germ layer and bran of Brown rice (BR) help maintain good health and prevent AD. Because the germ layer and bran absorb little water and are very hard and difficult to cook, they are often removed during processing. To solve these problems, in this study, we tried to use a high-pressure (HP) technique., Methods: We produced the highly water pressurized brown rice (HPBR) by pressurizing BR at 600 MPa, and then we fed it to an AD mouse model, senescence-accelerated mouse prone 8, to investigate the therapeutic effects of HPBR on cognitive dysfunction by Y-maze spatial memory test., Results: HP treatment increased the water absorbency of BR without nutrient loss. HPBR ameliorated cognitive dysfunction and reduced the levels of amyloid-β, which is a major protein responsible for AD, in the brain., Conclusions: These results suggest that HPBR is effective for preventing AD.

Aggregation of amyloid-β (Aβ) and tau plays a crucial role in the onset and progression of Alzheimer's disease (AD). Therefore, the inhibition of Aβ and tau aggregation may represent a potential therapeutic target for AD. Herein, we designed and synthesized both Aβ and tau dual aggregation inhibitors based on the structure of curcumin and developed the novel curcumin derivative PE859. In this study, we investigated the inhibitory activity of PE859 on Aβ aggregationin vitro and the therapeutic effects of PE859 on cognitive dysfunction via dual inhibition of Aβ and tau aggregation in vivo. PE859 inhibited Aβ aggregation in vitro and protected cultured cells from Aβ-induced cytotoxicity. Furthermore, PE859 ameliorated cognitive dysfunction and reduced the amount of aggregated Aβ and tau in brains of senescence-accelerated mouse prone 8 (SAMP8). These results warrant consideration of PE859 as a candidate drug for AD.

The abnormal production and deposition of amyloid-β (Aβ) peptides is a pathologic hallmark of Alzheimer's disease. Aβ is generated from amyloid-β protein precursor (AβPP) by two sequential proteolytic cleavage steps involving β- and γ-secretases in the trans-Golgi network and endosomes. Since direct inhibition of secretase could induce undesirable side-effects due to inadvertent inhibition of unrelated secretase substrates, it is important to establish methods for inhibiting Aβ production that do not affect secretase activity. It has been suggested that curcumin may have potent anti-amyloidogenic effect. In the present study, we evaluate the effect of curcumin derivatives on Aβ production in human neuroblastoma SH-SY5Y cells and in CHO cells which stably express human AβPP (CHO-AβPP). We found that the curcumin derivative CU6 was more effective than curcumin itself in reducing Aβ secretion. We further found that in SH-SY5Y cells CU6 inhibited neither β- nor γ-secretase activity, and that increased amounts of immature forms of AβPP accumulated in the endoplasmic reticulum (ER). We also found that CU6 induced expression of the ER chaperone glucose-regulated protein 78 (GRP78), and enhanced formation of the AβPP/GRP78 complex. These results suggest that CU6 downregulates intracellular AβPP trafficking, resulting in suppression of Aβ production independently of secretase activity.

Alzheimer's disease (AD) is the most common form of dementia. In an AD patient's brain, senile plaques and neurofibrillary tangles, the abnormal aggregates of amyloid β (Aβ) peptide and tau protein, are observed as the two major hallmarks of this disease. To develop a new drug for treatment of AD, we have designed and synthesized a series of curcumin derivatives and evaluated their inhibitory activities against both tau and Aβ aggregation. In this study, we describe the development of the more potent aggregation inhibitor 3-[(1E)-2-(1H-indol-6-yl)ethenyl]-5-[(1E)-2-[2-methoxy-4-(2-pyridylmethoxy) phenyl] ethenyl]-1H-pyrazole (compound 4, PE859). This compound has a better pharmacokinetic profile and pharmacological efficacy in vivo than curcumin, making it suitable as a drug for AD., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Neurofibrillary tangles, composed of hyperphosphorylated tau fibrils, are a pathological hallmark of Alzheimer's disease; the neurofibrillary tangle load correlates strongly with clinical progression of the disease. A growing body of evidence indicates that tau oligomer formation precedes the appearance of neurofibrillary tangles and contributes to neuronal loss. Here we show that tau oligomer formation can be inhibited by compounds whose chemical backbone includes 1,2-dihydroxybenzene. Specifically, we demonstrate that 1,2-dihydroxybenzene-containing compounds bind to and cap cysteine residues of tau and prevent its aggregation by hindering interactions between tau molecules. Further, we show that orally administered DL-isoproterenol, an adrenergic receptor agonist whose skeleton includes 1,2-dihydroxybenzene and which penetrates the brain, reduces the levels of detergent-insoluble tau, neuronal loss and reverses neurofibrillary tangle-associated brain dysfunction. Thus, compounds that target the cysteine residues of tau may prove useful in halting the progression of Alzheimer's disease and other tauopathies.

Neural stem/progenitor cells (NSPCs) proliferate and differentiate depending on their intrinsic properties and local environment. During the development of the mammalian nervous system, NSPCs generate neurons and glia sequentially. However, little is known about the mechanism that determines the timing of switch from neurogenesis to gliogenesis. In this study, we established a culture system in which the neurogenic potential of NSPCs is decreased in a time-dependent manner, so that short-term-cultured NSPCs differentiate into more neurons compared with long-term-cultured NSPCs. We found that short-term-cultured NSPCs express high levels of integrin-associated protein form 2 (IAP2; so-called CD47) mRNA using differential display analysis. Moreover, IAP2 overexpression in NSPCs induced neuronal differentiation of NSPCs. These findings reveal a novel mechanism by which IAP2 induces neuronal differentiation of NSPCs.

