Electrophilic Cyclopentenone Neuroprostanes Are Anti-inflammatory Mediators Formed from the Peroxidation of the ω -3 Polyunsaturated Fatty Acid Docosahexaenoic Acid.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) possesses potent anti-inflammatory properties and has shown therapeutic benefit in numerous inflammatory diseases. However, the molecular mechanisms of these anti-inflamma- tory properties are poorly understood. DHA is highly suscepti- ble to peroxidation, which yields an array of potentially bioac- tive lipid species. One class of compounds are cyclopentenone neuroprostanes (A4/J4-NPs), which are highly reactive and similar in structure to anti-inflammatory cyclopentenone prostaglandins. Here we show that a synthetic A4/J4-NP, 14-A4-NP (A4-NP), potently suppresses lipopolysaccharide- induced expression of inducible nitric-oxide synthase and cyclooxygenase-2 in macrophages. Furthermore, A4-NP blocks lipopolysaccharide-induced NF-KB activation via inhibition of I kinase-mediated phosphorylation of IKB. Mutation on Ik kinase b-cysteine 179 markedly diminishes the effect of A4-NP, suggesting that A4-NP acts via thiol mod- ification at this residue. Accordingly, the effects of A4-NP are independent of peroxisome proliferator-activated receptor-gamma and are dependent on an intact reactive cyclopentenone ring. Interestingly, free radical-mediated oxidation of DHA greatly enhances its anti-inflammatory potency, an effect that closely parallels the formation of A4/J4-NPs. Furthermore, chemical reduction or conjugation to glutathione, both of which elim- inate the bioactivity of A4-NP, also abrogate the anti-inflam- matory effects of oxidized DHA. Thus, we have demonstrated that A4/J4-NPs, formed via the oxidation of DHA, are potent inhibitors of NF-kB signaling and may contribute to the anti- inflammatory actions of DHA. These findings have implica- tions for understanding the anti-inflammatory properties of omega-3 fatty acids, and elucidate novel interactions between lipid peroxidation products and inflammation

Haploinsufficiency of the E3 Ubiquitin Ligase C-Terminus of Heat Shock Cognate 70 Interacting Protein (CHIP) Produces Specific Behavioral Impairments

The multifunctional E3 ubiquitin ligase CHIP is an essential interacting partner of HSP70, which together promote the proteasomal degradation of client proteins. Acute CHIP overexpression provides neuroprotection against neurotoxic mitochondrial stress, glucocorticoids, and accumulation of toxic amyloid fragments, as well as genetic mutations in other E3 ligases, which have been shown to result in familial Parkinson's disease. These studies have created a great deal of interest in understanding CHIP activity, expression and modulation. While CHIP knockout mice have the potential to provide essential insights into the molecular control of cell fate and survival, the animals have been difficult to characterize in vivo due to severe phenotypic and behavioral dysfunction, which have thus far been poorly characterized. Therefore, in the present study we conducted a battery of neurobehavioral and physiological assays of adult CHIP heterozygotic (HET) mutant mice to provide a better understanding of the functional consequence of CHIP deficiency. We found that CHIP HET mice had normal body and brain weight, body temperature, muscle tone and breathing patterns, but do have a significant elevation in baseline heart rate. Meanwhile basic behavioral screens of sensory, motor, emotional and cognitive functions were normative. We observed no alterations in performance in the elevated plus maze, light-dark preference and tail suspension assays, or two simple cognitive tasks: novel object recognition and spontaneous alternation in a Y maze. Significant deficits were found, however, when CHIP HET mice performed wire hang, inverted screen, wire maneuver, and open field tasks. Taken together, our data indicate a clear subset of behaviors that are altered at baseline in CHIP deficient animals, which will further guide whole animal studies of the effects of CHIP dysregulation on cardiac function, brain circuitry and function, and responsiveness to environmental and cellular stress

Enrichment of elevated plasma F2t-isoprostane levels in individuals with autism who are stratified by presence of gastrointestinal dysfunction.

