Ru(0)-Catalyzed Direct Coupling of Internal Alkynes with Conjugated Dienes: An Efficient Access to Conjugated Trienes

Ru­(0)-catalyzed direct coupling of internal alkynes with conjugated dienes enables a direct access to conjugated trienes, where the reaction is formally regarded as a stereoselective syn alkyne insertion into the terminal C–H bond in the conjugated diene. The reaction is catalyzed by Ru­(η⁶-naphthalene)­(η⁴-1,5-COD) (<b>1</b>; 3–10 mol %) with high regio- and stereoselectivities

Ru(0)-Catalyzed Direct Coupling of Internal Alkynes with Conjugated Dienes: An Efficient Access to Conjugated Trienes

Ru­(0)-catalyzed direct coupling of internal alkynes with conjugated dienes enables a direct access to conjugated trienes, where the reaction is formally regarded as a stereoselective syn alkyne insertion into the terminal C–H bond in the conjugated diene. The reaction is catalyzed by Ru­(η⁶-naphthalene)­(η⁴-1,5-COD) (<b>1</b>; 3–10 mol %) with high regio- and stereoselectivities

Effects of Oxidative Stress on the Solubility of HRD1, a Ubiquitin Ligase Implicated in Alzheimer’s Disease

<div><p>The E3 ubiquitin ligase HRD1 is found in the endoplasmic reticulum membrane of brain neurons and is involved in endoplasmic reticulum-associated degradation. We previously demonstrated that suppression of HRD1 expression in neurons causes accumulation of amyloid precursor protein, resulting in amyloid β production associated with endoplasmic reticulum stress and apoptosis. Furthermore, HRD1 levels are significantly decreased in the cerebral cortex of Alzheimer’s disease patients because of its insolubility. The mechanisms that affect HRD1 solubility are not well understood. We here show that HRD1 protein was insolubilized by oxidative stress but not by other Alzheimer’s disease-related molecules and stressors, such as amyloid β, tau, and endoplasmic reticulum stress. Furthermore, we raise the possibility that modifications of HRD1 by 4-hydroxy-2-nonenal, an oxidative stress marker, decrease HRD1 protein solubility and the oxidative stress led to the accumulation of HRD1 into the aggresome. Thus, oxidative stress-induced HRD1 insolubilization might be involved in a vicious cycle of increased amyloid β production and amyloid β-induced oxidative stress in Alzheimer’s disease pathogenesis.</p></div

Induction of HRD1 aggresome formation by oxidative stress.

<p>SH-SH5H cells treated with H₂O₂ (10 µM), rotenone (10 nM), and 4-HNE (15 µM) for 48 h and then subjected to immunofluorescence staining using HRD1 (red) and γ-tubulin (green) antibodies. Arrowheads, MTOC; arrows, HRD1 in aggresomes. Scale bars, 20 µm.</p

Induction of HRD1 and SEL1L expression by ER stress-inducing agents.

<p><i>A</i>. Real-time PCR analysis of gene expression in N2a cells treated for 24 h with or without tunicamycin (Tm; an inhibitor of <i>N</i>-linked glycosylation) and thapsigargin (Tg; an inhibitor of Ca²⁺ ATPase). Data are normalized to the amount of β-actin; results are expressed as a fold increase compared with the non-treated control (mean ± SEM; <i>n</i> = 3). Statistical analysis was performed with ANOVA, followed by Bonferroni correction (Control vs. Tm and Tg; *<i>p</i><0.05). <i>B-C.</i> Western blotting of (<i>B</i>) NP-40-soluble and (<i>C</i>) -insoluble fractions from N2a cells treated with or without Tm and Tg for 24 h using the indicated antibodies. The black arrow in soluble fraction indicates mature SEL1L, and the white arrow indicates immature SEL1L without glycosylation.</p

4-HNE modifications of HRD1 protein.

<p><i>A–B</i>. SH-SY5Y cells were treated with or without 4-HNE for 24 h. The total cell lysates of NP-40-soluble (A) and -insoluble (B) fractions were analyzed by western blotting with the indicated antibodies. <i>C</i>. Total cell lysates were immunoprecipitated (IP) with anti-HRD1/SYVN1 antibody. These IP samples were treated with or without 100 µM of 4-HNE at 37°C for 3 h and then analyzed by western blotting with the indicated antibodies. Black arrow, 4-HNE-modified HRD1; white arrow, unmodified HRD1 protein. Lane 1, total cell lysate; Lane 2, IP with anti-HRD1/SYVN1 antibody; Lane 3, IP with anti-HRD1/SYVN1 antibody treated with 4-HNE; Lane 4, IP with anti-mouse IgG.</p

Insolubilization of HRD1 and SEL1L proteins by oxidative stress-inducing agents.

