Loss of functionality during aging of cells and organisms is caused and accompanied by altered cell-to-cell communication and signalling. One factor thereby is the chronic accumulation of senescent cells and the concomitant senescence-associated secretory phenotype (SASP) that contributes to microenvironment remodelling and a pro-inflammatory status. While protein based SASP factors have been well characterized, little is known about small extracellular vesicles (sEVs) and their miRNA cargo. Therefore, we analysed secretion of sEVs from senescent human dermal fibroblasts and catalogued the therein contained miRNAs. We observed a four-fold increase of sEVs, with a concomitant increase of >80% of all cargo miRNAs. The most abundantly secreted miRNAs were predicted to collectively target mRNAs of pro-apoptotic proteins, and indeed, senescent cell derived sEVs exerted anti-apoptotic activity. In addition, we identified senescence-specific differences in miRNA composition of sEVs, with an increase of miR-23a-5p and miR-137 and a decrease of miR-625-3p, miR-766-3p, miR-199b-5p, miR-381-3p, miR-17-3p. By correlating intracellular and sEV-miRNAs, we identified miRNAs selectively retained in senescent cells (miR-21-3p and miR-17-3p) or packaged specifically into senescent cell derived sEVs (miR-15b-5p and miR-30a-3p). Therefore, we suggest sEVs and their miRNA cargo to be novel, members of the SASP that are selectively secreted or retained in cellular senescence.

There is increasing evidence that senescent cells are a driving force behind many age-related pathologies and that their selective elimination increases the life- and healthspan of mice. Senescent cells negatively affect their surrounding tissue by losing their cell specific functionality and by secreting a pro-tumorigenic and pro-inflammatory mixture of growth hormones, chemokines, cytokines and proteases, termed the senescence-associated secretory phenotype (SASP). Here we identified an extract from the plant Solidago virgaurea subsp. alpestris, which exhibited weak senolytic activity, delayed the acquisition of a senescent phenotype and induced a papillary phenotype with improved functionality in human dermal fibroblasts. When administered to stress-induced premature senescent fibroblasts, this extract changed their global mRNA expression profile and particularly reduced the expression of various SASP components, thereby ameliorating the negative influence on nearby cells. Thus, the investigated plant extract represents a promising possibility to block age-related loss of tissue functionality.

Extracellular vesicles (EVs) and their miRNA cargo are intercellular communicators transmitting their pleiotropic messages between different cell types, tissues, and body fluids. Recently, they have been reported to contribute to skin homeostasis and were identified as members of the senescence-associated secretory phenotype of human dermal fibroblasts. However, the role of EV-miRNAs in paracrine signaling during skin aging is yet unclear. Here we provide evidence for the existence of small EVs in the human skin and dermal interstitial fluid using dermal open flow microperfusion and show that EVs and miRNAs are transferred from dermal fibroblasts to epidermal keratinocytes in 2D cell culture and in human skin equivalents. We further show that the transient presence of senescent fibroblast derived small EVs accelerates scratch closure of epidermal keratinocytes, whereas long-term incubation impairs keratinocyte differentiation in vitro. Finally, we identify vesicular miR-23a-3p, highly secreted by senescent fibroblasts, as one contributor of the EV-mediated effect on keratinocytes in in vitro wound healing assays. To summarize, our findings support the current view that EVs and their miRNA cargo are members of the senescence-associated secretory phenotype and, thus, regulators of human skin homeostasis during aging.

Skin aging is driven by intrinsic and extrinsic factors impacting on skin functionality with progressive age. One factor of this multifaceted process is cellular senescence, as it has recently been identified to contribute to a declining tissue functionality in old age. In the skin, senescent cells have been found to markedly accumulate with age, and thus might impact directly on skin characteristics. Especially the switch from young, extracellular matrix-building fibroblasts to a senescence-associated secretory phenotype (SASP) could alter the microenvironment in the skin drastically and therefore promote skin aging. In order to study the influence of senescence in human skin, 3D organotypic cultures are a well-suited model system. However, only few "aged" skinequivalent (SE) models are available, requiring complex and long-term experimental setups. Here, we adapted a previously published full-thickness SE model by seeding increasing ratios of stress-induced premature senescent versus normal fibroblasts into the collagen matrix, terming these SE "senoskin". Immunohistochemistry stainings revealed a shift in the balance between proliferation (Ki67) and differentiation (Keratin 10 and Filaggrin) of keratinocytes within our senoskin equivalents, as well as partial impairment of skin barrier function and changed surface properties. Monitoring of cytokine levels of known SASP factors confirmedly showed an upregulation in 2D cultures of senescent cells and at the time of seeding into the skin equivalent. Surprisingly, we find a blunted response of cytokines in the senoskin equivalent over time during 3D differentiation.

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Defective DNA repair is widely acknowledged to negatively impact on healthy aging, since mutations in DNA repair factors lead to accelerated and premature aging. However, the opposite, namely if improved DNA repair will also increase the life or health span is less clear, and only few studies have tested if overexpression of DNA repair factors modulates life and health span in cells or organisms. Recently, we identified and characterized SNEVhPrp19/hPso4, a protein that plays a role in DNA repair and pre-mRNA splicing, and observed a doubling of the replicative life span upon ectopic overexpression, accompanied by lower basal DNA damage and apoptosis levels as well as an increased resistance to oxidative stress. Here we find that SNEVhPrp19/hPso4 is phosphorylated at S149 in an ataxia telangiectasia mutated protein (ATM)-dependent manner in response to oxidative stress and DNA double strand break inducing agents. By overexpressing wild-type SNEVhPrp19/hPso4 and a phosphorylation-deficient point-mutant, we found that S149 phosphorylation is necessary for mediating the resistance to apoptosis upon oxidative stress and is partially necessary for elongating the cellular life span. Therefore, ATM dependent phosphorylation of SNEVhPrp19/hPso4 upon DNA damage or oxidative stress might represent a novel axis capable of modulating cellular life span.

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