Strengths and opportunities in research into extracellular matrix ageing: A consultation with the ECMage research community.

Ageing causes progressive decline in metabolic, behavioural, and physiological functions, leading to a reduced health span. The extracellular matrix (ECM) is the three-dimensional network of macromolecules that provides our tissues with structure and biomechanical resilience. Imbalance between damage and repair/regeneration causes the ECM to undergo structural deterioration with age, contributing to age-associated pathology. The ECM 'Ageing Across the Life Course' interdisciplinary research network (ECMage) was established to bring together researchers in the United Kingdom, and internationally, working on the emerging field of ECM ageing. Here we report on a consultation at a joint meeting of ECMage and the Medical Research Council / Versus Arthritis Centre for Integrated Research into Musculoskeletal Ageing, held in January 2023, in which delegates analysed the key questions and research opportunities in the field of ECM ageing. We examine fundamental biological questions, enabling technologies, systems of study and emerging in vitro and in silico models, alongside consideration of the broader challenges facing the field.

Systems Biology of Ageing.

The ageing process is highly complex involving multiple processes operating at different biological levels. Systems Biology presents an approach using integrative computational and laboratory study that allows us to address such complexity. The approach relies on the computational analysis of knowledge and data to generate predictive models that may be validated with further laboratory experimentation. Our understanding of ageing is such that translational opportunities are within reach and systems biology offers a means to ensure that optimal decisions are made. We present an overview of the methods employed from bioinformatics and computational modelling and describe some of the insights into ageing that have been gained.

The DNA repair enzyme, aprataxin, plays a role in innate immune signaling.

Ataxia with oculomotor apraxia type 1 (AOA1) is a progressive neurodegenerative disorder characterized by a gradual loss of coordination of hand movements, speech, and eye movements. AOA1 is caused by an inactivation mutation in the APTX gene. APTX resolves abortive DNA ligation intermediates. APTX deficiency may lead to the accumulation of 5'-AMP termini, especially in the mitochondrial genome. The consequences of APTX deficiency includes impaired mitochondrial function, increased DNA single-strand breaks, elevated reactive oxygen species production, and altered mitochondrial morphology. All of these processes can cause misplacement of nuclear and mitochondrial DNA, which can activate innate immune sensors to elicit an inflammatory response. This study explores the impact of APTX knockout in microglial cells, the immune cells of the brain. RNA-seq analysis revealed significant differences in the transcriptomes of wild-type and APTX knockout cells, especially in response to viral infections and innate immune pathways. Specifically, genes and proteins involved in the cGAS-STING and RIG-I/MAVS pathways were downregulated in APTX knockout cells, which suggests an impaired immune response to cytosolic DNA and RNA. The clinical relevance of these findings was supported by analyzing publicly available RNA-seq data from AOA1 patient cell lines. Comparisons between APTX-deficient patient cells and healthy control cells also revealed altered immune responses and dysregulated DNA- and RNA-sensing pathways in the patient cells. Overall, this study highlights the critical role of APTX in regulating innate immunity, particularly in DNA- and RNA-sensing pathways. Our findings contribute to a better understanding of the underlying molecular mechanisms of AOA1 pathology and highlights potential therapeutic targets for this disease.

Systems modelling predicts chronic inflammation and genomic instability prevent effective mitochondrial regulation during biological ageing.

The regulation of mitochondrial turnover under conditions of stress occurs partly through the AMPK-NAD+-PGC1α-SIRT1 signalling pathway. This pathway can be affected by both genomic instability and chronic inflammation since these will result in an increased rate of NAD+ degradation through PARP1 and CD38 respectively. In this work we develop a computational model of this signalling pathway, calibrating and validating it against experimental data. The computational model is used to study mitochondrial turnover under conditions of stress and how it is affected by genomic instability, chronic inflammation and biological ageing in general. We report that the AMPK-NAD+-PGC1α-SIRT1 signalling pathway becomes less responsive with age and that this can prime for the accumulation of dysfunctional mitochondria.

Cytosolic Self-DNA-A Potential Source of Chronic Inflammation in Aging.

