Kenneth Hensley

Direct evidence of oxidative injury produced by the Alzheimer's β-Amyloid peptide (1–40) in cultured hippocampal neurons

The beta-Amyloid peptide (A beta) is hypothesized to mediate the neurodegeneration seen in Alzheimers disease. Recently, we proposed a new hypothesis to explain the toxicity of A beta based on the free-radical generating capacity of A beta. We have recently demonstrated using electron paramagnetic resonance (EPR) spectroscopy that A beta (1-40) generates free radicals in solution. It was therefore suggested that A beta radicals can attack cell membranes, initiate lipoperoxidation, damage membrane proteins, and compromise ion homeostasis resulting in neurodegeneration. To evaluate this hypothesis, the ability of A beta to induce neuronal oxidation, changes in calcium levels, enzyme inactivation, and neuronal death were compared with the ability of A beta to produce free-radicals. Using hippocampal neurons in culture, several methods for detection of oxidation were utilized such as the conversion of 2,7-dichlorofluorescin to 2,7-dichlorofluorescein, and a new fluorescence microscopic method for the detection of carbonyls. The ability of A beta to produce free-radicals was determined using EPR with the spin-trapping compound N-tert-butyl-alpha-phenylnitrone. Consistent with previous studies, we found that preincubation of A beta increased the toxicity of the peptide. There is a strong correlation between the intensity of radical generation by A beta and neurotoxicity. The highest neuronal oxidation and toxicity was seen at a time when A beta was capable of generating the most intense radical signal. Furthermore, little oxidation and toxicity was seen when cultures were treated with freshly dissolved A beta, which did not generate a detectable radical signal. These data are consistent with the hypothesis that free-radical-based oxidative damage induced by A beta contributes to the neurodegeneration of Alzheimers disease.

Direct detection of ototoxicant-induced reactive oxygen species generation in cochlear explants

The proposal that free-radical generation contributes to the ototoxicities of several chemical agents was studied utilizing electron paramagnetic resonance (EPR) spectrometry to detect directly ototoxicant-induced reactive oxygen species formation in cochlear tissue. Guinea pig cochlear explants in chelexed artificial perilymph (AP: 200 microliters) were exposed to an ototoxicant or AP for 10 min. Ototoxic agents included gentamicin sulfate (4.0 mM), kanamycin monosulfate (4.0 mM), ethacrynic acid (0.5 mM), furosemide (0.3 mM), cisplatin (0.1 mM), trimethyltin chloride (0.1 mM), and quinine HCl (3.0 mM). Following incubation, 20 microliters of AP/ototoxicant mixture was replaced by the filtered spin trap, 5,5-dimethylpyrroline-N-oxide (DMPO). After 10 min, the EPR spectrum of the mixture was obtained. Four line EPR spectra of relative intensities 1:2:2:1, associated with hydroxyl radical (OH)/DMPO adduct formation, were evidenced by reaction mixtures containing cochlear explants exposed to each ototoxicant. Cisplatin, quinine and the loop diuretics produced weak OH-associated EPR signals in the absence of a cochlear explant, which were amplified in its presence. Deferoxamine quenched all OH spectral peaks. Peroxide levels, assayed in parallel experiments, were diminished by each ototoxicant relative to those seen following AP exposure, suggesting possible H2O2 conversion to OH. These data support the proposal that various ototoxic agents are capable of reactive oxygen species generation or promotion in cochlear tissues.

Amyloid β Peptide (25–35) Inhibits Na+‐Dependent Glutamate Uptake in Rat Hippocampal Astrocyte Cultures

Abstract: Large numbers of neuritic plaques surrounded by reactive astrocytes are characteristic of Alzheimers disease (AD). There is a large body of research supporting a causal role for the amyloid β peptide (Aβ), a main constituent of these plaques, in the neuropathology of AD. Several hypotheses have been proposed to explain the toxicity of Aβ including free radical injury and excitotoxicity. It has been reported that treatment of neuronal/astrocytic cultures with Aβ increases the vulnerability of neurons to glutamate‐induced cell death. One mechanism that may explain this finding is inhibition of the astrocyte glutamate transporter by Aβ. The aim of the current study was to determine if Aβs inhibit astrocyte glutamate uptake and if this inhibition involves free radical damage to the transporter/astrocytes. We have previously reported that Aβ can generate free radicals, and this radical production was correlated with the oxidation of neurons in culture and inhibition of astrocyte glutamate uptake. In the present study, Aβ (25–35) significantly inhibited l‐glutamate uptake in rat hippocampal astrocyte cultures and this inhibition was prevented by the antioxidant Trolox. Decreases in astrocyte function, in particular l‐glutamate uptake, may contribute to neuronal degeneration such as that seen in AD. These results lead to a revised excitotoxicity/free radical hypothesis of Aβ toxicity involving astrocytes.

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.

