Synergistic induction of ER stress by homocysteine and beta-amyloid in SH-SY5Y cells

Clinical studies have raised the possibility that elevated plasma levels of homocysteine increase the risk of atherosclerosis, stroke and possibly neurodegenerative diseases such as Alzheimer's disease (AD); however, the direct impact of homocysteine on neuron cells and the mechanism by which it could induce neurodegeneration have yet to be clearly demonstrated. Here, we investigated the effect of homocysteine on endoplasmic reticulum (ER) stress, the suggested mechanism of neurotoxicity, in human neuroblastoma SH-SY5Y cells. The effect of homocysteine on amyloid-beta (Abeta)-induced neurotoxicity and the protective activity of folate were also investigated. Homocysteine led to increased expressions of the binding protein (BiP) and the spliced form of X-box-protein (XBP)-1 mRNAs, suggesting activation of the unfolded-protein response and an increase in apoptosis. When cells were cotreated with homocysteine and Abeta, caspase-3 activity was significantly increased, and expressions of BiP and the spliced form of XBP-1 mRNAs were significantly induced. The neurotoxicity of homocysteine was attenuated by the treatment of cells with folate, as determined by caspase-3 activity and apoptotic body staining. These findings indicate that homocysteine induces ER stress and, ultimately, apoptosis and sensitizes neurons to amyloid toxicity via the synergistic induction of ER stress. Furthermore, a neuroprotective effect of folate against homocysteine-induced toxicity was also observed. Therefore, the findings of our study suggest that ER stress-induced homocysteine toxicity may play an important physiological role in enhancing the pathogenesis of Abeta-induced neuronal degeneration.     

Cu（Ⅱ） Potentiation of Alzheimer Aβ1-40 Cytotoxicity and Transition on its Secondary Structure

Mounting evidence has shown that dyshomeostasis of the redox-active biometals such as Cuand Fe can lead to oxidative stress,which plays a key role in the neuropathology of Alzheimer's disease(AD).Here we demonstrate that with the formation of Cu(Ⅱ)·Aβ1-40 complexes,copper markedly potentiatesthe neurotoxicity exhibited by β-amyloid peptide (Aβ).A greater amount of hydrogen peroxide was releasedwhen Cu(Ⅱ)·Aβ1-40 complexes was added to the xanthine oxidase/xanthine system detected by potassiumiodide spectrophotometry.Copper bound to Aβ1-40 was observed by electron paramagnetic resonance(EPR) spectroscopy.Circular dichroism (CD) studies indicated that copper chelation could cause a structuraltransition of Aβ.The addition of copper to Aβ introduced an increase on β-sheet as well as α-helix,whichmay be responsible for the aggregation of Aβ.We hypothesized that Aβ aggregation induced by copper maybe responsible for local injury in AD.The interaction between Cu~(2 ) and Aβ also provides a possible mechanismfor the enrichment of metal ions in amyloid plaques in the AD brain.     

Exacerbation of copper toxicity in primary neuronal cultures depleted of cellular glutathione

Perturbations to glutathione (GSH) metabolism may play an important role in neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases. A primary function of GSH is to prevent the toxic interaction between free radicals and reactive transition metals such as copper (Cu). Due to the potential role of Cu in neurodegeneration, we examined the effect of GSH depletion on Cu toxicity in murine primary neuronal cultures. Depletion of cellular GSH with L-buthionine-[S,R]-sulfoximine resulted in a dramatic potentiation of Cu toxicity in neurons without effect on iron (Fe) toxicity. Similarly, inhibition of glutathione reductase (GR) activity with 1,3-bis(2-chloroethyl)-1-nitrosurea also increased Cu toxicity in neurons. To determine if the Alzheimer's amyloid-beta (Abeta) peptide can affect neuronal resistance to transition metal toxicity, we exposed cultures to nontoxic concentrations of Abeta25-35 in the presence or absence of Cu or Fe. Abeta25-35 pretreatment was found to deplete neuronal GSH and increase GR activity, confirming the ability of Abeta to perturb neuronal GSH homeostasis. Abeta25-35 pretreatment potently increased Cu toxicity but had no effect on Fe toxicity. These studies demonstrate an important role for neuronal GSH homeostasis in selective protection against Cu toxicity, a finding with widespread implications for neurodegenerative disorders.     

