Cellular senescence is a potent tumor-suppressive mechanism that arrests cell proliferation and has been linked to aging. However, studies of senescence have been impeded by the lack of simple, ...exclusive biomarkers of the senescent state. Senescent cells develop characteristic morphological changes, which include enlarged and often irregular nuclei and chromatin reorganization. Because alterations to the nuclear lamina can affect both nuclear morphology and gene expression, we examined the nuclear lamina of senescent cells. We show here than lamin B1 is lost from primary human and murine cell strains when they are induced to senesce by DNA damage, replicative exhaustion, or oncogene expression. Lamin B1 loss did not depend on the p38 mitogen-activated protein kinase, nuclear factor-κB, ataxia telangiectasia-mutated kinase, or reactive oxygen species signaling pathways, which are positive regulators of senescent phenotypes. However, activation of either the p53 or pRB tumor suppressor pathway was sufficient to induce lamin B1 loss. Lamin B1 declined at the mRNA level via a decrease in mRNA stability rather than by the caspase-mediated degradation seen during apoptosis. Last, lamin B1 protein and mRNA declined in mouse tissue after senescence was induced by irradiation. Our findings suggest that lamin B1 loss can serve as biomarker of senescence both in culture and in vivo.
Chronic inflammation is associated with aging and plays a causative role in several age-related diseases such as cancer, atherosclerosis and osteoarthritis. The source of this chronic inflammation is ...often attributed to the progressive activation of immune cells over time. However, recent studies have shown that the process of cellular senescence, a tumor suppressive stress response that is also associated with aging, entails a striking increase in the secretion of proinflammatory proteins and might be an important additional contributor to chronic inflammation. Here, we list the secreted factors that make up the proinflammatory phenotype of senescent cells and describe the impact of these factors on tissue homeostasis. We also summarize the cellular pathways/processes that are known to regulate this phenotype – namely, the DNA damage response, microRNAs, key transcription factors and kinases and chromatin remodeling.
Functional modeling of many adult epithelia is limited by the difficulty in maintaining relevant stem cell populations in culture. Here, we show that dual inhibition of SMAD signaling pathways ...enables robust expansion of primary epithelial basal cell populations. We find that TGFβ/BMP/SMAD pathway signaling is strongly activated in luminal and suprabasal cells of several epithelia, but suppressed in p63+ basal cells. In airway epithelium, SMAD signaling promotes differentiation, and its inhibition leads to stem cell hyperplasia. Using dual SMAD signaling inhibition in a feeder-free culture system, we have been able to expand airway basal stem cells from multiple species. Expanded cells can produce functional airway epithelium physiologically responsive to clinically relevant drugs, such as CFTR modulators. This approach is effective for the clonal expansion of single human cells and for basal cell populations from epithelial tissues from all three germ layers and therefore may be broadly applicable for modeling of epithelia.
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•SMAD activity is active in suprabasal cells but is weaker in basal epithelial cells•SMAD signaling activity correlates with mucociliary differentiation in the airway•Dual TGFβ/BMP inhibition prevents spontaneous differentiation in culture•Dual TGFβ/BMP inhibition allows prolonged culture of diverse epithelial basal cells
Mou et al. show that small-molecule-mediated SMAD signaling inhibition allows prolonged feeder-free culture of diverse functional epithelial basal stem cells in a 2D format. This methodology provides a facile patient-specific epithelial disease modeling platform, as shown by the expansion of airway epithelium from non-invasively obtained specimens from cystic fibrosis patients.
Telomere maintenance by telomerase is impaired in the stem cell disease dyskeratosis congenita and during human aging. Telomerase depends upon a complex pathway for enzyme assembly, localization in ...Cajal bodies, and association with telomeres. Here, we identify the chaperonin CCT/TRiC as a critical regulator of telomerase trafficking using a high-content genome-wide siRNA screen in human cells for factors required for Cajal body localization. We find that TRiC is required for folding the telomerase cofactor TCAB1, which controls trafficking of telomerase and small Cajal body RNAs (scaRNAs). Depletion of TRiC causes loss of TCAB1 protein, mislocalization of telomerase and scaRNAs to nucleoli, and failure of telomere elongation. DC patient-derived mutations in TCAB1 impair folding by TRiC, disrupting telomerase function and leading to severe disease. Our findings establish a critical role for TRiC-mediated protein folding in the telomerase pathway and link proteostasis, telomere maintenance, and human disease.