Bcl-2 Protects Isolated Plasma and Mitochondrial Membranes Against Lipid Peroxidation Induced by Hydrogen Peroxide and Amyloid β-Peptide

Abstract: The bcl‐2 protooncogene product possesses antiapoptotic properties in neuronal and nonneuronal cells. Recent data suggest that Bcl‐2s potency as a survival factor hinges on its ability to suppress oxidative stress, but neither the subcellular site(s) nor the mechanism of its action is known. In this report electron paramagnetic resonance (EPR) spectroscopy analyses were used to investigate the local effects of Bcl‐2 on membrane lipid peroxidation. Using hydrogen peroxide (H2O2) and amyloid β‐peptide (Aβ) as lipoperoxidation initiators, we determined the loss of EPR‐detectable paramagnetism of nitroxyl stearate (NS) spin labels 5‐NS and 12‐NS. In intact cell preparations and postnuclear membrane fractions, Aβ and H2O2 induced significant loss of 5‐NS and 12‐NS signal amplitude in control PC12 cells, but not PC12 cells expressing Bcl‐2. Cells were subjected to differential subcellular fractionation, yielding preparations of plasma membrane and mitochondria. In preparations derived from Bcl‐2‐expressing cells, both fractions contained Bcl‐2 protein. 5‐NS and 12‐NS signals were significantly decreased following Aβ and H2O2 exposure in control PC12 mitochondrial membranes, and Bcl‐2 largely prevented these effects. Plasma membrane preparations containing Bcl‐2 were also resistant to radical‐induced loss of spin label. Collectively, our data suggest that Bcl‐2 is localized to mitochondrial and plasma membranes where it can act locally to suppress oxidative damage induced by Aβ and H2O2, further highlighting the important role of lipid peroxidation in apoptosis.

Structural and Functional Changes in Proteins Induced by Free Radical‐mediated Oxidative Stress and Protective Action of the Antioxidants N‐tert‐Butyl‐α‐phenylnitrone and Vitamin Ea

ABSTRACT: The free radical theory of aging proposes that reactive oxygen species (ROS) cause oxidative damage over the lifetime of the subject. It is the cumulative and potentially increasing amount of accumulated damage that accounts for the dysfunctions and pathologies seen in normal aging. We have prevously demonstrated that both normal rodent brain aging and normal human brain aging are associated with an increase in oxidative modification of proteins and in changes in plasma membrane lipids. Several lines of investigation indicate that one of the likely sources of ROS is the mitochondria. There is an increase in oxidative damage to the mitochondrial genome in aging and a decreased expression of mitochondrial mRNA in aging. We have used a multidisciplinary approach to the characterization of the changes that occur in aging and in the modeling of brain aging, both in vitro and in vivo. Exposure of rodents to acute normobaric hyperoxia for up to 24 h results in oxidative modifications in cytosolic proteins and loss of activity for the oxidation‐sensitve enzymes glutamine synthetase and creatine kinase. Cytoskeletal protein spin labeling also reveals synaptosomal membrane protein oxidation following hyperoxia. These changes are similar to the changes seen in senescent brains, compared to young adult controls. The antioxidant spin‐trapping compound N‐tert‐butyl‐α‐phenylnitrone (PBN) was effective in preventing all of these changes. In a related study, we characterized the changes in brain protein spin labeling and cytosolic enzyme activity in a series of phenotypically selected senescence‐accelerated mice (SAMP), compared to a resistant line (SAMR1) that was derived from the same original parents. In general, the SAM mice demonstrated greater oxidative changes in brain proteins. In a sequel study, a group of mice from the SAMP8‐sensitive line were compared to the SAMR1‐resistant mice following 14 days of daily PBN treatment at a dose of 30 mg/kg. PBN treatment resulted in an improvement in the cytoskeletal protein labeling toward that of the normal control line (SAMR1). The results of these and related studies indicate that the changes in brain function seen in several different studies may be related to the progressive oxidation of critical brain proteins and lipids. These components may be critical targets for the beneficial effects of gerontotherapeutics both in normal aging and in disease of aging.

The arachidonic acid 5‐lipoxygenase inhibitor nordihydroguaiaretic acid inhibits tumor necrosis factor α 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 (TNFα) 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 TNFα antagonists. Walker EOC‐20 microglia cells were stimulated with recombinant TNFα, 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 μm). 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, TNFα antagonists and especially 5LOX inhibitors might offer new opportunities for treatment of ALS.

Electron paramagnetic resonance investigations of free radical-induced alterations in neocortical synaptosomal membrane protein infrastructure

Evidence is presented that free radical stress can directly induce physico-chemical alterations in rodent neocortical synaptosomal membrane proteins. Synaptosomes were prepared from gerbil cortical brain tissue and incubated with 3 mM ascorbate and various concentrations of exogenous Fe2+ for 30-240 min at 37 degrees C. Synaptosomes were then lysed and covalently labeled with the protein thiol-selective spin label MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperdin-1-oxyl) and subjected to electron paramagnetic resonance (EPR) spectrometry. In separate experiments, synaptosomal membranes were labeled with the thiol-specific spin label MTS ((1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl)-methanethiosulfonate), or the lipid-specific spin probe 5-NS (5-nitroxide stearate). Free radical stress induced by iron/ascorbate treatment has a rigidizing effect on the protein infrastructure of these membranes, as appraised by EPR analysis of membrane protein-bound spin label, but no change was detected in the lipid component of the membrane. These results are discussed with reference to potential oxidative mechanisms in aging and neurological disorders.