Etiology is unknown in the majority of individuals with autism spectrum disorder (ASD). One strategy to investigate pathogenesis is to stratify this heterogeneous disorder based on a prominent phenotypic feature that enriches for homogeneity within population strata. Co-occurring gastrointestinal dysfunction (GID) characterizes a subset of children with ASD. Our current objective was to investigate a potential pathophysiological measure to test the hypothesis that children with both ASD and GID have a more severe metabolic dysfunction than children with ASD-only, given that the highly metabolically active brain and gastrointestinal system may additively contribute measurable impairment. Plasma levels of F2t-Isoprostanes (F2-IsoPs), a gold standard biomarker of oxidative stress, were measured in 87 children in four groups: ASD-GID, ASD-only, GID-only and Unaffected. F2-IsoP levels were elevated in all 3 clinical groups compared to the Unaffected group, with the ASD-GID group significantly elevated above the ASD-only group (mean, SD in pg/mg: ASD-GID 53.6, 24.4; ASD-only 36.5, 13.3; p = 0.007). Adjusting for age, sex, and triglyceride levels, F2-IsoP levels remained significantly different between study groups, with a moderate effect size of η(p)(2) = 0.187 (p = 0.001). Elevation in peripheral oxidative stress is consistent with, and may contribute to, the more severe functional impairments in the ASD-GID group. With unique medical, metabolic, and behavioral features in children with ASD-GID, the present findings serve as a compelling rationale for both individualized approaches to clinical care and integrated studies of biomarker enrichment in ASD subgroups that may better address the complex etiology of ASD

C-Terminus of Heat Shock Cognate 70 Interacting Protein Increases Following Stroke and Impairs Survival Against Acute Oxidative Stress

The decision to remove or refold oxidized, denatured, or misfolded proteins by heat shock protein 70 and its binding partners is critical to determine cell fate under pathophysiological conditions. Overexpression of the ubiquitin ligase C-terminus of HSC70 interacting protein (CHIP) can compensate for failure of other ubiquitin ligases and enhance protein turnover and survival under chronic neurological stress. The ability of CHIP to alter cell fate after acute neurological injury has not been assessed. Using postmortem human tissue samples, we provide the first evidence that cortical CHIP expression is increased after ischemic stroke. Oxygen glucose deprivation in vitro led to rapid protein oxidation, antioxidant depletion, proteasome dysfunction, and a significant increase in CHIP expression. To determine if CHIP upregulation enhances neural survival, we overexpressed CHIP in vitro and evaluated cell fate 24 h after acute oxidative stress. Surprisingly, CHIP overexpressing cells fared worse against oxidative injury, accumulated more ubiquitinated and oxidized proteins, and experienced decreased proteasome activity. Conversely, using small interfering RNA to decrease CHIP expression in primary neuronal cultures improved survival after oxidative stress, suggesting that increases in CHIP observed after stroke like injuries are likely correlated with diminished survival and may negatively impact the neuroprotective potential of heat shock protein 70. Antioxid. Redox Signal. 14, 1787–1801

Analysis of Covariance — Age-, Sex-, and Triglyceride-Adjusted F_2t-Isoprostane Levels.

<p>Analysis of Covariance — Age-, Sex-, and Triglyceride-Adjusted F_2t-Isoprostane Levels.</p

Plasma F_2t-Isoprostane levels among four study groups.

<p>Data are plotted as mean (SD). Open circles are female participants; closed circles are male participants.</p

Basic Characteristics of Study Participants.

*<p>Note: Some children had more than one diagnosis, thus percentages sum to greater than 100%.</p

Dynamic Phosphorylation of Apoptosis Signal Regulating Kinase 1 (ASK1) in Response to Oxidative and Electrophilic Stress

Apoptosis signal-regulating kinase 1 (ASK1) is a critical cellular stress sensor that senses diverse reactive chemotypes and integrates these chemical signals into a single biological pathway response. It is unknown whether ASK1 senses all stressors in the same way or if unique stress-specific mechanisms detect distinct chemotypes. In order to answer this question, we treated ASK1-expressing cells with two distinct stress activators, H₂O₂ and 4-hydroxy-2-nonenal (HNE), and monitored the phosphorylation state of ASK1. Phosphorylation is an important regulator for the activity of ASK1, and we hypothesized that these two chemically distinct molecules may produce differences in the phosphorylation state of ASK1. Shotgun mass spectrometry and manual validation identified 12 distinct ASK1 phosphosites. Targeted parallel reaction monitoring assays were used to track the phosphorylation dynamics of each confirmed site in response to treatment. Eleven phosphosites exhibited dynamic response to one or both treatments. Six of these sites were identified in both H₂O₂- and HNE-treated cells, and four of these exhibited a consistent response between the two molecules. The results confirm that different chemotypes produce distinct phosphorylation patterns in concert with activation of a common MAPK pathway