Heterogeneity of increased biological age in type 2 diabetes correlates with differential tissue DNA methylation, biological variables, and pharmacological treatments.

Biological age (BA) closely depicts age-related changes at a cellular level. Type 2 diabetes mellitus (T2D) accelerates BA when calculated using clinical biomarkers, but there is a large spread in the magnitude of individuals' age acceleration in T2D suggesting additional factors contributing to BA. Additionally, it is unknown whether BA can be changed with treatment. We hypothesized that potential determinants of the heterogeneous BA distribution in T2D could be due to differential tissue aging as reflected at the DNA methylation (DNAm) level, or biological variables and their respective therapeutic treatments. Publicly available DNAm samples were obtained to calculate BA using the DNAm phenotypic age (DNAmPhenoAge) algorithm. DNAmPhenoAge showed age acceleration in T2D samples of whole blood, pancreatic islets, and liver, but not in adipose tissue or skeletal muscle. Analysis of genes associated with differentially methylated CpG sites found a significant correlation between eight individual CpG methylation sites and gene expression. Clinical biomarkers from participants in the NHANES 2017-2018 and ACCORD cohorts were used to calculate BA using the Klemera and Doubal (KDM) method. Cardiovascular and glycemic biomarkers associated with increased BA while intensive blood pressure and glycemic management reduced BA to CA levels, demonstrating that accelerated BA can be restored in the setting of T2D.

Spatial mapping of cellular senescence: emerging challenges and opportunities.

Cellular senescence is a well-established driver of aging and age-related diseases. There are many challenges to mapping senescent cells in tissues such as the absence of specific markers and their relatively low abundance and vast heterogeneity. Single-cell technologies have allowed unprecedented characterization of senescence; however, many methodologies fail to provide spatial insights. The spatial component is essential, as senescent cells communicate with neighboring cells, impacting their function and the composition of extracellular space. The Cellular Senescence Network (SenNet), a National Institutes of Health (NIH) Common Fund initiative, aims to map senescent cells across the lifespan of humans and mice. Here, we provide a comprehensive review of the existing and emerging methodologies for spatial imaging and their application toward mapping senescent cells. Moreover, we discuss the limitations and challenges inherent to each technology. We argue that the development of spatially resolved methods is essential toward the goal of attaining an atlas of senescent cells.

Expression of retrotransposons contributes to aging in Drosophila.

Retrotransposons are a class of transposable elements capable of self-replication and insertion into new genomic locations. Across species, the mobilization of retrotransposons in somatic cells has been suggested to contribute to the cell and tissue functional decline that occurs during aging. Retrotransposons are broadly expressed across cell types, and de novo insertions have been observed to correlate with tumorigenesis. However, the extent to which new retrotransposon insertions occur during normal aging and their effect on cellular and animal function remains understudied. Here, we use a single nucleus whole genome sequencing approach in Drosophila to directly test whether transposon insertions increase with age in somatic cells. Analyses of nuclei from thoraces and indirect flight muscles using a newly developed pipeline, Retrofind, revealed no significant increase in the number of transposon insertions with age. Despite this, reducing the expression of two different retrotransposons, 412 and Roo, extended lifespan, but did not alter indicators of health such as stress resistance. This suggests a key role for transposon expression and not insertion in regulating longevity. Transcriptomic analyses revealed similar changes to gene expression in 412 and Roo knockdown flies and highlighted changes to genes involved in proteolysis and immune function as potential contributors to the observed changes in longevity. Combined, our data show a clear link between retrotransposon expression and aging.

LINE-1 retrotransposon expression in cancerous, epithelial and neuronal cells revealed by 5' single-cell RNA-Seq.

LINE-1 retrotransposons are sequences capable of copying themselves to new genomic loci via an RNA intermediate. New studies implicate LINE-1 in a range of diseases, especially in the context of aging, but without an accurate understanding of where and when LINE-1 is expressed, a full accounting of its role in health and disease is not possible. We therefore developed a method-5' scL1seq-that makes use of a widely available library preparation method (10x Genomics 5' single cell RNA-seq) to measure LINE-1 expression in tens of thousands of single cells. We recapitulated the known pattern of LINE-1 expression in tumors-present in cancer cells, absent from immune cells-and identified hitherto undescribed LINE-1 expression in human epithelial cells and mouse hippocampal neurons. In both cases, we saw a modest increase with age, supporting recent research connecting LINE-1 to age related diseases.

Single-cell transcriptomics of peripheral blood in the aging mouse.