Aging is the consequence of a lifelong accumulation of stochastic damage to tissues and cellular components. Advancing age closely associates with elevated markers of innate immunity and low-grade chronic inflammation, probably reflecting steady increasing incidents of cellular and tissue damage over the life course. The DNA sensing cGAS-STING signaling pathway is activated by misplaced cytosolic self-DNA, which then initiates the innate immune responses. Here, we hypothesize that the stochastic release of various forms of DNA from the nucleus and mitochondria, e.g., because of DNA damage, altered nucleus integrity, and mitochondrial damage, can result in chronic activation of inflammatory responses that characterize the aging process. This cytosolic self-DNA-innate immunity axis may perturb tissue homeostasis and function that characterizes human aging and age-associated pathology. Proper techniques and experimental models are available to investigate this axis to develop therapeutic interventions.

Modelling the role of redox-related mechanisms in musculoskeletal ageing.

The decline in the musculoskeletal system with age is driven at the cellular level by random molecular damage. Cells possess mechanisms to repair or remove damage and many of the pathways involved in this response are regulated by redox signals. However, with ageing there is an increase in oxidative stress which can lead to chronic inflammation and disruption of redox signalling pathways. The complexity of the processes involved has led to the use of computational modelling to help increase our understanding of the system, test hypotheses and make testable predictions. This paper will give a brief background of the biological systems that have been modelled, an introduction to computational modelling, a review of models that involve redox-related mechanisms that are applicable to musculoskeletal ageing, and finally a discussion of the future potential for modelling in this field.

PyCoTools: a Python toolbox for COPASI.

Motivation: COPASI is an open source software package for constructing, simulating and analyzing dynamic models of biochemical networks. COPASI is primarily intended to be used with a graphical user interface but often it is desirable to be able to access COPASI features programmatically, with a high level interface. Results: PyCoTools is a Python package aimed at providing a high level interface to COPASI tasks with an emphasis on model calibration. PyCoTools enables the construction of COPASI models and the execution of a subset of COPASI tasks including time courses, parameter scans and parameter estimations. Additional 'composite' tasks which use COPASI tasks as building blocks are available for increasing parameter estimation throughput, performing identifiability analysis and performing model selection. PyCoTools supports exploratory data analysis on parameter estimation data to assist with troubleshooting model calibrations. We demonstrate PyCoTools by posing a model selection problem designed to show case PyCoTools within a realistic scenario. The aim of the model selection problem is to test the feasibility of three alternative hypotheses in explaining experimental data derived from neonatal dermal fibroblasts in response to TGF-ß over time. PyCoTools is used to critically analyze the parameter estimations and propose strategies for model improvement. Availability and implementation: PyCoTools can be downloaded from the Python Package Index (PyPI) using the command 'pip install pycotools' or directly from GitHub (https://github.com/CiaranWelsh/pycotools). Documentation at http://pycotools.readthedocs.io. Supplementary information: Supplementary data are available at Bioinformatics online.

Predominant Asymmetrical Stem Cell Fate Outcome Limits the Rate of Niche Succession in Human Colonic Crypts.

Stem cell (SC) dynamics within the human colorectal crypt SC niche remain poorly understood, with previous studies proposing divergent hypotheses on the predominant mode of SC self-renewal and the rate of SC replacement. Here we use age-related mitochondrial oxidative phosphorylation (OXPHOS) defects to trace clonal lineages within human colorectal crypts across the adult life-course. By resolving the frequency and size distribution of OXPHOS-deficient clones, quantitative analysis shows that, in common with mouse, long-term maintenance of the colonic epithelial crypt relies on stochastic SC loss and replacement mediated by competition for limited niche access. We find that the colonic crypt is maintained by ~5 effective SCs. However, with a SC loss/replacement rate estimated to be slower than once per year, our results indicate that the vast majority of individual SC divisions result in asymmetric fate outcome. These findings provide a quantitative platform to detect and study deviations from human colorectal crypt SC niche homeostasis during the process of colorectal carcinogenesis.

'Molecular habituation' as a potential mechanism of gradual homeostatic loss with age.

The ability of reactive oxygen species (ROS) to cause molecular damage has meant that chronic oxidative stress has been mostly studied from the point of view of being a source of toxicity to the cell. However, the known duality of ROS molecules as both damaging agents and cellular redox signals implies another perspective in the study of sustained oxidative stress. This is a perspective of studying oxidative stress as a constitutive signal within the cell. In this work, we adopt a theoretical perspective as an exploratory and explanatory approach to examine how chronic oxidative stress can interfere with signal processing by redox signalling pathways in the cell. We report that constitutive signals can give rise to a 'molecular habituation' effect that can prime for a gradual loss of biological function. This is because a constitutive signal in the environment has the potential to reduce the responsiveness of a signalling pathway through the prolonged activation of negative regulators. Additionally, we demonstrate how this phenomenon is likely to occur in different signalling pathways exposed to persistent signals and furthermore at different levels of biological organisation.

