Victoria Ayala

Pathological aspects of lipid peroxidation.

Abstract Lipid peroxidation (LPO) product accumulation in human tissues is a major cause of tissular and cellular dysfunction that plays a major role in ageing and most age-related and oxidative stress-related diseases. The current evidence for the implication of LPO in pathological processes is discussed in this review. New data and literature review are provided evaluating the role of LPO in the pathophysiology of ageing and classically oxidative stress-linked diseases, such as neurodegenerative diseases, diabetes and atherosclerosis (the main cause of cardiovascular complications). Striking evidences implicating LPO in foetal vascular dysfunction occurring in pre-eclampsia, in renal and liver diseases, as well as their role as cause and consequence to cancer development are addressed.

Proteins in Human Brain Cortex Are Modified by Oxidation, Glycoxidation, and Lipoxidation EFFECTS OF ALZHEIMER DISEASE AND IDENTIFICATION OF LIPOXIDATION TARGETS

Diverse oxidative pathways, such as direct oxidation of amino acids, glycoxidation, and lipoxidation could contribute to Alzheimer disease pathogenesis. A global survey for the amount of structurally characterized probes for these reactions is lacking and could overcome the lack of specificity derived from measurement of 2,4-dinitrophenylhydrazine reactive carbonyls. Consequently we analyzed (i) the presence and concentrations of glutamic and aminoadipic semialdehydes, Nϵ-(carboxymethyl)-lysine, Nϵ-(carboxyethyl)-lysine, and Nϵ-(malondialdehyde)-lysine by means of gas chromatography/mass spectrometry, (ii) the biological response through expression of the receptor for advanced glycation end products, (iii) the fatty acid composition in brain samples from Alzheimer disease patients and agematched controls, and (iv) the targets of Nϵ-(malondialdehyde)-lysine formation in brain cortex by proteomic techniques. Alzheimer disease was associated with significant, although heterogeneous, increases in the concentrations of all evaluated markers. Alzheimer disease samples presented increases in expression of the receptor for advanced glycation end products with high molecular heterogeneity. Samples from Alzheimer disease patients also showed content of docosahexaenoic acid, which increased lipid peroxidizability. In accordance, Nϵ-(malondialdehyde)-lysine formation targeted important proteins for both glial and neuronal homeostasis such as neurofilament L, α-tubulin, glial fibrillary acidic protein, ubiquinol-cytochrome c reductase complex protein I, and the β chain of ATP synthase. These data support an important role for lipid peroxidation-derived protein modifications in Alzheimer disease pathogenesis.

Evidence of oxidative stress in the neocortex in incidental Lewy body disease.

Oxidative stress has been well documented in the substantia nigra in Parkinson disease (PD), but little is known about oxidative damage, particularly lipoxidation, advanced glycation (AGE), and AGE receptors (RAGE) in other structures, including the cerebral cortex, in early stages of diseases with Lewy bodies. The present study was undertaken to analyze these parameters in the frontal cortex (area 8), amygdala, and substantia nigra in selected cases with no neurologic symptoms and with neuropathologically verified incidental Lewy body disease-related changes, comparing them with healthy age-matched individuals. Results of the present study have shown mass spectrometric and immunologic evidences of increased lipoxidative damage by the markers malondialdehyde-lysine (MDAL) and 4-hydroxynonenal-lysine (HNE), increased expression of AGE in the substantia nigra, amygdala, and frontal cortex, and increased and heterogeneous RAGE cellular expression in the substantia nigra and frontal cortex in cases with early stages of parkinsonian neuropathology. In addition, increased content of the highly peroxidizable docosahexaenoic acid in the amygdala and frontal cortex. These changes were not associated to α-synuclein aggregation in cortex, contrasting with aggregates found in SDS-soluble fractions of frontal cortex in dementia with Lewy bodies (DLB) cases. The pattern of lipidic abnormalities differed in DLB and incidental Lewy body disease. Furthermore, although AGE and RAGE expression were raised in DLB, no increase in the total amount of HNE and MDAL adducts was found in the cerebral cortex in DLB. Preliminary analyses have identified 2 proteins with lipoxidative damage, α-synuclein and manganese superoxide dismutase (SOD2), in incidentally Lewy body disease cortex. This study demonstrates abnormal fatty acid profiles, increased and selective lipoxidative damage, and increased AGE and RAGE expression in the frontal cortex in cases with early stages of parkinsonian neuropathology without treatment. These findings further support antioxidant therapy in the treatment of PD to reduce cortical damage associated with oxidative stress.

