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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.)

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.)

Preconditioning of sublethal ischemia exhibits neuroprotection against subsequent ischemia-induced neuronal death. It has been indicated that glutamate, an excitatory amino acid, is involved in the pathogenesis of ischemia-induced neuronal death or neurodegeneration. To elucidate whether prestimulation of glutamate receptor could counter ischemia-induced neuronal death or neurodegeneration, we examined the effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), an ionotropic subtype of glutamate receptor, on excess glutamate-induced excitotoxicity using primary cortical neuronal cultures. We found that AMPA exerted a neuroprotective effect in a time- and concentration-dependent manner. A blocker of phosphatidylinositol-3 kinase (PI3K), LY294002 (10 microM), significantly attenuated AMPA-induced protection. In addition, Ser473 of Akt/PKB, a downstream target of PI3K, was phosphorylated by AMPA administration (10 microM). Glycogen synthase kinase 3beta (GSK3beta), which has been reported to be inactivated by Akt, was phosphorylated at Ser9 by AMPA. Ser9-phosphorylated GSK3beta or inactivated form would be a key molecule for neuroprotection, insofar as lithium chloride (100 microM) and SB216763 (10 microM), inhibitors of GSK3beta, also induced phosphorylation of GSK3beta at Ser9 and exerted neuroprotection, respectively. Glutamate (100 microM) increased cleaved caspase-3, an apoptosis-related cysteine protease, and caspase-3 inhibitor (Ac-DEVD-CHO; 1 microM) blocked glutamate-induced excitotoxicity in our culture. AMPA (10 microM, 24 hr) and SB216763 (10 microM) prominently decreased glutamate-induced caspase-3 cleavage. These findings suggest that AMPA activates PI3K-Akt and subsequently inhibits GSK3beta and that inactivated GSK3beta attenuates glutamate-induced caspase-3 cleavage and neurotoxicity.

Myricetin (3,3',4',5,5',7-hexahydroxyflavone) is classified as a flavonoid with strong antioxidant effects. Oxidative stress plays a key role in various neurological diseases such as ischemia and Alzheimer's disease (AD). To elucidate whether myricetin could counter the progress of AD, we examined the effects of myricetin on neurotoxicity induced by beta-amyloid (A beta), a component of senile plaques in the AD brain. We found that cultured rat primary cortical neurons treated for 48 hr with A beta1-42 (1 microM) induced significant neuronal injury. Conformationally altered A beta1-42 caused apoptotic changes, such as nuclear fragmentation, as shown by DAPI staining. Pre- and simultaneous administration of myricetin and A beta1-42 reduced A beta neurotoxicity in a concentration-dependent manner. By using circular dichroism spectroscopy and a thioflavin T binding assay, we show that myricetin (10 microM, 48 hr) prevented structural changes in A beta1-42 from a random coil to a beta-sheet-rich structure. A beta1-42-induced apoptotic changes and caspase-3 activation were reduced by myricetin treatment. Furthermore, we determined that administration of myricetin significantly decreased A beta1-40 and A beta1-42 levels in culture media. These effects were based on two mechanisms: the activation and up-regulation of alpha-secretase (ADAM10) protein levels as indicated by fluorescence resonance energy transfer (FRET) assay and immunoblot analysis and the direct binding and inhibition of beta-secretase (BACE-1) indicated by cell-free FRET assays. Evidently, myricetin has multiple functions to counter the progress of AD by the reduction of A beta production and the detoxification of A beta through a structural change., ((c) 2007 Wiley-Liss, Inc.)

Mulberry leaf has been reported to possess medicinal properties, including hypoglycemic, hypotensive and diuretic effects. Little is known, however, about its medicinal properties for central nervous system disorders, including Alzheimer's disease. Accumulating evidence suggests that amyloid beta-peptide (1-42) plays an important role in the etiology of Alzheimer's disease. Here we show that mulberry leaf extract inhibits the amyloid beta-peptide (1-42) fibril formation by both the thioflavin T fluorescence assay and atomic force microscopy. Furthermore, mulberry leaf extract protected hippocampal neurons against amyloid beta-peptide (1-42)-induced cell death in a concentration-dependent manner. These results suggest that mulberry leaf extract provides a viable treatment for Alzheimer's disease through the inhibition of amyloid beta-peptide (1-42) fibril formation and attenuation of amyloid beta-peptide (1-42)-induced neurotoxicity.

