Evaluation of Apolipoprotein E Fragmentation as a Biomarker for Alzheimer's Disease.

Recent studies have supported a role for the proteolytic cleavage of apolipoprotein E4 (APOE4) as a potential mechanism for the enhanced dementia risk associated with Alzheimer's disease. To determine whether APOE4 fragmentation is correlated with AD, ELISA assays were performed with cerebral spinal fluid (CSF) and plasma samples utilizing an antibody that specifically detects a 17 kDa amino-terminal fragment (p17) of APOE (nApoECF antibody). In CSF samples, levels of APOE fragmentation were minimal in both neuropathological normals (NPNs) and AD cases and there were no significant differences between the two cohorts across APOE genotypes. Similar results were found in plasma samples where the p17 APOE fragment comprised only 8.4% of the total level of identified APOE. As with CSF, there were no significant differences found between NPNs and AD cases in terms of the amount of nApoECF quantified. Taken together, these results suggest that the p17 amino-terminal fragment of APOE is not correlated with AD or APOE genotype in the plasma or CSF.

Identification of a caspase-derived N-terminal tau fragment in cellular and animal Alzheimer's disease models.

Biochemical modifications of tau proteins have been proposed to be among the earliest neurobiological changes in Alzheimer's disease (AD) and correlate better with cognitive symptoms than do beta-amyloid plaques. We have recently reported that adenovirus-mediated overexpression of the NH2 26-230aa tau fragment evokes a potent NMDA-mediated neurotoxic effect in primary neuronal cultures. In order to assess whether such N-terminal tau fragment(s) are indeed produced during apoptosis or neurodegeneration in vivo, we attempted to ascertain their presence in cell and animal models using an anti-tau antibody directed against the N-terminal sequence of human protein located downstream of the caspase(s)-cleavage site DRKD(25)-QGGYTMHQDQ. We provide biochemical evidence that a caspase(s)-cleaved NH2-terminal tau fragment of 20-22 kDa, consistent with the size of the NH2 26-230aa neurotoxic fragment of tau, is generated in vitro in differentiated human SH-SY5Y cells undergoing apoptosis by BDNF withdrawal or following treatment with staurosporine. In addition this NH2-terminally cleaved tau fragment, whose expression correlates with a significant up-regulation of caspase(s) activity, is also specifically detected in vivo in the hippocampus of 15 month-old AD11 transgenic mice, a model in which a progressive AD-like neurodegeneration is induced by the expression of transgenic anti-NGF antibodies. The results support the idea that aberrant activation of caspase(s), following apoptotic stimuli or neurodegeneration insults, may produce one or more toxic NH2 tau fragments, that further contribute to propagate and increase cellular dysfunctions in AD.

Activation of caspase-8 in the Alzheimer's disease brain.

Recent studies support the activation of apoptotic pathways in the Alzheimer's disease (AD) brain. Neurons committed to apoptosis may do so by either activation of a receptor-mediated pathway employing caspase-8 or through an alternative mitochondrial pathway involving oxidative stress. In the present study, the role of caspase-8 in the AD brain was examined by designing a caspase-cleavage site-directed antibody to one of the active fragments of caspase-8. In vitro analysis with this antibody, termed CASP-8p18, demonstrated that it recognized the active 18-kDa fragment of caspase-8 but not the precursor protein. In vivo immunohistochemical analysis using hippocampal tissue sections from AD or aged-matched control brains demonstrated CASP-8p18 immunolabeling of neurons in all AD cases, whereas little staining was observed in controls. These results were confirmed using a commercially available antibody that, like the CASP-8p18 antibody reacts only with the 18-kDa fragment of caspase-8 and not full-length caspase-8. As with CASP-8p18 antibody, the commercial antibody-labeled neurons in all AD cases, while showing a relative paucity of staining in representative control cases. Labeling of CASP-8p18 within tangle-bearing neurons was observed in double-labeling studies with AT8 or PHF-1, both markers for neurofibrillary tangles (NFTs). In addition, using a caspase-cleavage site-directed antibody that recognizes cleavage products of caspase-3 showed colocalization of this antibody with the CASP-8p18 antibody within NFTs. These results suggest a role for caspase-8 and the receptor-mediated apoptotic pathway as a mechanism leading to the activation of caspase-3 within neurons of the AD brain.