In tauopathies, a neural microtubule-associated protein tau (MAPT) is abnormally aggregated and forms neurofibrillary tangle. Therefore, inhibition of the tau aggregation is one of the key approaches for the treatment of these diseases. Here, we have identified a novel tau aggregation inhibitor, PE859. An oral administration of PE859 resulted in the significant reduction of sarkosyl-insoluble aggregated tau along with the prevention of onset and progression of the motor dysfunction in JNPL3 P301L-mutated human tau transgenic mice. These results suggest that PE859 is useful for the treatment of tauopathies.

In addition to cognitive decline, Alzheimer's disease patients also exhibit non-cognitive symptoms commonly referred to as behavioral and psychological symptoms of dementia, or BPSD. These symptoms have a serious impact on the quality of life of these patients, as well as that of their caregivers, but there are currently no effective therapies. The amyloid β-peptide (Aβ) is suspected to play a central role in the cascade leading to Alzheimer's disease, but the precise mechanisms are still incompletely known. To assess the influence of Aβ pathology on cognitive and non-cognitive behaviors, we examined locomotor activity, motor coordination, and spatial memory in male and female APPswePS1dE9 mice (Alzheimer's disease model, double transgenic mice expressing an amyloid precursor protein with Swedish mutation and a presenilin-1 with deletion of exon 9) at 5 months of age, when the mice had subtle Aβ deposits, and again at 9 months of age, when the mice had numerous Aβ deposits. Compared to wild-type mice, the male and female APPswe/PS1dE9 mice showed normal motor coordination in the rotarod test at both 5 and 9 months. In the Morris water maze test, male and female APPswe/PS1dE9 mice showed impaired spatial memory at 9 months; however, no such deficits were found at 5 months. In a locomotor activity test, male APPswe/PS1dE9 mice exhibited locomotor hyperactivity at 9 months, while females exhibited locomotor hyperactivity at both 5 and 9 months compared to the control mice. Together, these results indicate that APPswe/PS1dE9 mice developed spatial memory impairment and BPSD-like behavioral alterations resulting from Aβ accumulation.

Aggregations of both amyloid-β (Aβ) and hyper-phosphorylated tau proteins are recognized as key pathological manifestations of Alzheimer's disease (AD). Agents that inhibit both those forms of aggregation show promise as drug candidates. Seventeen oligo heteroaromatic compounds were rapidly synthesized via a one-pot, 3- or 4-component coupling procedure. Evaluations showed that compounds E16 and E18 were the most potent inhibitors of Aβ and tau aggregations (E16: IC50s = 0.38, 0.29 μM against Aβ, tau, respectively, E18: IC50s = 0.55, 0.30 μM against Aβ, tau, respectively)., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Rotenone, a mitochondrial complex I inhibitor, has been used to generate animal and cell culture models of Parkinson's disease. Recent studies suggest that microtubule destabilization causes selective dopaminergic neuronal loss. In this study, we investigated glycogen synthase kinase-3β (GSK3β) involvement in rotenone-induced microtubule destabilization. Rotenone-induced cytotoxicity in SH-SY5Y cells was attenuated by the GSK3β inhibitor SB216763. Tau, a microtubule-associated protein and substrate for GSK3β, has been implicated in the pathogenesis of tauopathies such as Alzheimer's disease. Rotenone induced an increase in phosphorylated tau, the effect of which was attenuated by concomitant treatment with SB216763. Rotenone treatment also decreased tau expression in the microtubule fraction and increased tau expression in the cytosol fraction. These effects were suppressed by SB216763, which suggests that rotenone reduces the capacity of tau to bind microtubules. Rotenone treatment increased the amount of free tubulin and reduced the amount of polymerized tubulin, indicating that rotenone destabilizes microtubules. Rotenone-induced microtubule destabilization was suppressed by SB216763 and taxol, a microtubule stabilizer. Taxol prevented rotenone-induced cytotoxicity and morphological changes. Taken together, these results suggest that rotenone-induced cytotoxicity is mediated by microtubule destabilization via GSK3β activation, and that microtubule destabilization is caused by reduction in the binding capacity of tau to microtubules, which is a result of tau phosphorylation via GSK3β activation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Despite the importance of the production of new neurons in the adult hippocampus, the transcription network governing this process remains poorly understood. The High Mobility Group (HMG)-box transcription factor, Sox2, and the cell surface activated transcriptional regulator, Notch, play important roles in CNS stem cells. Here, we demonstrate that another member of the SoxB (Sox1/Sox2/Sox3) transcription factor family, Sox21, is also a critical regulator of adult neurogenesis in mouse hippocampus. Loss of Sox21 impaired transition of progenitor cells from type 2a to type 2b, thereby reducing subsequent production of new neurons in the adult dentate gyrus. Analysis of the Sox21 binding sites in neural stem/progenitor cells indicated that the Notch-responsive gene, Hes5, was a target of Sox21. Sox21 repressed Hes5 gene expression at the transcriptional level. Simultaneous overexpression of Hes5 and Sox21 revealed that Hes5 was a downstream effector of Sox21 at the point where the Notch and Sox pathways intersect to control the number of neurons in the adult hippocampus. Therefore, Sox21 controls hippocampal adult neurogenesis via transcriptional repression of the Hes5 gene.