Pervasive gene expression responses to a fluctuating diet in Drosophila melanogaster: The importance of measuring multiple traits to decouple potential mediators of life span and reproduction.

Phenotypic plasticity is an important concept in life-history evolution, and most organisms, including Drosophila melanogaster, show a plastic life-history response to diet. However, little is known about how these life-history responses are mediated. In this study, we compared adult female flies fed an alternating diet (yoyo flies) with flies fed a constant low (CL) or high (CH) diet and tested how whole genome expression was affected by these diet regimes and how the transcriptional responses related to different life-history traits. We showed that flies were able to respond quickly to diet fluctuations throughout life span by drastically changing their transcription. Importantly, by measuring the response of multiple life-history traits we were able to decouple groups of genes associated with life span or reproduction, life-history traits that often covary with a diet change. A coexpression network analysis uncovered which genes underpin the separate and shared regulation of these life-history traits. Our study provides essential insights to help unravel the genetic architecture mediating life-history responses to diet, and it shows that the flies' whole genome transcription response is highly plastic.

Systems modelling ageing: from single senescent cells to simple multi-cellular models.

Systems modelling has been successfully used to investigate several key molecular mechanisms of ageing. Modelling frameworks to allow integration of models and methods to enhance confidence in models are now well established. In this article, we discuss these issues and work through the process of building an integrated model for cellular senescence as a single cell and in a simple tissue context.

Explaining sex differences in lifespan in terms of optimal energy allocation in the baboon.

We provide a quantitative test of the hypothesis that sex role specialization may account for sex differences in lifespan in baboons if such specialization causes the dependency of fitness upon longevity, and consequently the optimal resolution to an energetic trade-off between somatic maintenance and other physiological functions, to differ between males and females. We present a model in which females provide all offspring care and males compete for access to reproductive females and in which the partitioning of available energy between the competing fitness-enhancing functions of growth, maintenance, and reproduction is modeled as a dynamic behavioral game, with the optimal decision for each individual depending upon his/her state and the behavior of other members of the population. Our model replicates the sexual dimorphism in body size and sex differences in longevity and reproductive scheduling seen in natural populations of baboons. We show that this outcome is generally robust to perturbations in model parameters, an important finding given that the same behavior is seen across multiple populations and species in the wild. This supports the idea that sex differences in longevity result from differences in the value of somatic maintenance relative to other fitness-enhancing functions in keeping with the disposable soma theory.

Growing more positive with age: The relationship between reproduction and survival in aging flies.

Populations of laboratory animals that are selected for increased lifespan often show negative correlated responses in early fecundity. However, late fecundity and/or total lifetime fecundity can be higher in the populations selected for increased lifespan. This has been interpreted by some as being at odds with the disposable soma theory, which predicts decreased lifespan to increase total reproductive output. Alternatively, the Y-model explores the effects of variation in resource allocation and acquisition on life histories. In this model, a negative relationship between lifespan and reproduction can be viewed as variation in allocation, whereas a positive relationship is the result of variation in acquisition. However, a frequently neglected complication of the Y-model is that older individuals often show a decline in resource acquisition. Therefore, differential allocation to maintenance and survival might affect this decline in late-life acquisition which will affect resource availability across the whole lifespan. In this paper we show that a model which incorporates the ideas of the Y-model, the disposable soma theory, and an age-related decrease in resource acquisition, i.e. feeding senescence, can explain how the relationship between fecundity and lifespan changes with age. Furthermore, by modeling environments with contrasting extrinsic mortality rates, we explored how the outcome of the model depended on the relative importance of early and late-life reproduction. In high mortality environments a relatively higher early fecundity, lower late fecundity, and lower lifespans were more optimal, whereas the opposite was true for low mortality environments. We applied predictions from the model to a cohort of individually-housed female Drosophila melanogaster flies for which we measured age specific fecundity and lifespan. Early fecundity was negatively associated with lifespan, while late fecundity related positively with lifespan in the same cohort. This verified that the mechanism of feeding senescence could explain patterns for age specific relationships between lifespan and fecundity.

A systems study reveals concurrent activation of AMPK and mTOR by amino acids.

Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational-experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes.

Modeling and gene knockdown to assess the contribution of nonsense-mediated decay, premature termination, and selenocysteine insertion to the selenoprotein hierarchy.