<p><i>A–C.</i> SH-SY5Y cells were treated with or without H₂O₂ (<i>A</i>) and rotenone (<i>B</i>) for 24 h. The total cell lysates of NP-40-soluble (left panel) and -insoluble (right panel) fractions were analyzed by western blotting with the indicated antibodies. <i>C</i>. Decrease in soluble HRD1 due to oxidative stress under inhibition of <i>de novo</i> protein synthesis. SH-SY5Y normal cells were treated with 25 µg/ml CHX. At 5 min after treatment, the cells were additionally treated with or without 100 µM hydrogen peroxide (H₂O₂) for 6 h. The total cell lysates of NP-40 soluble fractions were analyzed by western blotting using the indicated antibodies. Data are normalized to the amount of β-actin; results are expressed as a fold increase compared with the non-treated control (mean ± SEM; <i>n</i> = 6). Statistical analysis was performed with ANOVA, followed by Student’s <i>t</i>-test (CHX vs. CHX+H₂O₂; *<i>p</i><0.05). Abbreviation: NT, non-treated control.</p

Influence of Aβ or tau on HRD1 and SEL1L solubility in the cerebral cortex of Tg2576 or JNPL3 mice.

<p>HRD1 and SEL1L levels in the cerebral cortex of Tg2576 (hemizygous human Swedish double-mutated APP transgenic; APP_SWE) or JNPL3 (human mutated four-repeat tau_P301L transgenic; Tau_P301L) mice. The cerebral cortex of each mouse at 16 to 20 months of age was examined. The total lysates of (<i>A</i>) NP-40-soluble and (<i>B</i>) -insoluble fractions were analyzed by western blotting with the indicated antibodies; statistical analysis of these results are expressed as a dot plot (• wt, <i>n</i> = 7; • APP_SWE, <i>n</i> = 4; ○ Tau_P301L, <i>n</i> = 5). Data are normalized to the wt averages for each fraction. Statistical analysis was performed with ANOVA, followed by Bonferroni correction (Control vs. APP_SWE and Tau_P301L; *<i>p</i><0.05).</p

Effect of Aβ and tau on HRD1 solubility in N2a cells.

<p><i>A</i>. ELISA of Aβ peptides, Aβ40 and Aβ42, in N2a cells stably expressing PS2 24 h after transfection with <i>APP-FLAG</i> or an empty vector (mock). Results are expressed as a ratio of Aβ peptide (pg/ml) to whole cell protein extract (mg). <i>B</i>. Total cell lysates of NP-40-soluble and -insoluble fractions analyzed by western blotting with the indicated antibodies. <i>C</i>. Statistical analysis of (<i>B</i>) NP-40 soluble HRD1. Data are normalized to β-actin levels, and results are expressed as the fold increase compared with protein expression levels in untransfected control (mean ± SEM; <i>n</i> = 3). Statistical analysis was performed with ANOVA, followed by Student’s <i>t</i>-test (mock vs. APP; *<i>p</i><0.05). D-E. Western blotting of (<i>D</i>) NP-40-soluble and (<i>E</i>) NP-40-insoluble proteins in the total cell lysates of N2a cells 24 h after transfection with the four-repeat isoform of human tau (tau_0N4R; 0N4R), mutant tau (tau_P301L; P301L), or the empty vector (mock). Abbreviation: m, mock. A, APP. p.c., positive control, which was extracted by NP-40 from wild-type HRD1-expressing Flp-In-293 cell.</p

Metabolic Effect of 5HT on a High Fat Diet.

<p>A, The concentrations of blood NEFA, cholesterol, triglyceride, glucose, insulin, leptin and adiponectin were measured in mice aged between 21 and 26 weeks of age (n = 7–12). B and C, Glucose tolerance (B) and insulin tolerance (C) tests were performed in each group of mice at 21 weeks of age (n = 7). Circles, Ch+PBS; filled circles, Ch+5HT; squares, F+PBS; filled squares, F+5HT. Data are means ± s.e. Data in (A) were analyzed by two-way ANOVA. Data in (B) and (C) were analyzed by Student's t test. Columns with a different letter are significantly different (p<0.05). *p<0.05, **p<0.01 indicates significance in F+5HT mice against F+PBS mice.</p