15-deoxy-delta12,14-prostaglandin J2, a specific ligand for peroxisome proliferator-activated receptor-gamma, induces neuronal apoptosis.

Although considerable research has shown a role for peroxisome proliferator-activated receptors (PPAR) in adipose differentiation and in the regulation of inflammation, little is known about its possible functions in neurons. We investigated the role of PPARgamma in primary cultures of cortical neurons and human neuroblastoma SH-SYSY cells. Incubation of cortical neurons with the specific PPARgamma ligand 15-Deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) induced morphological changes including neurite degeneration and nuclear condensation that were consistent with neurons dying by apoptosis. The morphological changes associated with incubation of cortical neurons with 15d-PGJ2 were prevented following pretreatment of neurons with the general caspase inhibitor, Z-VAD. These results highlight a novel role for PPARgamma in neurons and suggest that unwarranted activation of PPARgamma may contribute to the neuronal apoptosis associated with certain neurodegenerative disorders including Alzheimer's disease (AD).

Correlation between caspase activation and neurofibrillary tangle formation in Alzheimer's disease.

Although evidence suggests that neurofibrillary tangles (NFTs) and neuronal cell loss are prominent features of Alzheimer's disease (AD), the relationship between the two remains unknown. In the present study, the relationship between the activation of apoptotic mechanisms and NFT formation in AD was investigated using a caspase-cleavage site-directed antibody to fodrin, an abundant neuronal cytoskeleton protein. This antibody recognized cleavage products of fodrin after digestion by caspase-3, but did not recognize full-length fodrin. In vitro analysis of this fodrin caspase-cleavage product (CCP) antibody demonstrates that it is a specific probe for the detection of apoptotic but not necrotic pathways in cultured neurons. To determine whether caspases cleave fodrin in vivo, tissue sections from controls and AD were immunostained for fodrin (CCPs). Although no staining was observed in control cases, labeling of neurons was observed in the hippocampus of all AD cases, which increased as a function of disease progression. To determine a possible relationship between caspase activation and NFT formation, double-labeling experiments with fodrin CCP and PHF-1 were performed. Co-localization of these markers was observed in many neurons, and quantitative analysis showed that as the extent of NFT formation increased, there was a significant corresponding increase in fodrin CCP immunolabeling (r = 0.84). Taken together, these results provide evidence for the activation of apoptotic mechanisms in neurons in the AD brain and suggest that there is an association between NFT formation and the activation of apoptotic pathways in AD.

A monoclonal antibody to amyloid precursor protein induces neuronal apoptosis.

Although there is considerable evidence suggesting that altered metabolism of beta-amyloid precursor protein (APP) and accumulation of its beta-amyloid fragment are key features of Alzheimer's disease (AD), the normal physiological function of APP remains elusive. We investigated the potential role of APP in neurons using the monoclonal antibody 22C11, which binds to the extracellular domain of the human, rat, or mouse APP. Exposure of cortical neurons to 22C11 induced morphological changes including neurite degeneration, nuclear condensation, and internucleosomal DNA cleavage that were consistent with neurons dying by apoptosis. Supporting a role for 22C11-mediated apoptosis occurring by binding to APP were data demonstrating that preincubation of 22C11 with either purified APP or a synthetic peptide (APP(66-81)) that contains the epitope for 22C11 significantly attenuated neuronal damage induced by 22C11. The specificity of 22C11 was further supported by data showing no apparent effects of either mouse IgG or the monoclonal antibody P2-1, which is specific for the aminoterminal end of human but not rat APP. In addition, biochemical features indicative of apoptosis were the formation of 120- and 150-kDa breakdown products of fodrin following treatment of cortical neurons with 22C11. Both the morphological and the biochemical changes induced by 22C11 were prevented following pretreatment of neurons with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl ketone. Prior incubation of cortical neurons with GSH ethyl ester (GEE), a cell-permeable form of GSH, resulted in complete protection from the 22C11 insult, thus implicating an oxidative pathway in 22C11-mediated neuronal degeneration. This was further supported by the observation that prior treatment of neurons with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteinyl synthetase, potentiated the toxic effects of 22C11. Finally, with use of compartmented cultures of hippocampal neurons, it was also demonstrated that selective application of 22C11 caused local neuritic degeneration that was prevented by the addition of GEE to the neuritic compartment. Thus, the binding of a monoclonal antibody to APP initially triggers neurite degeneration that is followed by caspase-dependent apoptosis in neuronal cultures and illustrates a novel property of this protein in neurons that may contribute to the profound neuronal cell death associated with AD.