The expression of selenoproteins, a specific group of proteins that incorporates selenocysteine, is hierarchically regulated by the availability of Se, with some, but not all selenoprotein mRNA transcripts decreasing in abundance with decreasing Se. Selenocysteine insertion into the peptide chain occurs during translation following recoding of an internal UGA stop codon. There is increasing evidence that this UGA recoding competes with premature translation termination, which is followed by nonsense-mediated decay (NMD) of the transcript. In this study, we tested the hypothesis that the susceptibility of different selenoprotein mRNAs to premature termination during translation and differential sensitivity of selenoprotein transcripts to NMD are major factors in the selenoprotein hierarchy. Selenoprotein transcript abundance was measured in Caco-2 cells using real-time PCR under different Se conditions and the data obtained fitted to mathematical models of selenoprotein translation. A calibrated model that included a combination of differential sensitivity of selenoprotein transcripts to NMD and different frequency of non-NMD related premature translation termination was able to fit all the measurements. The model predictions were tested using SiRNA to knock down expression of the crucial NMD factor UPF1 (up-frameshift protein 1) and selenoprotein mRNA expression. The calibrated model was able to predict the effect of UPF1 knockdown on gene expression for all tested selenoproteins, except SPS2 (selenophosphate synthetase), which itself is essential for selenoprotein synthesis. These results indicate an important role for NMD in the hierarchical regulation of selenoprotein mRNAs, with the exception of SPS2 whose expression is likely regulated by a different mechanism.

Genome-Wide MicroRNA and Gene Analysis of Mesenchymal Stem Cell Chondrogenesis Identifies an Essential Role and Multiple Targets for miR-140-5p.

microRNAs (miRNAs) are abundantly expressed in development where they are critical determinants of cell differentiation and phenotype. Accordingly miRNAs are essential for normal skeletal development and chondrogenesis in particular. However, the question of which miRNAs are specific to the chondrocyte phenotype has not been fully addressed. Using microarray analysis of miRNA expression during mesenchymal stem cell chondrogenic differentiation and detailed examination of the role of essential differentiation factors, such as SOX9, TGF-ß, and the cell condensation phase, we characterize the repertoire of specific miRNAs involved in chondrocyte development, highlighting in particular miR-140 and miR-455. Further with the use of mRNA microarray data we integrate miRNA expression and mRNA expression during chondrogenesis to underline the particular importance of miR-140, especially the -5p strand. We provide a detailed identification and validation of direct targets of miR-140-5p in both chondrogenesis and adult chondrocytes with the use of microarray and 3'UTR analysis. This emphasizes the diverse array of targets and pathways regulated by miR-140-5p. We are also able to confirm previous experimentally identified targets but, additionally, identify a novel positive regulation of the Wnt signaling pathway by miR-140-5p. Wnt signaling has a complex role in chondrogenesis and skeletal development and these findings illustrate a previously unidentified role for miR-140-5p in regulation of Wnt signaling in these processes. Together these developments further highlight the role of miRNAs during chondrogenesis to improve our understanding of chondrocyte development and guide cartilage tissue engineering.

Integrated Stochastic Model of DNA Damage Repair by Non-homologous End Joining and p53/p21-Mediated Early Senescence Signalling.

Unrepaired or inaccurately repaired DNA damage can lead to a range of cell fates, such as apoptosis, cellular senescence or cancer, depending on the efficiency and accuracy of DNA damage repair and on the downstream DNA damage signalling. DNA damage repair and signalling have been studied and modelled in detail separately, but it is not yet clear how they integrate with one another to control cell fate. In this study, we have created an integrated stochastic model of DNA damage repair by non-homologous end joining and of gamma irradiation-induced cellular senescence in human cells that are not apoptosis-prone. The integrated model successfully explains the changes that occur in the dynamics of DNA damage repair after irradiation. Simulations of p53/p21 dynamics after irradiation agree well with previously published experimental studies, further validating the model. Additionally, the model predicts, and we offer some experimental support, that low-dose fractionated irradiation of cells leads to temporal patterns in p53/p21 that lead to significant cellular senescence. The integrated model is valuable for studying the processes of DNA damage induced cell fate and predicting the effectiveness of DNA damage related medical interventions at the cellular level.

Detecting translational regulation by change point analysis of ribosome profiling data sets.

