Molecular Features of the Measles Virus Viral Fusion Complex That Favor Infection and Spread in the Brain

International audienceMeasles virus (MeV) infection can cause serious complications in immunocompromised individuals, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE), another severe central nervous system (CNS) complication, develop even in the face of a systemic immune response

Exposure to a northern contaminant mixture (NCM) alters hepatic energy and lipid metabolism exacerbating hepatic steatosis in obese JCR rats.

Non-alcoholic fatty liver disease (NAFLD), defined by the American Liver Society as the buildup of extra fat in liver cells that is not caused by alcohol, is the most common liver disease in North America. Obesity and type 2 diabetes are viewed as the major causes of NAFLD. Environmental contaminants have also been implicated in the development of NAFLD. Northern populations are exposed to a myriad of persistent organic pollutants including polychlorinated biphenyls, organochlorine pesticides, flame retardants, and toxic metals, while also affected by higher rates of obesity and alcohol abuse compared to the rest of Canada. In this study, we examined the impact of a mixture of 22 contaminants detected in Inuit blood on the development and progression of NAFLD in obese JCR rats with or without co-exposure to 10% ethanol. Hepatosteatosis was found in obese rat liver, which was worsened by exposure to 10% ethanol. NCM treatment increased the number of macrovesicular lipid droplets, total lipid contents, portion of mono- and polyunsaturated fatty acids in the liver. This was complemented by an increase in hepatic total cholesterol and cholesterol ester levels which was associated with changes in the expression of genes and proteins involved in lipid metabolism and transport. In addition, NCM treatment increased cytochrome P450 2E1 protein expression and decreased ubiquinone pool, and mitochondrial ATP synthase subunit ATP5A and Complex IV activity. Despite the changes in mitochondrial physiology, hepatic ATP levels were maintained high in NCM-treated versus control rats. This was due to a decrease in ATP utilization and an increase in creatine kinase activity. Collectively, our results suggest that NCM treatment decreases hepatic cholesterol export, possibly also increases cholesterol uptake from circulation, and promotes lipid accumulation and alters ATP homeostasis which exacerbates the existing hepatic steatosis in genetically obese JCR rats with or without co-exposure to ethanol

Protein Delivery of an Artificial Transcription Factor Restores Widespread Ube3a Expression in an Angelman Syndrome Mouse Brain

Angelman syndrome (AS) is a neurological genetic disorder caused by loss of expression of the maternal copy of UBE3A in the brain. Due to brain-specific genetic imprinting at this locus, the paternal UBE3A is silenced by a long antisense transcript. Inhibition of the antisense transcript could lead to unsilencing of paternal UBE3A, thus providing a therapeutic approach for AS. However, widespread delivery of gene regulators to the brain remains challenging. Here, we report an engineered zinc finger-based artificial transcription factor (ATF) that, when injected i.p. or s.c., crossed the blood–brain barrier and increased Ube3a expression in the brain of an adult mouse model of AS. The factor displayed widespread distribution throughout the brain. Immunohistochemistry of both the hippocampus and cerebellum revealed an increase in Ube3a upon treatment. An ATF containing an alternative DNA-binding domain did not activate Ube3a. We believe this to be the first report of an injectable engineered zinc finger protein that can cause widespread activation of an endogenous gene in the brain. These observations have important implications for the study and treatment of AS and other neurological disorders

Lipid contents and contaminant levels (ng/g wt) in the liver of obese JCR rats dosed with vehicle or NCM and treated with or without ethanol.

<p><i>n</i> = 7–8, means ± SEM, 2-way ANOVA with Tukey's post-hoc test.</p>a<p>denotes statistically significant difference between vehicle (V) control and high dose (H) groups at p<0.001.</p>b<p>denotes statistically significant difference between OWV and OEV, and between OWH and OEH groups at p<0.001.</p><p>Lipid contents and contaminant levels (ng/g wt) in the liver of obese JCR rats dosed with vehicle or NCM and treated with or without ethanol.</p

Pathways of hepatic cholesterol and Coenzyme Q₁₀ (CoQ10) biosynthesis in liver.

<p>Pathways of hepatic cholesterol and Coenzyme Q₁₀ (CoQ10) biosynthesis in liver.</p

Hepatic adenylate and TCA cycle metabolite levels in obese JCR rats dosed with vehicle or NCM and treated with or without ethanol.

<p><i>n</i> = 7–8, means ±SEM, 2-way ANOVA with Tukey's post-hoc test.</p>a<p>denotes statistical significance at P<0.001 between vehicle (V) control and high dose (H).</p><p>Hepatic adenylate and TCA cycle metabolite levels in obese JCR rats dosed with vehicle or NCM and treated with or without ethanol.</p

Contaminant levels (ng/g wt) in the sera of obese JCR rats treated with vehicle (V) or high dose (H) NCM with (E) or without (W) co-exposure to ethanol.

<p>n = 7–8, means ±SEM, 2-way ANOVA with Tukey's post-hoc test.</p>a<p>denotes statistically significant difference between vehicle (V) control and high dose (H) groups at p<0.05.</p><p>Contaminant levels (ng/g wt) in the sera of obese JCR rats treated with vehicle (V) or high dose (H) NCM with (E) or without (W) co-exposure to ethanol.</p

NCM treatment diminishes the activity and expression Complexes IV and V of the respiratory chain, which is associated with increased hepatic CYP2E1 protein levels.