Compositional and transcriptional changes in the hematopoietic system have been used as biomarkers of immunosenescence and aging. Here, we use single-cell RNA-sequencing to study the aging peripheral blood in mice and characterize the changes in cell-type composition and transcriptional profiles associated with age. We identified 17 clusters from a total of 14,588 single cells. We detected a general upregulation of antigen processing and presentation and chemokine signaling pathways and a downregulation of genes involved in ribosome pathways with age. In old peripheral blood, we also observed an increased percentage of cells expressing senescence markers (Cdkn1a, and Cdkn2a). In addition, we detected a cluster of activated T cells exclusively found in old blood, with lower expression of Cd28 and higher expression of Bcl2 and Cdkn2a, suggesting that the cells are senescent and resistant to apoptosis.

Single-cell analysis of the aging female mouse hypothalamus.

Alterations in metabolism, sleep patterns, body composition, and hormone status are all key features of aging. While the hypothalamus is a well-conserved brain region that controls these homeostatic and survival-related behaviors, little is known about the intrinsic features of hypothalamic aging. Here, we perform single nuclei RNA-sequencing of 40,064 hypothalamic nuclei from young and aged female mice. We identify cell type-specific signatures of aging in neuronal subtypes as well as astrocytes and microglia. We uncover changes in cell types critical for metabolic regulation and body composition, and in an area of the hypothalamus linked to cognition. Our analysis also reveals an unexpected female-specific feature of hypothalamic aging: the master regulator of X-inactivation, Xist, is elevated with age, particularly in hypothalamic neurons. Moreover, using machine learning, we show that levels of X-chromosome genes, and Xist itself, can accurately predict cellular age. This study identifies critical cell-specific changes of the aging hypothalamus in mammals, and uncovers a potential marker of neuronal aging in females.

The functional impact of nuclear reorganization in cellular senescence.

Cellular senescence is the irreversible cell cycle arrest in response to DNA damage. Because senescent cells accumulate with age and contribute to chronic inflammation, they are promising therapeutic targets for healthspan extension. The senescent phenotype can vary depending on cell type and on the specific insults that induce senescence. This variability is also reflected in the extensive remodeling of the genome organization within the nucleus of senescent cells. Here, we give an overview of the nuclear changes that occur in different forms of senescence, including changes to chromatin state and composition and to the three-dimensional organization of the genome, as well as alterations to the nuclear envelope and to the accessibility of repetitive genomic regions. Many of these changes are shared across all forms of senescence, implicating nuclear organization as a fundamental driver of the senescent state and of how senescent cells interact with the surrounding tissue.

Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner.

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease.

Toward a Three-Dimensional Chromosome Shape Alphabet.

The study of the three-dimensional (3D) structure of chromosomes-the largest macromolecules in biology-is one of the most challenging to date in structural biology. Here, we develop a novel representation of 3D chromosome structures, as sequences of shape letters from a finite shape alphabet, which provides a compact and efficient way to analyze ensembles of chromosome shape data, akin to the analysis of texts in a language by using letters. We construct a Chromosome Shape Alphabet from an ensemble of chromosome 3D structures inferred from Hi-C data-via SIMBA3D or other methods-by segmenting curves based on topologically associating domains (TADs) boundaries, and by clustering all TADs' 3D structures into groups of similar shapes. The median shapes of these groups, with some pruning and processing, form the Chromosome Shape Letters (CSLs) of the alphabet. We provide a proof of concept for these CSLs by reconstructing independent test curves by using only CSLs (and corresponding transformations) and comparing these reconstructions with the original curves. Finally, we demonstrate how CSLs can be used to summarize shapes in an ensemble of chromosome 3D structures by using generalized sequence logos.

Whole-body senescent cell clearance alleviates age-related brain inflammation and cognitive impairment in mice.

Cellular senescence is characterized by an irreversible cell cycle arrest and a pro-inflammatory senescence-associated secretory phenotype (SASP), which is a major contributor to aging and age-related diseases. Clearance of senescent cells has been shown to improve brain function in mouse models of neurodegenerative diseases. However, it is still unknown whether senescent cell clearance alleviates cognitive dysfunction during the aging process. To investigate this, we first conducted single-nuclei and single-cell RNA-seq in the hippocampus from young and aged mice. We observed an age-dependent increase in p16Ink4a senescent cells, which was more pronounced in microglia and oligodendrocyte progenitor cells and characterized by a SASP. We then aged INK-ATTAC mice, in which p16Ink4a -positive senescent cells can be genetically eliminated upon treatment with the drug AP20187 and treated them either with AP20187 or with the senolytic cocktail Dasatinib and Quercetin. We observed that both strategies resulted in a decrease in p16Ink4a exclusively in the microglial population, resulting in reduced microglial activation and reduced expression of SASP factors. Importantly, both approaches significantly improved cognitive function in aged mice. Our data provide proof-of-concept for senolytic interventions' being a potential therapeutic avenue for alleviating age-associated cognitive impairment.

