Cysteine and mercapturate conjugates of oxidized dopamine are in human striatum but only the cysteine conjugate impedes dopamine trafficking in vitro and in vivo.

Recent results have suggested that some products of mercapturic acid pathway (MAP) metabolism of oxidized dopamine (DA) may contribute to mesostriatal dopaminergic neurodegeneration, and that at least one product, 5-S-cysteinyldopamine (Cys-DA), is elevated in patients with advanced Parkinson's disease (PD) who have been treated with L-DOPA. Here we investigated MAP enzymes and products in the midbrain and striatum of control individuals and patients with dementia with Lewy bodies (DLB) who had less severe dopaminergic degeneration than PD patients and who were not treated with L-DOPA. We also determined the biological activity of MAP metabolites of oxidized DA using primary rat mesencephalic cultures, rat cerebral synaptosomes, and rat striatum in vivo microdialysis. Our results showed that the human mesostriatal dopaminergic pathway generates Cys-DA but has limited enzymatic capacity for mercapturate formation, that striatal levels of MAP products of oxidized DA are not elevated in DLB patients compared with controls, and that Cys-DA interferes with trafficking of DA in vitro and in vivo. These results indicate that while Cys-DA is not increased in striatum of patients with mild dopaminergic neurodegeneration, it may interfere with uptake of DA in patients with advanced PD.

Dopamine thioethers in neurodegeneration.

Dopamine oxidation is proposed to be a significant contributor to mesostriatal dopaminergic neurodegeneration, although the mechanisms are not fully resolved. Recent results from in vitro and in vivo models have suggested that some products from mercapturic acid pathway (MAP) metabolism of oxidized dopamine (DA) may contribute to dopaminergic neurodegeneration, and that at least one product of this pathway, 5-S-cysteinyldopamine (Cys-DA), is elevated in patients with advanced Parkinson's disease (PD). Here we review recent findings on MAP enzymes and their products in rodent brain and in diseased regions of brain from patients with mesostriatal dopaminergic neurodegeneration. We also review the current data and our recent findings on the neurobiological activity of MAP metabolites of oxidized DA. We conclude that human striatum has limited enzymatic capacity for mercapturate formation, that levels of MAP products of oxidized DA are significantly elevated in PD patients with advanced dopaminergic neurodegeneration but not in patients with less severe degeneration, and that Cys-DA interferes with trafficking of DA in vitro and in vivo. These results indicate that Cys-DA may interfere with DAtrafficking in patients with advanced dopaminergic neurodegeneration.

Dopamine mercapturate can augment dopaminergic neurodegeneration.

Pathological and biochemical studies have consistently associated endogenous catechol oxidation with dopaminergic neurodegeneration in Parkinson's disease (PD). Recently, it has been proposed that products of catechol oxidation, the catechol thioethers, may contribute to dopaminergic neurodegeneration. In other organ systems, thioether cytotoxicity is influenced profoundly by the mercapturic acid pathway. We have pursued the hypothesis that endogenous catechol thioethers produced in the mercapturic acid pathway contribute to dopaminergic neurodegeneration. Our results showed that the extent of in vitro metal-catalyzed oxidative damage by catechol thioethers varied with the structures of the parent catechol and thioether adduct. Catechol mercapturates uniquely produced more oxidative damage than their parent catechols. In dopaminergic cell cultures, dopamine induced apoptosis in a concentration-dependent manner from 5 to 50 microM. The apoptotic effect of dopamine was greatly enhanced by subcytotoxic concentrations of the mitochondrial inhibitor, N-methyl-4-phenylpyridinium (MPP+). Similarly, subcytotoxic levels of the mercapturate or homocysteine conjugate of dopamine significantly augmented dopamine-induced apoptosis. Finally, microsomal fractions of substantia nigra from PD patients or age-matched controls had comparable cysteine-S-conjugate N-acetyltransferase activity. These data indicate that the mercapturate conjugate of dopamine may augment dopaminergic neurodegeneration and that the mercapturate pathway exists in human substantia nigra.

Divergence of brain prostaglandin H synthase activity and oxidative damage in mice with encephalitis.

Several lines of evidence point to inflammation and increased oxidant injury in brain regions of patients with Alzheimer disease (AD). Prostaglandin H synthase (PGHS) catalyzes the limiting step in prostaglandin synthesis and generates a potent oxidizing agent as by-product. One form of PGHS, PGHS-2, is induced by pro-inflammatory signals; thus leading to the 2-step hypothesis that pro-inflammatory signals in AD brain induce PGHS-2 that in turn contributes to brain oxidant injury. Here we have tested directly this 2-step hypothesis in a murine reovirus type 3 encephalitis model by measuring cerebral PGHS activity and quantifying oxidant injury. Our results showed a robust chronic inflammatory infiltrate and a 2-fold increase in PGHS activity in encephalitic mice compared with controls. Despite these changes, there was no significant increase in F2-isoprostanes or F4-neuroprostanes, accurate in vivo biomarkers of oxidant injury, and only minimal accumulation of protein adducts from the lipid peroxidation product 4-hydroxy-2-nonenal in the most intensely inflamed brain regions. These results challenge the proposal of others that pro-inflammatory induction of PGHS activity significantly contributes to oxidant injury in brain.

Elevated CSF prostaglandin E2 levels in patients with probable AD.