Glutamate excitotoxicity is mediated by intracellular Ca(2+) overload, caspase-3 activation, and ROS generation. Here, we show that curcumin, tannic acid (TA) and (+)-catechin hydrate (CA) all inhibited glutamate-induced excitotoxicity. Curcumin inhibited PKC activity, and subsequent phosphorylation of NR1 of the NMDA receptor. As a result, glutamate-mediated Ca(2+) influx was reduced. TA attenuated glutamate-mediated Ca(2+) influx only when simultaneously administered, directly interfering with Ca(2+). Both curcumin and TA inhibited glutamate-induced caspase-3 activation. Although Ca(2+) influx was not attenuated by CA, caspase-3 was reduced by direct inhibition of the enzyme. All polyphenols reduced glutamate-induced generation of ROS.

We have previously reported that cyclic guanosine-3',5'-monophosphate (cGMP) protects spinal motor neurons against acute reactive oxygen species (ROS)-induced toxicity but not against chronic ROS-induced or glutamate (Glu)-induced toxicity. In this study, we investigated the effects of phosphodiesterase (PDE) inhibitors on the survival of cultured spinal motor neurons. Selective PDE5 inhibitors (dipyridamole, T-1032, and zaprinast) as well as a nonselective PDE inhibitor (aminophylline) protected motor and nonmotor neurons against both acute ROS-induced and chronic Glu-induced neurotoxicity, whereas selective inhibitors of PDE1-4 offered no protection. 8-Bromo-cGMP (8br-cGMP), a cGMP analogue, protected both motor and nonmotor neurons against acute ROS-induced toxicity but protected only nonmotor neurons against chronic Glu-induced toxicity. This neuroprotection was blocked by KT5823, a cGMP-dependent protein kinase (PKG) inhibitor. Immunohistochemical staining confirmed that PDE5 and PKG are located in almost all rat lumbar spinal neurons. Furthermore, semiquantitative analysis of the immunostaining intensity revealed that PDE5 was more abundant in motor neurons than in nonmotor neurons. Our results suggest that this difference in the amount of PDE5 may be responsible for the vulnerability of motor neurons to chronic excitotoxicity. In addition, the results of this study raise the possibility that PDE5 inhibitors might be used as a treatment for amyotrophic lateral sclerosis., (Copyright 2002 Wiley-Liss, Inc.)

Glutamate, one of the excitatory neurotransmitters, contributes to the neuronal death associated with neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, and with ischemia. In Alzheimer's disease brains, there is a decreased number of dopamine D2 receptors, which might cause neuronal dysfunction or death. In the present study, bromocriptine exerted a protective effect against glutamate-induced cytotoxicity in rat cortical neurons. This neuroprotective effect was mediated via D2 receptors, because it was attenuated by domperidone, a D2 dopaminergic receptor antagonist. Another dopamine D2 agonist, quinpirole, also protected cells against glutamate toxicity. D2 agonists protected cells from calcium influx, nitric oxide, and peroxynitrite toxicity, which are thought to be the mediators of glutamate toxicity. The phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) inhibited this neuroprotective effect of bromocriptine, in contrast to the mitogen-activated protein kinase kinase (MAPKK) inhibitor (PD98059), which did not counter the protective effect. Furthermore, Akt protein kinase, which is an effector of PI3K, was activated by bromocriptine, and the antiapoptotic protein Bcl-2 was up-regulated by bromocriptine treatment. These results suggest that D2 dopaminergic receptor activation plays an important role in neuroprotection against glutamate cytotoxicity and that the up-regulation of Bcl-2 expression via the PI3K cascade is, at least partially, involved in this effect., (Copyright 2002 Wiley-Liss, Inc.)