The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a cellular defense system against oxidative stress. Activation of this pathway increases expression of antioxidant enzymes. Epidemiological studies have demonstrated that the consumption of fruits and vegetables is associated with reduced risk of contracting a variety of human diseases. The aim of this study is to find Nrf2-ARE activators in dietary fruits and vegetables. We first attempted to compare the potency of ARE activation in six fruit and six vegetables extracts. Green perilla (Perilla frutescens var. crispa f. viridis) extract exhibited high ARE activity. We isolated the active fraction from green perilla extract through bioactivity-guided fractionation. Based on nuclear magnetic resonance and mass spectrometric analysis, the active ingredient responsible for the ARE activity was identified as 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC). DDC induced the expression of antioxidant enzymes, such as γ-glutamylcysteine synthetase (γ-GCS), NAD(P)H: quinone oxidoreductase-1 (NQO1), and heme oxygenase-1. DDC inhibited the formation of intracellular reactive oxygen species and the cytotoxicity induced by 6-hydroxydopamine. Inhibition of the p38 mitogen-activated protein kinase pathway abolished ARE activation, the induction of γ-GCS and NQO1, and the cytoprotective effect brought about by DDC. Thus, this study demonstrated that DDC contained in green perilla enhanced cellular resistance to oxidative damage through activation of the Nrf2-ARE pathway., (Copyright © 2012 Elsevier Inc. All rights reserved.)

FE65 is reported to act as an adaptor protein with several protein-interaction domains, including one WW domain and two phosphotyrosine interaction/binding domains. Through these binding domains, FE65 was considered to recruit various binding partners together to form functional complexes in a certain cellular compartment. In this study, we demonstrated that Rac1, a member of the Rho family GTPases, bound with FE65. We also elucidated that Rac1 inhibitor significantly suppressed FE65 expression, and Rac1 small interfering RNA transduction significantly decreased FE65 expression. FE65 small interfering RNA, however, did not influence Rac1 expression and its activity. Taken together, our results reveal that Rac1 interacts with FE65, and Rac1 activity regulates FE65 expression.

Invasiveness, cellular atypia, and proliferation are hallmarks of malignant gliomas. To effectively target each of these characteristics, it is important to understand their sequence during tumorigenesis. However, because most gliomas are diagnosed at an advanced stage, the chronology of gliomagenesis milestones is not well understood. The aim of the present study was to determine the onset of these characteristics during tumor development. Brain tumor-initiating cells (BTICs) were established by overexpressing H-Ras(V12) in normal neural stem/progenitor cells isolated from the subventricular zone of adult mice harboring a homozygous deletion of the Ink4a/Arf locus. High-grade malignant brain tumors were then created by orthotopic implantation of 10(5) BTICs into the forebrain of 6-week-old wild-type mice. Micewere killed every week for 5 weeks, and tumors were assessed for cellular atypia, proliferation, hemorrhage, necrosis, and invasion. All mice developed highly invasive, hypervascular glioblastoma-like tumors. A 100% penetrance rate and a 4-week median survival were achieved. Tumor cell migration along fiber tracts started within days after implantation and was followed by perivascular infiltration of tumor cells with marked recruitment of reactive host cells. Next, cellular atypia became prominent. Finally, mass proliferation and necrosis were observed in the last stage of the disease. Video monitoring of BTICs in live brain slices confirmed the early onset of migration, as well as the main cell migration patterns. Our results showed that perivascular and intraparenchymal tumor cell migration precede tumor mass formation in the adult brain, suggesting the need for an early and sustained anti-invasion therapy.

Cellular modulator of immune recognition (c-MIR) is an E3 ubiquitin ligase that ubiquitinates MHC class II and CD86 for their endocytosis and subsequent lysosomal degradation. In accordance with their importance in antigen presentation, systemic c-MIR over-expression downmodulates adaptive immune responses. Rheumatoid arthritis (RA) is a chronic synovitis driven by autoimmunity in the joints. Since antigen-presenting cells, such as macrophages, dendritic cells (DCs) and rheumatoid factor-positive B cells are abundant in the rheumatoid synovial tissues, autoantigens released by tissue damage should be presented locally, leading to amplification of systemic arthritogenic immune responses. Assuming that inhibition of the antigen presentation in the synovial tissues should suppress systemic arthritis, we transferred the c-MIR gene to the hind leg synovial tissues from mice with type II collagen (CII)-induced arthritis, an animal model of RA. The gene was transferred adenovirally because adenoviruses can infect DC and macrophages in vivo. Unexpectedly, therapeutic effect was observed only in the treated joints. Splenocyte responses and serum antibodies against CII were not suppressed. Moreover, in vitro studies disclosed that c-MIR gene transfer suppressed IL-6 production from synovial fibroblasts stimulated with tumor necrosis factor (TNF)-α or IL-1β. Bone marrow-derived macrophages and DC from c-MIR transgenic mice were impaired in IL-6 and TNF-α production when stimulated with LPS. This suppression was controlled at the post-transcriptional level since their mRNA was not affected. These results have disclosed a new function of c-MIR, inhibition of inflammatory cytokine production. Induction of c-MIR in the joints could be a new therapeutic approach to the treatment of RA.

Oxidative stress plays pivotal roles in aging, neurodegenerative disease, and pathological conditions such as ischemia. We investigated the effect of sulforaphane and 6-(methysulfinyl) hexyl isothiocyanate (6-HITC), a naturally occurring isothiocyanate, on oxidative stress-induced cytotoxicity using primary neuronal cultures of rat striatum. Pretreatment with sulforaphane and 6-HITC significantly protected against H(2)O(2)- and paraquat-induced cytotoxicity in a concentration-dependent manner. Sulforaphane and 6-HITC induced the translocation of nuclear factor E2-related factor 2 (Nrf2) into the nucleus and increased the expression of γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme in glutathione synthesis, and the intracellular glutathione content. Treatment with reduced glutathione (GSH) and N-acetyl-L-cysteine, a substance for glutathione synthesis, significantly prevented the cytotoxicity induced by H(2)O(2) and paraquat. Moreover, exposure to L-buthionine-sulfoximine, an irreversible inhibitor of γ-GCS, suppressed the protective effects of sulforaphane and 6-HITC. In contrast, sulforaphane and 6-HITC increased heme oxygenase-1 (HO-1) expression in neurons. However, zinc-protophorphyrin IX, a competitive inhibitor of HO-1, did not influence the protective effects of sulforaphane and 6-HITC. These results suggest that sulforaphane and 6-HITC prevent oxidative stress-induced cytotoxicity in rat striatal cultures by raising the intracellular glutathione content via an increase in γ-GCS expression induced by the activation of the Nrf2-antioxidant response element pathway.