Neuronal apoptosis induced by beta-amyloid is mediated by caspase-8.

The Alzheimer disease-associated beta-amyloid peptide has been shown to induce apoptotic neuronal death. In the present study, we test the hypothesis that the apoptotic pathway activated by beta-amyloid is similar to the pathway activated by the Fas/TNFR family of death receptors, which requires caspase-8 activity and adaptor proteins such as FADD. We demonstrate that the selective caspase-8 inhibitor IETD-fmk blocks neuronal death induced by beta-amyloid. Furthermore, using viral-mediated gene delivery, we show that neurons expressing dominant-negative FADD are protected from apoptosis induced by beta-amyloid. Together these results indicate that the apoptotic pathway activated by beta-amyloid requires both caspase-8 activity and FADD. These findings further support the hypothesis that beta-amyloid might initiate apoptosis by cross-linking death receptors of the Fas/TNFR family.

Inhibition of GTP binding to Rac2 by peroxynitrite: potential role for tyrosine modification.

Peroxynitrite is a potent oxidant generated by the reaction of nitric oxide (*NO) and superoxide anion (O2*-), and both can be produced in inflammatory tissues. In the present studies, we analyzed the effects of peroxynitrite treatment on the GTP-binding activity of Rac2, a low molecular weight GTP-binding protein important in regulating a number of cellular functions. Using a fluorescent analog of GTP (methylanthraniloyl guanosine triphosphate or mant-GTP) as a reporter group, we found that treatment of Rac2 with peroxynitrite inhibited the binding of mant-GTP to Rac2 in a dose-dependent manner. Peroxynitrite was also able to react directly with free mant-GTP, resulting in a significant decrease in mant-GTP fluorescence; however, the mechanism of peroxynitrite-mediated damage to mant-GTP was different than with Rac2. In the case of mant-GTP, protection from peroxynitrite-mediated oxidation was observed in the presence of the free radical scavengers, mannitol and DMTU. In contrast, DMTU was unable to prevent peroxynitrite-mediated inhibition of mant-GTP binding to Rac2. Instead, our data demonstrates a role for peroxynitrite-mediated tyrosine modification in the inhibition of mant-GTP binding to Rac2, and we were able to demonstrate the formation of a significant level of nitrotyrosine formation in Rac2 exposed to peroxynitrite. Thus, our studies support the premise that oxidative modification of key cellular proteins, such as Rac2, plays an important role in the cytotoxic effects observed for peroxynitrite and other reactive oxidants.

Priming of human neutrophils by peroxynitrite: potential role in enhancement of the local inflammatory response.

Peroxynitrite is a potent oxidant generated from the reaction of nitric oxide (NO) and superoxide anion (O2-), both of which can be produced in inflammatory tissues. In these studies, we analyzed what direct effect peroxynitrite had on neutrophil (PMN) function. We found that peroxynitrite was an effective priming agent for PMNs, as demonstrated by enhanced O2- production on subsequent activation with low doses of PMA or N-formyl-methionine-leucine-phenylalanine (fMLF), changes in the expression of PMN surface markers (L-selectin, Mac-1, flavocytochrome b, and fMLF receptor), and increased intracellular calcium levels. Analysis of the mechanism of PMN priming by peroxynitrite demonstrated that peroxynitrite resulted in minimal oxidation of protein sulfhydryl groups and subsequent protein cross-linking. In contrast, treatment of PMNs with peroxynitrite resulted in significant nitration of tyrosine residues on neutrophil proteins. In addition, inhibition of tyrosine nitration with a pyrrolopyrimidine antioxidant blocked the majority of peroxynitrite-induced priming effects, further suggesting that PMN priming was mediated primarily by nitration of tyrosine residues on PMN proteins. The ability of peroxynitrite to serve as an effective priming agent for PMNs at sites of inflammation may play a key role in modulating the host-defense process.