Methionine restriction decreases mitochondrial oxygen radical generation and leak as well as oxidative damage to mitochondrial DNA and proteins

Previous studies have consistently shown that caloric restriction (CR) decreases mitochondrial reactive oxygen species (ROS) (mitROS) generation and oxidative damage to mtDNA and mitochondrial proteins, and increases maximum longevity, although the mechanisms responsible for this are unknown. We recently found that protein restriction (PR) also produces these changes independent of energy restriction. Various facts link methionine to aging, and methionine restriction (MetR) without energy restriction increases, like CR, maximum longevity. We have thus hypothesized that MetR is responsible for the decrease in mitROS generation and oxidative stress in PR and CR. In this investigation we subjected male rats to exactly the same dietary protocol of MetR that is known to increase their longevity. We have found, for the first time, that MetR profoundly decreases mitROS production, decreases oxidative damage to mtDNA, lowers membrane unsaturation, and decreases all five markers of protein oxidation measured in rat heart and liver mitochondria. The concentration of complexes I and IV also decreases in MetR. The decrease in mitROS generation occurs in complexes I and III in liver and in complex I in heart mitochondria, and is due to an increase in efficiency of the respiratory chain in avoiding electron leak to oxygen. These changes are strikingly similar to those observed in CR and PR, suggesting that the decrease in methionine ingestion is responsible for the decrease in mitochondrial ROS production and oxidative stress, and possibly part of the decrease in aging rate, occurring during caloric restriction.—Sanz, A., Caro, P., Ayala, V., Portero‐Otin, M., Pamplona, R., Barja, G. Methionine restriction decreases mitochondrial oxygen radical generation and leak as well as oxidative damage to mitochondrial DNA and proteins. FASEB J. 20, 1064–1073 (2006)

Methylglyoxal induces advanced glycation end product (AGEs) formation and dysfunction of PDGF receptor-β: implications for diabetic atherosclerosis

Purpose: Low molecular weight carbonyl compounds, such as the α‐ketoaldehydes methylglyoxal (MGO) and glyoxal (GO), are formed under hypergly‐cemic conditions and behave as advanced glycation end product (AGE) precursors. They form adducts on proteins, thereby inducing cellular dysfunctions involved in chronic complications of diabetes. Methods and main findings: Nontoxic concentrations of GO or MGO altered the PDGF‐induced PDGFRβ‐phosphorylation, ERKl/2‐activation, and nuclear translocation, and the subsequent proliferation of mesenchymal cells (smooth muscle cells and skin fibroblasts). This resulted mainly from inhibition of the intrinsic tyrosine kinase of PDGFRβ and in part from altered PDGF‐BB binding to PDGFRβ. Concomitantly, the formation of AGE adducts (Nεcarboxyrnethyl‐lysine and Nεcarboxyethyl‐ly‐sine) was observed on immunoprecipitated PDGFRβ. Arginine and aminoguanidine, used as carbonyl scavengers, reversed the inhibitory effect and the formation of AGE adducts on PDGFRβ. AGE‐PDGFRβ adducts were also detected by anti‐AGE antibodies in PDGFRβ immunopurified from aortas of diabetic (streptozoto‐cin‐treated) compared to nondiabetic apolipoprotein E‐null mice. Mass spectrometry analysis of aortas demonstrated increased AGE formation in diabetic specimens. Conclusions: these data indicate that MGO and GO induce desensitization of PDGFRβ that helps to reduce mesenchymal cell proliferation.—Cantero, A.‐V., Portero‐Otín, M., Ayala, V., Auge, N., Sanson, M., Elbaz, M., Thiers, J.‐C., Pamplona, R., Salvayre, R., Negre‐Salvayre, A. Methylglyoxal induces advanced gly‐cation end product (AGEs) formation and dysfunction of PDGF receptor‐β: implications for diabetic atherosclerosis. FASEB J. 21, 3096–3106 (2007)

