The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses, and activation of p53 can trigger apoptosis in many cell types, including neurons. We found that this nuclear protein was significantly phosphorylated when human neuroblastoma SH-SY5Y cells were exposed to in vitro oxidized polyunsaturated fatty acids. To identify an oxidized lipid that induces p53 phosphorylation, we conducted a screening of lipid peroxidation products in human neuroblastoma SH-SY5Y cells and identified 4-oxo-2-nonenal (ONE), a recently identified aldehyde originating from the peroxidation of 6 polyunsaturated fatty acids, as a potential inducer of the p53 phosphorylation. We also found that ONE induced the phosphorylation of ataxia telangiectasia-mutated, which plays an essential role in transmitting DNA damage signals by the phosphorylation of p53. In addition, exposure of the cells to ONE resulted in an accumulation of ubiquitinated proteins and in a significant inhibition of proteasome activities, suggesting that ONE acted on the ubiquitin-proteasome pathway, a regulatory mechanism of p53 turnover. In addition, the observation that the ONE-induced p53 response was associated with the induction of apoptosis suggested that ONE activated the p53-dependent apoptosis mechanism via activation of the p53 signaling pathway and down-regulation of the p53 turnover. Finally, we observed that the ONE-2-deoxyguanosine adduct, 7-(2-oxo-heptyl)-substituted 1,N 2 -etheno-2-deoxyguanosine, was accumulated in the spinal cord motor neurons of patients with sporadic amyotrophic lateral sclerosis. These data may suggest the potential critical role for ONE in the induction of a neuronal apoptosis program during oxidative processes.

Emerging evidence suggests the involvement of programmed cell death and inflammation in amyotrophic lateral sclerosis (ALS). To assess molecular pathological effects of the anti-inflammatory peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist pioglitazone in ALS, we verified changes in the population of neurons, astrocytes, and microglia in the ventral horns of spinal cord lumbar segments from the pioglitazone-treated and non-treated groups of mice carrying a transgene for G93A mutant human superoxide dismutase-1 (SOD1) (ALS mice) and non-transgenic littermates (control mice), performed immunohistochemical and immunoblot analyses of PPARgamma, active form of phosphorylated p38 mitogen-activated protein kinase (p-p38) and inhibitor of nuclear factor-kappaB (NF-kappaB)-alpha (IkappaBalpha) in the spinal cords, and compared the results between the different groups. Image analysis revealed that optical density of NeuN-immunoreactive neurons was significantly lower in the non-treated groups of presymptomatic and advanced ALS mice than in the non-treated groups of age-matched control mice and was recovered with pioglitazone treatment, and that optical densities of GFAP-immunoreactive astrocytes and Iba1-immunoreactive microglia were significantly higher in the non-treated group of advanced ALS mice than in the non-treated group of control mice and were recovered with pioglitazone treatment. Immunohistochemical analysis demonstrated that immunoreactivities for PPARgamma and p-p38 were mainly localized in neurons, and that IkappaBalpha immunoreactivity was mainly localized in astrocytes and microglia. Immunoblot analysis showed that pioglitazone treatment resulted in no significant change in nuclear PPARgamma-immunoreactive density, a significant decrease in cytosolic p-p38-immunoreactive density, and a significant increase in cytosolic IkappaBalpha-immunoreactive density. Our results suggest that pioglitazone protects motor neurons against p38-mediated neuronal death and NF-kappaB-mediated glial inflammation via a PPARgamma-independent mechanism.

