Sod2 haploinsufficiency does not accelerate aging of telomere dysfunctional mice

Telomere shortening represents a causal factor of cellular senescence. At the same time, several lines of evidence indicate a pivotal role of oxidative DNA damage for the aging process in vivo. A causal connection between the two observations was suggested by experiments showing accelerated telomere shorting under conditions of oxidative stress in cultured cells, but has never been studied in vivo. We therefore have analysed whether an increase in mitochondrial derived oxidative stress in response to heterozygous deletion of superoxide dismutase (Sod2+/-) would exacerbate aging phenotypes in telomere dysfunctional (mTerc-/-) mice. Heterozygous deletion of Sod2 resulted in reduced SOD2 protein levels and increased oxidative stress in aging telomere dysfunctional mice, but this did not lead to an increase in basal levels of oxidative nuclear DNA damage, an accumulation of nuclear DNA breaks, or an increased rate of telomere shortening in the mice. Moreover, heterozygous deletion of Sod2 did not accelerate the depletion of stem cells and the impairment in organ maintenance in aging mTerc-/- mice. In agreement with these observations, Sod2 haploinsufficiency did not lead to a further reduction in lifespan of mTerc-/- mice. Together, these results indicate that a decrease in SOD2-dependent antioxidant defence does not exacerbate aging in the context of telomere dysfunction

European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.The EU-ROS consortium (COST Action BM1203) was supported by the European Cooperation in Science and Technology (COST). The present overview represents the final Action dissemination summarizing the major achievements of COST Action BM1203 (EU-ROS) as well as research news and personal views of its members. Some authors were also supported by COST Actions BM1005 (ENOG) and BM1307 (PROTEOSTASIS), as well as funding from the European Commission FP7 and H2020 programmes, and several national funding agencies

University of Wollongong Campus News 12 August 1983

Stromal remodeling, in particular fibroblast-to-myofibroblast differentiation, is a hallmark of benign prostatic hyperplasia (BPH) and solid tumors, including prostate cancer (PCa). Increased local production of TGF?1 is considered the inducing stimulus. Given that stromal remodeling actively promotes BPH/PCa development, there is considerable interest in developing stromal-targeted therapies. Microarray and quantitative PCR analysis of primary human prostatic stromal cells induced to undergo fibroblast-to-myofibroblast differentiation with TGF?1 revealed up-regulation of the reactive oxygen species (ROS) producer reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and down-regulation of the selenium-containing ROS-scavenging enzymes glutathione peroxidase 3, thioredoxin reductase 1 (TXNRD1), and the selenium transporter selenoprotein P plasma 1. Consistently, NOX4 expression correlated specifically with the myofibroblast phenotype in vivo, and loss of selenoprotein P plasma 1 was observed in tumor-associated stroma of human PCa biopsies. Using lentiviral NOX4 short hairpin RNA-mediated knockdown, pharmacological inhibitors, antioxidants, and selenium, we demonstrate that TGF?1 induction of NOX4-derived ROS is required for TGF?1-mediated phosphorylation of c-jun N-terminal kinase, which in turn is essential for subsequent downstream cytoskeletal remodeling. Significantly, selenium supplementation inhibited differentiation by increasing ROS-scavenging selenoenzyme biosynthesis because glutathione peroxidase 3 and TXNRD1 expression and TXNRD1 enzyme activity were restored. Consistently, selenium depleted ROS levels downstream of NOX4 induction. Collectively, this work demonstrates that dysregulated redox homeostasis driven by elevated NOX4-derived ROS signaling underlies fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma. Further, these data indicate the potential clinical value of selenium and/or NOX4 inhibitors in preventing the functional pathogenic changes of stromal cells in BPH and PCa

Monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (GFP)-based reporter protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)-dgn fusion protein (GFP-dgn, unstable) and a variant carrying a frameshift mutation (GFP-dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP-dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry

Methionine restriction slows down senescence in human diploid fibroblasts

Methionine restriction (MetR) extends lifespan in animal models including rodents. Using human diploid fibroblasts (HDF), we report here that MetR significantly extends their replicative lifespan, thereby postponing cellular senescence. MetR significantly decreased activity of mitochondrial complex IV and diminished the accumulation of reactive oxygen species. Lifespan extension was accompanied by a significant decrease in the levels of subunits of mitochondrial complex IV, but also complex I, which was due to a decreased translation rate of several mtDNA-encoded subunits. Together, these findings indicate that MetR slows down aging in human cells by modulating mitochondrial protein synthesis and respiratory chain assembly

<i>fahd-1(tm5005) C</i>. <i>elegans</i> have a locomotion deficit.

