Coumarin derivatives protection against ROS production in cellular models of Abeta toxicities

The role of oxidative stress and free radicals in the development of Alzheimer's disease (AD) has been the focus of many recent studies. The role of hydrogen peroxide (H(2)O(2)) in AD is thought to be associated with Abeta (amyloid - beta) damage in cells. A number of coumarin derivatives were previously found to be potent anti-inflammatory and antioxidant agents. Herein, these coumarin derivatives were tested as H(2)O(2) scavengers with the DCF assay using two types of neuronal cells: (a) wild type (N2a) neuroblastoma cells and (b) APP/PS1 transgenic cell line expressing Abeta. Their scavenging activity was varied between the types of cell cultures and it was found to be concentration and time dependent in the mutant cells. Their protective role against cell death further supports this notion. These results suggest that these compounds could be used as a template in the design of new molecules with a possible role in AD.     

Abeta mediated diminution of MTT reduction--an artefact of single cell culture?

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) reduction assay is a frequently used and easily reproducible method to measure beta-amyloid (Abeta) toxicity in different types of single cell culture. To our knowledge, the influence of Abeta on MTT reduction has never been tested in more complex tissue. Initially, we reproduced the disturbed MTT reduction in neuron and astroglia primary cell cultures from rats as well as in the BV2 microglia cell line, utilizing four different Abeta species, namely freshly dissolved Abeta (25-35), fibrillar Abeta (1-40), oligomeric Abeta (1-42) and oligomeric Abeta (1-40). In contrast to the findings in single cell cultures, none of these Abeta species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Abeta to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Abeta also did not influence the MTT reduction in the respective tissue. Failure of Abeta penetration into the tissue cannot explain the differences between single cells and the more complex brain tissue. Thus electrophysiological investigations disclosed an impairment of long-term potentiation (LTP) in the CA1 region of hippocampal slices from rat by application of oligomeric Abeta (1-40), but not by freshly dissolved Abeta (25-35) or fibrillar Abeta (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Abeta species on MTT reduction. Particularly, the differential effect of oligomeric versus other Abeta forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Abeta oligomer effect on synaptic function reflected by LTP impairment precedes changes in formazane formation rate or that cells embedded in a more natural environment in the tissue are less susceptible to damage by Abeta, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies.     

Decreased thioredoxin and increased thioredoxin reductase levels in Alzheimer's disease brain

Increasing evidence supports the role of reactive oxygen species (ROS) in the pathogenesis of Alzheimer's disease (AD). Both in vivo and in vitro studies demonstrate that thioredoxin (Trx) and thioredoxin reductase (TR), the enzyme responsible for reduction of oxidized Trx, have protective roles against cytotoxicity mediated by the generation of ROS. The present study measured levels of Trx protein and activities of TR in the brain in AD compared with control subjects, and evaluated the possible protective role of TR and Trx against amyloid beta-peptide (Abeta) toxicity in neuronal cultures. Analysis of Trx protein levels in 10 AD and 10 control subjects demonstrated a general decrease in all AD brain regions studied, with statistically significant decreases in the amygdala (p <.05), hippocampus/parahippocampal gyrus (p <.05), and marginally significant (p <.10) depletions in the superior and middle temporal gryi. Thioredoxin reductase activity levels were increased in all AD brain regions studied with statistically significant increases occurring in AD amygdala (p =.01) and cerebellum (p =.007). To investigate the protective effects of Trx and TR against Abeta-induced toxicity, primary hippocampal cultures were treated with Trx or TR in combination with toxic doses of Abeta. Treatment of cultures with Trx led to a statistically significant concentration-dependent enhancement in cell survival against Abeta-mediated toxicity as did treatment with TR. Together, these data suggest that, although TR is protective against Abeta-mediated toxicity, the increase observed in AD brain offers no protection due to the significant decrease in Trx levels. This decrease in the antioxidant Trx-TR system may contribute to the increased oxidative stress and subsequent neurodegeneration observed in the brain in AD.     

