In vivo characterization of several rodent glioma models by 1H MRS.

The assessment of metabolites by (1)H MRS can provide information regarding glioma growth, and may be able to distinguish between different glioma models. Rat C6, 9 L/LacZ, F98 and RG2, and mouse GL261, cells were intracerebrally implanted into the respective rodents, and human U87 MG cells were implanted into athymic rats. Ethyl-nitrosourea induction was also used. Glioma metabolites [e.g. total choline (tCho), total creatine (tCr), N-acetylaspartate (NAA), lactate (Lac), glutamine (Gln), glutamate (Glu), aspartate (Asp), guanosine (Gua), mobile lipids and macromolecules (MMs)] were assessed from (1)H MRS using point-resolved spectroscopy (PRESS) [TE = 24 ms; TR = 2500 ms; variable pulse power and optimized relaxation delay (VAPOR) water suppression; 27-µL and 8-µL voxels in rats and mice, respectively] at 7 T. Alterations in metabolites (Totally Automatic Robust Quantitation in NMR, TARQUIN) in tumors were characterized by increases in lipids (Lip1.3: 8.8-54.5 mM for C6 and GL261) and decreases in NAA (1.3-2.0 mM for RG2, GL261 and C6) and tCr (0.8-4.0 mM for F98, RG2, GL261 and C6) in some models. F98, RG2, GL261 and C6 models all showed significantly decreased (p < 0.05) tCr, and RG2, GL261 and C6 models all exhibited significantly decreased (p < 0.05) NAA. The RG2 model showed significantly decreased (p < 0.05) Gln and Glu, the C6 model significantly decreased (p < 0.05) Asp, and the F98 and U87 models significantly decreased (p < 0.05) Gua, compared with controls. The GL261 model showed the greatest alterations in metabolites. (1)H MRS was able to differentiate the metabolic profiles in many of the seven rodent glioma models assessed. These models are considered to resemble certain characteristics of human glioblastomas, and this study may be helpful in selecting appropriate models.

Inhalation of environmental stressors & chronic inflammation: autoimmunity and neurodegeneration.

Human life expectancy and welfare has decreased because of the increase in environmental stressors in the air. An environmental stressor is a natural or human-made component present in our environment that upon reaching an organic system produces a coordinated response. This response usually involves a modification of the metabolism and physiology of the system. Inhaled environmental stressors damage the airways and lung parenchyma, producing irritation, recruitment of inflammatory cells, and oxidative modification of biomolecules. Oxidatively modified biomolecules, their degradation products, and adducts with other biomolecules can reach the systemic circulation, and when found in higher concentrations than normal they are considered to be biomarkers of systemic oxidative stress and inflammation. We classify them as metabolic stressors because they are not inert compounds; indeed, they amplify the inflammatory response by inducing inflammation in the lung and other organs. Thus the lung is not only the target for environmental stressors, but it is also the source of a number of metabolic stressors that can induce and worsen pre-existing chronic inflammation. Metabolic stressors produced in the lung have a number of effects in tissues other than the lung, such as the brain, and they can also abrogate the mechanisms of immunotolerance. In this review, we discuss recent published evidence that suggests that inflammation in the lung is an important connection between air pollution and chronic inflammatory diseases such as autoimmunity and neurodegeneration, and we highlight the critical role of metabolic stressors produced in the lung. The understanding of this relationship between inhaled environmental pollutants and systemic inflammation will help us to: (1) understand the molecular mechanism of environment-associated diseases, and (2) find new biomarkers that will help us prevent the exposure of susceptible individuals and/or design novel therapies.

Phenyl-tert-butylnitrone induces tumor regression and decreases angiogenesis in a C6 rat glioma model.