<p><b>A</b>) Measurement of the specific activities of Complex I (NADH:ubiquinone oxidoreductase) and Complex IV (cytochrome C oxidase) in liver homogenate. For Complex I, activities were ascertained by measuring the consumption of NADH. Complex IV activities were determined by measuring the consumption of Cyt C. <i>n</i> = 5, means ±SEM. <b>B</b>) Immunoblot analysis of respiratory complex protein content. Different respiratory complex subunits were detected simultaneously using OXPHOS MitoProfile antibodies. Membranes were then stripped and probed for GAPDH. Blots were quantified using ImageJ software and values were normalized to GAPDH loading control levels. <i>n</i> = 3, means ±SEM. <b>C</b>) Immunoblot analysis of CYP2E1 protein levels. Membranes were stripped and probed for GAPDH. Blots were quantified using ImageJ software and values were normalized to GAPDH loading control levels. <i>n</i> = 3, means ±SEM. Two-way ANOVA with Tukey's post-hoc test. *, or ^# and **** or ^#### denotes P<0.05, P<0.0001 respectively. * denotes statistical comparison between vehicle control (V) and high dose (H) and ^# denotes statistical comparison between water (W) and ethanol (E) treated groups. OWV; obese water vehicle, OWH; obese water high dose, OEV; obese ethanol vehicle, OEH; obese ethanol high dose.</p

NCM exposure increases hepatic creatine kinase activity and total ATPase activity, which is associated with decreases in hepatic ABCA1, CD36, and L-FABP protein expression and circulating levels of cholesterol and triglycerides.

<p><b>A</b>). Measurement of the specific activities of pyruvate kinase, creatine kinase, and total MDR ATPases in liver homogenate. For pyruvate kinase, activities were ascertained by measuring the consumption of NADH. Creatine kinase activities were measured by ADP production. <i>n</i> = 5, means ±SEM. Total MDR ATPases activities were measured using BD Gentest ATPase Assay kit. <b>B</b>) Immunoblot analysis of ABCA1 and ApoB-100 protein levels. Membranes were stripped and probed for GAPDH. Blots were quantified using ImageJ software and values were normalized to GAPDH loading control levels. <i>n</i> = 3, means ±SEM. <b>C</b>) Hepatic CD36 and L-FABP levels were measured using ELISA kits from MyBioSource. <i>n</i> = 6, means±SEM. <b>D</b>) Serum cholesterol and triglycerides levels were measured using the Hitachi Model 917 Multichannel Analyzer. <i>n</i> = 5, means ±SEM. Two-way ANOVA with Tukey's post-hoc test. *, **, and *** denotes P<0.05, 0.01 and 0.001 respectively. * denotes statistical comparison between vehicle control (V) and high dose (H) and ^# denotes statistical comparison between water (W) and ethanol (E) treated groups. OWV; obese water vehicle, OWH; obese water high dose, OEV; obese ethanol vehicle, OEH; obese ethanol high dose.</p

Impact of NCM treatment on liver physiology and fatty acid profile in obese JCR rats treated with or without ethanol.

<p>A) Involvement of mitochondria in hepatic lipid biosynthesis and degradation. <i>1</i>. pyruvate enters into mitochondria and is converted to acetyl-CoA in the matrix. <i>2</i>. Following the synthesis of citric acid it is exported to the cytosol from the matrix and cleaved to reproduce acetyl-CoA. <i>3</i>. Acetyl-CoA is carboxylated and then utilized for the synthesis of fatty acids which then esterify glycerol to produce triglycerides. <i>4</i>. Lipids enter the matrix and are oxidized to acetyl-CoA. <i>5</i>. Acetyl-CoA enters the TCA cycle and produces NADH which then drives oxidative phosphorylation. E: electron transport flavoprotein-ubiquinone oxidoreductase, CTP: citrate transport protein, MCT: monocarboxylate transporter, CACT: carnitine:acyl-carntine transporter. B) Liver weights, <i>n</i> = 8, means ±SEM, and pathological grading of liver hypertrophy. C) Oil red staining of lipid droplets in liver section. D) Total number of lipid vacuoles and quantification of lipid vacuole diameter. Lipid droplets number and diameter were determined using Axion Vision software. <i>n</i> = 6, means ±SEM. E) Hepatic saturated fatty acid, monounsaturated fatty acid, and polyunsaturated fatty acid levels. Fatty acids were extracted and measured by GC-MS. <i>n</i> = 8, means ±SEM. Two-way ANOVA with Tukey's post-hoc test. * denotes statistical comparison between vehicle control (V) and high dose (H) and ^# denotes statistical comparison between water (W) and ethanol (E) treated groups. * or ^#, **, and *** indicate significant difference at p<0.05, 0.02, and 0.001, respectively. OWV; obese water vehicle, OWH; obese water high dose, OEV; obese ethanol vehicle, OEH; obese ethanol high dose.</p