AGING AND CALORIC RESTRICTION AFFECT MITOCHONDRIAL RESPIRATION AND LIPID MEMBRANE STATUS: AN ELECTRON PARAMAGNETIC RESONANCE INVESTIGATION

Previous studies have indicated that reactive oxygen species (ROS) are likely involved in the pathogenesis of neurodegenerative diseases including Alzheimers disease (AD). ROS, generated by succinate-stimulated mitochondria, have been reported to be spin trapped and detected by electron paramagnetic resonance (EPR). Our aim in the current study was to study the impact of aging on the effect of increased metabolic stimuli on mitochondrial respiration in terms of oxy-radical generation and possible lipid peroxidative changes in brain neocortical membranes. A mixed population of brain synaptosomes and mitochondria from brown norway male rats of differing ages being fed either ad lib (AL) or a caloric-restricted diet (DR) was prepared and labeled with 5-nitroxyl stearate (5-NS), a membrane lipid-specific spin label. The changes in anisotropic motion of the intercalated 5-NS spin probe also allows one to evaluate the status of the membrane fluidity in the lipid microenvironment via the order parameter. Upon succinate stimulation of mitochondria, the ROS generated resulted in a decrease in the EPR signal amplitude of the 5-NS reporter molecule indicative of the flux of oxy-radicals produced and possible peroxidation-induced changes in the synaptosomal lipid membrane. The line width remained constant, indicating that the overall intensity was reduced. The results showed a significant overall age effect in the ability to generate oxygen-derived radicals following metabolic stimulation (p < .0001). Stimulation of state 4 mitochondrial respiration with 20 mM succinate resulted in greater oxy-radical production in 25-month-old animals as compared to younger animals, suggesting increased mitochondrial leakage with age. Free radical stress induced by metabolic stimulation also causes a concomitant increase in membrane fluidity (p < .0001). There was also a significant age effect (p < .0007) on the order parameter of the mixed population of membranes. Although caloric restriction attenuated the membrane rigidization caused by aging, it was found to play a role in limiting the oxy-radical production following metabolic stimulation of mitochondria. The overall effect of age on membrane spin-label intensities EPR signal upon succinate stimulation suggests that progressive mitochondrial dysfunction may be a key factor in the aging process and in development of age-associated diseases.

Nitrone Inhibition of Age-Associated Oxidative Damage

Abstract: The mechanistic basis of the neuroprotective activity of the nitrone‐based free radical trap PBN (α‐phenyl‐N‐tert‐butyl nitrone) has been investigated extensively. Key observations exclude its simple mass action spin trapping of free radicals activity as the key mechanism of action. These include: A) the fact that it protects in experimental stroke even if administered several hours after the event and B) the fact that its chronic low‐level administration to old experimental animals reverses their age‐enhanced susceptibility to stroke even several days after the last dosage. PBN was found to inhibit gene induction in several models including stroke and an LPS‐mediated septic shock model. Stoke causes inducible nitric oxide synthase (iNOS) to be expressed. High levels of nitric oxide and peroxynitrite (formed from nitric oxide), produced by iNOS, is particularly neurotoxic. PBN inhibits iNOS induction. Therefore, it seems that prevention of the formation of neurotoxic products is a rational mechanism of action of PBN in the stroke model. There is strong rationale to consider that there is an enhanced propensity for a “smoldering” neuro‐inflammatory state in the old brain. Reversal of this state by PBN may explain its action in preventing age‐enhanced stroke susceptibility in old experimental animals. Significant new findings underscore the importance of neuro‐inflammatory processes in neuronal death or dysfunction in Alzheimers disease. Neuro‐inflammatory processes implicate enhanced signal transduction processes. Strong evidence for this is the enhanced p38 kinase activation in neurons near plaques and tangles of the Alzheimers brain in contrast to normal aged‐matched control brain which did not show p38 activation. In rat primary astrocytes p38 activation by the pro‐inflammatory cytokine IL‐1β, as well as by H2O2, was significantly suppressed by PBN. Mechanistically it was shown that PBN suppresses the amount of reactive oxygen species (ROS) produced in mitochondrial respiration. Much evidence indicates that ROS are signaling molecules and that they also are involved in maintaining brain phosphatases in an inactive state. We argue that finding a specific high affinity site mechanism for the neuroprotective action of PBN is unlikely based on the complexity of the system reflecting ROS generation and signal transduction processes that have apparently evolved to maintain adaptive responses. The promising pharmacological activity of molecules like PBN is not diminished by this however, for only excessive amounts of ROS is considered detrimental. The action of PBN in suppressing signal transduction processes, most likely by suppressing ROS production in mitochondrial respiration, effectively controls excessive oxidative damage and prevents induction of genes that form neurotoxic products.