Cellular Dysfunction in Diabetes as Maladaptive Response to Mitochondrial Oxidative Stress

Oxidative stress has been implicated in diabetes long-term complications. In this paper, we summarize the growing evidence suggesting that hyperglycemia-induced overproduction of superoxide by mitochondrial electron transport chain triggers a maladaptive response by affecting several metabolic and signaling pathways involved in the pathophysiology of cellular dysfunction and diabetic complications. In particular, it is our goal to describe physiological mechanisms underlying the mitochondrial free radical production and regulation to explain the oxidative stress derived from a high intracellular glucose concentration and the resulting maladaptive response that leads to a cellular dysfunction and pathological state. Finally, we outline potential therapies for diabetes focused to the prevention of mitochondrial oxidative damage.

Forty Percent Methionine Restriction Decreases Mitochondrial Oxygen Radical Production and Leak at Complex I During Forward Electron Flow and Lowers Oxidative Damage to Proteins and Mitochondrial DNA in Rat Kidney and Brain Mitochondria

Eighty percent dietary methionine restriction (MetR) in rodents (without calorie restriction), like dietary restriction (DR), increases maximum longevity and strongly decreases mitochondrial reactive oxygen species (ROS) production and oxidative stress. Eighty percent MetR also lowers the degree of membrane fatty acid unsaturation in rat liver. Mitochondrial ROS generation and the degree of fatty acid unsaturation are the only two known factors linking oxidative stress with longevity in vertebrates. However, it is unknown whether 40% MetR, the relevant methionine restriction degree to clarify the mechanisms of action of standard (40%) DR can reproduce these effects in mitochondria from vital tissues of strong relevance for aging. Here we study the effect of 40% MetR on ROS production and oxidative stress in rat brain and kidney mitochondria. Male Wistar rats were fed during 7 weeks semipurified diets differing only in their methionine content: control or 40% MetR diets. It was found that 40% MetR decreases mitochondrial ROS production and percent free radical leak (by 62-71%) at complex I during forward (but not during reverse) electron flow in both brain and kidney mitochondria, increases the oxidative phosphorylation capacity of brain mitochondria, lowers oxidative damage to kidney mitochondrial DNA, and decreases specific markers of mitochondrial protein oxidation, lipoxidation, and glycoxidation in both tissues. Forty percent MetR also decreased the amount of respiratory complexes I, III, and IV and apoptosis-inducing factor (AIF) in brain mitochondria and complex IV in kidney mitochondria, without changing the degree of mitochondrial membrane fatty acid unsaturation. Forty percent MetR, differing from 80% MetR, did not inhibit the increase in rat body weight. These changes are very similar to the ones previously found during dietary and protein restriction in rats. We conclude that methionine is the only dietary factor responsible for the decrease in mitochondrial ROS production and oxidative stress, and likely for part of the longevity extension effect, occurring in DR.

Cell stress induces TDP-43 pathological changes associated with ERK1/2 dysfunction: implications in ALS

TDP-43 has been implicated in the pathogenesis of amyotrophic lateral sclerosis and other neurodegenerative diseases. Here we demonstrate, using neuronal and spinal cord organotypic culture models, that chronic excitotoxicity, oxidative stress, proteasome dysfunction and endoplasmic reticulum stress mechanistically induce mislocalization, phosphorylation and aggregation of TDP-43. This is compatible with a lack of function of this protein in the nucleus, specially in motor neurons. The relationship between cell stress and pathological changes of TDP-43 also includes a dysfunction in the survival pathway mediated by mitogen-activated protein kinase/extracellular signal-regulated kinases (ERK1/2). Thus, under stress conditions, neurons and other spinal cord cells showed cytosolic aggregates containing ERK1/2. Moreover, aggregates of abnormal phosphorylated ERK1/2 were also found in the spinal cord in amyotrophic lateral sclerosis (ALS), specifically in motor neurons with abnormal immunoreactive aggregates of phosphorylated TDP-43. These results demonstrate that cellular stressors are key factors in neurodegeneration associated with TDP-43 and disclose the identity of ERK1/2 as novel players in the pathogenesis of ALS.

