Probes for CDKN2A

The placenta is essential for reproductive success. The murine placenta includes polyploid giant cells that are crucial for its function. Polyploidy occurs broadly in nature but the regulators and significance in the placenta are unknown. We discovered that many murine placental cell types are polyploid. We identified factors that license polyploidy using single-cell RNA seq. c-Myc is a key regulator of polyploidy and placental development and is required for multiple rounds of DNA replication, likely via endocycles, in trophoblast giant cells. Furthermore, c-MYC supports the expression of DNA replication and nucleotide biosynthesis genes along with ribosomal RNA. Increased DNA damage and senescence occur in trophoblast giant cells without c-Myc, accompanied by senescence in the neighboring maternal decidua. These data reveal c-Myc is essential for polyploidy to support normal placental development, thereby preventing premature senescence. Our study combined with the literature suggests c-Myc is an evolutionarily conserved regulator of polyploidy.

The contribution of cellular senescence to the behavioral changes observed in the elderly remains elusive. Here, we observed that aging is associated with a decline in protein phosphatase 2A (PP2A) activity in the brains of zebrafish and mice. Moreover, drugs activating PP2A reversed age-related behavioral changes. We developed a transgenic zebrafish model to decrease PP2A activity in the brain through knockout of the ppp2r2c gene encoding a regulatory subunit of PP2A. Mutant fish exhibited the behavioral phenotype observed in old animals and premature accumulation of neural cells positive for markers of cellular senescence, including senescence-associated β-galactosidase, elevated levels cdkn2a/b, cdkn1a, senescence-associated secretory phenotype gene expression, and an increased level of DNA damage signaling. The behavioral and cell senescence phenotypes were reversed in mutant fish through treatment with the senolytic ABT263 or diverse PP2A activators as well as through cdkn1a or tp53 gene ablation. Senomorphic function of PP2A activators was demonstrated in mouse primary neural cells with downregulated Ppp2r2c. We conclude that PP2A reduction leads to neural cell senescence thereby contributing to age-related behavioral changes and that PP2A activators have senotherapeutic properties against deleterious behavioral effects of brain aging.

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by the cell cycle inhibitors Cdkn2a, Cdkn1a, and Trp53. Senescent cells are implicated in chronic diseases and tissue repair through their increased secretion of pro-inflammatory factors known as the senescence-associated secretory phenotype (SASP). Here, we use spatial transcriptomics and single-cell RNA sequencing (scRNAseq) to demonstrate that cells displaying senescent characteristics are "transiently" present within regenerating skeletal muscle and within the muscles of D2-mdx mice, a model of Muscular Dystrophy. Following injury, multiple cell types including macrophages and fibrog-adipogenic progenitors (FAPs) upregulate senescent features such as senescence pathway genes, SASP factors, and senescence-associated beta-gal (SA-β-gal) activity. Importantly, when these cells were removed with ABT-263, a senolytic compound, satellite cells are reduced, and muscle fibers were impaired in growth and myonuclear accretion. These results highlight that an "acute" senescent phenotype facilitates regeneration similar to skin and neonatal myocardium.

Progressive loss of muscle mass and function due to muscle fiber atrophy and loss in the elderly and chronically ill is now defined as sarcopenia. It is a major contributor to loss of independence, disability, need of long-term care as well as overall mortality. Sarcopenia is a heterogenous disease and underlying mechanisms are not completely understood. Here, we newly identified and used Tmem158, alongside Cdkn1a, as relevant senescence and denervation markers (SDMs), associated with muscle fiber atrophy. Subsequent application of laser capture microdissection (LCM) and RNA analyses revealed age- and disease-associated differences in gene expression and alternative splicing patterns in a rodent sarcopenia model. Of note, genes exhibiting such differential alternative splicing (DAS) are mainly involved in the contractile function of the muscle. Many of these splicing events are also found in a mouse model for myotonic dystrophy type 1 (DM1), underscoring the premature aging phenotype of this disease. We propose to add differential alternative splicing to the hallmarks of aging.

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by cell cycle inhibitory proteins such as p16, p21 and p53. When cells enter senescence, they secrete a host of proinflammatory factors known as the senescence associated secretory phenotype which has deleterious effects on surrounding cells and tissues. Little is known of the role of senescence in Duchenne Muscular Dystrophy (DMD), the fatal X-linked neuromuscular disorder typified by chronic inflammation, extracellular matrix remodeling and a progressive loss in muscle mass and function. Here, we demonstrate using C57-mdx (8-week-old) and D2-mdx mice (4-week and 8-week-old), two mouse models of DMD, that cells displaying canonical markers of senescence are found within skeletal muscle. 8-week-old D2-mdx mice, which display severe muscle pathology, had greater numbers of senescent cells associated with areas of inflammation which were mostly Cdkn1a-positive macrophages while in C57-mdx muscle, senescent populations were endothelial cells and macrophages localized to newly regenerated myofibers. Interestingly, this pattern was similar to cardiotoxin (CTX)-injured wildtype (WT) muscle which experienced a transient senescent response. Dystrophic muscle demonstrated significant upregulations in senescence pathway genes (Cdkn1a (p21), Cdkn2a (p16INK4A), Trp53 (p53)) which correlated with the quantity of SA-b-Gal-positive cells. These results highlight an underexplored role for cellular senescence in murine dystrophic muscle.