The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the difficulty of an early and accurate diagnosis and the lack of currently efficient therapeutic compounds. The efficacy of phenyl-tert-butylnitrone (PBN) as a potential anti-glioma therapeutic drug was assessed by magnetic resonance (MR) imaging (T(1)/T(2)-weighted imaging) and MR angiography (time-of-flight imaging, in conjunction with a Mathematica-based program) methods by monitoring morphologic properties, growth patterns, and angiogenic behaviors of a moderately aggressive rat C6 glioma model. MR results from untreated rats showed the diffusive invasiveness of C6 gliomas, with some associated angiogenesis. PBN administration as a pretreatment was found to clearly induce a decrease in growth rate and tumor regression as well as preventing angiogenesis. This compound even had a 40% efficiency in reducing well-established tumors. MR findings rivaled those from histology and angiogenesis marker immunostaining evaluations. In this study we demonstrated the efficiency of PBN as a potential anti-glioma drug and found it to inhibit tumor cell proliferation and prevent vascular alterations in early stages of glioma progression. The MR methods that we used also proved to be particularly suitable in following the angiogenic behavior and treatment response of a potential anti-glioma agent in a rat C6 glioma model.

Antioxidants in central nervous system diseases: preclinical promise and translational challenges.

Oxidative damage is strongly implicated in the pathogenesis of neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease and stroke (brain ischemia/reperfusion injury). The availability of transgenic and toxin-inducible models of these conditions has facilitated the preclinical evaluation of putative antioxidant agents ranging from prototypic natural antioxidants such as vitamin E (alpha-tocopherol) to sophisticated synthetic free radical traps and catalytic oxidants. Literature review shows that antioxidant therapies have enjoyed general success in preclinical studies across disparate animal models, but little benefit in human intervention studies or clinical trials. Recent high-profile failures of vitamin E trials in Parkinson's disease, and nitrone therapies in stroke, have diminished enthusiasm to pursue antioxidant neuroprotectants in the clinic. The translational disappointment of antioxidants likely arises from a combination of factors including failure to understand the drug candidate's mechanism of action in relationship to human disease, and failure to conduct preclinical studies using concentration and time parameters relevant to the clinical setting. This review discusses the rationale for using antioxidants in the prophylaxis or mitigation of human neurodiseases, with a critical discussion regarding ways in which future preclinical studies may be adjusted to offer more predictive value in selecting agents for translation into human trials.

A survey of sesamin and composition of tocopherol variability from seeds of eleven diverse sesame (Sesamum indicum L.) genotypes using HPLC-PAD-ECD.

The objective of this study was to determine the composition and content of sesamin and desmethyl tocopherols such as alpha-tocopherol (alphaT), delta-tocopherol (deltaT) and gamma-tocopherol (gammaT) in seeds of sesame (Sesamum indicum L.) for 11 genotypes conserved in the United States Department of Agriculture (USDA), Agricultural Research Service (ARS) and Plant Genetic Resources Conservation Unit (PGRCU) in Griffin, Georgia, USA. Seed accessions studied were collections from eight countries worldwide, including one landrace from Thailand and two cultivars from Texas, USA. Novel methodologies and analytical techniques described herein consisted of reverse-phase high-performance liquid chromatography (HPLC) connected in series with two detection systems specific for each analyte class. Photodiode array detection was employed for sesamin analysis and electrochemical array detection was used in the determination of tocopherols. A preliminary study was conducted to assess sesamin levels in 2003 and tocopherol levels in 2004 from sesame seed samples conserved at the USDA, ARS and PGRCU. In 2005, sesame seed samples were grown, harvested and evaluated for sesamin as well as tocopherol levels. The overall results (n = 3) showed that sesamin, alphaT, deltaT and gammaT levels were 0.67-6.35 mg/g, 0.034-0.175 microg/g, 0.44-3.05 microg/g and 56.9-99.3 microg/g respectively, indicating that the sesame seed accessions contained higher levels of sesamin and gammaT compared with alphaT and deltaT. Statistical analysis was conducted and significant differences were observed among the 11 different sesame genotypes. This suggests that genetic, environmental and geographical factors influence sesamin and desmethyl tocopherol content.

On the relation of oxidative stress to neuroinflammation: lessons learned from the G93A-SOD1 mouse model of amyotrophic lateral sclerosis.

