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Structure-activity analysis of diffusible lipid electrophiles associated with phospholipid peroxidation: 4-hydroxynonenal and 4-oxononenal analogues.

ABSTRACT: Electrophile-mediated disruption of cell signal-ing is involved in the pathogenesis of several diseases including atherosclerosis and cancer. Diffusible and membrane bound lipid electrophiles are known to modify DNA and protein substrates and modulate cellular pathways including ER stress, antioxidant response, DNA damage, heat shock, and apoptosis. Herein we report on a structure-activity relationship for several electrophilic analogues of 4-hydroxynonenal (HNE) and 4-oxononenal (ONE) with regard to toxicity and anti-inflammatory activity. The analogues studied were the oxidation products of HNE and ONE, HNEA/ONEA, the in vivo hydrolysis products of oxidized phosphatidylcholine, COOH-HNE/COOH-ONE, and their methyl esters, COOMe-HNE/ONE. The reactivity of each compound toward N-acetylcysteine was determined and compared to the toxicity toward a human colorectal carcinoma cell line (RKO) and a human monocytic leukemia cell line (THP-1). Further analysis was performed in differentiated THP-1 macrophages to assess changes in macrophage activation and pro-inflammatory signaling in response to each lipid electrophile. HNE/ONE analogues inhibited THP-1 macrophage production of the pro-inflammatory cytokines, IL-6, IL-1β, and TNFα, after lipopolysaccharide (LPS)/IFNγ activation. Inhibition of cytokine production was observed at submicromolar concentrations of several analogues with as little as 30 min of exposure. Phagocytosis of fluorescent beads was also inhibited by lipid electrophile treatment. Lipid electrophiles related to HNE/ONE are both toxic and anti-inflammatory, but the anti-inflammatory effects in human macrophages are observed at nontoxic concentrations. Neither toxicity nor anti-inflammatory activity are strongly correlated to the reactivity of the model nucleophile, N-acetylcysteine.

SUBMITTER: McGrath CE

PROVIDER: S-EPMC3062932 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Parkinson's disease (PD) is characterized by accumulations of toxic α-synuclein aggregates in vulnerable neuronal populations in the brainstem, midbrain, and cerebral cortex. Recent findings suggest that α-synuclein pathology can be propagated transneuronally, but the underlying molecular mechanisms are unknown. Advances in the genetics of rare early-onset familial PD indicate that increased production and/or reduced autophagic clearance of α-synuclein can cause PD. The cause of the most common late-onset PD is unclear, but may involve metabolic compromise and oxidative stress upstream of α-synuclein accumulation. As evidence, the lipid peroxidation product 4-hydroxynonenal (HNE) is elevated in the brain during normal aging and moreso in brain regions afflicted with α-synuclein pathology. Here, we report that HNE increases aggregation of endogenous α-synuclein in primary neurons and triggers the secretion of extracellular vesicles (EVs) containing cytotoxic oligomeric α-synuclein species. EVs released from HNE-treated neurons are internalized by healthy neurons which as a consequence degenerate. Levels of endogenously generated HNE are elevated in cultured cells overexpressing human α-synuclein, and EVs released from those cells are toxic to neurons. The EV-associated α-synuclein is located both inside the vesicles and on their surface, where it plays a role in EV internalization by neurons. On internalization, EVs harboring pathogenic α-synuclein are transported both anterogradely and retrogradely within axons. Focal injection of EVs containing α-synuclein into the striatum of wild-type mice results in spread of synuclein pathology to anatomically connected brain regions. Our findings suggest a scenario for late-onset PD in which lipid peroxidation promotes intracellular accumulation and then extrusion of EVs containing toxic α-synuclein species; the EVs are then internalized by adjacent neurons, so propagating the neurodegenerative process.

Project description:Free radical-mediated oxidative damage to proteins, lipids, and DNA occurs in neurons during acute brain injuries and in neurodegenerative disorders. Membrane lipid peroxidation contributes to neuronal dysfunction and death, in part by disrupting neuronal ion homeostasis and cellular bioenergetics. Emerging findings suggest that 4-hydroxynonenal (HNE), an aldehyde produced during lipid peroxidation, impairs the function of various proteins involved in neuronal homeostasis. Here we tested the hypothesis that HNE impairs the cellular system that removes damaged proteins and organelles, the autophagy-lysosome pathway in rat primary cortical neurons. We found that HNE, at a concentration that causes apoptosis over a 48-72 h period, increases protein levels of LC3 II and p62 and within 1 and 4 h of exposure, respectively; LC3 II and p62 immunoreactive puncta were observed in the cytoplasm of HNE-treated neurons at 6 h. The extent of up-regulation of p62 and LC3 II in response to HNE was not affected by co-treatment with the lysosome inhibitor bafilomycin A1, suggesting that the effects of HNE on autophagy were secondary to lysosome inhibition. Indeed, we found that neurons exposed to HNE exhibit elevated pH levels, and decreased protein substrate hydrolysis and cathepsin B activity. Neurons exposed to HNE also exhibited the accumulation of K63-linked polyubiquitinated proteins, which are substrates targeted for lysosomal degradation. Moreover, we found that the levels of LAMP2a and constitutively active heat-shock protein 70, and numbers of LAMP2a-positive lysosomes, are decreased in neurons exposed to HNE. Our findings demonstrate that the lipid peroxidation product HNE causes early impairment of lysosomes which may contribute to the accumulation of damaged and dysfunctional proteins and organelles and consequent neuronal death. Because impaired lysosome function is increasingly recognized as an early event in the neuronal death that occurs in neurodegenerative disorders, our findings suggest a role for HNE in such lysosomal dysfunction.

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Structure-activity analysis of diffusible lipid electrophiles associated with phospholipid peroxidation: 4-hydroxynonenal and 4-oxononenal analogues.

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