Metal swap between Zn7-metallothionein-3 and amyloid-beta-Cu protects against amyloid-beta toxicity

Aberrant interactions of copper and zinc ions with the amyloid-beta peptide (Abeta) potentiate Alzheimer's disease (AD) by participating in the aggregation process of Abeta and in the generation of reactive oxygen species (ROS). The ROS production and the neurotoxicity of Abeta are associated with copper binding. Metallothionein-3 (Zn(7)MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain and downregulated in AD. This protein protects, by an unknown mechanism, cultured neurons from the toxicity of Abeta. Here, we show that a metal swap between Zn(7)MT-3 and soluble and aggregated Abeta(1-40)-Cu(II) abolishes the ROS production and the related cellular toxicity. In this process, copper is reduced by the protein thiolates forming Cu(I)(4)Zn(4)MT-3, in which an air-stable Cu(I)(4)-thiolate cluster and two disulfide bonds are present. The discovered protective effect of Zn(7)MT-3 from the copper-mediated Abeta(1-40) toxicity may lead to new therapeutic strategies for treating AD.     

The A beta peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction.

Oxidative stress markers characterize the neuropathology both of Alzheimer's disease and of amyloid-bearing transgenic mice. The neurotoxicity of amyloid A beta peptides has been linked to peroxide generation in cell cultures by an unknown mechanism. We now show that human A beta directly produces hydrogen peroxide (H2O2) by a mechanism that involves the reduction of metal ions, Fe(III) or Cu(II), setting up conditions for Fenton-type chemistry. Spectrophotometric experiments establish that the A beta peptide reduces Fe(III) and Cu(II) to Fe(II) and Cu(I), respectively. Spectrochemical techniques are used to show that molecular oxygen is then trapped by A beta and reduced to H2O2 in a reaction that is driven by substoichiometric amounts of Fe(II) or Cu(I). In the presence of Cu(II) or Fe(III), A beta produces a positive thiobarbituric-reactive substance (TBARS) assay, compatible with the generation of the hydroxyl radical (OH.). The amounts of both reduced metal and TBARS reactivity are greatest when generated by A beta 1-42 > A beta 1-40 > rat A beta 1-40, a chemical relationship that correlates with the participation of the native peptides in amyloid pathology. These findings indicate that the accumulation of A beta could be a direct source of oxidative stress in Alzheimer's disease.     

Peroxidase activity of cyclooxygenase-2 (COX-2) cross-links beta-amyloid (Abeta) and generates Abeta-COX-2 hetero-oligomers that are increased in Alzheimer's disease

Oxidative stress is associated with the neuropathology of Alzheimer's disease. We have previously shown that human Abeta has the ability to reduce Fe(III) and Cu(II) and produce hydrogen peroxide coupled with these metals, which is correlated with toxicity against primary neuronal cells. Cyclooxygenase (COX)-2 expression is linked to the progression and severity of pathology in AD. COX is a heme-containing enzyme that produces prostaglandins, and the enzyme also possesses peroxidase activity. Here we investigated the possibility of direct interaction between human Abeta and COX-2 being mediated by the peroxidase activity. Human Abeta formed dimers when it was reacted with COX-2 and hydrogen peroxide. Moreover, the peptide formed a cross-linked complex directly with COX-2. Such cross-linking was not observed with rat Abeta, and the sole tyrosine residue specific for human Abeta might therefore be the site of cross-linking. Similar complexes of Abeta and COX-2 were detected in post-mortem brain samples in greater amounts in AD tissue than in age-matched controls. COX-2-mediated cross-linking may inhibit Abeta catabolism and possibly generate toxic intracellular forms of oligomeric Abeta.     