U-101033E (2,4-diaminopyrrolopyrimidine), a potent inhibitor of membrane lipid peroxidation as assessed by the production of 4-hydroxynonenal, malondialdehyde, and 4-hydroxynonenal--protein adducts.

4-Hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) are major lipid peroxidation products generated by free radical attack on membranes and appear to contribute to the cytotoxic effects of oxidative stress by a mechanism involving adduct formation with cellular proteins. In the present studies, we investigated the relationship between lipid peroxidation and eventual inactivation of plasma membrane proteins using a model system consisting of purified red blood cell membranes and Fe2+/EDTA. Using this system, we also analyzed the ability of a novel antioxidant, U-101033E (2,4-diaminopyrrolopyrimidine), to inhibit lipid peroxidation and associated protein damage. Our results demonstrated that significant levels of MDA and 4-HNE are generated in this model system, and that both aldehydes are capable of cross-linking membrane proteins. In addition, we used a monoclonal antibody to demonstrate the presence of 4-HNE-protein adducts in this system. The generation of 4-HNE-protein adducts closely paralleled the time course of lipid peroxidation and membrane protein cross-linking, suggesting that 4-HNE may contribute to membrane protein cross-linking. Analysis of U-101033E in this system showed that this antioxidant inhibited lipid peroxidation, prevented the appearance of 4-HNE-protein adducts, and strongly reduced membrane protein cross-linking, with an EC50 of 0.5 microM. We also show that these antioxidant effects were not due to the scavenging of superoxide anion. Thus, these studies demonstrate the potential usefulness of U-101033E for treating certain disease processes where lipid peroxidation plays a role in disease pathogenesis.

Inhibition of peroxynitrite-mediated tyrosine nitration by a novel pyrrolopyrimidine antioxidant.

Peroxynitrite is a cytotoxic, free radical species that is formed by the combination of superoxide and nitric oxide. The goal of the present study was to examine the ability of a novel antioxidant, U-101033E, to prevent peroxynitrite-mediated oxidative damage of red blood cell membrane proteins. Treatment of red blood cell membranes with peroxynitrite resulted in oxidative damage as evidenced by the presence of both membrane protein cross-linking and nitration of tyrosine residues. Membrane protein cross-linking was the result of oxidation of sulfhydryl groups and was completely blocked by the addition of dithiothreitol. Dithiothreitol also prevented peroxynitrite-mediated nitration of tyrosine red blood cell proteins. U-101033E prevented nitrotyrosine formation in peroxynitrite-treated red blood cell membrane proteins in a concentration-dependent manner, with maximal protection observed at 100 microM U-101033E. However, at a similar concentration where U-101033E prevented tyrosine nitration, it had little or no effect on membrane protein cross-linking. Our results suggest that U-101033E may be intercepting a peroxynitrite-derived reactive nitrogen species that is capable of nitrating tyrosine residues. The ability of U-101033E to prevent tyrosine nitration by peroxynitrite represents a new role for this class of antioxidants and suggests that the pyrrolopyrimidines may be useful in the treatment of diseases where peroxynitrite-mediated injury is implicated.

Xanthine affects [Ca2+]i and contractile responses of ventricular cardiocytes to electrical stimulation.