<p>(A) 4-day old wild-type and <i>fahd-1(tm5005)</i> animals were placed on an empty NGM plate (without bacterial lawn) and the number of body bends was counted for one minute. <i>fahd-1(tm5005)</i> animals show fewer and more uncoordinated body bends compared to wild-type animals (28.53 ±SD 1.12 for wt vs. 11.57 ±SD 0.42 for <i>fahd-1(tm5005)</i>; p = 1.60E-05; N = 3 experiments (30 worms total)). (B) 4-day old wild type and <i>fahd-1(tm5005)</i> worms were put in the center of an NGM plate seeded with <i>E</i>. <i>coli</i> OP50. The radial dispersal rate (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134161#sec002" target="_blank">Materials and Methods</a>) was determined for three different time points (30 sec, 1 min, 2 min). The radial dispersal rate is decreased in <i>fahd-1(tm5005) C</i>. <i>elegans</i> compared to wild-type controls (3.41 ±SD 0.76 zones after two minutes for wt vs. 1.69 ±SD 0.11 zones after two minutes for <i>fahd-1(tm5005)</i>; p < 0.03 in all cases; N = 3 experiments (90 worms total)). (C) 4-day old wild-type and <i>fahd-1(tm5005)</i> hermaphrodites were screened for their swimming endurance performance in M9 buffer for up to 10 h. Wild-type worms are able to swim significantly longer than <i>fahd-1(tm5005)</i> worms (94.56% ±SD 4.72% swimming after 5 hours for wild-type vs. 23.72% ±SD 6.18 swimming after 10 hours for <i>fahd-1(tm5005)</i>; p = 0.001; N = 3 experiments (72 worms total)).</p

FAH Domain Containing Protein 1 (FAHD-1) Is Required for Mitochondrial Function and Locomotion Activity in <i>C</i>. <i>elegans</i>

<div><p>The fumarylacetoacetate hydrolase (FAH) protein superfamily of metabolic enzymes comprises a diverse set of enzymatic functions, including ß-diketone hydrolases, decarboxylases, and isomerases. Of note, the FAH superfamily includes many prokaryotic members with very distinct functions that lack homologs in eukaryotes. A prokaryotic member of the FAH superfamily, referred to as Cg1458, was shown to encode a soluble oxaloacetate decarboxylase (ODx). Based on sequence homologies to Cg1458, we recently identified human FAH domain containing protein-1 (FAHD1) as the first eukaryotic oxaloacetate decarboxylase. The physiological functions of ODx in eukaryotes remain unclear. Here we have probed the function of <i>fahd-1</i>, the nematode homolog of FAHD1, in the context of an intact organism. We found that mutation of <i>fahd-1</i> resulted in reduced brood size, a deregulation of the egg laying process and a severe locomotion deficit, characterized by a reduced frequency of body bends, reduced exploratory movements and reduced performance in an endurance exercise test. Notably, mitochondrial function was altered in the <i>fahd-1(tm5005)</i> mutant strain, as shown by a reduction of mitochondrial membrane potential and a reduced oxygen consumption of <i>fahd-1(tm5005)</i> animals. Mitochondrial dysfunction was accompanied by lifespan extension in worms grown at elevated temperature; however, unlike in mutant worms with a defect in the electron transport chain, the mitochondrial unfolded protein response was not upregulated in worms upon inactivation of <i>fahd-1</i>. Together these data establish a role of <i>fahd-1</i> to maintain mitochondrial function and consequently physical activity in nematodes.</p></div

Expression and subcellular localization of FAHD-1 in nematodes.