Hydrogen peroxide can be generated by tau in the presence of Cu(II)

Alzheimer's disease has been closely related with oxidative stress, which might be responsible for the dysfunction or death of neuronal cells that contributes to disease pathogenesis. Impaired copper homeostasis makes contribution to the oxidative stress and consequently to several neurodegenerative conditions. Inappropriate binding of Cu(II) to cellular proteins are currently being explored as sources of pathological oxidative stress in several neurodegenerative disorders. Here we report that a fragment of tau protein possesses copper reduction activity and initiates the copper-mediated generation of hydrogen peroxide. The tau peptide was found to be oxidized to form disulfide bond-linked dimer. The hydrogen peroxide generated was quantified by TCEP/DTNB (tris(2-carboxyethyl) phosphine hydrochloride/5,5'-dithio-bis(2-nitrobenzoic acid). Since the copper reduction capacity and the generation of hydrogen peroxide were believe to be a major toxicological pathway of Abeta peptide, the functional similarity shared by tau and Abeta implies a new perspective of tau pathology.     

Iron inhibits neurotoxicity induced by trace copper and biological reductants

The extracellular microenvironment of the brain contains numerous biological redox agents, including ascorbate, glutathione, cysteine and homocysteine. During ischemia/reperfusion, aging or neurological disease, extracellular levels of reductants can increase dramatically owing to dysregulated homeostasis. The extracellular concentrations of transition metals such as copper and iron are also substantially elevated during aging and in some neurodegenerative disorders. Increases in the extracellular redox capacity can potentially generate neurotoxic free radicals from reduction of Cu(II) or Fe(III), resulting in neuronal cell death. To investigate this in vitro, the effects of extracellular reductants (ascorbate, glutathione, cysteine, homocysteine or methionine) on primary cortical neurons was examined. All redox agents except methionine induced widespread neuronal oxidative stress and subsequent cell death at concentrations occurring in normal conditions or during neurological insults. This neurotoxicity was totally dependent on trace Cu (0.4 M) already present in the culture medium and did not require addition of exogenous Cu. Toxicity involved generation of Cu(I) and H₂O₂, while other trace metals did not induce toxicity. Surprisingly, administration of Fe(II) or Fe(III) (2.5 M) completely abrogated reductant-mediated neurotoxicity. The potent protective activity of Fe correlated with Fe inhibiting reductant-mediated Cu(I) and H₂O₂ generation in cell-free assays and reduced cellular Cu uptake by neurons. This demonstrates a novel role for Fe in blocking Cu-mediated neurotoxicity in a high reducing environment. A possible pathogenic consequence for these phenomena was demonstrated by abrogation of Fe neuroprotection after pre-exposure of cultures to the Alzheimers amyloid beta peptide (A). The loss of Fe neuroprotection against reductant toxicity was greater after treatment with human A1–42 than with human A1–40 or rodent A1–42, consistent with the central role of A1–42 in Alzheimers disease. These findings have important implications for trace biometal interactions and free radical-mediated damage during neurodegenerative illnesses such as Alzheimers disease and old-age dementia.Abbreviations A amyloid beta - AD Alzheimers disease - Asc ascorbate - BC bathocuproine disulfonate - Cys cysteine - DCF 2,7-dichlorofluorescein - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - FCS fetal calf serum - Glut glutamate - GSH reduced glutathione - GSSG oxidized glutathione - Hcys homocysteine - ICP-MS inductively coupled plasma mass spectrometry - MEM minimal essential media - Met methionine - MnTMPyP manganese tetrakis(1-methyl-4-pyridyl)porphyrin - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide     

Probing copper/tau protein interactions electrochemically

Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by peptide and protein misfolding and aggregation, in part due to the presence of excess metal ions such as copper(II) [Cu(II)]. Recently, the brain levels of Cu(II) complexes in vivo were linked to the oxidative stress in neurodegenerative disorders, including AD. Amyloid β-peptide (Aβ), found outside neuronal cells, has been investigated extensively in connection with Cu(II) ion toxicity; however, the effects of metallation on tau are less known. Normal tau protein binds and stabilizes the microtubules in neurons, but in diseased cells tau hyperphosphorylation and aggregation are evident and compromise tau function. There is increasing evidence that the Cu(II) ion may play an important role in tau biochemistry. Here, we present an electrochemical study of the interactions between full-length tau-410 and Cu(II) ions. The coordination of Cu(II) ions to tau immobilized on gold surfaces induces an electrochemical signal at approximately 140 ± 5 mV versus Ag/AgCl due to the Cu(II)/Cu(I) redox couple. Redox potentials and current intensities of Cu(II)-containing nonphosphorylated tau (nTau) and phosphorylated tau (pTau) films were determined at different pH conditions. Greater Cu(II) uptake by pTau over nTau films was observed at low pH. Competitive zinc(II) [Zn(II)] ion binding studies revealed significant Cu(II) ion displacement in pTau films. X-ray photoelectron spectroscopy analysis indicated the presence of Cu 2p and Zn 2p binding energies in protein samples, further supporting metal ion coordination to protein films. The surface-based electrochemical technique requires a minimal protein amount (a few microliters) and allows monitoring the bound Cu(II) ions and the redox activities of the resulting metalloprotein films.     