Xanthine, a major purine by-product of ATP, accumulates during myocardial ischemia. In the present study, we show that xanthine (0.5-1 mM) impaired the occurrence of cytosolic Ca2+ concentration ([Ca2+]i) transients, visualized in fura 2-loaded cells, and twitches of contraction in ventricular cardiocytes in response to electrical stimulation. This effect of xanthine was independent of superoxide anion production. That it was a result of decreased membrane excitability was supported by the following: 1) it was reversed by increasing either the amplitude of the stimulus voltage required to stimulate cardiocytes or the extracellular concentration of NaCl; and 2) xanthine reversed the depolarization following electrical stimulation in cardiocytes loaded with the voltage-sensitive dye bis-oxonol. P2 purinergic-agonists, including ATP (10 microM), but not P1 purinergic agonists reproduced the effects seen with xanthine. In addition, a lack of additivity between xanthine and ATP at maximal concentrations was observed. We conclude that xanthine, through activation of a P2 purinoceptor, may contribute to myocardial arrhythmia occurring during ischemia-reperfusion injury.

Inhibition of Ca2+-pump ATPase and the Na+/K+-pump ATPase by iron-generated free radicals. Protection by 6,7-dimethyl-2,4-DI-1- pyrrolidinyl-7H-pyrrolo[2,3-d] pyrimidine sulfate (U-89843D), a potent, novel, antioxidant/free radical scavenger.

Preincubation of red blood cell (RBC) membranes with a model system known to generate reactive oxygen species (ROS) and free radicals (200 microM ferrous sulfate and 200 microM EDTA, Fe2+/EDTA) resulted inhibition of the Na+/K+ -pump ATPases was also associated with membrane protein crosslinking and lipid peroxidation, the latter as monitored by the formation of thiobarbituric acid reactive substances (TBARS). Inhibition of the ion transport ATPases, protein cross-linking and formation of TBARS were prevented by U-89843D in a concentration-dependent manner, with half-maximal protection seen at 0.3 microM. U-89843D was more potent than the classical antioxidant butylated hydroxytoluene. Neither U-89843D nor the solvent DMSO had any effect on the assay of TBARS. U-89843D exerted only minimal inhibitory activity on ATPase activities. Thus, U-89843D was potent in vitro in preventing a variety of membrane-damaging reactions mediated by ROS. It is suggested that protection of membranes from ROS-mediated damage is of potential usefulness in the prevention and treatment of certain disease processes.

Inhibition by activated neutrophils of the Ca2+ pump ATPase of intact red blood cells.

Human neutrophils, activated by phorbol myristate acetate in the presence of intact red blood cells (RBCs), caused inhibition of the Ca2+ pump ATPase of the RBCs and fragmentation of the enzyme as well as other membrane proteins. Inhibition of the Ca2+ pump ATPase of intact RBCs was directly related to the neutrophil concentration and the time of incubation. Ca2+ pump ATPase activity was partially protected by the addition of exogenous glutathione-glutathione peroxidase, but not by superoxide dismutase. The addition of sodium azide, a potent inhibitor of endogenous RBC catalase, enhanced inhibition of the Ca2+ pump ATPase of intact RBCs. Examination by SDS-polyacrylamide gel electrophoresis of membrane proteins isolated from RBCs preincubated with activated neutrophils showed gross changes in banding patterns as compared to controls. Thus, a significant amount of methemoglobin appeared to be associated with the membrane proteins, and, in general, protein bands appeared to be more diffuse and less defined than proteins in control lanes. In addition, there was an increase in the low molecular weight protein bands. Using a monoclonal antibody to the Ca2+ pump ATPase, it was shown that the 140 kDa band representing the Ca2+ pump ATPase decreased, with concomitant appearance of two low molecular weight bands running at 8.2 and 6.8 kDa in the membrane proteins from RBCs preincubated with activated neutrophils. The data are interpreted to suggest that inhibition of the Ca2+ pump ATPase in intact RBCs under these conditions occurred as a result of: neutrophil-derived superoxide, dismutation of superoxide, to H2O2, diffusion of H2O2 into RBCs, a Fenton type reaction between oxyhemoglobin, and H2O2 producing hydroxyl radical and/or a ferryl radical capable of promoting protein fragmentation of RBC membrane proteins, including the plasma membrane Ca2+ pump ATPase.