The central nervous system (CNS) presents both challenges and opportunities to researchers of redox biochemistry. The CNS is sensitive to oxidative damage during aging or disease; excellent transgenic models of specific neurodegenerative diseases have been created that reproduce oxidative stress components of the corresponding human disorder. Mouse models of familial amyotrophic lateral sclerosis (ALS) based on overexpressed mutant human Cu, Zn-superoxide dismutase (SOD1) are cases in point. These animals experience predictably staged, age-dependent motor neuron degeneration with profound cellular and biochemical damage to nerve fibers and spinal cord tissue. Severe protein and lipid oxidation occurs in these animals, apparently as an indirect consequence of protein aggregation or cytopathic protein-protein interactions, as opposed to aberrant redox catalysis by the mutant enzyme. Recent studies of G93A-SOD1 mice and rats suggest that oxidative damage is part of an unmitigated neuroinflammatory reaction, possibly arising in combination from mitochondrial dysfunction plus pathophysiologic activation of both astrocytes and microglia. Lesions to redox signal-transduction pathways in mutant SOD1+ glial cells may stimulate broad-spectrum upregulation of proinflammatory genes, including arachidonic acid-metabolizing enzymes [e.g., cyclooxygenase-II (COX-II) and 5-lipoxygenase (5LOX)]; nitric oxide synthase (NOS) isoforms; cytokines (particularly tumor necrosis factor alpha, TNF-alpha); chemokines; and immunoglobulin Fc receptors (FcgammaRs). The integration of these processes creates a paracrine milieu inconsistent with healthy neural function. This review summarizes what has been learned to date from studies of mutant SOD1 transgenic animals and demonstrates that the G93A-SOD1 mouse in particular is a robust laboratory for the study of neuroinflammation and redox biochemistry.

Primary glia expressing the G93A-SOD1 mutation present a neuroinflammatory phenotype and provide a cellular system for studies of glial inflammation.

Detailed study of glial inflammation has been hindered by lack of cell culture systems that spontaneously demonstrate the "neuroinflammatory phenotype". Mice expressing a glycine --> alanine substitution in cytosolic Cu, Zn-superoxide dismutase (G93A-SOD1) associated with familial amyotrophic lateral sclerosis (ALS) demonstrate age-dependent neuroinflammation associated with broad-spectrum cytokine, eicosanoid and oxidant production. In order to more precisely study the cellular mechanisms underlying glial activation in the G93A-SOD1 mouse, primary astrocytes were cultured from 7 day mouse neonates. At this age, G93A-SOD1 mice demonstrated no in vivo hallmarks of neuroinflammation. Nonetheless astrocytes cultured from G93A-SOD1 (but not wild-type human SOD1-expressing) transgenic mouse pups demonstrated a significant elevation in either the basal or the tumor necrosis alpha (TNFalpha)-stimulated levels of proinflammatory eicosanoids prostaglandin E2 (PGE2) and leukotriene B4 (LTB4); inducible nitric oxide synthase (iNOS) and *NO (indexed by nitrite release into the culture medium); and protein carbonyl products. Specific cytokine- and TNFalpha death-receptor-associated components were similarly upregulated in cultured G93A-SOD1 cells as assessed by multiprobe ribonuclease protection assays (RPAs) for their mRNA transcripts. Thus, endogenous glial expression of G93A-SOD1 produces a metastable condition in which glia are more prone to enter an activated neuroinflammatory state associated with broad-spectrum increased production of paracrine-acting substances. These findings support a role for active glial involvement in ALS and may provide a useful cell culture tool for the study of glial inflammation.

Modification of lupus-associated 60-kDa Ro protein with the lipid oxidation product 4-hydroxy-2-nonenal increases antigenicity and facilitates epitope spreading.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibodies as a near universal feature of the disease. The Ro ribonucleoprotein particle, composed of a 60-kDa protein noncovalently associated with human cytoplasmic RNA, is the target of antibodies in 25-40% of lupus patients. Purified human 60-kDa Ro was found to be oxidatively modified. Earlier investigations from our laboratory revealed increased oxidative damage in SLE patients. Therefore we hypothesized that oxidation by-products, such as 4-hydroxy-2-nonenal (HNE), could lead to neoantigens like HNE-modified 60-kDa Ro, which could in turn initiate autoimmunity or drive epitope spreading. To test this hypothesis we immunized rabbits with either HNE-modified 60-kDa Ro or the unmodified Ro. Intramolecular epitope spreading within the Ro molecule and intermolecular epitope spreading to La, double-stranded DNA, nRNP, and Sm occurred preferentially in HNE-Ro-immunized animals. Nonspecific anti-HNE antibody, generated by immunization with HNE-keyhole limpet hemocyanin conjugate, did not significantly bind to these autoantigens. These data may suggest a hitherto unappreciated mechanism by which oxidative stress facilitates epitope spreading in SLE.