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•Telomere maintenance requires assembly and trafficking of telomerase•Proteostasis factor TRiC folds the telomerase cofactor TCAB1•Loss of TRiC causes telomerase mislocalization and telomere elongation failure•TCAB1 mutations in patients disrupt TRiC-mediated folding, leading to disease
The assembly and localization of telomerase and the consequent control of telomere elongation are found to be dependent upon the chaperonin TRiC. Mutations in the telomerase cofactor TCAB1 that impair its folding by TRiC cause dyskeratosis congenita, thus highlighting a proteostatic level of control of telomere function.
Telomere synthesis in cancer cells and stem cells involves trafficking of telomerase to Cajal bodies, and telomerase is thought to be recruited to telomeres through interactions with telomere-binding ...proteins. Here, we show that the OB-fold domain of the telomere-binding protein TPP1 recruits telomerase to telomeres through an association with the telomerase reverse transcriptase TERT. When tethered away from telomeres and other telomere-binding proteins, the TPP1 OB-fold domain is sufficient to recruit telomerase to a heterologous chromatin locus. Expression of a minimal TPP1 OB-fold inhibits telomere maintenance by blocking access of telomerase to its cognate binding site at telomeres. We identify amino acids required for the TPP1-telomerase interaction, including specific loop residues within the TPP1 OB-fold domain and individual residues within TERT, some of which are mutated in a subset of pulmonary fibrosis patients. These data define a potential interface for telomerase-TPP1 interaction required for telomere maintenance and implicate defective telomerase recruitment in telomerase-related disease.
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► Enforced expression of telomerase forms neo-Cajal bodies at telomeres ► TPP1 OB-fold domain recruits telomerase to a heterologous chromatin locus ► TPP1-OB alone sequesters telomerase within Cajal bodies and causes telomere shortening ► OB-fold mutations and some disease mutations in TERT block telomerase recruitment
The shelterin component TPP1 recruits telomerase from storage in Cajal bodies via a direct interaction with the enzyme’s catalytic subunit TERT. Mutations in TERT found associated with an idiopathic lung disease disrupt the interaction and impair telomerase mobilization.
Cellular senescence is a tumor suppressive mechanism that can paradoxically contribute to aging pathologies. Despite evidence of immune clearance in mouse models, it is not known how senescent cells ...(SnCs) persist and accumulate with age or in tumors in individuals. Here, we identify cooperative mechanisms that orchestrate the immunoevasion and persistence of normal and cancer human SnCs through extracellular targeting of natural killer receptor signaling. Damaged SnCs avoid immune recognition through MMPs-dependent shedding of NKG2D-ligands reinforced via paracrine suppression of NKG2D receptor-mediated immunosurveillance. These coordinated immunoediting processes are evident in residual, drug-resistant tumors from cohorts of >700 prostate and breast cancer patients treated with senescence-inducing genotoxic chemotherapies. Unlike in mice, these reversible senescence-subversion mechanisms are independent of p53/p16 and exacerbated in oncogenic RAS-induced senescence. Critically, the p16INK4A tumor suppressor can disengage the senescence growth arrest from the damage-associated immune senescence program, which is manifest in benign nevi lesions where indolent SnCs accumulate over time and preserve a non-pro-inflammatory tissue microenvironment maintaining NKG2D-mediated immunosurveillance. Our study shows how subpopulations of SnCs elude immunosurveillance, and reveals secretome-targeted therapeutic strategies to selectively eliminate -and restore the clearance of- the detrimental SnCs that actively persist after chemotherapy and accumulate at sites of aging pathologies.
Understanding how genetic variation shapes a complex trait relies on accurately quantifying both the additive genetic and genotype–environment interaction effects in an age-dependent manner. We used ...a linear mixed model to quantify diet-dependent genetic contributions to body weight measured through adulthood in diversity outbred female mice under five diets. We observed that heritability of body weight declined with age under all diets, except the 40% calorie restriction diet. We identified 14 loci with age-dependent associations and 19 loci with age- and diet-dependent associations, with many diet-dependent loci previously linked to neurological function and behavior in mice or humans. We found their allelic effects to be dynamic with respect to genomic background, age, and diet, identifying several loci where distinct alleles affect body weight at different ages. These results enable us to more fully understand and predict the effectiveness of dietary intervention on overall health throughout age in distinct genetic backgrounds.
