SenNet recommendations for detecting senescent cells in different tissues.

Once considered a tissue culture-specific phenomenon, cellular senescence has now been linked to various biological processes with both beneficial and detrimental roles in humans, rodents and other species. Much of our understanding of senescent cell biology still originates from tissue culture studies, where each cell in the culture is driven to an irreversible cell cycle arrest. By contrast, in tissues, these cells are relatively rare and difficult to characterize, and it is now established that fully differentiated, postmitotic cells can also acquire a senescence phenotype. The SenNet Biomarkers Working Group was formed to provide recommendations for the use of cellular senescence markers to identify and characterize senescent cells in tissues. Here, we provide recommendations for detecting senescent cells in different tissues based on a comprehensive analysis of existing literature reporting senescence markers in 14 tissues in mice and humans. We discuss some of the recent advances in detecting and characterizing cellular senescence, including molecular senescence signatures and morphological features, and the use of circulating markers. We aim for this work to be a valuable resource for both seasoned investigators in senescence-related studies and newcomers to the field.

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.

Hydroxycarbamide effects on DNA methylation and gene expression in myeloproliferative neoplasms.

Hydroxycarbamide (HC, hydroxyurea) is a cytoreductive drug inducing cell cycle blockade. However, emerging evidence suggests that HC plays a role in the modulation of transcription through the activity of transcription factors and DNA methylation. Examining the global mechanism of action of HC in the context of myeloproliferative neoplasms (MPNs), for which HC is the first-line treatment, will provide a better understanding of its molecular effects. To explore the effects of HC genome-wide, transcriptomic analyses were performed on two clinically relevant cell types at different stages of differentiation treated with HC in a murine MPN model. This study was replicated in MPN patients by profiling genome-wide gene expression and DNA methylation using patient blood samples collected longitudinally, before and following HC exposure. The effects of HC on the transcriptome were not only associated with cell cycle interruption but also with hematopoietic functions. Moreover, a group of genes were restored to normal expression levels in murine hematopoietic stem cells (HSCs) following drug treatment, including the master regulator of hematopoiesis, RUNX1 In humans, HC significantly modifies DNA methylation levels in HSCs at several distal regulatory regions, which we show to be associated with SPI1 binding sites and at the SPI1 locus itself. We have identified novel targets of HC that include pivotal transcription factors involved in hematopoiesis, and for the first time we report abnormal methylation patterns in MPN patients at the master regulator gene SPI1 and its distal binding sites, which HC is able to restore to normal levels.

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.

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.