Cell aging and cellular senescence in skin aging — Recent advances in fibroblast and keratinocyte biology

Journal Pre-proof Cell aging and cellular senescence in skin aging — Recent advances in fibroblast and keratinocyte biology

Florian Gruber, Christopher Kremslehner, Leopold Eckhart, Erwin Tschachler PII:

S0531-5565(19)30575-3

DOI:

https://doi.org/10.1016/j.exger.2019.110780

Reference:

EXG 110780

To appear in:

Experimental Gerontology

Received date:

28 August 2019

Revised date:

7 November 2019

Accepted date:

10 November 2019

Please cite this article as: F. Gruber, C. Kremslehner, L. Eckhart, et al., Cell aging and cellular senescence in skin aging — Recent advances in fibroblast and keratinocyte biology, Experimental Gerontology(2018), https://doi.org/10.1016/j.exger.2019.110780

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© 2018 Published by Elsevier.

Journal Pre-proof Title page information for EXG 110780

Title “Cell aging and cellular senescence in skin aging – recent advances in fibroblast and keratinocyte biology.” Authors: Florian Grubera,b,*,Christopher Kremslehnera,b, Leopold Eckharta, Erwin Tschachlera a) Division for Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria b) Christian Doppler Laboratory for the Biotechnology of Skin Aging, Vienna, Austria

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* Corresponding author at: Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, Leitstelle 7J, 1090 Vienna, Austria

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Email address: [email protected]

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Cell aging and cellular senescence in skin aging – recent advances in fibroblast and keratinocyte biology.

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The aging of the skin is the most visible and obvious manifestation of organismal aging and may serve as a predictor of life expectancy and health. It is, however, also the human desire for long-lasting beauty that further raises interests in the topic, and thus considerable means and efforts are put into studying the mechanisms of skin aging in basic and applied research. Both medical und non-medical interests are of benefit for skin research in general because the results from these studies help to deepen our understanding of the complex molecular, biological, cell signaling, developmental and immunological processes in this organ. In fact, the skin is an ideal organ to observe and analyze the impact of extrinsic and intrinsic drivers of aging. Within the past five years technological advances like lineage tracing of cells in model organisms, intra-vital microscopy, nucleic acid sequencing at the single cell level, and high resolution mass spectrometry have allowed to study aging and senescence of individual skin cells within the tissue context, their signaling and communication, and to derive new hypotheses for experimental studies in vitro. In this short review we will discuss very recent developments that promise to extend the existing knowledge on cell aging and senescence of dermal fibroblasts and epidermal keratinocytes in skin aging.

1 Introduction

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The skin consists of an outer epidermal layer and an inner dermal layer which are connected by the basement membrane. Cooperation of epithelial cells with mesenchymal cells of the dermis forms the skin appendages such as the hair follicles, sebaceous glands, sweat glands and nails. Beneath the dermis is a layer of white adipose tissue, the hypodermis, which contains adipocytes that massively accumulate lipids upon their maturation.

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The epidermis is a multi-layered epithelium that consists mainly of keratinocytes. These maintain a dynamic tissue equilibrium by proliferating in the basal layer where they are attached to the basement membrane, but as soon as they detach, stop proliferation and undergo a terminal differentiation program that strengthens the cytoskeleton, establishes an intercellular diffusion barrier and results in a specialized form of programmed cell death, known as cornification. The cornifying keratinocytes provide an efficient and continuously renewing barrier to the environment (Eckhart and Zeeuwen 2018). The keratinocyte population of the basal layer is maintained by stem cells residing in the bulge region of hair follicles and the inter-follicular epidermis (Page et al. 2013). The most obvious effects of aging on the epidermis are its thinning and an impairment of epidermal barrier recovery after damage which suggests that the re-supplementation chain from the stem cell pool becomes less efficient over time. It is becoming increasingly evident that the microenvironment or niche of stem cells is important for the homeostasis of the epidermis. The epidermal niche was defined as the interplay of cell-extrinsic factors (matrix composition, growth factors, cell-cell interactions) with cell-intrinsic factors like epigenetics and metabolic state both acting on the stem cells and amplifying cells (Rognoni and Watt 2018), and these parameters change upon both chronologic and extrinsic aging. Besides keratinocytes, the epidermis houses organized networks of melanocytes that produce pigment which protects from ultraviolet radiation, and networks of the dendritic antigen presenting cells of the immune system, the Langerhans cells. Apart from being exposed to the aging process as individual cell types, these networks rely on homeostasis of the keratinocyte component of the epidermis to function properly. Disturbance of intercellular coordination between the different cell types contributes to the age-related decline in epidermal immune function and pigment regularity.