Mitochondrial dysfunction in choline deficiency-induced apoptosis in cultured rat hepatocytes.

Our recent studies have demonstrated that generation of ROS is associated with choline deficiency (CD)-induced apoptosis in CWSV-1 cells, an immortalized rat hepatocyte that becomes tumorigenic by stepwise culturing in decreasing levels of choline. In the present study, we investigated the effect of CD on loss of mitochondrial membrane potential (MMP), using the JC-1 probe by FASCAN assay. Our data demonstrate that MMP in CD-cultured cells was decreased in a time- and dose-dependent manner and that significant disruption occurred at 24 h, relative to high choline (HC, 70 microM) cultured cells. In order to investigate further the relationship among the CD-induced ROS, MMP collapse, and apoptosis, we examined the effects of different inhibitors on ROS production, MMP disruption, and apoptosis in CD or HC-cultured CWSV-1 cells. These data indicate that the disruption of MMP is an upstream event in CD-induced apoptosis, and mitochondrial dysfunction plays a key role in mediating CD-induced apoptosis in CWSV-1 cells.

Defining the catalytic activity of nanoceria in the P23H-1 rat, a photoreceptor degeneration model.

PURPOSE: Inorganic catalytic nanoceria or cerium oxide nanoparticles (CeNPs) are bona fide antioxidants that possess regenerative radical scavenging activities in vitro. Previously, we demonstrated that CeNPs had neuroprotective and anti-angiogenic properties in rodent retinal degeneration and neovascularization models. However, the cellular mechanisms and duration of the catalytic activity of CeNPs in preventing photoreceptor cell loss are still unknown. In this study, we sought to answer these questions using the P23H-1 rat, an autosomal dominant retinitis pigmentosa (adRP) model. METHODS: A single dose of either saline or CeNPs was delivered intravitreally into the eyes of P23H-1 rats at 2-3 weeks of age. Retinal functions were examined at 3 to 7 weeks post injection. We quantified retinal proteins by Western blot analyses and counted the number of apoptotic (TUNEL+) profiles in the outer nuclear layer (ONL) of retinal sections. We measured free 8-isoprostanes to quantify lipid peroxidation in retinal tissues. RESULTS: We observed increased rod and cone cell functions up to three weeks post injection. Apoptotic cells were reduced by 46%, 56%, 21%, and 24% at 3, 7, 14, 21 days, respectively, after CeNPs injection compared to saline. Additionally, reduction of lipid peroxidation in the retinas of CeNPs-treated vs saline-treated animals was detected 14 days post injection. CONCLUSIONS: We validated that CeNPs were effective in delaying loss of photoreceptor cell function in an adRP rat model. This represents the fourth rodent retinal disease model that shows delay in disease progression after a single application of CeNPs. We further demonstrated that CeNPs slowed the rate of photoreceptor cell death. We deduced that the catalytic activity of CeNPs in vivo in this rat model to be undiminished for at least 7 days and then declined over the next 14 days after CeNPs administration.

Reactive oxygen species in choline deficiency-induced apoptosis in rat hepatocytes.