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Objective-A diverse range of lipid oxidation products detected in oxidized low-density lipoprotein (oxLDL) and atherosclerotic lesions are capable of eliciting biological responses in vascular cells. We performed DNA microarray experiments to explore novel responses of human umbilical vein endothelial cells (HUVECs) to oxLDL and its components. Methods and Results-cDNA microarray analysis showed that oxLDL, lysophosphatidylcholine (LysoPC), 4-hydroxy-2-nonenal, and oxysterols altered gene expression specifically, but some genes were commonly induced in HUVECs. Solute carrier family 3 member 2 and family 7 member 5, encoding the heavy chain of the cell surface antigen 4F2 (4F2hc) and the L-type amino acid transporter 1 (LAT1), respectively, were induced by oxLDL and many oxidation products. LAT1 requires 4F2hc to form a heterodimeric functional complex to transport neutral amino acids into the cell. LysoPC increased membrane protein levels of LAT1 confirmed by Western blot analysis and also uptake of L-[ 14 C]leucine, which was inhibited by a competitive inhibitor for LAT1. The release of interleukin 6 (IL-6) and IL-8 was increased in LysoPC-treated cells and was attenuated by the LAT1 inhibitor. Key Words: amino acid transporter Ⅲ atherosclerosis Ⅲ cytokine Ⅲ HUVEC Ⅲ LysoPC A therosclerosis, which leads to coronary heart disease and stroke, is the most common cause of death in industrialized nations. It has been suggested that oxidative modification of low-density lipoprotein (LDL) is a key initial event in atherosclerosis pathogenesis, 1 and a wide variety of oxidized lipids have been detected in atherosclerotic lesions. 2 LDL is composed of a cholesteryl ester (CE) and triglyceride core with an outer monolayer composed of phosphatidylcholine (PC) and free cholesterol solubilized in blood by 1 molecule of apolipoprotein. 3 The esterified fatty acids of PC and CE are oxidized enzymatically and nonenzymatically to yield lipid hydroperoxides as the primary products, 4 followed by secondary reactions to form lipid hydroxides and aldehydes such as malondialdehyde, acrolein, 5 and 4-hydroxy-2-nonenal (4HNE). Acrolein and 4HNE are known to be highly reactive and to form adducts with proteins and nucleic acids. 6 In particular, many studies have shown that 4HNE regulates cell-signaling pathways through activation protein 1 (AP-1). [7][8][9] Cholesterol is also oxidized to give several classes of oxysterols: 7-ketocholesterol, which induces monocyte differentiation and promotes foam cell formation; 10 22(R)-hydroxycholesterol, which is a ligand for the liver X receptor and regulates the expression of genes involved in cholesterol and fatty acid homeostasis; 11 and 25-hydroxycholesterol, which regulates cholesterol synthesis via the sterol regulatory element-binding protein (SREBP)/SREBP cleavage-activating protein regulatory pathway. 12 Lysophosphatidylcholine (LysoPC) is present at high concentrations in oxidized LDL (oxLDL) and formed via the reaction of phospholipase A2. ␣-Palmitoyl-LysoPC (16:0) is known to induc...

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Purpose: To compare the effect of cilostazol plus aspirin versus aspirin alone on the progression of intracranial arterial stenosis (IAS), and to compare ischemic and hemorrhagic events in patients with symptomatic IAS, an investigator-driven, nationwide multicenter cooperative randomized controlled trial (CATHARSIS; ClinicalTrials.gov Identifier 00333164) was conducted. Methods: 165 noncardioembolic ischemic stroke patients with >50% stenosis in the responsible intracranial artery after 2 weeks to 6 months from the onset were randomly allocated to receive either cilostazol 200 mg/day plus aspirin 100 mg/day (n = 83, CA group) or aspirin 100 mg/day alone (n = 82, A group). The primary endpoint was the progression of IAS on magnetic resonance angiography at 2 years after randomization. Secondary endpoints were any vascular events, any cause of death, serious adverse events, new silent brain infarcts, and worsening of the modified Rankin Scale score. Results: Progression of IAS was observed in 9.6% of the CA group patients and in 5.6% of the A group patients, with no significant intergroup difference (p = 0.53). The incidence of the secondary endpoints tended to be lower in the CA group compared with the A group, although the differences were not significant. By using exploratory logistic regression analysis adjusted for patient background characteristics, it was shown that the risk for certain combinations of secondary endpoints was lower in the CA group than in the A group [all vascular events and silent brain infarcts: odds ratio (OR) = 0.37, p = 0.04; stroke and silent brain infarcts: OR = 0.34, p = 0.04; all vascular events, worsening of modified Rankin Scale scores and silent brain infracts: OR = 0.41, p = 0.03]. Major hemorrhage was observed in 4 patients of the CA group and in 3 of the A group. Conclusion: Progression ofIAS during the 2-year observation period appears to be less frequent than previously reported in stroke patients on antiplatelet agents after the acute phase, which could be due to the adequate control of risk factors, and because patients with stroke within 2 weeks after the onset were excluded. The results of the CATHARSIS trial suggest a potential utility of pharmacotherapies with cilostazol plus aspirin as well as of strict control of risk factors for the management of symptomatic IAS. Larger studies with higher statistical power are required to obtain conclusive results.

