Carlo De Marco

Elevated protein-bound levels of the lipid peroxidation product, 4-hydroxy-2-nonenal, in brain from persons with mild cognitive impairment

Oxidative damage is a feature of many age-related neurodegenerative diseases, including Alzheimers disease (AD). 4-Hydroxy-2-nonenal (HNE) is a highly reactive product of the free radical-mediated lipid peroxidation of unsaturated lipids, particularly arachidonic acid, in cellular membranes. In the present study we show for the first time in brain obtained at short postmortem intervals that the levels of HNE are elevated in mild cognitive impairment (MCI) hippocampus and inferior parietal lobules compared to those of control brain. Thus, increased levels of HNE in MCI brain implicate lipid peroxidation as an early event in AD pathophysiology and also suggest that the pharmacologic intervention to prevent lipid peroxidation at the MCI stage or earlier may be a promising therapeutic strategy to delay or prevent progression to AD.

Elevated levels of 3-nitrotyrosine in brain from subjects with amnestic mild cognitive impairment: Implications for the role of nitration in the progression of Alzheimer's disease

A number of studies reported that oxidative and nitrosative damage may be important in the pathogenesis of Alzheimers disease (AD). However, whether oxidative damage precedes, contributes directly, or is secondary to AD pathogenesis is not known. Amnestic mild cognitive impairment (MCI) is a clinical condition that is a transition between normal aging and dementia and AD, characterized by a memory deficit without loss of general cognitive and functional abilities. Analysis of nitrosative stress in MCI could be important to determine whether nitrosative damage directly contributes to AD. In the present study, we measured the level of total protein nitration to determine if excess protein nitration occurs in brain samples from subjects with MCI compared to that in healthy controls. We demonstrated using slot blot that protein nitration is higher in the inferior parietal lobule (IPL) and hippocampus in MCI compared to those regions from control subjects. Immunohistochemistry analysis of hippocampus confirmed this result. These findings suggest that nitrosative damage occurs early in the course of MCI, and that protein nitration may be important for conversion of MCI to AD.

Proteomic Analysis of Protein Expression and Oxidative Modification in R6/2 Transgenic Mice A Model of Huntington Disease

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by motor, psychiatric, and cognitive symptoms. The genetic defect responsible for the onset of the disease, expansion of CAG repeats in exon 1 of the gene that codes for huntingtin on chromosome 4, has been unambiguously identified. On the other hand, the mechanisms by which the mutation causes the disease are not completely understood yet. However, defects in energy metabolism of affected cells may cause oxidative damage, which has been proposed as one of the underlying molecular mechanisms that participate in the etiology of the disease. In our effort to investigate the extent of oxidative damage occurring at the protein level, we used a parallel proteomic approach to identify proteins potentially involved in processes upstream or downstream of the disease-causing huntingtin in a well established HD mouse model (R6/2 transgenic mice). We have demonstrated that the expression levels of dihydrolipoamide S-succinyltransferase and aspartate aminotransferase increase consistently over the course of disease (10-week-old mice). In contrast, pyruvate dehydrogenase expression levels were found to be decreased in 10-week-old HD transgenic mice compared with young (4-week-old) mice. Our experimental approach also led to the identification of oxidatively modified proteins. Six proteins were found to be significantly oxidized in old R6/2 transgenic mice compared with either young transgenic mice or non-transgenic mice. These proteins are α-enolase, γ-enolase (neuron-specific enolase), aconitase, the voltage-dependent anion channel 1, heat shock protein 90, and creatine kinase. Because oxidative damage has proved to play an important role in the pathogenesis and the progression of Huntington disease, our results for the first time identify specific oxidatively modified proteins that potentially contribute to the pathogenesis of Huntington disease.

The copper catalyzed oxidation of cysteine to cystine

Abstract The addition of cysteine to an alkaline solution of CuII produces the immediate appearance of a yellow color. The spectrum of this color is characterized by a broad peak at 330 mμ and a shoulder at 400 mμ. By chemical analysis and EPR spectrometry, it has been demonstrated that this yellow compound is to be identified with a cysteine-copperII complex, in which the cysteine to copper ratio is 2:1. This complex accounts for nearly all the copper present in solution; remains unchanged during the cysteine oxidation by molecular oxygen, and quickly disappears at the end of the reaction. At this time no more cysteine is present in solution in its reduced form and all the copper may be recovered as CuI if immediately trapped by neocuproine. The yellow cysteine-CuII complex is also immediately produced in the absence of oxygen, however, under this condition, it disappears slowly, CuII being reduced by excess of cysteine. Some kinetic approaches indicate that both the formation of the yellow complex, and its anaerobic reduction, are very complex reactions. The results obtained indicate that the cysteine-CuII complex represents the real intermediate catalyst in the copper-catalyzed oxidation of cysteine.