Choline deficiency (CD) is involved in hepatocellular carcinoma and CD-induced apoptosis may be implicated in cellular malignant transformation. In this report, we studied the effects of choline deficiency on generation of reactive oxygen species (ROS) using the fluorescent probe dichlorodihydrofluorescein diacetate and the possible role of ROS on CD-induced apoptosis in cultured CWSV-1 cells, an immortalized rat hepatocyte. This cell line is reported to become tumorigenic by step-wise culturing in lower levels of choline. Our data demonstrate that CD induces a time- and dose-dependent increase in ROS in CWSV-1 cells. The increase in ROS production may be related to dysfunction of the mitochondrial respiratory chain. Our data also demonstrated that ROS generation occurred before CD-induced apoptosis, suggesting ROS may play a key role in signaling CD-induced apoptosis in CWSV-1 cells.

New perspectives on vitamin E: gamma-tocopherol and carboxyelthylhydroxychroman metabolites in biology and medicine.

Vitamin E (alpha-tocopherol or alphaT) has long been recognized as a classic free radical scavenging antioxidant whose deficiency impairs mammalian fertility. In actuality, alpha-tocopherol is one member of a class of phytochemicals that are distinguished by varying methylation of a chroman head group. Early studies conducted between 1922 and 1950 indicated that alpha-tocopherol was specific among the tocopherols in allowing fertility of laboratory animals. The unique vitamin action of alphaT, combined with its prevalence in the human body and the similar efficiency of tocopherols as chain-breaking antioxidants, led biologists to almost completely discount the "minor" tocopherols as topics for basic and clinical research. Recent discoveries have forced a serious reconsideration of this conventional wisdom. New and unexpected biological activities have been reported for the desmethyl tocopherols, such as gamma-tocopherol, and for specific tocopherol metabolites, most notably the carboxyethyl-hydroxychroman (CEHC) products. The activities of these other tocopherols do not map directly to their chemical antioxidant behavior but rather reflect anti-inflammatory, antineoplastic, and natriuretic functions possibly mediated through specific binding interactions. Moreover, a nascent body of epidemiological data suggests that gamma-tocopherol is a better negative risk factor for certain types of cancer and myocardial infarction than is a alpha-tocopherol. The potential public health implications are immense, given the extreme popularity of alphaT supplementation which can unintentionally deplete the body of gamma-tocopherol. These findings may or may not signal a major paradigm shift in free radical biology and medicine. The data argue for thorough experimental and epidemiological reappraisal of desmethyl tocopherols, especially within the contexts of cardiovascular disease and cancer biology.

Catalytic nanoceria are preferentially retained in the rat retina and are not cytotoxic after intravitreal injection.

Cerium oxide nanoparticles (nanoceria) possess catalytic and regenerative radical scavenging activities. The ability of nanoceria to maintain cellular redox balance makes them ideal candidates for treatment of retinal diseases whose development is tightly associated with oxidative damage. We have demonstrated that our stable water-dispersed nanoceria delay photoreceptor cell degeneration in rodent models and prevent pathological retinal neovascularization in vldlr mutant mice. The objectives of the current study were to determine the temporal and spatial distributions of nanoceria after a single intravitreal injection, and to determine if nanoceria had any toxic effects in healthy rat retinas. Using inductively-coupled plasma mass spectrometry (ICP-MS), we discovered that nanoceria were rapidly taken up by the retina and were preferentially retained in this tissue even after 120 days. We also did not observe any acute or long-term negative effects of nanoceria on retinal function or cytoarchitecture even after this long-term exposure. Because nanoceria are effective at low dosages, nontoxic and are retained in the retina for extended periods, we conclude that nanoceria are promising ophthalmic therapeutics for treating retinal diseases known to involve oxidative stress in their pathogeneses.

Identification of lanthionine synthase C-like protein-1 as a prominent glutathione binding protein expressed in the mammalian central nervous system.

Proteomic experiments were performed to identify novel glutathione (GSH) binding proteins expressed in the mammalian central nervous system. Bovine brain lysate was affinity purified using an immobilized glutathione-Sepharose column. Proteins that bound the immobilized glutathione were eluted with free glutathione and identified by one- and two-dimensional electrophoresis coupled with mass spectrometric analysis of tryptic fragments. Major proteins purified by this technique were glutathione S-transferase-mu (GST-mu) and GST-pi and lanthionine synthase C-like protein-1 (LanCL1). LanCL1 is a mammalian homologue of a prokaryotic enzyme responsible for the synthesis of thioether (lanthionine) cross-links within nascent polypeptide chains, yielding macrocyclic proteins with potent microbicidal activity. An antibody against LanCL1 was generated and applied to immunochemical studies of spinal cord tissue from SOD1G93A transgenic mice, a model for amyotrophic lateral sclerosis (ALS), wherein LanCL1 expression was found to be increased at presymptomatic stages of the disease. These results indicate LanCL1 is a glutathione binding protein possibly significant to neurodegenerative disease.