ADAM metallopeptidase domain 22 (ADAM22) is a neuronal membrane-spanning protein that is a potential receptor for leucine-rich, glioma-inactivated 1 (LGI1), and leucine-rich repeat LGI family, member 4 (LGI4). Several lines of study have shown a direct interaction between ADAM22 and LGI1, a mutation which is responsible for inherited epilepsy in humans. Both ADAM22-deficient mice and claw paw mice, congenitally deficient in LGI4, show hypomyelination in the peripheral nerves, suggesting that these molecules are involved in myelination processes. These findings mark ADAM22 as a potential target molecule for epilepsy or demyelination diseases. To investigate the relationship between ADAM22 mutation and its biological character, we designed and examined several ADAM22 variants. We discovered that the ADAM22 P81R variant, the most common polymorphic variation, works as well as the wild-type ADAM22. We also showed that mutations in the disintegrin domain cause a marked decrease in the processing of ADAM22 preproteins, and result in reduced LGI4-binding abilities. Our findings provide valuable information for mutation screening of the ADAM22 gene in patients suffering from epilepsy or demyelinating diseases.

Currently, there are five anti-Alzheimer's disease drugs approved. These are tacrine, donepezil, rivastigmine, galantamine, and memantine. The mechanism of the first four drugs is acetylcholinesterase inhibition, while memantine is an NMDA-receptor antagonist. However, these drugs do not cure Alzheimer's, but are only symptomatic treatments. Therefore, a cure for Alzheimer's disease is truly needed. Alzheimer's disease is a progressive neurodegenerative disease characterized by cognitive deficits. The cause of the disease is not well understood, but research indicates that the aggregation of beta-amyloid is the fundamental cause. This theory suggests that beta-amyloid aggregation causes neurotoxicity. Therefore, development of the next anti-Alzheimer's disease drug is based on the beta-amyloid theory. We are now studying natural products, such as mulberry leaf extracts and curcumin derivatives, as potential cure for Alzheimer's disease. In this report, we describe some data about these natural products and derivatives.

We previously indicated that amyloid beta (Abeta) augments protein levels of beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) through oxidative stress. In this study, we revealed that BACE-1 is involved in the cleavage of membrane-bound prostaglandin E2 synthase-2 (mPGES-2) in its N-terminal portion, which, in turn, enhanced the generation of prostaglandin E2 (PGE2). PGE2 results in increased Abeta production, initiating a cell-injuring cycle. Using rat primary cortical neurons, a 48 h treatment with Abeta 1-42 (5 microM) resulted in the enhanced extracellular PGE2 levels up to about 1 ng/mL, which was attenuated by treatment with a BACE-1 inhibitor (200 nM). A synthetic peptide sequence of 20-amino acids that included the cleavage site of mPGES-2 (HTARWHL RAQDLHERS AAQLSLSS) was cleaved by recombinant BACE-1, confirmed using reverse-phase high-performance liquid chromatography. Cleaved or activated mPGES-2 augments the generation of PGE2. In addition, mPGES-2 was determined to be colocalized with BACE-1 and cyclooxygenase-2 in the perinuclear region in cells after exposure to Abeta. Exposure of neurons to PGE2 led to cell death, and Abeta production was enhanced by PGE2 (1 ng/mL, 48 h). Collectively, these results suggest that Abeta might cause neuroinflammation that aggravates Alzheimer's disease pathogenesis., (Copyright 2010 Elsevier Inc. All rights reserved.)

Reduction in or dysfunction of glutamate transporter 1 (GLT1) is linked to several neuronal disorders such as stroke, Alzheimer's disease, and amyotrophic lateral sclerosis. However, the detailed mechanism underlying GLT1 regulation has not been fully elucidated. In the present study, we first demonstrated the effects of mammalian target of rapamycin (mTOR) signaling on GLT1 regulation. We prepared astrocytes cultured in astrocyte-defined medium (ADM), which contains several growth factors including epidermal growth factor (EGF) and insulin. The levels of phosphorylated Akt (Ser473) and mTOR (Ser2448) increased, and GLT1 levels were increased in ADM-cultured astrocytes. Treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor or an Akt inhibitor suppressed the phosphorylation of Akt (Ser473) and mTOR (Ser2448) as well as decreased ADM-induced GLT1 upregulation. Treatment with the mTOR inhibitor rapamycin decreased GLT1 protein and mRNA levels. In contrast, rapamycin did not affect Akt (Ser473) phosphorylation. Our results suggest that mTOR is a downstream target of the PI3K/Akt pathway regulating GLT1 expression., (2010 Elsevier Inc. All rights reserved.)

We synthesized carbamate-modified (-)-N(1)-phenethylnorphysostigmine derivatives 3a-u and evaluated their anti-cholinesterase activities. In vitro evaluation showed that cyclohexylmethylcarbamate derivative 3u potently and selectively inhibits butyrylcholinesterase., (Copyright 2010 Elsevier Ltd. All rights reserved.)

We synthesized a series of N(1)-substituted norcymserine derivatives 7a-p and evaluated their anti-cholinesterase activities. In vitro evaluation showed that the pyridinylethyl derivatives 7m-o and the piperidinylethyl derivative 7p improved the anti-butyrylcholinesterase activity by approximately threefold compared to N(1)-phenethylnorcymserine (PEC, 2). A quantitative structure-activity relationship (QSAR) study indicated that logS might be a key feature of the improved compounds., (Copyright 2010 Elsevier Ltd. All rights reserved.)