Body weight is one trait influenced by genes, age and environmental factors. Both internal and external environmental pressures are known to affect genetic variation over time. However, it is largely unknown how all factors – including age – interact to shape metabolism and bodyweight.
Wright et al. set out to quantify the interactions between genes and diet in ageing mice and found that the effect of genetics on mouse body weight changes with age. In the experiments, Wright et al. weighed 960 female mice with diverse genetic backgrounds, starting at two months of age into adulthood. The animals were randomized to different diets at six months of age. Some mice had unlimited food access, others received 20% or 40% less calories than a typical mouse diet, and some fasted one or two days per week.
Variations in their genetic background explained about 80% of differences in mice’s weight, but the influence of genetics relative to non-genetic factors decreased as they aged. Mice on the 40% calorie restriction diet were an exception to this rule and genetics accounted for 80% of their weight throughout adulthood, likely due to reduced influence from diet and reduced interactions between diet and genes. Several genes involved in metabolism, neurological function, or behavior, were associated with mouse weight.
The experiments highlight the importance of considering interactions between genetics, environment, and age in determining complex traits like body weight. The results and the approaches used by Wright et al. may help other scientists learn more about how the genetic predisposition to disease changes with environmental stimuli and age.
Behavior and physiology are essential readouts in many studies but have not benefited from the high-dimensional data revolution that has transformed molecular and cellular phenotyping. To address ...this, we developed an approach that combines commercially available automated phenotyping hardware with a systems biology analysis pipeline to generate a high-dimensional readout of mouse behavior/physiology, as well as intuitive and health-relevant summary statistics (resilience and biological age). We used this platform to longitudinally evaluate aging in hundreds of outbred mice across an age range from 3 months to 3.4 years. In contrast to the assumption that aging can only be measured at the limits of animal ability via challenge-based tasks, we observed widespread physiological and behavioral aging starting in early life. Using network connectivity analysis, we found that organism-level resilience exhibited an accelerating decline with age that was distinct from the trajectory of individual phenotypes. We developed a method, Combined Aging and Survival Prediction of Aging Rate (CASPAR), for jointly predicting chronological age and survival time and showed that the resulting model is able to predict both variables simultaneously, a behavior that is not captured by separate age and mortality prediction models. This study provides a uniquely high-resolution view of physiological aging in mice and demonstrates that systems-level analysis of physiology provides insights not captured by individual phenotypes. The approach described here allows aging, and other processes that affect behavior and physiology, to be studied with improved throughput, resolution, and phenotypic scope.
Research on aging requires the ability to measure aging, and therein lies a challenge: it is impossible to measure every molecular, cellular, and physiological change that develops over time, but it ...is difficult to prioritize phenotypes for measurement because it is unclear which biological changes should be considered aspects of aging and, further, which species and environments exhibit “real aging.” Here, I propose a strategy to address this challenge: rather than classify phenotypes as “real aging” or not, conceptualize aging as the set of all age-dependent phenotypes and appreciate that this set and its underlying mechanisms may vary by population. Use automated phenotyping technologies to measure as many age-dependent phenotypes as possible within individuals over time, prioritizing organism-level (i.e., physiological) phenotypes in order to enrich for health relevance. Use those high-dimensional phenotypic data to construct dynamic networks that allow aging to be studied with unprecedented sophistication and rigor.
In this Perspective, Adam Freund focuses on the challenge of measuring aging and demystifies the myth of “real aging.” He re-conceptualizes aging as a set of related phenotypes that may vary by population and advocates approaching their characterization with systems-level tools and analyses. Automated phenotyping technologies enable the measurement of functional aging phenotypes simultaneously and repeatedly, and construction of dynamic networks allows us to interpret how relationships between those phenotypes change over time.
The objective of this paper is to present an investigation of resistance welded thermoplastic joints using stainless steel wire mesh and polyethylene terephthalate (PET) with unidirectional E-glass ...fiber reinforcement. Five weld pressures and six heating element types were investigated to achieve high quality welds. Scanning electron microscope (SEM) images of welded heating element cross-sections were analyzed to provide a phenomenological description of the weld. Welded joints were also subjected to experimental lap shear evaluation and joint strengths were compared with values from the literature to assess the overall quality of welded joints. Characteristic B-basis strengths and corresponding SEM images are compared to lead to a recommended weld pressure of 345 kPa with a stainless steel wire mesh with 100 openings per inch.
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