FOXO3 regulates a common genomic program in aging and glioblastoma stem cells.

Background: Glioblastoma (GBM) is an aggressive, age-associated malignant glioma that contains populations of cancer stem cells. These glioma stem cells (GSCs) evade therapeutic interventions and repopulate tumors due to their existence in a slowly cycling quiescent state. Although aging is well known to increase cancer initiation, the extent to which the mechanisms supporting GSC tumorigenicity are related to physiological aging remains unknown. Aims: Here, we investigate the transcriptional mechanisms by which Forkhead Box O3 (FOXO3), a transcriptional regulator that promotes healthy aging, affects GSC function and the extent to which FOXO3 transcriptional networks are dysregulated in aging and GBM. Methods and results: We performed transcriptome analysis of clinical GBM tumors and observed that high FOXO3 activity is associated with gene expression signatures of stem cell quiescence, reduced oxidative metabolism, and improved patient outcomes. Consistent with these findings, we show that elevated FOXO3 activity significantly reduces the proliferation of GBM-derived GSCs. Using RNA-seq, we find that functional ablation of FOXO3 in GSCs rewires the transcriptional circuitry associated with metabolism, epigenetic stability, quiescence, and differentiation. Since FOXO3 has been implicated in healthy aging, we then investigated the extent to which it regulates common transcriptional programs in aging neural stem cells (NSCs) and GSCs. We uncover a shared transcriptional program and, most strikingly, find that FOXO3-regulated pathways are associated with altered mitochondrial functions in both aging and GBM. Conclusions: This work identifies a FOXO-associated transcriptional program that correlates between GSCs and aging NSCs and is enriched for metabolic and stemness pathways connected with GBM and aging.

Inflammaging in Endemic Areas for Infectious Diseases.

Immunosenescence is marked by a systemic process named inflammaging along with a series of defects in the immunological activity that results in poor responses to infectious agents and to vaccination. Inflammaging, a state of low-grade chronic inflammation, usually leads to chronic inflammatory diseases and frailty in the elderly. However, some elderly escape from frailty and reach advanced age free of the consequences of inflammaging. This process has been called immunological remodeling, and it is the hallmark of healthy aging as described in the studies of centenarians in Italy. The biological markers of healthy aging are still a matter of debate, and the studies on the topic have focused on inflammatory versus remodeling processes and molecules. The sub-clinical inflammatory status associated with aging might be a deleterious event for populations living in countries where chronic infectious diseases are not prevalent. Nevertheless, in other parts of the world where they are, two possibilities may occur. Inflammatory responses may have a protective effect against these infectious agents. At the same time, the long-term consequences of protective immune responses during chronic infections may result in accelerated immunosenescence in these individuals. Therefore, the biological markers of healthy aging can vary according to environmental, cultural, and geographical settings that reflect worldwide, and in a non-biased, non-westernized perspective, the changes that we experience regarding our contacts with microorganisms and the outcomes of such contacts.

Author Correction: L1 drives IFN in senescent cells and promotes age-associated inflammation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bayesian Estimation of Three-Dimensional Chromosomal Structure from Single-Cell Hi-C Data.

The problem of three-dimensional (3D) chromosome structure inference from Hi-C data sets is important and challenging. While bulk Hi-C data sets contain contact information derived from millions of cells and can capture major structural features shared by the majority of cells in the sample, they do not provide information about local variability between cells. Single-cell Hi-C can overcome this problem, but contact matrices are generally very sparse, making structural inference more problematic. We have developed a Bayesian multiscale approach, named Structural Inference via Multiscale Bayesian Approach, to infer 3D structures of chromosomes from single-cell Hi-C while including the bulk Hi-C data and some regularization terms as a prior. We study the landscape of solutions for each single-cell Hi-C data set as a function of prior strength and demonstrate clustering of solutions using data from the same cell.

Single cell RNA-seq in the sea urchin embryo show marked cell-type specificity in the Delta/Notch pathway.

Sea urchin embryos are excellent for in vivo functional studies because of their transparency and tractability in manipulation. They are also favorites for pharmacological approaches since they develop in an aquatic environment and addition of test substances is straightforward. A concern in many pharmacological tests though is the potential for pleiotropic effects that confound the conclusions drawn from the results. Precise cellular interpretations are often not feasible because the impact of the perturbant is not known. Here we use single-cell mRNA (messenger RNA) sequencing as a metric of cell types in the embryo and to determine the selectivity of two commonly used inhibitors, one each for the Wnt and the Delta-Notch pathways, on these nascent cell types. We identified 11 distinct cell types based on mRNA profiling, and that the cell lineages affected by Wnt and Delta/Notch inhibition were distinct from each other. These data support specificity and distinct effects of these signaling pathways in the embryo and illuminate how these conserved pathways selectively regulate cell lineages at a single cell level. Overall, we conclude that single cell RNA-seq analysis in this embryo is revealing of the cell types present during development, of the changes in the gene regulatory network resulting from inhibition of various signaling pathways, and of the selectivity of these pathways in influencing developmental trajectories.