We have previously shown that chronic donepezil treatment induces nicotinic acetylcholine receptor up-regulation and enhances the sensitivity of the neurons to the neuroprotective effect of donepezil. Further analyses revealed that the nicotinic receptor is involved in this enhancement. In this study, we examined whether nicotinic receptor stimulation is sufficient to make neurons more sensitive to donepezil. We treated primary cultures of rat cortical neurons with nicotine and confirmed that chronic nicotine treatment induced nicotinic receptor up-regulation and made the neurons more sensitive to the neuroprotective effects of donepezil. Analyses with receptor antagonists and kinase inhibitors revealed that the effects of chronic nicotine treatment are mediated by nicotinic receptors and their downstream effectors including phosphatidylinositol 3-kinase. In contrast to chronic donepezil treatment that enhanced the level of nicotine-induced Ca(2+) influx, chronic nicotine treatment did not significantly alter the level of Ca(2+) influx.

We recently demonstrated that microglia as multipotential stem cells give rise to microtubule-associated protein 2 (MAP2)-positive and glial fibrillary acidic protein (GFAP)-positive cells and that microglia-derived MAP2-positive cells possess properties of functional neurons. In this study, we investigated the role of fibroblast growth factor (FGF) signaling in the molecular mechanism underlying the generation of microglia-derived MAP2-positive and GFAP-positive cells. Real-time quantitative PCR analyses demonstrated that mRNA levels of a family of three FGF receptors, Fgfr1-3, were upregulated in microglia treated with 70% fetal bovine serum (FBS). Immunocytochemical analyses demonstrated that basic FGF (bFGF) promoted the generation of microglia-derived MAP2-positive and GFAP-positive cells, and the FGF receptor tyrosine kinase inhibitor SU5402 and the MEK inhibitor PD98059 both inhibited this process. Western blot analyses demonstrated that bFGF increased phosphorylated ERK1/2 levels without altering total ERK1/2 levels. These results suggest that bFGF promotes the generation of microglia-derived MAP2-positive and GFAP-positive cells via FGF receptors and the ERK-MAP kinase pathway.

Amyloid-beta peptide (Abeta) is thought to be linked to the pathogenesis of Alzheimer's disease. Recent studies suggest that Abeta has important physiological roles in addition to its pathological roles. We recently demonstrated that Abeta42 protects hippocampal neurons from glutamate-induced neurotoxicity, but the relationship between Abeta42 assemblies and their neuroprotective effects remains largely unknown. In this study, we prepared non-fibrillar and fibrillar Abeta42 based on the results of the thioflavin T assay, Western blot analysis, and atomic force microscopy, and examined the effects of non-fibrillar and fibrillar Abeta42 on glutamate-induced neurotoxicity. Non-fibrillar Abeta42, but not fibrillar Abeta42, protected hippocampal neurons from glutamate-induced neurotoxicity. Furthermore, non-fibrillar Abeta42 decreased both neurotoxicity and increases in the intracellular Ca(2+) concentration induced by N-methyl-d-aspartate (NMDA), but not by alpha-amino-3-hydrozy-5-methyl-4-isoxazole propionic acid (AMPA). Our results suggest that non-fibrillar Abeta42 protects hippocampal neurons from glutamate-induced neurotoxicity through regulation of the NMDA receptor.

Fibrillar aggregates of amyloid beta-peptides are major constituents of the plaques found in the brains of patients with Alzheimer's disease, and have been implicated in the neurotoxicity of Alzheimer's. We previously reported that the methanol extract of mulberry leaves inhibits the formation of amyloid beta-peptide (1-42)-fibrils in vitro, and protects hippocampal neurons from amyloid beta-peptide (1-42)-induced cell death. In this study, we identified antiamyloidogenic substances, pheophorbide a, kaempferol -3-O-glucoside, and kaempferol -3-O-(6-malonyl) glucoside, from the methanol extract of mulberry leaves. We also compared the antiamyloidogenic activity of pheophorbide a with that of other porphyrin-related compounds.

Rac1, a member of the Rho family GTPases, participates in a variety of cellular functions including lamellipodia formation, actin cytoskeleton organization, cell growth, apoptosis, and neuronal development. Recent studies have implicated Rac1 in cytoskeletal abnormalities, production of reactive oxygen species, and generation of the amyloid beta-peptide (Abeta) observed in Alzheimer's disease. In this study, we examined the relationship between Rac1 and amyloid precursor protein (APP), because the abnormal proteolytic processing of APP is a pathologic feature of Alzheimer's disease. In primary hippocampal neurons, the Rac1-specific inhibitor NSC23766 decreased both Rac1 activity and APP protein levels in a concentration-dependent manner. To elucidate how NSC23766 decreases APP protein levels, we examined the effects of NSC23766 on APP processing, degradation, and biosynthesis. NSC23766 did not increase the levels of the proteolytic products of APP, sAPPalpha, Abeta40, and Abeta42. The proteasome inhibitor lactacystin did not reverse the NSC23766-induced decrease in APP protein levels. NSC23766 did, however, decrease the levels of both APP mRNA and APP protein. Decreased levels of APP mRNA and protein were also observed when HEK293 cells were transfected with an expression vector containing a dominant-negative Rac1 mutant or with siRNA targeting Rac1. By overexpressing progressively deleted fragments of the APP promoter in HEK293 cells, we identified a Rac1 response site at positions -233 to -41 bp in the APP promoter. Taken together, our results suggest that Rac1 regulates transcription of the APP gene in primary hippocampal neurons., ((c) 2009 Wiley-Liss, Inc.)