Coumarin derivatives protection against ROS production in cellular models of Aβ toxicities

The role of oxidative stress and free radicals in the development of Alzheimer's disease (AD) has been the focus of many recent studies. The role of hydrogen peroxide (H₂O₂) in AD is thought to be associated with Aβ (amyloid – β) damage in cells. A number of coumarin derivatives were previously found to be potent anti-inflammatory and antioxidant agents. Herein, these coumarin derivatives were tested as H₂O₂ scavengers with the DCF assay using two types of neuronal cells: (a) wild type (N2a) neuroblastoma cells and (b) APP/PS1 transgenic cell line expressing Aβ. Their scavenging activity was varied between the types of cell cultures and it was found to be concentration and time dependent in the mutant cells. Their protective role against cell death further supports this notion. These results suggest that these compounds could be used as a template in the design of new molecules with a possible role in AD.     

Docosahexaenoic acid: one molecule diverse functions

Docosahexaenoic acid (DHA, C22:6, ω-3) is a highly polyunsaturated omega-3 fatty acid. It is concentrated in neuronal brain membranes, for which reason it is also referred to as a “brain food”. DHA is essential for brain development and function. It plays an important role in improving antioxidant and cognitive activities of the brain. DHA deficiency occurs during aging and dementia, impairs memory and learning, and promotes age-related neurodegenerative diseases, including Alzheimer’s disease (AD). For about two decades, we have reported that oral administration of DHA increases spatial memory acquisition, stimulates neurogenesis, and protects against and reverses memory impairment in amyloid β peptide-infused AD rat models by decreasing amyloidogenesis and protects against age-related cognitive decline in the elderly. These results demonstrate a robust link between DHA and cognitive health. Rodents that were fed a diet low in ω-3 polyunsaturated fatty acids, particularly those that were DHA-deficient, frequently suffered from anxiety, depression and memory impairment. Although the exact mechanisms of action of DHA in brain functions are still elusive, a host of mechanisms have been proposed. For example, DHA, which inherently has a characteristic three-dimensional structure, increases membrane fluidity, strengthens antioxidant activity and enhances the expression of several proteins that act as substrates for improving memory functions. It reduces the brain amyloid burden and inhibits in vitro fibrillation and amyloid-induced neurotoxicity in cell-culture model. In this review, we discuss how DHA acts as a molecule with diverse functions.     

Neuroprotective effects of non-steroidal anti-inflammatory drugs by direct scavenging of nitric oxide radicals

Recently, it has been reported that inflammatory processes are associated with the pathophysiology of Alzheimer's disease and that treatment of non-steroidal anti-inflammatory drugs reduce the risk for Alzheimer's disease. In the present study, we examined nitric oxide radical quenching activity of non-steroidal anti-inflammatory drugs and steroidal drugs using our established direct in vitro nitric oxide radical detecting system by electron spin resonance spectrometry. The non-steroidal anti-inflammatory drugs, aspirin, mefenamic acid, indomethacin and ketoprofen directly and dose-dependently scavenged generated nitric oxide radicals. In experiments of nitric oxide radical donor, NOC18-induced neuronal damage, these four non-steroidal drugs significantly prevented the NOC18-induced reduction of cell viability and apoptotic nuclear changes in neuronal cells without affecting the induction of inducible nitric oxide synthase-like immunoreactivity. However, ibuprofen, naproxen or steroidal drugs, which had less or no scavenging effects in vitro, showed almost no protective effects against NOC18-induced cell toxicity. These results suggest that the protective effects of the former four non-steroidal anti-inflammatory drugs against apoptosis might be mainly due to their direct nitric oxide radical scavenging activities in neuronal cells. These direct NO. quenching activities represent novel effects of non-steroidal anti-inflammatory drugs. Our findings identified novel pharmacological mechanisms of these drugs to exert not only their anti-inflammatory, analgesic, antipyretic activities but also neuroprotective activities against neurodegeneration.     

