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The leading hypothesis of why organisms age is the “Free Radical Theory of Aging”, which states that the accumulation of reactive oxygen species (ROS), such as superoxide (O2•-) and hydrogen peroxide (H2O2), causes protein, lipid and DNA damage and leads to the observed age-related decline of cells and tissues. A major obstacle in analyzing the role of oxidative stress in aging organisms is the inability to precisely localize and quantify the oxidants, to identify proteins and pathways that might be affected, and ultimately, to correlate changes in oxidant levels with the lifespan of the organism. To directly monitor the onset and extent of oxidative stress during the lifespan of Caenorhabditis elegans, we utilized the fluorescent H2O2 sensor protein HyPer, which enabled us to quantify endogenous peroxide levels in different tissues of living animals in real time. We made the surprising observation that wildtype C. elegans is exposed to very high peroxide levels during development. Peroxide levels drop rapidly as the animals mature, and low peroxide levels then prevail throughout the reproductive age, after which an age-accompanying increase of peroxide level is observed. These results were in excellent agreement with findings obtained by using the highly quantitative redox proteomic technique OxICAT, which monitors the oxidation status of redox-sensitive proteins as read-out for onset, localization, and protein targets of oxidative stress. By using OxICAT, we detected increased protein thiol oxidation during the development of C. elegans and in aging animals. Many processes in C. elegans might potentially contribute to the elevated peroxide levels observed during development, including cuticle formation, apoptosis, proliferation, gametogenesis, or ROS signaling. The finding that all investigated C. elegans mutants regardless of their lifespan are exposed to high developmental peroxide levels argues for ROS accumulation to be a universal and necessary event. Yet, recovery from the early oxidative boost might determine the subsequent adult lifespan, as we found that long-lived daf-2 mutants transition faster to reducing conditions than short-lived daf-16 mutants, which retain higher peroxide levels throughout their mature life. These results suggest that changes in the cellular oxidant homeostasis, encountered at a very early stage in life, might determine subsequent redox levels and potentially the lifespan of organisms. Manipulation of developmental oxidant levels using glucose restriction or a short bolus of superoxide caused a disruption in developmental growth, a delay in reproduction, and a shortened lifespan. These results suggest that developmental oxidant levels are fine-tuned and optimized. Future experiments are aimed to investigate the sources of developmental hydrogen peroxide, and to elucidate whether active down-regulation of antioxidant enzymes during the larval period might foster peroxide accumulation. Preliminary results indicate that this might indeed be the case for peroxiredoxin 2, whose expression was significantly lower during development than at later stages in life. Finally, we investigated whether the observed variances in the developmental peroxide levels of individual worms within a synchronized wildtype population might be responsible for the observed significant variances in lifespan, and hence could serve as a predictor for adult lifespan. Preliminary results revealed that neither too low nor too high peroxide levels during development are beneficial for the lifespan of wildtype worms, suggesting that ROS level during development might be optimized for maximized lifespan. Future experiments aim to reveal the processes that are affected by ROS and which might influence the individual’s lifespan early in life.
Approaches to the Analysis of Proteomics and Transcriptomics Data based on Statistical Methodology
(2014)
Recent developments in genomics and molecular biology led to the generation of an enormous amount of complex data of different origin. This is demonstrated by a number of published results from microarray experiments in Gene Expression Omnibus. The number was growing in exponential pace over the last decade. The challenge of interpreting these vast amounts of data from different technologies led to the development of new methods in the fields of computational biology and bioinformatics. Researchers often want to represent biological phenomena in the most detailed and comprehensive way. However, due to the technological limitations and other factors like limited resources this is not always possible. On one hand, more detailed and comprehensive research generates data of high complexity that is very often difficult to approach analytically, however, giving bioinformatics a chance to draw more precise and deeper conclusions. On the other hand, for low-complexity tasks the data distribution is known and we can fit a mathematical model. Then, to infer from this mathematical model, researchers can use well-known and standard methodologies. In return for using standard methodologies, the biological questions we are answering might not be unveiling the whole complexity of the biological meaning. Nowadays it is a standard that a biological study involves generation of large amounts of data that needs to be analyzed with a statistical inference. Sometimes data challenge researchers with low complexity task that can be performed with standard and popular methodologies as in Proteomic analysis of mouse oocytes reveals 28 candidate factors of the "reprogrammome". There, we established a protocol for proteomics data that involves preprocessing of the raw data and conducting Gene Ontology overrepresentation analysis utilizing hypergeometric distribution. In cases, where the data complexity is high and there are no published frameworks a researcher could follow, randomization can be an approach to exploit. In two studies by The mouse oocyte proteome escapes maternal aging and CellFateScout - a bioinformatics tool for elucidating small molecule signaling pathways that drive cells in a specific direction we showed how randomization can be performed for distinct complex tasks. In The mouse oocyte proteome escapes maternal aging we constructed a random sample of semantic similarity score between oocyte transcriptome and random transcriptome subset of oocyte proteome size. Therefore, we could calculate whether the proteome is representative of the trancriptome. Further, we established a novel framework for Gene Ontology overrepresentation that involves randomization testing. Every Gene Ontology term is tested whether randomly reassigning all gene labels of belonging to or not belonging to this term will decrease the overall expression level in this term. In CellFateScout - a bioinformatics tool for elucidating small molecule signaling pathways that drive cells in a specific direction we validated CellFateScout against other well-known bioinformatics tools. We stated the question whether our plugin is able to predict small molecule effects better in terms of expression signatures. For this, we constructed a protocol that uses randomization testing. We assess here if the small molecule effect described as a (set of) active signaling pathways, as detected by our plugin or other bioinformatics tools, is significantly closer to known small molecule targets than a random path.