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) is known to activate the ER, which is termed ER stress. Here, we demonstrated that amyloid precursor protein (APP) is a novel mediator of ER stress-induced apoptosis through the C/EBP homologous protein (CHOP) pathway. Expression of APP mRNA was elevated by tunicamycin- or dithiothreitol-induced ER stress. The levels of C83 and APP intracellular domain (AICD) fragments, which are cleaved from APP, were significantly increased under ER stress, although the protein level of full-length APP was decreased. Cellular viability was reduced in APP-over-expressing cells, which was attenuated by treatment with a gamma-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Cellular viability was also reduced in AICD-FLAG-over-expressing cells. The mRNA and protein levels of CHOP, an ER stress-responsive gene, were remarkably increased by APP over-expression, which was attenuated by treatment with DAPT. CHOP mRNA induction was also found in AICD-FLAG-over-expressing cells. Cell death and CHOP up-regulation by ER stress were attenuated by APP knockdown. Data obtained with a luciferase assay and chromatin immunoprecipitation assay indicated that AICD associates with the promoter region of the CHOP gene. In conclusion, ER stress-induced APP undergoes alpha- and gamma-secretase cleavage and subsequently induces CHOP-mediated cell death.

Protection of neurons from neuronal damage and cell death in neurodegenerative disease is a major challenge in neuroscience research. Donepezil, galantamine and tacrine are acetylcholinesterase inhibitors used for the treatment of Alzheimer's disease, and were believed to be symptomatic drugs whose therapeutic effects are achieved by slowing the hydrolysis of acetylcholine at synaptic termini. However, recent accumulated evidence strongly suggests that these acetylcholinesterase inhibitors also possess neuroprotective properties whose mechanism is independent of acetylcholinesterase inhibition. We have shown that acetylcholinesterase inhibitors protect neurons from glutamate-induced neurotoxicity in the primary culture of rat cortical neurons. It was also found that acetylcholinesterase inhibitor treatment induces up-regulation of nicotinic receptor expression levels, a property which may also have some bearing on their therapeutic effects. We next showed that alpha4 and alpha7-nicotinic receptors play important roles in acetylcholinesterase inhibitor-induced neuroprotection and nicotinic receptor up-regulation. Our results also demonstrate the important roles of the phosphatidylinositol 3-kinase pathway downstream of nicotinic receptors in protecting neurons from death and up-regulating nicotinic receptors. This review summarizes recent findings on the roles of the nicotinic receptor in acetylcholinesterase inhibitor-induced neuroprotection and nicotinic receptor up-regulation.

We recently demonstrated that, as a type of multipotential stem cells, microglia give rise to microtubule-associated protein 2 (MAP2)-positive and glial fibrillary acidic protein (GFAP)-positive cells. In this study, we investigated the role of SOX2, a high-mobility group DNA binding domain transcription factor, in the generation of microglia-derived MAP2-positive and GFAP-positive cells. Western blot analysis demonstrated that expression of SOX2 was upregulated by treatment with 70% fetal bovine serum treatment. Immunocytochemical analyses demonstrated that SOX2 expression was evident in the nuclei of microglia-derived MAP2-positive and GFAP-positive cells, whereas it was not present in the nuclei of microglia. These assays also showed that Sox2 siRNA inhibited the generation of MAP2-positive and GFAP-positive cells from microglia. Interestingly, this activity was also inhibited by Smad4 siRNA, which reduces SOX2 expression. These results indicate that SOX2 upregulation is involved in the generation of microglia-derived MAP2-positive and GFAP-positive cells through SMAD4.

Background: It is well known that patients with chronic kidney disease, including diabetic nephropathy, often develop cardiovascular diseases. In case of radiographic procedures, reduced renal function may be deteriorated by the use of iodinated contrast medium (CM). This is known as CM-induced nephropathy. In this study, we have focused on the mechanisms of this type of injury in diabetic nephropathy and the preventive effects of serofendic acid., Methods: We evaluated the cytotoxicity of CM and high glucose on tubular epithelial cells using an LLC-PK1 cell line, and measured cell viability with an alamarBlue assay. We further evaluated superoxide production levels measured by dihydroethidium. We also examined the protective effects of serofendic acid on cytotoxicity with superoxide production of CM and high glucose., Results: CM reduced cell numbers in a dose-dependent and time-dependent manner in LLC-PK1 cells. Furthermore, cytotoxicity of CM in diluted concentration was additively influenced by high glucose. CM and high glucose increased superoxide production, which was evaluated by the response to dihydroethidium, and was suppressed by serofendic acid. Cytotoxicity of CM, high glucose, and H(2)O(2) was suppressed by serofendic acid, as well as the suppression by N-acetylcysteine on CM toxicity. Interestingly, the recovery by serofendic acid in H(2)O(2)- and high glucose-induced cellular injury was to the basal level, in contrast with the partial recovery from CM-induced injury. Finally, serofendic acid suppressed CM-induced injury and high glucose-induced apoptosis., Conclusions: These results suggest that CM and high glucose induce cytotoxicity and oxidative stress in LLC-PK1 cells and that serofendic acid protects the injury probably from superoxide generation.

Emerging evidence has suggested that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) protects neurons from glutamate-induced neurotoxicity. In this study, we examined the effect of AMPA on the cell surface expression of the N-methyl-D-aspartate (NMDA) receptor, a central player in glutamate-induced neurotoxicity, using rat cortical neurons. AMPA (10 microM, 24 h) attenuated the expression of cell surface NR1, an NMDA receptor subunit, and also inhibited glutamate-induced increases in intracellular Ca2+. SB216763, an inhibitor of glycogen synthase kinase 3beta (GSK3beta), had effects similar to those of AMPA. We have earlier shown that AMPA treatment attenuated GSK3beta activity. Our data suggest that AMPA reduces the cell surface expression of NMDA receptors through the regulation of GSK3beta and, consequently, reduces the amount of intracellular Ca2+.