Notch Signaling Mediates Secondary Senescence.

Oncogene-induced senescence (OIS) is a tumor suppressive response to oncogene activation that can be transmitted to neighboring cells through secreted factors of the senescence-associated secretory phenotype (SASP). Currently, primary and secondary senescent cells are not considered functionally distinct endpoints. Using single-cell analysis, we observed two distinct transcriptional endpoints, a primary endpoint marked by Ras and a secondary endpoint marked by Notch activation. We find that secondary oncogene-induced senescence in vitro and in vivo requires Notch, rather than SASP alone, as previously thought. Moreover, Notch signaling weakens, but does not abolish, SASP in secondary senescence. Global transcriptomic differences, a blunted SASP response, and the induction of fibrillar collagens in secondary senescence point toward a functional diversification between secondary and primary senescence.

LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation.

Mice deficient for SIRT6 exhibit a severely shortened lifespan, growth retardation, and highly elevated LINE1 (L1) activity. Here we report that SIRT6-deficient cells and tissues accumulate abundant cytoplasmic L1 cDNA, which triggers strong type I interferon response via activation of cGAS. Remarkably, nucleoside reverse-transcriptase inhibitors (NRTIs), which inhibit L1 retrotransposition, significantly improved health and lifespan of SIRT6 knockout mice and completely rescued type I interferon response. In tissue culture, inhibition of L1 with siRNA or NRTIs abrogated type I interferon response, in addition to a significant reduction of DNA damage markers. These results indicate that L1 activation contributes to the pathologies of SIRT6 knockout mice. Similarly, L1 transcription, cytoplasmic cDNA copy number, and type I interferons were elevated in the wild-type aged mice. As sterile inflammation is a hallmark of aging, we propose that modulating L1 activity may be an important strategy for attenuating age-related pathologies.

L1 drives IFN in senescent cells and promotes age-associated inflammation.

Retrotransposable elements are deleterious at many levels, and the failure of host surveillance systems for these elements can thus have negative consequences. However, the contribution of retrotransposon activity to ageing and age-associated diseases is not known. Here we show that during cellular senescence, L1 (also known as LINE-1) retrotransposable elements become transcriptionally derepressed and activate a type-I interferon (IFN-I) response. The IFN-I response is a phenotype of late senescence and contributes to the maintenance of the senescence-associated secretory phenotype. The IFN-I response is triggered by cytoplasmic L1 cDNA, and is antagonized by inhibitors of the L1 reverse transcriptase. Treatment of aged mice with the nucleoside reverse transcriptase inhibitor lamivudine downregulated IFN-I activation and age-associated inflammation (inflammaging) in several tissues. We propose that the activation of retrotransposons is an important component of sterile inflammation that is a hallmark of ageing, and that L1 reverse transcriptase is a relevant target for the treatment of age-associated disorders.

The three-dimensional organization of the genome in cellular senescence and age-associated diseases.

Recent advances in genomics and imaging technologies have increased our ability to interrogate the 3D conformation of chromosomes and to better understand principles of organization and dynamics, as well as how their alteration can lead to disease. In this review we describe how these technologies have shed new light into the role of the 3D organization of the genome in defining cellular states in aging and age-associated diseases. We compare the genomic organization in cellular senescence and cancer, discuss the role of the lamina in maintaining the structural and functional integrity of the genome, and we highlight the recent findings on how this organization breaks down in disease states.

Cell-free DNA as a biomarker of aging.

Cell-free DNA (cfDNA) is present in the circulating plasma and other body fluids and is known to originate mainly from apoptotic cells. Here, we provide the first in vivo evidence of global and local chromatin changes in human aging by analyzing cfDNA from the blood of individuals of different age groups. Our results show that nucleosome signals inferred from cfDNA are consistent with the redistribution of heterochromatin observed in cellular senescence and aging in other model systems. In addition, we detected a relative cfDNA loss at several genomic locations, such as transcription start and termination sites, 5'UTR of L1HS retrotransposons and dimeric AluY elements with age. Our results also revealed age and deteriorating health status correlate with increased enrichment of signals from cells in different tissues. In conclusion, our results show that the sequencing of circulating cfDNA from human blood plasma can be used as a noninvasive methodology to study age-associated changes to the epigenome in vivo.