<p>(A) Young adult and gravid adult hermaphrodites of the transcriptional reporter N2;<i>Ex</i>[p_{<i>fahd-1</i>}GFP, pRF4] were monitored by confocal microscopy. <i>fahd-1</i> seems to be expressed in most tissues, including pharynx, canal cell, neurons, vulva, intestine, and stomato-intestinal muscle. Representative pictures are shown. Size bar = 100 μm. (B) Confocal images of young adult <i>C</i>. <i>elegans</i> of the transcriptional reporter N2;<i>Ex</i>[p_{<i>fahd-1</i>}GFP, pRF4]. Representative images are shown. Size bar = 10 μm. <i>Upper left panel</i>: Detailed picture of <i>fahd-1</i> expression in the peri-vulvar region; vulva and uterine muscles are stained positive. <i>Upper right panel</i>: Detailed picture of <i>fahd-1</i> expression in the pharyngeal region; buccal cavity, procorpus, metacorpus, isthmus, posterior bulb, and pharyngeal-intestinal valve, as well as sensory neurons in the head were stained positive. <i>Lower left panel</i>: Detailed picture of <i>fahd-1</i> expression in the mid-body region; body wall muscles, lateral ganglion, dorsal chord (DC), and ventral nerve chord (VNC) were stained positive. <i>Lower right panel</i>: Detailed picture of <i>fahd-1</i> expression in the anus region; the stomato-intestinal muscles, the anus and the intestine were stained positive. (C) Temporal expression pattern of <i>fahd-1</i>. The translational reporter N2;<i>Ex</i>[p_{<i>fahd-1</i>}FAHD-1::GFP, pRF4] was examined (and pictured) by confocal fluorescence microscopy from the time point the eggs were laid at gastrula stage to 10-day old post fertile adults. GFP fluorescence was overlaid with phase-contrast microscopy. The FAHD1-1::GFP fusion protein is present in all stages from gastrulation to old worms. Representative images are shown. Size bar = 50 μm. (D) Young adult hermaphrodites of the translational reporter N2;<i>Ex</i>[p_{<i>fahd-1</i>}FAHD-1::GFP, pRF4] expressing the FAHD-1::GFP fusion protein (shown in green) were stained with 100 nM MitoTracker Red (shown in red) overnight. Shown here are confocal pictures of the pharynx region of the worm recorded in the green and red channel, as well as the merged pictures. Regions of overlapping staining (shown in yellow) are indicated by arrows. Representative pictures are shown. Size bar = 10 μm.</p

Characterization of <i>fahd-1</i> deletion mutant animals.

<p>(A) <i>Left panel</i>: Single worm duplex PCR was performed on ten animals of the <i>fahd-1(tm5005)</i> mutant strain, to confirm the deletion on DNA level. Three primers were used, generating PCR products of 220 bp for the wild-type allele and 337 bp for the mutant allele. M = size marker; 1–10 = 10 different backcrossed <i>fahd-1(tm5005) C</i>. <i>elegans</i>; mut = mutant allele; wt = wild-type allele; b = <i>E</i>. <i>coli</i> OP50 (<i>C</i>. <i>elegans</i> bacterial food; to identify bands derived from bacterial contamination). <i>Right panel</i>: To confirm the deletion of the protein, protein lysates were prepared from wild-type and <i>fahd-1(tm5005)</i> mutant animals, as indicated. FAHD-1 protein was detected by Western blot using a peptide-specific rabbit polyclonal antibody against FAHD-1. β-actin served as a loading control. (B) Overall brood size was compared by counting progeny in wild-type and <i>fahd-1(tm5005) C</i>. <i>elegans</i> animals raised at 20°C and 25°C throughout the reproductive period. In <i>fahd-1(tm5005) C</i>. <i>elegans</i> the total brood size is reduced compared to wild-type controls when raised at 20°C (left panel; 281 ±SD 28.11 for wt vs. 238 ±SD 63.96 for <i>fahd-1(tm5005)</i>; p = 0.05; N = 9) and even more severe when raised at 25°C (right panel; 199 ±SD 71.30 for wt vs. 98.8 ±SD 49.23 for <i>fahd-1(tm5005)</i>; p = 0.0013; N = 10). (D) Egg laying patterns were determined for wild-type and <i>fahd-1(tm5005) C</i>. <i>elegans</i>. <i>Left panel</i>: 4-day old wild-type and <i>fahd-1(tm5005)</i> animals were kept in M9 buffer (without bacteria) for four hours and the number of eggs that were laid by each genotype during this time was counted. While wild-type worms do not lay their eggs under these unfavorable conditions, <i>fahd-1(tm5005)</i> worms ‘lose’ them over the course of time (2.08 ±SD 1.75 for wt vs. 10.26 ±SD 0.32; p = 0.0598 for 60 min, p < 0.002 in all other cases; N = 3 experiments (72 worms total)). <i>Right panel</i>: Micrographs were prepared from 4-day old, well-fed and unstressed hermaphrodites of the wild-type and <i>fahd-1(tm5005)</i> genotype. While wild-type worms carry between 18 and 20 eggs, <i>fahd-1(tm5005)</i> animals show great variety in the number of eggs, tending to accumulate them in the uterus. Representative pictures are shown.</p