Increased vulnerability of hippocampal neurons from presenilin-1 mutant knock-in mice to amyloid beta-peptide toxicity: central roles of superoxide production and caspase activation

Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. Overexpression of PS1 mutations in cultured PC12 cells increases their vulnerability to apoptosis-induced trophic factor withdrawal and oxidative insults. We now report that primary hippocampal neurons from PS1 mutant knock-in mice, which express the human PS1M146V mutation at normal levels, exhibit increased vulnerability to amyloid beta-peptide toxicity. The endangering action of mutant PS1 was associated with increased superoxide production, mitochondrial membrane depolarization, and caspase activation. The peroxynitrite-scavenging antioxidant uric acid and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone protected hippocampal neurons expressing mutant PS1 against cell death induced by amyloid beta-peptide. Increased oxidative stress may contribute to the pathogenic action of PS1 mutations, and antioxidants may counteract the adverse property of such AD-linked mutations.     

A humanin derivative reduces amyloid beta accumulation and ameliorates memory deficit in triple transgenic mice

Humanin (HN), a 24-residue peptide, was identified as a novel neuroprotective factor and shows anti-cell death activity against a wide spectrum of Alzheimer's disease (AD)-related cytotoxicities, including exposure to amyloid beta (Abeta), in vitro. We previously demonstrated that the injection of S14G-HN, a highly potent HN derivative, into brain ameliorated memory loss in an Abeta-injection mouse model. To fully understand HN's functions under AD-associated pathological conditions, we examined the effect of S14G-HN on triple transgenic mice harboring APP(swe), tau(P310L), and PS-1(M146V) that show the age-dependent development of multiple pathologies relating to AD. After 3 months of intranasal treatment, behavioral analyses showed that S14G-HN ameliorated cognitive impairment in male mice. Moreover, ELISA and immunohistochemical analyses showed that Abeta levels in brains were markedly lower in S14G-HN-treated male and female mice than in vehicle control mice. We also found the expression level of neprilysin, an Abeta degrading enzyme, in the outer molecular layer of hippocampal formation was increased in S14G-HN-treated mouse brains. NEP activity was also elevated by S14G-HN treatment in vitro. These findings suggest that decreased Abeta level in these mice is at least partly attributed to S14G-HN-induced increase of neprilysin level. Although HN was identified as an anti-neuronal death factor, these results indicate that HN may also have a therapeutic effect on amyloid accumulation in AD.     

Bcl-2 protects against Abeta(25-35)-induced oxidative PC12 cell death by potentiation of antioxidant capacity

A substantial body of data indicates that reactive oxygen intermediates (ROIs) are implicated in pathogenesis of diverse human diseases. Oxidative stress induced by ROIs often causes cell death via apoptosis that is regulated by a plenty of functional genes and their protein products. Bcl-2 is one such protein that blocks apoptosis induced by various death stimuli. In spite of extensive research, the molecular mechanisms underlying antiapoptotic function of Bcl-2 are not fully clarified. In the present work, we have investigated the role of bcl-2 in protecting against beta-amyloid (Abeta)-induced oxidative death in rat pheochromocytoma (PC12) cells. Transfection with the antiapoptotic bcl-2 gene rescued PC12 cells from apoptotic death induced by Abeta. Addition of an NF-kappaB inhibitor, such as pyrrolidine dithiocarbamate or N-tosyl-l-phenylalanine chloromethyl ketone, to the media aggravated Abeta-induced PC12 cell death. PC12 cells overexpressing bcl-2 exhibited higher levels of constitutively activated NF-kappaB compared with vector-transfected controls, which appear to be mediated by the elevated activation of Akt/protein kinase B. The ectopic expression of bcl-2 enhanced both the expression and the activity of catalase, which were attenuated by NF-kappaB blockers. These results suggest that NF-kappaB plays a role in bcl-2-mediated protection against Abeta-induced apoptosis in PC12 cells through augmentation of cellular antioxidant capacity.     