Parkinson's disease is characterized by the mesencephalic dopaminergic neuronal loss, possibly by apoptosis, and the prevalence is higher in males than in females. The estrogen receptor (ER) subtype in the mesencephalon is exclusively ER beta, a recently cloned novel subtype. Bound with estradiol, it enhances gene transcription through the estrogen response element (ERE) or inhibits it through the activator protein-1 (AP-1) site. We demonstrated that 17beta-estradiol provided protection against nigral neuronal apoptosis caused by exposure to either bleomycin sulfate (BLM) or buthionine sulfoximine (BSO). BLM and BSO-induced nigral apoptosis was blocked by inhibitors for caspase-3 or c-Jun/AP-1. The antiapoptotic effect by estradiol was blocked by ICI 182,780, an antagonist for ER, but not by a synthesized peptide that inhibits binding of the ER to the ERE. Estradiol had no effects on caspase-3 activation and c-Jun NH(2)-terminal kinase (JNK), which were activated by BLM. It also suppressed apoptosis by serum deprivation, which was independent of caspase-3 activation. Therefore, the antiapoptotic neuroprotection by estradiol is mediated by transcription through AP-1 site downstream from JNK and caspase-3 activation. Furthermore, 17alpha-estradiol, a stereoisomer without female hormone activity, also provided an antiapoptotic effect. Therefore, the antiapoptotic effect is independent of female hormone activity.

It has been shown that estrogen replacement in menopausal women is effective in slowing down the progression of cognitive impairment in Alzheimer's disease. Although recent studies have demonstrated the neuroprotective effects of estrogen, the precise mechanism of neuroprotection has not been elucidated. In the present study, we show that the phosphatidylinositol 3-kinase (PI3-K) cascade is involved in the neuroprotective mechanism stimulated by estrogen. Exposure to glutamate reduced the viability of rat primary cortical neurons. Pretreatment with 10 nM 17beta-estradiol significantly attenuated the glutamate-induced toxicity. This neuroprotective effect of 17beta-estradiol was blocked by co-administration with LY294002, a selective PI3-K inhibitor, but not by co-administration with PD98059, a selective mitogen activated protein kinase kinase inhibitor. Pretreatment with ICI182780, a specific estrogen receptor antagonist, also blocked the neuroprotection. Immunoblotting assay revealed that treatment with 17beta-estradiol induced the phosphorylation of Akt/PKB, an effector immediately downstream of PI3-K. These results suggest that PI3-K mediates the neuroprotective effect of 17beta-estradiol against glutamate-induced neurotoxicity., (Copyright 2000 Wiley-Liss, Inc.)

omega-Agatoxin IVA-sensitive Ca(2+) channels have been thought to be involved in physiological excitatory amino acid glutamate release and these channels may also contribute to the development of ischemic brain injury. Recently, we demonstrated that alpha-eudesmol from Juniperus virginiana Linn. (Cupressaceae) inhibits potently the presynaptic omega-agatoxin IVA-sensitive Ca(2+) channels. In the present study, we investigated the effects of alpha-eudesmol on brain edema formation and infarct size determined after 24 h of reperfusion following 1 h of middle cerebral artery occlusion in rats. We first found that alpha-eudesmol concentration-dependently inhibited glutamate release from rat brain synaptosomes and that its inhibitory effect was Ca(2+)-dependent. In the middle cerebral artery occlusion study, intracerebroventricular (i.c.v.) treatment with alpha-eudesmol significantly attenuated the post-ischemic increase in brain water content. alpha-Eudesmol also significantly reduced the size of the infarct area determined by triphenyltetrazolium chloride staining after 24 h of reperfusion. Using a microdialysis technique, we further demonstrated that alpha-eudesmol inhibits the elevation of the extracellular concentration of glutamate during ischemia. From these results, we suggest that alpha-eudesmol displays an ability to inhibit exocytotic glutamate release and to attenuate post-ischemic brain injury.

Glial cell line-derived neurotrophic factor (GDNF) provides neuroprotection, but its neuroprotective mechanism has not been resolved. We investigated the neuroprotective mechanism of GDNF using primary culture of the rat mesencephalon. Bleomycin sulfate (BLM) and L-buthionine-[S,R]-sulfoximine (BSO) caused apoptosis in both dopaminergic and nondopaminergic neurons, as revealed by the presence of chromatin condensation, and positive staining by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL). GDNF preincubation blocked the neurotoxicity and reduced the number of the TUNEL-positive cells caused by BLM and BSO exposure. In contrast, GDNF did not provide neuroprotection against glutamate toxicity, which was not accompanied by these apoptotic features. The neuroprotection was mediated by phosphatidylinositol 3-kinase, an effector downstream from c-Ret, because it was blocked by LY294002. GDNF pretreatment caused up-regulation of Bcl-2 and Bcl-x. Furthermore, GDNF suppressed oxygen radical accumulation caused by BLM. Apoptosis induced by BLM and BSO was blocked by a caspase-3 inhibitor. Caspase-3 activity was elevated by BLM and suppressed by GDNF pretreatment. These findings indicate that GDNF has no effect on necrosis but exerts protection against apoptosis by activation of phosphatidylinositol 3-kinase and the subsequent up-regulation of Bcl-2 and Bcl-x, which suppresses accumulation of oxygen radicals followed by caspase-3 activation.