In vivo protective effects of ferulic acid ethyl ester against amyloid-beta peptide 1-42-induced oxidative stress.

Alzheimers disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid‐beta peptide (Aβ), a peptide that as both oligomers and fibrils is believed to play a central role in the development and progress of AD by inducing oxidative stress in brain. Therefore, treatment with antioxidants might, in principle, prevent propagation of tissue damage and neurological dysfunction. The aim of the present study was to investigate the in vivo protective effect of the antioxidant compound ferulic acid ethyl ester (FAEE) against Aβ‐induced oxidative damage on isolated synaptosomes. Gerbils were injected intraperitoneally (i.p.) with FAEE or with dimethylsulfoxide, and synaptosomes were isolated from the brain. Synaptosomes isolated from FAEE‐injected gerbils and then treated ex vivo with Aβ1–42 showed a significant decrease in oxidative stress parameters: reactive oxygen species levels, protein oxidation (protein carbonyl and 3‐nitrotyrosine levels), and lipid peroxidation (4‐hydroxy‐2‐nonenal levels). Consistent with these results, both FAEE and Aβ1–42 increased levels of antioxidant defense systems, evidenced by increased levels of heme oxygenase 1 and heat shock protein 72. FAEE led to decreased levels of inducible nitric oxide synthase. These results are discussed with potential therapeutic implications of FAEE, a brain accessible, multifunctional antioxidant compound, for AD involving modulation of free radicals generated by Aβ.

Heme oxygenase and cyclooxygenase in the central nervous system: A functional interplay

Heme oxygenase (HO) and cyclooxygenase (COX) are two hemeproteins involved in the regulation of several functions in the nervous system. Heme oxygenase is the enzyme responsible for the degradation of heme into ferrous iron, carbon monoxide (CO), and biliverdin, the latter being further reduced in bilirubin (BR) by biliverdin reductase. Heme oxygenase‐derived CO is a gaseous neuromodulator and plays an important role in the synaptic plasticity, learning and memory processes, as well as in the regulation of hypothalamic neuropeptide release, whereas BR is an endogenous molecules with antioxidant and anti‐nitrosative activities. Cyclooxygenase is considered a pro‐inflammatory enzyme as free radicals and prostaglandins (PGs) are produced during its catalytic cycle. Although PGs are also involved in a variety of physiologic conditions including angiogenesis, hemostasis, or regulation of kidney function, upregulation of COX and increase in PGs levels are a common feature of neuroinflammation. In the brain, a functional interplay exists between HO and COX. Heme oxygenase regulates COX activity by reducing the intracellular heme content or by generating CO, which stimulates PGE2 release. Increased levels of PGs, free radicals, and the associated oxidative stress serve in the brain as a trigger for the induction of HO isoforms which increases cellular antioxidant defenses to counteract oxidative damage. The importance of the interaction between HO and COX in the regulation of physiologic brain functions, and its relevance to neuroprotective or neurodegenerative mechanisms are discussed.

In vivo induction of heat shock proteins in the substantia nigra following L‐DOPA administration is associated with increased activity of mitochondrial complex I and nitrosative stress in rats: regulation by glutathione redox state

Increasing evidence suggests a critical role for oxidative and nitrosative stress in the pathogenesis of most important neurodegenerative disorders. Parkinson’s disease (PD) is a neurodegenerative disease characterized by a severe depletion in number of dopaminergic cells of the substantia nigra (SN). Administration of L‐DOPA (LD) is the more effective treatment for patients with PD. However, the vast majority of patients suffer LD‐related complications, which represent the major problem in the clinical management of PD. In the present study, LD administration to rats resulted in a significant dose‐dependent increase in Hsp70 synthesis which was specific for the SN. The amount of 70 kDa protein increased after 6 h treatment reaching the maximal induction after 24–48 h. Induction of Hsp70 in the SN was associated with a significant increase in constitutive Hsc70 and mitochondrial Hsp60 stress proteins, and with increased expression of mitochondrial complex I whereas no significant changes were found in the activity of complex IV. In the same experimental conditions, a significant decrease in reduced glutathione was observed, which was associated with an increased content of oxidized glutathione content as well as nitric oxide (NO) synthase activity, NO metabolites and nitrotyrosine immunoreactivity. Interestingly, Hsp70 induction, iNOS up‐regulation and nitrotyrosine formation have been confirmed also in SN and striatum of rats treated with LD and carbidopa, this latter being an inhibitor of the peripheral DOPA decarboxylase. Our data are in favor of the importance of the heat shock signal pathway as a basic mechanism of defense against neurotoxicity elicited by free radical oxygen and nitrogen species produced in aging and neurodegenerative disorders.