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The dermis has a lower cellular density than the epidermis and contains large amounts of extracellular matrix composed of proteins and specialized carbohydrates. It can be roughly divided into a papillary layer that starts at the epidermal basement membrane and is more densely populated by fibroblasts than the lower, reticular dermis, which contains thicker collagen fibers. With increasing age, the ECM becomes less organized due to altered ECM protein turnover or accumulating post-translational modifications. The dominant dermal cells are fibroblasts which derive in the development of the mouse dermis from at least two cell lineages (Driskell et al. 2013) and dynamically change their identity during aging, as we will discuss below. The dermis houses also nerve endings and Schwann cells, endothelial cells organized in vessels, pericytes, mast cells, tissue macrophages and other cells of the immune system (McGrath and Uitto 2016). The adult dermis is regarded a post-mitotic tissue settled by specialized, rarely proliferating cells that are expected to rely more on functional damage repair and stress response mechanisms, whereas in the epidermis keratinocytes are continuously proliferating and renewing the outer skin barrier which acts as “first line of defense” against extrinsic stressors including those which promote aging (Tigges et al. 2014). Chronologically aged, photo-protected skin appears thin, has fine wrinkles and appears dry, whereas photo-aged skin has a leathery, coarsely wrinkled appearance. Wrinkles and sagging of skin are mainly due to changes in the dermal compartments in aging, such as decreased synthesis and increased remodeling or degradation of dermal matrix components. In chronologically aged skin, the fibrillar ordering of dermal elastin is impaired (Rittie and Fisher 2015). Similarly, type I collagen, the most abundant dermal extracellular matrix protein is a target of both chronological- and photo-aging which both promote degradation, lack of ordering and reduced production of this protein (Rittie and Fisher 2015). Several mechanisms likely contribute to extrinsically driven dermal matrix modification. Firstly, although UVB radiation does not penetrate into the dermal compartment, it prompts keratinocytes to release matrix metalloproteinases, which degrade the dermal matrix (Fisher et al. 1997;Quan et al. 2009). The resulting accumulation fragmented and crosslinked elastin in sunexposed upper dermis, termed “solar elastosis”, is one hallmark of photo-aging that causes loss of elasticity. Further extrinsic causes for damage to the dermal matrix are UVA- mediated elastin modifications by reactive carbonyls (Larroque-Cardoso et al. 2015) and the UV-induced (Schuch et al. 2017) or chronologic accumulation of senescent cells (Velarde et al. 2015). The latter produce matrixdegrading enzymes (Malaquin et al. 2013) and secrete lipids (Ni et al. 2016) that can chemically modify proteins (Bochkov et al. 2017). Skin aging, like aging of other organs, is characterized by progressive loss of functionality and regenerative potential. More than in other organs, the aging process of the skin is determined by extrinsic factors depending on lifestyle and exposure to environmental noxious agents in addition to intrinsic drivers of aging (Gilchrest and Krutmann 2006) and (Rittie and Fisher 2015). The most significant extrinsic aging factor is ultraviolet radiation which causes DNA damage and oxidative damage both promotors of cellular aging and/or senescence. Which cell type and compartment is reached (penetration depth) and the type of aging-relevant damage elicited by UV, depend on the wavelength (reviewed in (Cavinato and Jansen-Durr 2017;Schuch et al. 2017). Generally, the systematic hallmarks of aging (Lopez-Otin et al. 2013) also apply to the various compartments and cell types of the skin (Tigges et al. 2014), but cells which reside for a long timespan in the tissue appear to be affected more severely by loss of cellular maintenance and repair mechanisms than highly proliferative cells that are replaced frequently (Sukseree et al. 2018a;Sukseree et al. 2018b). In this review we discuss recent experimental studies on cell-specific aging and senescent phenotypes of fibroblasts and keratinocytes and the impact of new analytical technologies on the mechanistic characterization of skin aging.