We investigated the effect of serofendic acid, a neuroprotective substance derived from fetal calf serum, on the morphological changes in cultured cortical astrocytes. Cultured astrocytes developed a stellate morphology with several processes following exposure to dibutylyl cAMP (dbcAMP), a membrane-permeable cAMP analog; 8-Br-cGMP, a membrane-permeable cGMP analog; or phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator. Serofendic acid significantly accelerated the stellation induced by dbcAMP- and 8-Br-cGMP. In contrast, the PMA-induced stellation was not affected by serofendic acid. Next, we attempted to elucidate the mechanism underlying the dbcAMP-induced stellation and explore the site of action of serofendic acid. Both the stellation induced by dbcAMP and the promotional effect of serofendic acid were partially inhibited by KT5720, a specific protein kinase A (PKA) inhibitor. Furthermore, serofendic acid failed to facilitate the stellation induced by Y-27632, an inhibitor of Rho-associated kinase (ROCK). These results indicate that serofendic acid promotes dbcAMP- and 8-Br-cGMP-induced stellation and the promotional effect on dbcAMP-induced stellation is mediated at least partly by the regulation of PKA activity and not by controlling ROCK activity.

In neurons, Presenilin 1(PS1)/gamma-secretase is located at the synapses, bound to N-cadherin. We have previously reported that N-cadherin-mediated cell-cell contact promotes cell-surface expression of PS1/gamma-secretase. We postulated that N-cadherin-mediated trafficking of PS1 might impact synaptic PS1-amyloid precursor protein interactions and Abeta generation. In the present report, we evaluate the effect of N-cadherin-based contacts on Abeta production. We demonstrate that stable expression of N-cadherin in Chinese hamster ovary cells, expressing the Swedish mutant of human amyloid precursor protein leads to enhanced secretion of Abeta in the medium. Moreover, N-cadherin expression decreased Abeta(42/40) ratio. The effect of N-cadherin expression on Abeta production was accompanied by the enhanced accessibility of PS1/gamma-secretase to amyloid precursor protein as well as a conformational change of PS1, as demonstrated by the fluorescence lifetime imaging technique. These results indicate that N-cadherin-mediated synaptic adhesion may modulate Abeta secretion as well as the Abeta(42/40) ratio via PS1/N-cadherin interactions.

Abnormal proteolytic processing of amyloid precursor protein (APP) is a pathologic feature of Alzheimer's disease. Recent studies have demonstrated that serine/threonine phosphorylation specifically at amino-acid residue Thr668 (APP695 numbering) regulates APP processing. In this study, we investigated the possibility that tyrosine phosphorylation of APP regulates APP processing. A tyrosine kinase inhibitor decreased expression of the C83 fragment which is a cleaved product of APP by alpha-secretase. By overexpressing APP mutant proteins, Tyr687 was found to be the major tyrosine kinase phosphorylation site. Expression of the C83 fragment was decreased in APPY687A-expressing cells relative to APP wild-type (APPWT)-expressing cells, which likely reflects the different cellular localization patterns of these two proteins. Expression of APP intracellular domain (AICD) which is a cleaved product of the C83 fragment by gamma-secretase was decreased in C83Y687A-expressing cells. These results suggest that phosphorylation of APP at Tyr687 regulates APP processing by alpha- and gamma-secretases, determining the expression level of AICD.

Previously, we showed that in rat cortical neurons, chronic donepezil treatment (10 microM, 4 days) up-regulates nicotinic receptors (nAChR) and makes neurons more sensitive to the neuroprotective effect of donepezil. Here we examined the mechanism of donepezil-induced neuroprotection in neurons chronically treated with donepezil. The mechanism of neuroprotection was examined under different conditions of exposure to glutamate, acute and moderate, that induce cell death associated with necrotic and apoptotic cell death, respectively. Concomitant treatment with antagonists of nAChRs but not muscarinic receptors inhibited donepezil pretreatment-induced neuroprotection against acute glutamate treatment-induced death. Donepezil pretreatment prevented acute glutamate- and ionomycin-induced neurotoxicity, but not S-nitrosocysteine-induced neurotoxicity, suggesting that donepezil protects neurons via nAChR at levels before nitric oxide synthase activation against acute glutamate neurotoxicity. Concomitant treatment with antagonists of nAChR or phosphatidylinositol 3-kinase (PI3K) signaling inhibitors significantly inhibited neuroprotection against moderate glutamate neurotoxicity and decreased the phosphorylation level of Akt. Neuroprotection was also inhibited by treatment with inhibitor of mitogen-activated protein kinase (MAPK) kinase. These results suggest that donepezil protects neurons against moderate glutamate neurotoxicity via nAChR-PI3K-Akt and MAPK signaling pathways. This study provides novel insight into the mechanism of donepezil-induced neuroprotection that involves nAChR up-regulation., ((c) 2008 Wiley-Liss, Inc.)

A wide range of evidences show that cholinesterase (ChE) inhibitors can interfere with the progression of Alzheimer's disease (AD). The earliest known ChE inhibitors, namely, physostigmine and tacrine, showed modest improvement in the cognitive function of AD patients. However, clinical studies show that physostigmine has poor oral activity, brain penetration and pharmacokinetic parameters while tacrine has hepatotoxic liability. Studies were then focused on finding a new type of acetylcholinesterase (AChE) inhibitor that would overcome the disadvantages of these two compounds. During the study, by chance we found a seed compound. We then conducted a structure-activity relationship (SAR) study of this compound. After four years of exploratory research, we found donepezil hydrochloride (donepezil). Recently, acetylcholinesterase inhibitors (AChEIs) have been studied for other mechanisms of action, such as neuroprotective action and lowering of beta-amyloid (beta-amyloid). Donepezil also reduced beta-amyloid plaque in in vitro. The amyloid hypothesis is believed to be the most promising approach in the development of anti-AD drugs. We speculate the mechanism of lowering beta-amyloid by donepezil implicate alpha-secretase (alpha-secretase) enhancer.