Ribo-Seq maps the location of translating ribosomes on mature mRNA transcripts. While during normal translation, ribosome density is constant along the length of the mRNA coding region, this can be altered in response to translational regulatory events. In the present study, we developed a method to detect translational regulation of individual mRNAs from their ribosome profiles, utilizing changes in ribosome density. We used mathematical modeling to show that changes in ribosome density should occur along the mRNA at the point of regulation. We analyzed a Ribo-Seq data set obtained for mouse embryonic stem cells and showed that normalization by corresponding RNA-Seq can be used to improve the Ribo-Seq quality by removing bias introduced by deep-sequencing and alignment artifacts. After normalization, we applied a change point algorithm to detect changes in ribosome density present in individual mRNA ribosome profiles. Additional sequence and gene isoform information obtained from the UCSC Genome Browser allowed us to further categorize the detected changes into different mechanisms of regulation. In particular, we detected several mRNAs with known post-transcriptional regulation, e.g., premature termination for selenoprotein mRNAs and translational control of Atf4, but also several more mRNAs with hitherto unknown translational regulation. Additionally, our approach proved useful for identification of new transcript isoforms.

Dynamic modelling of pathways to cellular senescence reveals strategies for targeted interventions.

Cellular senescence, a state of irreversible cell cycle arrest, is thought to help protect an organism from cancer, yet also contributes to ageing. The changes which occur in senescence are controlled by networks of multiple signalling and feedback pathways at the cellular level, and the interplay between these is difficult to predict and understand. To unravel the intrinsic challenges of understanding such a highly networked system, we have taken a systems biology approach to cellular senescence. We report a detailed analysis of senescence signalling via DNA damage, insulin-TOR, FoxO3a transcription factors, oxidative stress response, mitochondrial regulation and mitophagy. We show in silico and in vitro that inhibition of reactive oxygen species can prevent loss of mitochondrial membrane potential, whilst inhibition of mTOR shows a partial rescue of mitochondrial mass changes during establishment of senescence. Dual inhibition of ROS and mTOR in vitro confirmed computational model predictions that it was possible to further reduce senescence-induced mitochondrial dysfunction and DNA double-strand breaks. However, these interventions were unable to abrogate the senescence-induced mitochondrial dysfunction completely, and we identified decreased mitochondrial fission as the potential driving force for increased mitochondrial mass via prevention of mitophagy. Dynamic sensitivity analysis of the model showed the network stabilised at a new late state of cellular senescence. This was characterised by poor network sensitivity, high signalling noise, low cellular energy, high inflammation and permanent cell cycle arrest suggesting an unsatisfactory outcome for treatments aiming to delay or reverse cellular senescence at late time points. Combinatorial targeted interventions are therefore possible for intervening in the cellular pathway to senescence, but in the cases identified here, are only capable of delaying senescence onset.

Computational modelling of the regulation of Insulin signalling by oxidative stress.

BACKGROUND: Existing models of insulin signalling focus on short term dynamics, rather than the longer term dynamics necessary to understand many physiologically relevant behaviours. We have developed a model of insulin signalling in rodent adipocytes that includes both transcriptional feedback through the Forkhead box type O (FOXO) transcription factor, and interaction with oxidative stress, in addition to the core pathway. In the model Reactive Oxygen Species are both generated endogenously and can be applied externally. They regulate signalling though inhibition of phosphatases and induction of the activity of Stress Activated Protein Kinases, which themselves modulate feedbacks to insulin signalling and FOXO. RESULTS: Insulin and oxidative stress combined produce a lower degree of activation of insulin signalling than insulin alone. Fasting (nutrient withdrawal) and weak oxidative stress upregulate antioxidant defences while stronger oxidative stress leads to a short term activation of insulin signalling but if prolonged can have other effects including degradation of the insulin receptor substrate (IRS1) and FOXO. At high insulin the protective effect of moderate oxidative stress may disappear. CONCLUSION: Our model is consistent with a wide range of experimental data, some of which is difficult to explain. Oxidative stress can have effects that are both up- and down-regulatory on insulin signalling. Our model therefore shows the complexity of the interaction between the two pathways and highlights the need for such integrated computational models to give insight into the dysregulation of insulin signalling along with more data at the individual level.A complete SBML model file can be downloaded from BIOMODELS (https://www.ebi.ac.uk/biomodels-main) with unique identifier MODEL1212210000.Other files and scripts are available as additional files with this journal article and can be downloaded from https://github.com/graham1034/Smith2012_insulin_signalling.