Tanshinone IIA protects PC12 cells from β-amyloid<Subscript>25–35</Subscript>-induced apoptosis via PI3K/Akt signaling pathway

For the aging populations of any nation, Dementia is becoming a primary problem and Alzheimer’s dementia (AD) is the most common type. However, until now, there is no effective treatment for AD. Tanshinone IIA (Tan IIA) has been reported for neuroprotective potential to against amyloid β peptides (Aβ)-induced cytotoxicity in the rat pheochromocytoma cell line PC-12, which is widely used as AD research model, but the mechanism still remains unclear. To investigate the effect of Tan IIA and the possible molecular mechanism in the apoptosis of PC12 cells, we induced apoptosis in PC12 cells with β-amyloid(25-35), and treated cells with Tan IIA. After 24 h treatment, we found that Tan IIA increased the cell viability and reduced the number of apoptotic cells induced by Aβ(25-35). However, neuroprotection of Tan IIA was abolished by PI3K inhibitor LY294002. Meanwhile, Treatment with lithium chloride, a phosphorylation inhibitor of GSK3β, which is a downstream target of PI3K/Akt, can block Aβ(25-35)-induced cell apoptosis in a Tan IIA-like manner. Our findings suggest that Tan IIA is an effective neuroprotective agent and a viable candidate in AD therapy and PI3K/Akt activation and GSK3β phosphorylation are involved in the neuroprotection of Tan IIA.     

Protective effects of a Rhodiola crenulata extract and salidroside on hippocampal neurogenesis against streptozotocin-induced neural injury in the rat

Previously we have demonstrated that a Rhodiola crenulata extract (RCE), containing a potent antioxidant salidroside, promotes neurogenesis in the hippocampus of depressive rats. The current study was designed to further investigate the protective effect of the RCE on neurogenesis in a rat model of Alzheimer's disease (AD) induced by an intracerebroventricular injection of streptozotocin (STZ), and to determine whether this neuroprotective effect is induced by the antioxidative activity of salidroside. Our results showed that pretreatment with the RCE significantly improved the impaired neurogenesis and simultaneously reduced the oxidative stress in the hippocampus of AD rats. In vitro studies revealed that (1) exposure of neural stem cells (NSCs) from the hippocampus to STZ strikingly increased intracellular reactive oxygen species (ROS) levels, induced cell death and perturbed cell proliferation and differentiation, (2) hydrogen peroxide induced similar cellular activities as STZ, (3) pre-incubation of STZ-treated NSCs with catalase, an antioxidant, suppressed all these cellular activities induced by STZ, and (4) likewise, pre-incubation of STZ-treated NSCs with salidroside, also an antioxidant, suppressed all these activities as catalase: reduction of ROS levels and NSC death with simultaneous increases in proliferation and differentiation. Our findings indicated that the RCE improved the impaired hippocampal neurogenesis in the rat model of AD through protecting NSCs by its main ingredient salidroside which scavenged intracellular ROS.     

Protection of PC12 cells from hydrogen peroxide-induced cytotoxicity by salvianolic acid B, a new compound isolated from Radix Salviae miltiorrhizae

A number of studies indicate that reactive oxygen species (ROS) are involved in neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). The neuroprotective effects of salvianolic acid B (SalB) from Radix Salviae miltiorrhizae (RSM) against hydrogen peroxide (H2O2)-induced rat pheochromocytoma line PC12 injury were evaluated in the present study. Vitamin E, a potent antioxidant, was employed as a positive control agent. Following exposure of cells to H2O2 (150 microM), a marked decrease in cell survival and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), as well as increased levels of malondialdehyde (MDA) production and lactate dehydrogenase (LDH) release were observed. In parallel, H2O2 caused significant elevation in intracellular Ca2+ level and caspase-3 activity, and induced apoptotic death as determined by flow cytometric assay. However, pretreatment of the cells with SalB (0.1-10 microM) prior to H2O2 exposure blocked these H2O2-induced cellular events noticeably. Moreover, SalB exhibited significantly higher potency as compared to Vitamin E. The present findings indicated that SalB exerts neuroprotective effects against H2O2 toxicity, which might be of importance and contribute to its clinical efficacy for the treatment of neurodegenerative diseases.     