Neurogenesis occurs in two neurogenic regions of the adult mammalian brain: the subgranular zone and the subventricular zone. We have recently demonstrated that the number of bromodeoxyuridine-positive and doublecortin-positive cells is decreased in the subgranular zone of amyloid precursor protein with a Swedish mutation and presenilin-1 with a deletion of exon 9 transgenic mice, an animal model of Alzheimer's disease. In this study, we characterized neurogenesis in the subventricular zone of amyloid precursor protein with a Swedish mutation and presenilin-1 with a deletion of exon 9 transgenic mice at 9 months of age and compared it with neurogenesis in the subgranular zone. In the subventricular zone, the number of proliferating cell nuclear antigen-positive and bromodeoxyuridine-positive cells were normal. In the subgranular zone, the number of proliferating cell nuclear antigen-positive cells was normal; however, the number of bromodeoxyuridine-positive cells was significantly decreased. These results suggest that neurogenesis, probably reflecting the survival of neural progenitor cells, differs between the subgranular zone and the subventricular zone.

Beta-site APP cleaving enzyme-1 (BACE-1), is a rate-limiting enzyme for beta amyloid production. Beta amyloid induces the production of radical oxygen species and neuronal injury. Oxidative stress plays a key role in various neurological diseases such as ischemia and Alzheimer's disease. Recent studies suggest that oxidative stress induces BACE-1 protein upregulation in neuronal cells. Here, we demonstrate that naturally occurring compounds (-)-epigallocatechin-3-gallate and curcumin suppress beta amyloid-induced BACE-1 upregulation. Exposure of beta amyloid 1-42 to neuronal culture increased BACE-1 protein levels. (-)-Epigallocatechin-3-gallate or curcumin significantly attenuated beta amyloid-induced radical oxygen species production and beta-sheet structure formation. These two compounds have novel pharmacological effects that may be beneficial for Alzheimer's disease treatment.

alpha7-nicotinic acetylcholine receptors are one of the most abundant subtypes of nicotinic receptors in the brain and have been shown to be involved in the neuroprotective effect of donepezil. Recently, we showed that in primary culture of rat cortical neurons, chronic donepezil treatment (10 muM, 4 days) (1) induces the up-regulation of alpha7-nicotinic receptors, (2) enhances the nicotine-induced increase in [Ca(2+)](i) and (3) enhances the sensitivity to the neuroprotective effect of donepezil. Here we demonstrate the involvement of alpha7-nicotinic receptors in these three effects. Concomitant treatment with nicotinic receptor antagonist inhibited the up-regulation of alpha7-nicotinic receptor, enhancement of the increase in [Ca(2+)](i) induced by nicotine, and enhancement of sensitivity to the neuroprotective effect of donepezil. Next, using inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways, we demonstrate the involvement of these pathways in the up-regulation of alpha7-nicotinic receptors and in making the neurons more sensitive to the neuroprotective effects of donepezil. Concomitant chronic donepezil treatment with inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways inhibited nicotinic receptor up-regulation and enhancement of the response to nicotine, and enhanced the sensitivity to donepezil. This study increases understanding of the less-studied mechanism of chronic donepezil treatment-induced nicotinic receptor up-regulation and increased sensitivity to donepezil.

The excitatory neurotransmitter glutamate can accumulate in the brain and is thought to be involved in the etiology of many neurodegenerative disorders, including ischemia and Alzheimer disease. Therefore, it is important to search for compounds that reduce glutamate neurotoxicity. This glutamate-mediated excitotoxicity is caused by intracellular Ca2+ overload via the N-methyl-D-aspartate receptor NMDAR), reactive oxygen species (ROS) generation, and caspase-3 activation. Here we show that the natural flavonoid myricetin inhibited glutamate-induced excitotoxicity and protected neurons by multiple, distinct pathways. First, myricetin affected modulation of the NMDAR by phosphorylation, causing a subsequent reduction in glutamate-induced intracellular Ca2+ overload. Second, myricetin inhibited the ROS production caused by glutamate. Finally, glutamate-induced activation of caspase-3 was reduced by myricetin treatment. Moreover, myricetin directly interacted with the active site of caspase-3 via three hydrogen bonds and inhibited its activity. We conclude that myricetin inhibited glutamate-induced neuronal toxicity by multiple biochemical pathways. These results show that myricetin is a potent antineurodegenerative compound and may contribute to the discovery of a drug with which to combat neurodegeneration., ((c) 2008 Wiley-Liss, Inc.)

We previously reported that a sulfur-containing neuroprotective substance named serofendic acid purified and isolated from fetal calf serum prevented glutamate neurotoxicity in rat cortical cultured neurons. In the present study, we investigated the effect of serofendic acid on ischemic injury induced by a transient occlusion of the middle cerebral artery in rats. Serofendic acid was intracerebroventricularly administered 30 min after the onset of the occlusion. Serofendic acid (30 nmol) significantly reduced total infarct volume, similar to edaravone (30 nmol), a free radical scavenger. Treatment with serofendic acid (1-30 nmol) reduced the infarct volume in a dose-dependent manner. Moreover, serofendic acid (30 nmol) improved neurological deficit scores. These results suggest that intracerebroventricular administration of serofendic acid prevents the neurodegeneration induced by a transient focal cerebral ischemia and reperfusion.