Protective Effects of Vitamin E against Oxidative Damage Induced by Aβ1-40Cu（Ⅱ） Complexes

β-amyloid peptide (Aβ) is considered to be responsible for the formation of senile plaques,which is the hallmark of Alzheimer's disease (AD).Oxidative stress,manifested by protein oxidation andlipid peroxidation,among other alterations,is a characteristic of AD brain.A growing body of evidence hasbeen presented in support of Aβ_(1-40) forming an oligomeric complex that binds copper at a CuZn superoxidedismutase-like binding site. Aβ_(1-40)Cu(Ⅱ) complexes generate neurotoxic hydrogen peroxide (H_2O_2) from O_2via Cue reduction,though the precise reaction mechanism is unclear.The toxicity of Aβ_(1-40) or the Aβ_(1-40)Cu(Ⅱ)complexes to cultured primary cortical neurons was partially attenuated when ( )-α-tocopherol (vitamin E)as free radical antioxidant was added at a concentration of 100 μM.The data derived from lactate dehydro-genase (LDH) release and the formation of H_2O_2 confirmed the results from the MTT assay.These findingsindicate that copper binding to Aβ_(1-40) can give rise to greater production of H_2O_2, which leads to a break-down in the integrity of the plasma membrane and subsequent neuronal death.Groups treated with vitaminE exhibited much slighter damage,suggesting that vitamin E plays a key role in protecting neuronal cellsfrom dysfunction or death.     

Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction

Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Abeta amyloid deposits in Alzheimer's disease. The neurotoxicity of Abeta in cell culture has been linked to H(2)O(2) generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by Abeta in cell culture. The potentiation of toxicity is greatest for Abeta1-42 > Abeta1-40 > mouse/rat Abeta1-40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H(2)O(2) in cell-free assays and to exhibit amyloid pathology. The copper complex of Abeta1-42 has a highly positive formal reduction potential ( approximately +500-550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Abeta-mediated oxidative damage in Alzheimer's disease.     

Involvement of PI3K/PKG/ERK1/2 signaling pathways in cortical neurons to trigger protection by cotreatment of acetyl-L-carnitine and alpha-lipoic acid against HNE-mediated oxidative stress and neurotoxicity: implications for Alzheimer's disease

Oxidative stress has been shown to underlie neuropathological aspects of Alzheimer's disease (AD). 4-Hydroxy-2-nonenal (HNE) is a highly reactive product of lipid peroxidation of unsaturated lipids. HNE-induced oxidative toxicity is a well-described model of oxidative stress-induced neurodegeneration. GSH plays a key role in antioxidant defense, and HNE exposure causes an initial depletion of GSH that leads to gradual toxic accumulation of reactive oxygen species. In the current study, we investigated whether pretreatment of cortical neurons with acetyl-L-carnitine (ALCAR) and alpha-lipoic acid (LA) plays a protective role in cortical neuronal cells against HNE-mediated oxidative stress and neurotoxicity. Decreased cell survival of neurons treated with HNE correlated with increased protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (HNE) accumulation. Pretreatment of primary cortical neuronal cultures with ALCAR and LA significantly attenuated HNE-induced cytotoxicity, protein oxidation, lipid peroxidation, and apoptosis in a dose-dependent manner. Additionally, pretreatment of ALCAR and LA also led to elevated cellular GSH and heat shock protein (HSP) levels compared to untreated control cells. We have also determined that pretreatment of neurons with ALCAR and LA leads to the activation of phosphoinositol-3 kinase (PI3K), PKG, and ERK1/2 pathways, which play essential roles in neuronal cell survival. Thus, this study demonstrates a cross talk among the PI3K, PKG, and ERK1/2 pathways in cortical neuronal cultures that contributes to ALCAR and LA-mediated prosurvival signaling mechanisms. This evidence supports the pharmacological potential of cotreatment of ALCAR and LA in the management of neurodegenerative disorders associated with HNE-induced oxidative stress and neurotoxicity, including AD.     

Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson's disease.

Although the cause of Parkinson's disease (PD) is unknown, data suggest roles for environmental factors that may sensitize dopaminergic neurons to age-related dysfunction and death. Based upon epidemiological data suggesting roles for dietary factors in PD and other age-related neurodegenerative disorders, we tested the hypothesis that dietary folate can modify vulnerability of dopaminergic neurons to dysfunction and death in a mouse model of PD. We report that dietary folate deficiency sensitizes mice to MPTP-induced PD-like pathology and motor dysfunction. Mice on a folate-deficient diet exhibit elevated levels of plasma homocysteine. When infused directly into either the substantia nigra or striatum, homocysteine exacerbates MPTP-induced dopamine depletion, neuronal degeneration and motor dysfunction. Homocysteine exacerbates oxidative stress, mitochondrial dysfunction and apoptosis in human dopaminergic cells exposed to the pesticide rotenone or the pro-oxidant Fe(2+). The adverse effects of homocysteine on dopaminergic cells is ameliorated by administration of the antioxidant uric acid and by an inhibitor of poly (ADP-ribose) polymerase. The ability of folate deficiency and elevated homocysteine levels to sensitize dopaminergic neurons to environmental toxins suggests a mechanism whereby dietary folate may influence risk for PD.     

Copper and Alzheimer's disease

Copper is essential for some of the enzymes that have a role in brain metabolism. Sophisticated mechanisms balance copper import and export to ensure proper nutrient levels (homeostasis) while minimizing toxic effects. Several neurodegenerative diseases including Alzheimer's disease (AD) are characterized by modified copper homeostasis. This change seems to contribute either directly or indirectly to increased oxidative stress, an important factor in neuronal toxicity. When coupled to misfolded proteins, this modified copper homeostasis appears to be an important factor in the pathological progression of AD.     

Correlation between maternal plasma homocysteine and zinc levels in preeclamptic women

Women with preeclampsia have been shown to have elevated blood levels of the metabolite homocysteine, and alterations in blood levels of zinc and copper have also been reported. This study measured plasma levels of zinc, copper, and homocysteine in women with preeclampsia and in women with healthy, normotensive pregnancies. For the patients with preeclampsia compared with controls, significantly higher mean plasma levels were found of homocysteine (16.39 vs 9.45 nmol/mL; p≤0.001), zinc (15.53 vs 11.93 μg/g protein; p < 0.05), and copper (47.90 vs 31.60 μg/g protein; p=0.001). The ratio of plasma Cu/Zn levels tended to be higher in preeclamptic women and could be taken as an index of inflammatory reaction, but the difference was not significant. Homocysteine concentrations correlated positively with plasma zinc concentrations in women with preeclampsia (r=0.588, p=0.003) but not in women with healthy pregnancies. No correlations were observed between plasma levels of homocysteine and copper. Thus, the present study found evidence that preeclampsia might be associated with hyperhomocysteinemia and elevated blood levels of zinc and copper. Furthermore, elevated blood levels of zinc were significantly associated with hyperhomocysteinemia in preeclampsia. More studies are warranted to investigate further any relationship between altered homocysteine metabolism and levels of zinc and copper in preeclampsia.     

Vitamin B6 suppresses apoptosis of NM-1 bovine endothelial cells induced by homocysteine and copper

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B(6)in vivo. In the present study, we examined effects of vitamin B(6) on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.     