Abstract
Caveolae position CaV3.2 (T‐type Ca2+ channel encoded by the α‐3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca2+ influx to trigger Ca2+ sparks and large‐conductance Ca2+‐activated K+ channel feedback in vascular smooth muscle. We hypothesize that this mechanism of Ca2+ spark generation is affected by age. Using smooth muscle cells (VSMCs) from mouse mesenteric arteries, we found that both Cav3.2 channel inhibition by Ni2+ (50 µM) and caveolae disruption by methyl‐ß‐cyclodextrin or genetic abolition of Eps15 homology domain‐containing protein (EHD2) inhibited Ca2+ sparks in cells from young (4 months) but not old (12 months) mice. In accordance, expression of Cav3.2 channel was higher in mesenteric arteries from young than old mice. Similar effects were observed for caveolae density. Using SMAKO Cav1.2−/− mice, caffeine (RyR activator) and thapsigargin (Ca2+ transport ATPase inhibitor), we found that sufficient SR Ca2+ load is a prerequisite for the CaV3.2‐RyR axis to generate Ca2+ sparks. We identified a fraction of Ca2+ sparks in aged VSMCs, which is sensitive to the TRP channel blocker Gd3+ (100 µM), but insensitive to CaV1.2 and CaV3.2 channel blockade. Our data demonstrate that the VSMC CaV3.2‐RyR axis is down‐regulated by aging. This defective CaV3.2‐RyR coupling is counterbalanced by a Gd3+ sensitive Ca2+ pathway providing compensatory Ca2+ influx for triggering Ca2+ sparks in aged VSMCs.
Age-related deterioration in white and gray matter is linked to cognitive deficits. Reduced microstructure of the fornix, the major efferent pathway of the hippocampus, and volume of the dentate gyrus (DG), may cause age-associated memory decline. However, the linkage between these anatomical determinants and memory retrieval in healthy aging are poorly understood. In 30 older adults, we acquired diffusion tensor and T1-weighted images for individual deterministic tractography and volume estimation. A memory task, administered outside of the scanner to assess retrieval of learned associations, required discrimination of previously acquired picture-word pairs. The results showed that fornix fractional anisotropy (FA) and left DG volumes were related to successful retrieval. These brain-behavior associations were observed for correct rejections, but not hits, indicating specificity of memory network functioning for detecting false associations. Mediation analyses showed that left DG volume mediated the effect of fornix FA on memory (48%), but not vice versa. These findings suggest that reduced microstructure induces volume loss and thus negatively affects retrieval of learned associations, complementing evidence of a pivotal role of the fornix in healthy aging. Our study offers a neurobehavioral model to explain variability in memory retrieval in older adults, an important prerequisite for the development of interventions to counteract cognitive decline.
Aging is an independent risk factor for hypertension, cardiovascular morbidity, and mortality. However, detailed mechanisms linking aging to cardiovascular disease are unclear. We studied the aging effects on the role of perivascular adipose tissue and downstream vasoconstriction targets, voltage-dependent KV7 channels, and their pharmacological modulators (flupirtine, retigabine, QO58, and QO58-lysine) in a murine model. We assessed vascular function of young and old mesenteric arteries in vitro using wire myography and membrane potential measurements with sharp electrodes. We also performed bulk RNA sequencing and quantitative reverse transcription-polymerase chain reaction tests in mesenteric arteries and perivascular adipose tissue to elucidate molecular underpinnings of age-related phenotypes. Results revealed impaired perivascular adipose tissue-mediated control of vascular tone particularly via KV7.3–5 channels with increased age through metabolic and inflammatory processes and release of perivascular adipose tissue-derived relaxation factors. Moreover, QO58 was identified as novel pharmacological vasodilator to activate XE991-sensitive KCNQ channels in old mesenteric arteries. Our data suggest that targeting inflammation and metabolism in perivascular adipose tissue could represent novel approaches to restore vascular function during aging. Furthermore, KV7.3–5 channels represent a promising target in cardiovascular aging.