Journal Pre-proof 2 Cell aging and senescence in fibroblasts

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Fibroblasts are the most abundant cell type of the dermis and contribute, either directly or via interactions with other cells, to most features of skin aging. Dermal fibroblasts that undergo in vitro aging protocols accumulate double strand breaks, oxidative DNA damage, chromosomal and epigenetic aberrations, shortening or oxidation of telomeres and the impairment of DNA repair mechanisms. Aged and especially senescent fibroblasts lose proteostasis (quantitative and qualitative homeostasis of the cellular proteome) due to aberrant synthesis, folding and degradation of proteins together with aberrant post-translational modifications like oxidation and crosslinking (Nowotny et al. 2014;Tigges et al. 2014). The damage itself or persistent danger signaling (Rodier et al. 2009) cause fibroblasts to become senescent, a state of usually irreversible cell cycle arrest going in hand with major changes in synthesis and secretion or vesicular release of specific proteins, lipids, metabolites and nucleic acids. In vivo, senescence is an important mechanism to prevent damaged cells from transforming into tumor cells (Baker et al. 2016) and has an important physiological role in wound healing (Demaria et al. 2014). Prolonged presence of senescent cells within tissues and their secretome contribute to aging related tissue decline and can even act as pro-tumorigenic factors (Tchkonia et al. 2013). In cultured fibroblasts, senescence is most reliably induced by either exhaustion of their proliferative potential or UVB irradiation. Yet, both protocols are somewhat artificial and results obtained by these protocols require confirmation in vivo (Waaijer et al. 2018).

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2.1 Novel findings on damage, dysfunction and repair of dermal fibroblasts in aging. (Fig 1 I-V) Among many interesting reports on the role of fibroblasts in skin aging, we want to highlight some publications which we consider particularly noteworthy in the context of this review. In a recent study, Wei and colleagues applied RNA sequencing and advanced bioinformatics to identify senescence factors detectable in various in vitro models that were then validated in senescent fibroblasts from aged human donors. They identified the p53 dependent gene Ras-related associated with diabetes (RRAD) as novel positively associated marker for senescence. Deletion of this gene exacerbated the sensitivity of non-senescent fibroblasts to hydrogen peroxide induced senescence. (Wei et al. 2019). In another recent study, Cavinato and coworkers demonstrated that dermal fibroblast senescence induced in vitro by UVB irradiation impaired the proteasome, which in turn, supposedly as compensatory mechanism for the degradation of oxidized proteins, induced autophagy (Cavinato and Jansen-Durr 2017). When additionally autophagy, the cellular bulk degradation mechanism that controls metabolism and proteostasis was blocked, the stressed cells underwent apoptosis. Autophagic capacity of dermal fibroblasts is, however, itself reduced in advanced age, as a recent study demonstrated (Bejarano et al. 2018) when comparing skin fibroblasts from juvenile and aged mice. In this study on aged but not senescent dermal fibroblasts, the expression of motor proteins, that move autophagosomes, was impaired, resulting in their decreased fusion with lysosomes and impaired cargo removal from the perinuclear area. Reduced autophagic removal of dysfunctional mitochondria (mitophagy) further amplifies oxidative stress in senescent cells (Tigges et al. 2014). Based on these and other reports, reviewed in (Sample and He 2017), we have recently proposed a concept how skin aging is affected by the cell type-specific and context-dependent impact of autophagic activity and its age-related decline (Eckhart et al. 2019). Expression of the immunoproteasome, a variant of the 20S proteasome is another important factor to control the levels of oxidatively modified proteins, and it was recently shown that in dermal fibroblasts from non-human primates the longevity of the species was reflected by the activity of the immuno-proteasome. Drugs that enhance lifespan in mice (primates have not yet been studied), including rapamycin, augmented the immuno-proteasome activity in cells of primates. Besides the proposed benefits of proteolysis of oxidized protein, immunoproteasome activity was also correlated with expression of antigen presentation genes and, of note, the interferon gamma receptor 2 and its

Journal Pre-proof signaling (Pickering et al. 2015). Thus, a loss of immunoproteasome activity may link age-related loss of fibroblast proteostasis with impairment of immune signaling. Hiebert and colleagues found that, surprisingly, constitutive activation of the redox regulator Nrf2 induced fibroblast senescence in vitro and in vivo (Hiebert et al. 2018). Nrf2 overexpressing fibroblasts produced an aberrant extracellular matrix (ECM) which did not only promote wound healing but acted pro-tumorigenic, again hinting that beneficial effects of senescent cells, such as their essential role in wound healing, are contextdependent, and that detrimental effects dominate when senescent cells are not cleared from the tissue (Demaria et al. 2015).

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2.2. Novel findings on communication, plasticity and identity of fibroblasts in aging. (Fig 1 4-9) One of the most interesting developing fields is the physical, paracrine and systemic communication of aging and of senescent cells with their environment. The secretome of senescent cells, the socalled senescence associated secretory phenotype (SASP) (Tchkonia et al. 2013), is a cocktail of immunomodulatory and proteolytic mediators including, as our group has recently shown, lipids (Ni et al. 2016). The SASP is considered the main effector of physiologic and detrimental effects of senescent cells in skin and in other tissues (Ghosh and Capell 2016). Meyer and colleagues recently presented a computational model that predicted transcriptional regulators of the SASP in various cellular contexts. They could then prove experimentally that, NEMO, a predicted transcriptional regulator of SASP (Fig. 1 V), was responsible for DDR-mediated SASP regulation in murine dermal fibroblasts (Meyer et al. 2017). As persistent presence of senescent cells and SASP secretion promotes aging-related tissue decline, the clearance of senescent cells is a promising approach to reduce age-related damage and disease (Burton and Stolzing 2018). The SASP appears to play a central role in directing cells of the innate and adaptive immune system towards senescent cells also in the skin and promotes their clearance (Ghosh and Capell 2016).