LIPOXYGENASE/H2O2-CATALYZED OXIDATION OF DIHYDROXYINDOLES : SYNTHESIS OF MELANIN PIGMENTS AND STUDY OF THEIR ANTIOXIDANT PROPERTIES

5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), which are important intermediates in melanogenesis, can be converted into the corresponding melanin pigments by the action of the lipoxygenase/H2O2 system. Kinetic and HPLC analyses indicate that both DHI and DHICA are good substrates for this enzymatic system. Enzyme activity on both substrates was measured in comparison with peroxidase and tyrosinase; the oxidizing behaviour of lipoxygenase is more similar to that of peroxidase rather than that of tyrosinase. The antioxidant properties of DHI- and DHICA-melanins have been investigated in comparison with other kinds of melanins. DHICA-melanin shows a more pronounced antioxidant effect than that of DHI-melanin and this behaviour can be ascribed to the different structure and solubility of the two pigments. The mixed polymer synthesized from DHI and DHICA is the most effective one. Some implications about the possible explanation of the above mentioned behaviour are discussed.

Luminol chemiluminescence studies of the oxidation of cysteine and other thiols to disulfides.

Abstract The luminol chemiluminescence excited by autooxidizing thiols has been investigated. When the copper-catalyzed thiol oxidation in alkaline medium is allowed to take place in the presence of luminol, a sharp light flash is observed at the end of the reaction. It has been demonstrated that the luminol chemiluminescence is due to the sudden degradation of the H2O2 accumulated during the reaction. By a chemical method, H2O2 production during thiol oxidation has been demonstrated. H2O2 is accumulated during the reaction and is immediately destroyed when all the thiol is oxidized to the disulfide. A comparison of the degradation of H2O2 and the excitement of luminol chemiluminescence shows that both reactions are strongly dependent upon pH. At high pH values H2O2 is suddenly destroyed by cupric ions, and chemiluminescence appears. At neutral pH values it is more stable and cannot excite luminol chemiluminescence. It has been shown that, under the experimental conditions described, the degradation of H2O2 requires the presence of a free metal, and only in these conditions may it excite the chemiluminescence of luminol. The results obtained indicate that during thiol oxidation the metal is strongly complexed by the thiol itself. At the end of the reaction, when thiol is no longer present in solution, the metal is again free and immediately decomposes the accumulated H2O2, thereby exciting the luminol chemiluminescence.

Spectroscopic features of native and bleached opio-melanins

Opioid peptides can be converted by tyrosinase into melanin-like compounds, in which the peptide moiety is retained. Such pigments, named opio-melanins, exhibit a characteristic absorption spectrum with a maximum at about 330 nm and a different solubility behaviour with respect to dopa-melanin, being completely soluble in hydrophylic solvents at neutral and basic pH. Opio-melanins precipitate in aqueous solutions below pH 5.0, and show apparent pKa values of 3.1, 3.6 and 4.4 for Tyr-Gly-melanin, Tyr-Gly-Gly-melanin and leuenk-melanin, respectively. The concomitant oxidation of dopa and opioid peptides by tyrosinase produces mixed polymers, showing the distinctive absorption peak at 330 nm. In the dark, in the pH range 5.5-7.0 the pigments are completely stable, whereas H2O2 addition provokes a slight degradation. At higher pH values or under simulated solar illumination with or without hydrogen peroxide, bleaching occurs more rapidly than in dopa-melanin. Upon photoirradiation the absorption spectrum of opio-melanins undergoes a marked variation, the peak at 330 nm being replaced by a broad shoulder in the range 280-350 nm. The absorption spectra of native and bleached pigments and the extent of opio-melanins degradation by bleaching agents, confirm the hypothesis that the different initial structure of the precursors accounts for a final diverse polymeric architecture of these pigments with respect to dopa-melanin.