Inhibition of the Ca pump of intact red blood cells by t-butyl hydroperoxide: importance of glutathione peroxidase.

Incubation of human red blood cells (RBCs) with t-butyl hydroperoxide (tBHP) resulted in inhibition of the Ca-pump ATPase. This was demonstrated using an assay of the Ca-pump ATPase activity in intact RBCs. In this assay, activity of the Ca-pump ATPase is expressed as the rate constant of the initial loss of ATP in RBCs exposed to Ca and A23187. Pseudo-first-order rate constants (Ca-pump ATPase rate constants) were lower in the presence of tBHP versus controls. Incubation of RBCs with tBHP resulted in both a time- and concentration-dependent inhibition of the Ca-pump ATPase (IC50 approximately 1 mM). Incubation of RBCs with tBHP also resulted in decreased oxyhemoglobin, increased methemoglobin and increased thiobarbituric acid reactive substances (TBARS). GSH levels were significantly lower in the presence of tBHP. GSH fell from a control value of 2.2 mmol/l RBC to 0.46 mmol/l RBC after incubation with 0.25 mM tBHP for 15 min. Both butylated hydroxytoluene and stobadine prevented the formation of TBARS and were partially effective in protecting the Ca-pump ATPase from tBHP-induced inhibition. Dithiothreitol was completely effective in preventing the tBHP-induced formation of TBARS as well as inhibition of the Ca-pump ATPase. However, when added after exposure to tBHP, dithiothreitol was unable to restore Ca-pump ATPase activity completely. An activity of dithiothreitol independent of enzymic thiol group reduction was apparent. In the presence of mercaptosuccinate, a potent inhibitor of glutathione peroxidase, the ability of dithiothreitol to protect the Ca-pump ATPase from tBHP-induced inhibition was abolished. Therefore, protection by dithiothreitol may be afforded by its ability to replenish GSH from oxidized glutathione, thus allowing glutathione peroxidase to metabolize tBHP. These results may be interpreted to suggest that inhibition of the Ca-pump ATPase in intact RBCs occurs as a result of tBHP-induced oxidant stress and subsequent lipid peroxidation which can be prevented by certain antioxidants including butylated hydroxytoluene, stobadine, and thiol-containing compounds such as dithiothreitol. These findings provide further insight into the mode of action of hydroperoxides and certain reactive oxygen species that have been implicated in oxidative stress associated with various pathological conditions. The importance of the GSH/glutathione peroxidase system in metabolizing organic hydroperoxides is also demonstrated.

Ion transport ATPases as targets for free radical damage. Protection by an aminosteroid of the Ca2+ pump ATPase and Na+/K+ pump ATPase of human red blood cell membranes.

Preincubation of red blood cell membranes in the presence of ferrous sulfate and EDTA resulted in both a concentration- and time-dependent inhibition of the Na+/K+ pump ATPase, basal Ca2+ pump ATPase, and the calmodulin- (CaM) activated Ca2+ pump ATPase. The IC50 for all three ATPases was approximately 2.5 x 10(-5) M iron. The addition to membranes of ferrous iron and EDTA in an approximately 1:1 ratio resulted in conversion to the ferric iron form in several minutes. However, inhibition of the ion pump ATPases and cross-linking of membrane proteins occurred over the course of several hours. The time course of formation of thiobarbituric acid-reactive substances (TBARS) closely paralleled inhibition of the ion pump ATPases. Inhibition of the ion pump ATPases was prevented by the addition of deferoxamine or superoxide dismutase but not by mannitol, or catalase. Both butylated hydroxytoluene and tirilazad mesylate (U74006F) prevented the formation of TBARS, limited the inhibition of the ion pump ATPases, and reduced cross-linking of membrane proteins. These data may be interpreted to suggest that inhibition of ion pump ATPases in plasma membranes may occur as a result of iron-promoted formation of superoxide and subsequent lipid peroxidation, which can be prevented by free-radical scavengers including butylated hydroxytoluene and U74006F.