Accumulating evidence suggests that the neurotrophin receptors, Trks and p75, play distinct roles in regulating cells survival and death, with Trks important for cell survival, and p75 acting to induce cell death. Here, we provide evidence that, in neuronal cultures from rat cerebral cortex, nerve growth factor (NGF) exerts neuroprotective actions via p75. Incubating cultures with NGF for 1-24 h protected cortical neurons from delayed cytotoxicity induced by brief exposure to glutamate. Delayed neurotoxicity induced by a calcium ionophore, ionomycin, or nitric oxide (NO) donors such as S-nitrosocysteine (SNOC) and 3-morpholinosydnonimine (SIN-1), was also attenuated by pretreatment with NGF. RT-PCR analysis revealed the presence of p75 and trkB transcripts in cortical cultures, but did not detect transcripts of trkA, a high-affinity receptor for NGF. Brain-derived neurotrophic factor (BDNF), but not NGF, induced tyrosine phosphorylation of Trks, indicating that NGF does not activate Trks in cortical neurons. Concurrent application of anti-p75 neutralizing antibody markedly reduced the neuroprotective effect of NGF, but resulted in only a modest reduction of that of BDNF. BDNF-induced neuroprotection, but not NGF-induced neuroprotection, was inhibited by a protein synthesis inhibitor cycloheximide. Distinct signaling pathways mobilized by NGF and BDNF were also revealed in that NGF but not BDNF stimulated significant production of ceramides, whereas BDNF but not NGF caused persistent activation of mitogen-activated protein kinases. These results indicate that, although NGF and BDNF both protect cortical neurons from excitotoxicity, the mechanisms involved in their effects are totally different. The present results are, to our knowledge, the first to demonstrate the principal involvement of p75 in cytoprotective actions of neurotrophins.

Oxidative stress is important in the process of dopaminergic neuronal degeneration in Parkinson's disease. Recent studies suggest that estrogens have neuroprotective effects in neurodegenerative disorders, including Alzheimer's disease. In the present study, we investigated neuroprotection against oxidative stress afforded by estradiol using primary neuronal culture of the rat ventral mesencephalon. Oxidative stress induced by glutamate, superoxide anions, and hydrogen peroxide caused significant neuronal death. Although simultaneous administration of 17beta-estradiol and glutamate did not show any significant effects, preincubation with 17beta-estradiol provided significant neuroprotection against glutamate-induced neurotoxicity (ED50 was 50 microM for dopaminergic and 15 microM for nondopaminergic neurons). Neuroprotection occurred even after a brief preincubation with 17beta-estradiol and was not significantly blocked by either an estrogen receptor antagonist or a protein synthesis inhibitor. These findings indicate that the neuroprotection against glutamate neurotoxicity is mediated by neither estrogen receptors nor activation of genome transcription. Other steroids (corticosterone, testosterone, and cholesterol) did not provide significant neuroprotection against glutamate-induced neurotoxicity. Furthermore, preincubation with 17beta-estradiol provided neuroprotection against neuronal death induced by both superoxide anions and hydrogen peroxide. Dichlorofluorescin diacetate, a marker of oxygen radicals, revealed that preincubation with 17beta-estradiol suppressed intracellular oxygen radicals induced by hydrogen peroxide. The biologically inactive stereoisomer of estradiol, 17alpha-estradiol, provided neuroprotection against glutamate-induced toxicity in dopaminergic neurons, as well as the 17beta isoform. 17Alpha-estradiol may be a potential therapeutic agent used to prevent dopaminergic neuronal death induced by oxidative stress in Parkinson's disease.