Overexpression of glutathione peroxidase increases the resistance of neuronal cells to Abeta-mediated neurotoxicity

Senile plaques are neuropathological manifestations in Alzheimer's disease (AD) and are composed mainly of extracellular deposits of amyloid beta-peptide (Abeta). Various data suggest that the accumulation of Abeta may contribute to neuronal degeneration and that Abeta neurotoxicity could be mediated by oxygen free radicals. Removal of free radicals by antioxidant scavengers or enzymes was found to protect neuronal cells in culture from Abeta toxicity. However, the nature of the free radicals involved is still unclear. In this study, we investigated whether the neuronal overexpression of glutathione peroxidase (GPx), the major hydrogen peroxide (H2O2)-de-grading enzyme in neurons, could increase their survival in a cellular model of Abeta-induced neurotoxicity. We infected pheochromocytoma (PC12) cells and rat embryonic cultured cortical neurons with an adenoviral vector encoding GPx (Ad-GPx) prior to exposure to toxic concentrations of Abeta(25-35) or (1-40). Both PC12 and cortical Ad-GPx-infected cells were significantly more resistant to Abeta-induced injury. These data strengthen the hypothesis of a role of H2O2 in the mechanism of Abeta toxicity and highlight the potential of Ad-GPx to reduce Abeta-induced damage to neurons. These findings may have applications in gene therapy for AD.     

A spectroscopic study of some of the peptidyl radicals formed following hydroxyl radical attack on beta-amyloid and alpha-synuclein

There is clear evidence implicating oxidative stress in the pathology of many neurodegenerative diseases. Reactive oxygen species (ROS) are the primary mediators of oxidative stress, and hydrogen peroxide, a key ROS, is generated during aggregation of the amyloid proteins associated with some of these diseases. Hydrogen peroxide is catalytically converted to the aggressive hydroxyl radical in the presence of Fe(II) and Cu(I), which renders amyloidogenic proteins such as beta-amyloid and alpha-synuclein (implicated in Alzheimer's disease (AD) and Parkinson's disease (PD), respectively) vulnerable to self-inflicted hydroxyl radical attack. Here, we report some of the peptide-derived radicals, detected by electron spin resonance spectroscopy employing sodium 3,5-dibromo-4-nitrosobenzenesulfonate as a spin-trap, following hydroxyl radical attack on Abeta(1-40), alpha-synuclein and some other related peptides. Significantly, we found that sufficient hydrogen peroxide was self-generated during the early stages of aggregation of Abeta(1-40) to produce detectable peptidyl radicals, on addition of Fe(II). Our results support the hypothesis that oxidative damage to Abeta (and surrounding molecules) in the brain in AD could be due, at least in part, to the self-generation of ROS. A similar mechanism could operate in PD and some other "protein conformational" disorders.     

Protective effect of isoflavones against homocysteine-mediated neuronal degeneration in SH-SY5Y cells

Previously, we reported that isoflavones exert a protective effect against the endoplasmic reticulum (ER) stress-mediated neuronal degeneration, and ER stress-mediated homocysteine toxicity may play an important role in the pathogenesis of neurodegeneration. Therefore, in this study we investigated the effects of isoflavones (genistein and daidzein) against homocysteine-mediated neurotoxicity in SH-SY5Y human neuroblastoma cells. The treatment of cells with either 17β-estradiol or isoflavones significantly protected the cells against homocysteine-mediated apoptosis. Isoflavones repressed homocysteine-mediated ER stress, reflected in the reduced expression of the immunoglobin heavy chain-binding protein mRNA, spliced X-box-protein-1 mRNA and the phosphorylated form of eukaryotic translation initiation factor 2α protein. Homocysteine caused significant increases in intracellular S-adenosylhomocysteine (SAH) and DNA damage. Isoflavones significantly alleviated DNA damage, but did not change SAH levels. Furthermore, the treatment of cells with isoflavones significantly reduced the microtubule-associated protein tau hyperphosphorylation by inactivating glycogen synthase kinase-3β and activating serine/threonine-protein phosphatase 2A. These results clearly demonstrate that isoflavones alleviate the ER stress- and DNA damage-mediated neurodegeneration caused by homocysteine.     

Vitamin B6 suppresses apoptosis of NM-1 bovine endothelial cells induced by homocysteine and copper

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B₆in vivo. In the present study, we examined effects of vitamin B₆ on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.