OBJECTIVE: To determine CSF eicosanoid concentrations and brain cyclo-oxygenase activity in AD patients and age-matched control subjects. BACKGROUND: Nonsteroidal anti-inflammatory drugs may benefit AD patients by inhibiting cyclo-oxygenases and thereby reducing prostaglandin (PG) production or oxidant stress in the CNS. METHODS: CSF eicosanoid and F2-isoprostane (IsoP) levels were determined in seven probable AD patients and seven age-matched control subjects. Cyclo-oxygenase activity was determined in microsomes prepared from the hippocampus of 10 definite AD patients and 8 age-matched control subjects. All measurements were made using gas chromatography/mass spectrometry. RESULTS: CSF concentrations of prostaglandin (PG) E2 were increased fivefold (p < 0.01) and 6-keto-PGF1alpha was decreased fourfold (p < 0.01) in probable AD patients. There was no change in total CSF eicosanoid concentration in probable AD patients. CSF F2-IsoP, a quantitative marker of lipid peroxidation in vivo, was increased in probable AD patients (p < 0.05). Cyclo-oxygenase activity in the hippocampus from definite AD patients was not different from age-matched control subjects. CONCLUSIONS: These data suggest that cyclo-oxygenase activity may not contribute significantly to CNS oxidative damage in AD. Increased CSF PGE2 concentration in probable AD patients suggest that cyclo-oxygenase inhibitors may benefit AD patients by limiting PG production.

The lipid peroxidation product 4-hydroxynonenal inhibits neurite outgrowth, disrupts neuronal microtubules, and modifies cellular tubulin.

Oxidative stress is believed to be an important factor in the development of age-related neurodegenerative diseases such as Alzheimer's disease (AD). The CNS is enriched in polyunsaturated fatty acids and is therefore particularly vulnerable to lipid peroxidation. Indeed, accumulation of lipid peroxidation products has been demonstrated in affected regions in brains of AD patients. Another feature of AD is a change in neuronal microtubule organization. A possible causal relationship between lipid peroxidation products and changes in neuronal cell motility and cytoskeleton has not been investigated. We show here that 4-hydroxy-2(E)-nonenal (HNE), a major product of lipid peroxidation, inhibits neurite outgrowth and disrupts microtubules in Neuro 2A cells. The effect of HNE on microtubules was rapid, being observed after incubation times as short as 15 min. HNE can react with target proteins by forming either Michael adducts or pyrrole adducts. 4-Oxononanal, an HNE analogue that can form only pyrrole adducts but not Michael adducts, had no effect on the microtubules. This suggests that the HNE-induced disruption of microtubules occurs via Michael addition. We also show that cellular tubulin is one of the major proteins modified by HNE and that the HNE adduction to tubulin occurs via Michael addition. Inhibition of neurite outgrowth, disruption of microtubules, and tubulin modification were observed at pathologically relevant HNE concentrations and were not accompanied by cytotoxicity. Our results show that these are proximal effects of HNE that may contribute to cytoskeletal alterations that occur in AD.

Cerebrospinal fluid lipoproteins are more vulnerable to oxidation in Alzheimer's disease and are neurotoxic when oxidized ex vivo.

Brain regional oxidative damage is thought to be a central mechanism in the pathogenesis of Alzheimer's disease (AD). Recent studies of cerebrospinal fluid (CSF) have suggested that increased lipid peroxidation of CSF and CSF lipoproteins also may occur in AD patients. In the present study, we determined the susceptibility of human CSF to ex vivo lipid peroxidation and tested the hypothesis that oxidized CSF lipoproteins may be neurotoxic. Whole CSF or a CSF lipoprotein fraction (d < 1.210 g/mL) was oxidized with 2,2'-azobis(2-amidino-propane)dihydrochloride (AAPH), a hydrophilic free-radical generator. Kinetics of CSF lipid peroxidation were followed by a standard fluorescence product accumulation assay. Oxidation of AD CSF yielded significantly shorter fluorescent lag times than controls, indicating reduced antioxidant capacity. Electrophoretic mobilities of CSF apolipoproteins were specifically reduced upon oxidation of CSF with AAPH, suggesting that lipoproteins are primary targets of CSF lipid peroxidation. Cultured neuronal cells were exposed to physiological concentrations of isolated CSF lipoproteins oxidized with increasing concentrations of AAPH; the resulting neurotoxicity showed a significant linear AAPH concentration-response relationship. These results suggest that oxidized CSF lipoproteins may contribute to the pathogenesis of neurodegeneration in AD.

Distribution of reducible 4-hydroxynonenal adduct immunoreactivity in Alzheimer disease is associated with APOE genotype.

Two major risk factors for late-onset familial and sporadic Alzheimer disease (AD), a leading cause of dementia worldwide, are increasing age and inheritance of the epsilon4 allele of the apolipoprotein E gene (APOE4). Several isoform-specific effects of apoE have been proposed; however, the mechanisms by which apoE isoforms influence the pathogenesis of AD are unknown. Also associated with AD is increased lipid peroxidation in the regions of the brain most damaged by disease. 4-hydroxynonenal (HNE), the most potent neurotoxic product of lipid peroxidation, is thought to be deleterious to cells through reactions with protein nucleophiles. We tested the hypothesis that accumulation of the most common forms of HNE-protein adducts, borohydride-reducible adducts, is associated with AD and examined whether there was a relationship to APOE. Our results demonstrated that reducible HNE adducts were increased in the hippocampus, entorhinal cortex, and temporal cortex of patients with AD. Furthermore, our data showed that the pattern of reducible HNE adduct accumulation was related to APOE genotype; AD patients homozygous for APOE4 had pyramidal neuron cytoplasmic accumulation of reducible HNE adducts, while AD APOE3 homozygotes had both pyramidal neuron and astrocyte accumulation of reducible HNE adducts. This is in contrast to our previous observations that a distinct HNE protein adduct, the pyrrole adduct, accumulates on neurofibrillary tangles in AD patients. We conclude that APOE genotype influences the cellular distribution of increased reducible HNE adduct accumulation in AD.