Oxidative stress, a process in which neurotoxic oxygen free radicals cause dopaminergic neuronal degeneration, has been implicated in the degenerative process in Parkinson's disease. Glutamate-induced neurotoxicity is a model of oxidative stress. We demonstrated that preincubation with D2-type dopamine agonists bromocriptine and quinpirole provides neuroprotection against glutamate-induced neurotoxicity in cultured rat mesencephalic neurons. Simultaneous administration of D2 agonists, however, did not provide neuroprotection. The protective effects were dependent on the duration of preincubation and were blocked by a D2 antagonist and a protein synthesis inhibitor. Furthermore, preincubation with D2 agonists provided neuroprotection against toxicity induced by calcium overload and exposure to superoxide anions. Confocal microscopic analysis, using 2,7-dichlorofluorescin diacetate, revealed that bromocriptine preincubation suppressed the action of radicals on neurons. These findings indicate that dopamine D2 agonists provide protection mediated not only by the inhibition of dopamine turnover but also via D2-type dopamine receptor stimulation and the subsequent synthesis of proteins that scavenge free radicals.

beta-Amyloid (A beta), a major constituent of senile plaques in Alzheimer's disease (AD), is thought to contribute to the neurodegeneration. We examined the effects of nicotinic receptor agonists on A beta cytotoxicity in cultured rat cortical neurons. The number of viable neurons decreased significantly when cultures were exposed to synthetic A beta peptides (25-35). Concomitant administration of nicotine with A beta markedly reduced the number of dead cells. This nicotine-induced neuroprotection was dependent on the concentration. When hexamethonium or mecamylamine, nicotinic antagonist, was added, neuroprotective effect of nicotine was blocked, which indicates that effect of nicotine was mediated by nicotinic receptors. In addition, a selective alpha7-receptor antagonist, alpha-bungarotoxin (alpha-BTX), blocked the neuroprotective effect of nicotine. Furthermore, incubation with 3-(2,4)-dimethoxybenzylidene anabaseine (DMXB), a selective alpha7-receptor agonist, protected against A beta-induced neuronal death. These results suggest that alpha7-receptor activation plays an important role in neuroprotection against A beta cytotoxicity. This study suggests that nicotinic receptor stimulation, especially alpha7-receptor activation, may be able to protect neurons from degeneration induced by A beta and may have effects that counter the progress of AD.

Background and Purpose: Glutamate excitotoxicity may be involved in ischaemic injury to the CNS and some neurodegenerative diseases, such as Alzheimer's disease. Donepezil, an acetylcholinesterase (AChE) inhibitor, exerts neuroprotective effects. Here we demonstrated a novel mechanism underlying the neuroprotection induced by donepezil.Experimental Approach: Cell damage in primary rat neuron cultures was quantified by lactate dehydrogenase release. Morphological changes associated with neuroprotective effects of nicotine and AChE inhibitors were assessed by immunostaining. Cell surface levels of the glutamate receptor sub-units, NR1 and NR2A, were analyzed using biotinylation. Immunoblot was used to measure protein levels of cleaved caspase-3, total NR1, total NR2A and phosphorylated NR1. Immunoprecipitation was used to measure association of NR1 with the post-synaptic protein, PSD-95. Intracellular Ca(2+) concentrations were measured with fura 2-acetoxymethylester. Caspase 3-like activity was measured using enzyme substrate, 7-amino-4-methylcoumarin (AMC)-DEVD.Key Results: Levels of NR1, a core subunit of the NMDA receptor, on the cell surface were significantly reduced by donepezil. In addition, glutamate-mediated Ca(2+) entry was significantly attenuated by donepezil. Methyllycaconitine, an inhibitor of alpha7 nicotinic acetylcholine receptors (nAChR), inhibited the donepezil-induced attenuation of glutamate-mediated Ca(2+) entry. LY294002, a phosphatidyl inositol 3-kinase (PI3K) inhibitor, had no effect on attenuation of glutamate-mediated Ca(2+) entry induced by donepezil.Conclusions and Implications: Decreased glutamate toxicity through down-regulation of NMDA receptors, following stimulation of alpha7 nAChRs, could be another mechanism underlying neuroprotection by donepezil, in addition to up-regulating the PI3K-Akt cascade or defensive system. [ABSTRACT FROM AUTHOR]