Lactuca capensis reverses memory deficits in Aβ1‐42‐induced an animal model of Alzheimer's disease

We investigated the neuropharmacological effects of the methanolic extract from Lactuca capensis Thunb. leaves (100 and 200 mg/kg) for 21 days on memory impairment in an Alzheimer's disease (AD) rat model produced by direct intraventricular delivery of amyloid‐β1‐42 (Aβ1‐42). Behavioural assays such as Y‐maze and radial arm maze test were used for assessing memory performance. Aβ1‐42 decreased cognitive performance in the behavioural tests which were ameliorated by pre‐treatment with the methanolic extract. Acetylcholinesterase activity and oxidant–antioxidant balance in the rat hippocampus were abnormally altered by Aβ1‐42 treatment while these deficits were recovered by pre‐treatment with the methanolic extract. In addition, rats were given Aβ1‐42 exhibited in the hippocampus decreased brain‐derived neurotrophic factor (BDNF) mRNA copy number and increased IL‐1β mRNA copy number which was reversed by the methanolic extract administration. These findings suggest that the methanolic extract could be a potent neuropharmacological agent against dementia via modulating cholinergic activity, increasing of BDNF levels and promoting antioxidant action in the rat hippocampus.     

Sulindac inhibits activation of the NF-kappaB pathway.

Sulindac is a non-steroidal anti-inflammatory agent that is related both structurally and pharmacologically to indomethacin. In addition to its anti-inflammatory properties, sulindac has been demonstrated to have a role in the prevention of colon cancer. Both its growth inhibitory and anti-inflammatory properties are due at least in part to its ability to decrease prostaglandin synthesis by inhibiting the activity of cyclooxygenases. Recently, we demonstrated that both aspirin and sodium salicylate, but not indomethacin, inhibited the activity of an IkappaB kinase beta (IKKbeta) that is required to activate the nuclear factor-kappaB (NF-kappaB) pathway. In this study, we show that sulindac and its metabolites sulindac sulfide and sulindac sulfone can also inhibit the NF-kappaB pathway in both colon cancer and other cell lines. Similar to our previous results with aspirin, this inhibition is due to sulindac-mediated decreases in IKKbeta kinase activity. Concentrations of sulindac that inhibit IKKbeta activity also reduce the proliferation of colon cancer cells. These results suggest that the growth inhibitory and anti-inflammatory properties of sulindac may be regulated in part by inhibition of kinases that regulate the NF-kappaB pathway.     

Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations

A cell-based assay system for monitoring NF-kappaB activity was developed to determine the influence of activated NF-kappaB in human HaCaT cells. The pNF-kappaB-SEAP-NPT plasmid that permits expression of the secretory alkaline phosphatase (SEAP) reporter gene in response to the NF-kappaB activity and contains the neomycin phosphotransferase (NPT) gene for geneticin resistance in host cells was constructed and transfected into the human keratinocyte cell line HaCaT. Human HaCaT transfectant cells were demonstrated to secrete the SEAP enzyme into the culture medium in a time-dependent manner until 72 h. NF-kappaB activities were measured by the SEAP reporter gene assay using a fluorescence detection method. HaCaT cell transfectants treated with antioxidants [e.g., N-acetyl-l-cysteine and vitamin C] showed reduction of NF-kappaB activity in a time- and concentration-dependent manner, whereas phorbol 12-myristate 13-acetate known as a stimulator of NF-kappaB expression increased NF-kappaB activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-kappaB activity in the human skin and allow the screening of anti-inflammatory agents for dermatological purpose from various synthetic chemicals and natural products.     

Neuronal membrane cholesterol loss enhances amyloid peptide generation

Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimer's disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its beta-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1-APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.