Lipidomic and metabolomic analyses reveal potential plasma biomarkers of early atheromatous plaque formation in hamsters

AIMS Atherosclerosis is the main pathological process contributing to cardiovascular disease, with diet being the most important factor involved. Although the lipidome of atheromatous plaque has been studied previously, the use of comparative lipidomics and metabolomics in plasma in early atherogenesis could lead to the discovery of plasma biomarkers that allow not only disease prediction but also measurement of disease progression. METHODS AND RESULTS High-throughput techniques, such as liquid chromatography/mass spectrometry, allowed us to compare the circulating and aortic lipidome and plasma metabolome in order to look for new molecular targets involved in atherogenesis. To achieve this objective, we chose the hamster (Mesocricetus auratus) as the best small animal model for diet-induced early atherosclerosis, because its lipoprotein metabolism is similar to that of humans. The results revealed the existence of several, previously unreported, changes in lipid and amino-acid metabolism, the peroxisome proliferator-activated receptor γ pathway, and oxidative and endoplasmic reticulum stress, also involving cell senescence. Furthermore, as a proof of concept in the modelling of dietary influences in atherogenesis, we have measured the effect of a potential anti-atherogenic polyphenol extract on the reported pathways. Our results support a previously unknown role for taurocholic acid as a potential plasma biomarker of early atheromatous plaque formation. CONCLUSION The use of comparative liquid chromatography/mass spectrometry-based lipidomics and metabolomics allows the discovery of novel pathways in atherogenesis, as well as new potential plasma biomarkers, which could allow us to predict disease in its early stages and measure its progression.

Protein and lipid oxidative damage and complex I content are lower in the brain of budgerigar and canaries than in mice. Relation to aging rate

What are the mechanisms determining the rate of animal aging? Of the two major classes of endothermic animals, bird species are strikingly long-lived compared to mammals of similar body size and metabolic rate. Thus, they are ideal models to identify longevity-related characteristics not linked to body size or low metabolic rates. Since oxidative stress seems to be related to the basic aging process, we measured specific markers of different kinds of oxidative damage to proteins, like glutamic and aminoadipic semialdehydes (GSA and AASA, specific protein carbonyls), Nɛ-(carboxyethyl)lysine (CEL), Nɛ-(carboxymethyl)lysine (CML), and Nɛ-(malondialdehyde)lysine (MDAL), as well as mitochondrial Complex I content and amino acid and membrane fatty acyl composition, in the brain of short-lived mice (maximum life span [MLSP] 3.5 years) compared with those of long-lived budgerigar ‘parakeets’ (MLSP, 21 years) and canaries (MLSP, 24 years). The brains of both bird species had significantly lower levels of compounds formed as a result of oxidative (GSA and AASA), glycoxidative (CEL and CML), and lipoxidative (CML and MDAL) protein modifications, as well as a lower levels of mitochondrial complex I protein. Although it is known that fatty acid unsaturation is lower in many tissues of long-lived compared to short-lived mammals, this is not true in the particular case of brain. In agreement with this, we also found that the brain tissue of bugerigars and canaries contains no fewer double bonds than that of mice. Amino acid composition analyses revealed that bird proteins have a significantly lower content of His, Leu and Phe, as well as, interestingly, of methionine, whereas Asp, Glu, Ala, Val, and Lys contents were higher than in the mammals. These results, together with those previously described in other tissues of pigeons (MLSP, 35 years) compared to rats (MLSP, 4 years), indicate that oxidative damage to proteins, lipids and mitochondrial DNA are lower in birds (very long-lived species) than in short-lived mammals of similar body size. The lower degree of oxidative modification of bird brain proteins was not due to decreases in the target amino acids (lysine for CEL, CML, MDAL, and AASA; and arg and pro for GSA), since these were present in bird brain proteins at higher or similar levels than in those of mice. These results are consistent with the possibility that decreases in oxidative protein modification are caused at least in part by the low rate of mitochondrial oxygen radical generation in these birds, as in all long-lived homeothermic vertebrates investigated so far.