Temporal patterns of cytokine and apoptosis-related gene expression in spinal cords of the G93A-SOD1 mouse model of amyotrophic lateral sclerosis.

Familial amyotrophic lateral sclerosis (FALS) is often caused by gain-of-function mutations in Cu,Zn-superoxide dismutase (SOD1). Multiprobe ribonuclease protection assays (RPAs) were used to investigate expression of 36 different cytokines and apoptosis-related genes in spinal cords of mice that ubiquitously express human SOD1 bearing a glycine (r) alanine substitution at residue 93 (G93A-SOD1). Mice were studied at late presymptomatic stage (80 days), and at 120 days when the animals experience severe hindlimb paralysis and accumulation of oxidatively modified proteins. Spinal cord tissue from G93A-SOD1 mice expressed a selective subset of macrophage-typical cytokines (monokines) including interleukin (IL)1alpha, IL1beta and IL1RA at 80 days increasing by 120 days. Contrastingly, T-cell derived cytokines (lymphokines) including IL2, IL3 and IL4 were detected at low levels in non-transgenic mice but these were not elevated in G93A-SOD1 mice even at 120 days. Apoptosis-related genes were generally unaffected at 80 days but multiple caspases and death receptor components were up-regulated at 120 days; the only exceptions being FADD and the tumor necrosis factor (TNF)alpha receptor p55 which was up-regulated at 80 days and increased further at 120 days. These data indicate that in the G93A-SOD1 mouse: (i) cytokine expression changes precede bulk protein oxidation and apoptosis gene expression; (ii) lymphocyte contributions to cytokine expression in FALS are likely minor; and (iii) TNFalpha and its receptors may link inflammation to apoptosis in ALS.

Anti-inflammatory effects of tocopherol metabolites.

Our objective was to assess the anti-inflammatory effects of alpha-tocopherol, gamma-tocopherol, and their metabolites 2,5,7,8-tetramethyl-2-(beta-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC) in defined cell culture systems. Rat aortic endothelial cells and mouse microglial cultures were treated with tumor necrosis factor TNFalpha or bacterial lipopolysaccharide (LPS) and nitrite and prostaglandin E(2) (PGE(2)) were measured. alpha-CEHC suppressed TNFalpha-stimulated nitrite production in both cell types, whereas both CEHC derivatives inhibited LPS-stimulated microglial nitrite efflux. Both alpha-CEHC and gamma-CEHC inhibited microglial PGE(2) production, but neither alpha- nor gamma-tocopherol was effective at inhibiting cytokine-stimulated inflammatory processes. These results show that the anti-inflammatory effects of tocopherols are highly cell type-, stimulus-, and endpoint-dependent.

The arachidonic acid 5-lipoxygenase inhibitor nordihydroguaiaretic acid inhibits tumor necrosis factor alpha activation of microglia and extends survival of G93A-SOD1 transgenic mice.