A trial fibrillation (AF) is a strong risk factor for ischemic stroke and a leading cause of cardioembolic stroke. 1In particular, patients with AF with previous stroke are at high risk of recurrence.2 Because anticoagulant therapy can remarkably reduce the risk of recurrence, 3 early identification of AF is crucial for targeted secondary prevention.Persistent AF is usually easy to diagnose using the standard 12-lead ECG. However, paroxysmal AF (PAF) is often difficult to detect in patients with acute ischemic stroke (AIS) because such patients are frequently asymptomatic or present with sinus rhythm on ECGs, 4 because of which the prevalence of PAF is possibly underestimated and anticoagulants are underused. Repetitive and extended cardiac monitoring, including standard 12-lead ECG, 24-hour Holter ECG, and inpatient telemetry monitoring, are recommended to detect occult PAF in patients with AIS. 5 However, the optimal timing, duration, and method to detect PAF remain to be clarified, and the detection rate of PAF after stroke is limited. Therefore, it would be helpful to determine a factor predicting covert PAF in patients with sinus rhythms on ECGs.The QT interval corrected for heart rate (QTc)-which represents the ventricular action potential duration-has long been established as a predictor of cardiac morbidity and mortality. 6,7 Several large population-based studies have recently shown that a prolonged QTc interval is associated with an increased risk of AF development. [8][9][10] Moreover, small studies have suggested that patients with congenital long-QT syndrome (LQTS) have a greater risk of developing AF than the general population.11,12 Thus, we hypothesized that the QTc interval is potentially a good predictor of occult PAF in patients with AIS. In the present study, we aimed to assess the predictive value of a prolonged QTc interval for the detection of poststroke PAF, using data from our observational stroke registry system. MethodsThe ethics committee at our institution approved the protocol of this study. The Tokyo Women's Medical University Stroke Registry Retrospective Cohort is an observational study including 1038 consecutive patients with AIS hospitalized at the Department of Neurology, Tokyo Women's Medical University Hospital, between April 2003 and November 2013. After excluding 66 patients with Background and Purpose-Paroxysmal atrial fibrillation (PAF) is often difficult to detect in patients with acute ischemic stroke. We aimed to assess the predictive value of a prolonged QT interval corrected for heart rate (QTc) in PAF detection after acute ischemic stroke. Methods-We enrolled 972 patients with acute ischemic stroke consecutively extracted from our observational stroke registry system. Exclusion criteria were as follows: (1) AF on the initial 12-lead ECG (n=171); (2) previously diagnosed PAF (n=47); and (3) the use of a cardiac pacemaker (n=10). Of the 972 patients, 744 (mean age, 67.6 years; men, 62.6%) were eligible for analysis. The clinical characteristics and 12-lead ECG findin...

Recent studies have suggested implications for α-synuclein cytotoxicity in the pathomechanism of multiple system atrophy (MSA). Given in vitro evidence that α-synuclein generates oxidative stress, it is proposed that lipid peroxidation may be accelerated in MSA. To address this issue, we performed an immunohistochemical analysis of protein-bound 4-hydroxy-2-nonenal (P-HNE) in sections of archival, formalin-fixed, paraffin-embedded pontine materials of eight sporadic MSA patients and eight age-matched control subjects. In the MSA cases, P-HNE immunoreactivity was localized in all of the neuronal cytoplasmic inclusions and glial cytoplasmic inclusions, both of them identified with α-synuclein and ubiquitin. It was also detectable in reactive astrocytes and phagocytic microglia but undetectable in activated microglia. By contrast, P-HNE immunoreactivity in the control cases was only very weak or not at all in the parenchyma including neurons and glia. The present results provide in vivo evidence that HNE participates in α-synuclein-induced cytotoxicity and neuroinflammation in MSA.