Over the past decades, the human life span has dramatically increased, and therefore, a steady increase in diseases associated with age (such as Alzheimer’s disease and Parkinson’s disease) is expected. In these neurodegenerative diseases, there is a cognitive decline and memory loss, which accompany increased systemic inflammation, the inflamm-aging, and the insulin resistance. Despite numerous studies of age-related pathologies, data on the contribution of brain insulin resistance and innate immunity components to aging are insufficient. Recently, much research has been focused on the consequences of nutrients and adiposity- and nutrient-related signals in brain aging and cognitive decline. Moreover, given the role of metainflammation in neurodegeneration, lifestyle interventions such as calorie restriction may be an effective way to break the vicious cycle of metainflammation and have a role in social behavior. The various effects of calorie restriction on metainflammation, insulin resistance, and neurodegeneration have been described. Less attention has been paid to the social determinants of aging and the possible mechanism by which calorie restriction might influence social behavior. The purpose of this review is to discuss current knowledge in the interdisciplinary field of geroscience—immunosenescence, inflamm-aging, and metainflammation—which makes a significant contribution to aging. A substantial part of the review is devoted to frontiers in the brain insulin resistance in relation to neuroinflammation. In addition, we summarize new data on potential mechanisms of calorie restriction that influence as a lifestyle intervention on the social brain. This knowledge can be used to initiate successful aging and slow the onset of neurodegenerative diseases.
Endothelial dysfunction (ED) comes with age, even without overt vessel damage such as that which occurs in atherosclerosis and diabetic vasculopathy. We hypothesized that aging would affect the downstream signalling of the endothelial nitric oxide (NO) system in the vascular smooth muscle (VSM). With this in mind, resistance mesenteric arteries were isolated from 13-week (juvenile) and 40-week-old (aged) mice and tested under isometric conditions using wire myography. Acetylcholine (ACh)-induced relaxation was reduced in aged as compared to juvenile vessels. Pretreatment with L-NAME, which inhibits nitrix oxide synthases (NOS), decreased ACh-mediated vasorelaxation, whereby differences in vasorelaxation between groups disappeared. Endothelium-independent vasorelaxation by the NO donor sodium nitroprusside (SNP) was similar in both groups; however, SNP bolus application (10−6 mol L−1) as well as soluble guanylyl cyclase (sGC) activation by runcaciguat (10−6 mol L−1) caused faster responses in juvenile vessels. This was accompanied by higher cGMP concentrations and a stronger response to the PDE5 inhibitor sildenafil in juvenile vessels. Mesenteric arteries and aortas did not reveal apparent histological differences between groups (van Gieson staining). The mRNA expression of the α1 and α2 subunits of sGC was lower in aged animals, as was PDE5 mRNA expression. In conclusion, vasorelaxation is compromised at an early age in mice even in the absence of histopathological alterations. Vascular smooth muscle sGC is a key element in aged vessel dysfunction.
Respiratory infection caused by Streptococcus pneumoniae is a leading cause of morbidity and mortality in older adults. Acquired CD4+ T cell mechanism are essential for the protection against colonization and subsequent development of infections by S. pneumoniae. In this study, we hypothesized that age-related changes within the CD4+ T-cell population compromise CD4+ T-cell specific responses to S. pneumoniae, thereby contributing to increased susceptibility at older age. To this end, we interrogated the CD4+ T-cell response against the immunogenic pneumococcal protein AliB, part of the unique oligopeptide ABC transporter system responsible for the uptake of nutrients for the bacterium and crucial for the development of pneumococcal meningitis, in healthy young and older adults. Specifically, proliferation of CD4+ T cells as well as concomitant cytokine profiles and phenotypic markers implied in immunosenescence were studied. Older adults showed decreased AliB-induced CD4+ T-cell proliferation that is associated with an increased frequency of regulatory T cells and lower levels of active CD25+CD127+CTLA-4−TIGIT-CD4+T cells. Additionally, levels of pro-inflammatory cytokines IFNy and IL-17F were decreased at older age. Our findings indicate that key features of a pneumococcal-specific CD4+ T-cell immune response are altered at older age, which may contribute to enhanced susceptibility for pneumococcal infections.