Familial forms of amyotrophic lateral sclerosis (ALS) can be caused by mutations in copper, zinc-superoxide dismutase (SOD1). Mice expressing SOD1 mutants demonstrate a robust neuroinflammatory reaction characterized, in part, by up-regulation of tumor necrosis factor alpha (TNFalpha) and its primary receptor TNF-RI. In an effort to identify small molecule inhibitors of neuroinflammation useful in treatment of ALS, a microglial culture system was established to identify TNFalpha antagonists. Walker EOC-20 microglia cells were stimulated with recombinant TNFalpha, with or without inhibitors, and the cell response was indexed by NO2- output. Three hundred and fifty-five rationally selected compounds were included in this bioassay. The arachidonic acid 5-lipoxygenase (5LOX) and tyrosine kinase inhibitor nordihydroguaiaretic acid (NDGA), a natural dicatechol, was one of the most potent non-cytotoxic antagonists tested (IC50 8 +/- 3 microm). Investigation of the G93A-SOD1 mouse model for ALS revealed increased message and protein levels of 5LOX at 120 days of age. Oral NDGA (2500 p.p.m.) significantly extended lifespan and slowed motor dysfunction in this mouse, when administration was begun relatively late in life (90 days). NDGA extended median total lifespan of G93A-SOD1 mice by 10%, and life expectancy following start of treatment was extended by 32%. Disease-associated gliosis and cleaved microtubule-associated tau protein, an indicator of axon damage, were likewise reduced by NDGA. Thus, TNFalpha antagonists and especially 5LOX inhibitors might offer new opportunities for treatment of ALS.

The nitration product 5-nitro-gamma-tocopherol is increased in the Alzheimer brain.

Oxidative stress and quasi-inflammatory processes recently have been recognized as contributing factors in the pathogenesis of Alzheimer's disease (AD). Reactive nitrating species have specifically been implicated in AD based on immunochemical and instrumental detection of nitrotyrosine in AD brain protein. The significance of lipid-phase nitration has not been investigated in AD. This study documents a significant two- to threefold increase in the lipid nitration product 5-nitro-gamma-tocopherol in affected regions of the AD brain as determined by high-performance liquid chromatography with electrochemical detection. In a bioassay to compare the relative potency of alpha-tocopherol and gamma-tocopherol against nitrative stress, rat brain mitochondria were exposed to the peroxynitrite-generating compound SIN-1. The oxidation-sensitive Kreb's cycle enzyme alpha-ketoglutarate dehydrogenase was inactivated by SIN-1, in a manner that could be significantly attenuated by gamma-tocopherol (at <10 microM) but not by alpha-tocopherol. These data indicate that nitric oxide-derived species are significant contributors to lipid oxidation in the AD brain. The findings are discussed in reference to the neuroinflammatory hypothesis of AD and the possible role of gamma-tocopherol as a major lipid-phase scavenger of reactive nitrogen species.

Message and protein-level elevation of tumor necrosis factor alpha (TNF alpha) and TNF alpha-modulating cytokines in spinal cords of the G93A-SOD1 mouse model for amyotrophic lateral sclerosis.

Recent data indicate that certain pro-inflammatory cytokines are transcriptionally upregulated in the spinal cords of G93A-SOD1 mice, a model of amyotrophic lateral sclerosis (ALS). We previously showed that the receptor for tumor necrosis factor alpha (TNF-R1) was notably elevated at late presymptomatic as well as symptomatic phases of disease (J. Neurochem. 82 (2002) 365). We now extend these findings by showing that message for TNFalpha, as well as mRNA for interferon gamma (IFNgamma) and transforming growth factor beta1/2 (TGFbeta1, TGFbeta2), is simultaneously increased. Furthermore, TNFalpha protein is significantly increased in G93A-SOD1 mouse spinal cords, as are protein levels for interleukin-6 (IL6), IFNgamma, and the chemokines RANTES (CCL5) and KC. The interaction of TNFalpha, IL6, and IFNgamma proteins was modeled in vitro using Walker EOC-20 murine microglia with nitrite (NO(2)(-)) efflux as a quantitative index of cell response. TNFalpha alone caused robust NO(2)(-) flux, while IL6 had a lesser effect and neither IFNgamma nor IL1beta was active when applied singly. The TNFalpha stimulus was potently magnified in the presence of IL6 or IFNgamma. When applied in combination at very low concentrations, IFNgamma co-synergized with IL6 to produce a multiplicative increase in NO(2)(-) after stimulation with TNFalpha. Taken together, these data suggest that modest increases in multiple synergistic cytokines could produce a disproportionately severe activation of microglia within the degenerating spinal cord. Our data support a model wherein TNFalpha acts as a principal driver for neuroinflammation, while several co-stimulating cytokines and chemokines act to potentiate the TNFalpha effects.