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Aged tissues nevertheless accumulate senescent cells which then contribute to aging-related tissue deterioration (reviewed in (Kirkland and Tchkonia 2017) and (Burton and Krizhanovsky 2014)). Pereira and colleagues recently reported that senescent dermal fibroblasts express the non-classical MHC molecule HLA-E which activates the inhibitory receptor NKG2A on natural killer cells (NK) and CD8+ T-cells and thereby evade elimination (Pereira et al. 2019). Another evolving concept of communication of senescent cells to the environment or niche is “bystander senescence”, the spreading of senescence features to cells in spatial proximity of senescent cells which was recently reported in vivo for dermal fibroblasts (da Silva et al. 2019). We have recently discovered another type of senescent fibroblast communication with neighboring cells, i.e. they are able to transfer extracellular vesicles containing bioactive microRNAs to keratinocytes in organotypic models, and the receiving keratinocytes in turn display phenotypes of geriatric skin keratinocytes. These vesicles can be detected in dermal interstitial fluid in vivo (Terlecki-Zaniewicz et al. 2019). Besides yielding detailed and novel findings on canonical hallmarks of aging in the dermal compartment, research of the past years has uncovered exciting data on the functional plasticity that the dermal fibroblasts display during aging. Especially the advent of deep sequencing, single cell sequencing and the application of lineage tracing has proven very useful for advancement of the field. Salzer and colleagues could identify roughly 1000 transcripts that differed between young and aged Pdgfra/CD34 double positive mouse fibroblasts (Salzer et al. 2018). As expected, the genes for ECM synthesis and secretion were most strongly reduced in the older age group. In addition to an increased expression of pro-inflammatory and stress fiber genes, they found a massive induction of genes regulating adipogenesis and lipid metabolism. Upon bioinformatic analysis of single cell RNA sequencing data, they discovered that transcriptomes of fibroblasts from newborn mice cluster into groups that correspond to papillary and reticular fibroblasts and identified proliferative and quiescent subclusters among the papillary cells. Using lineage tracing they found that progeny of

Journal Pre-proof Lrig+ cells, which define newborn papillary fibroblasts (Driskell et al. 2013) were already distributed throughout the whole dermis by two months of age. Strikingly, some fibroblasts differentiated into mature adipocytes and overall, the identity (based on transcriptomic signature) of the fibroblasts was increasingly blurred with increasing age. Setting mice on a caloric restriction diet during aging prevented the loss of papillary fibroblast identity and the acquisition of adipocytic features, demonstrating that cellular identity can be modulated by an accepted regimen that slows agerelated deterioration. The loss of cellular density in the murine dermis in aging is not a random event but rather represents accumulated loss of localized cell clusters. The loss was not compensated by proliferation of activated quiescent or stem cells but by membrane extensions of positionally stable neighboring cells, as Marsh and colleagues (Marsh et al. 2018) could show with an extensive intravital imaging approach.

3. Cell aging and senescence in epidermal keratinocytes

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Maintenance of the epidermis in homeostasis and after disturbance, like wounding, requires a dynamic equilibrium of precursor cells that retain the ability to yield further precursors as well as cells that commit to terminal differentiation. One widely accepted concept on such a mechanism is a pool of transiently amplifying epidermal cells that form a level of hierarchy between the epidermal stem cells and the differentiating cells. Lately, this concept has been challenged, as in vitro studies identified that a single keratinocyte progenitor cell type may provide the functions needed for maintaining epidermal homeostasis. This is, according to the model, achieved by switching bidirectionally between a “balanced” proliferation mode that maintains the basal and the differentiating populations in homeostasis, and an “expanding”, highly proliferative mode that replenishes substantial epithelial loss, as is required in wound healing (Roshan et al. 2016). This concept of a single type of uncommitted stem cells that can either proliferate or differentiate is largely supported by lineage tracing studies in vivo in the mouse epidermis (Rompolas et al. 2016). However, other lineage tracing studies identify functionally and molecularly distinct stem or progenitor cells in the epidermis (Sada et al. 2016) and such heterogeneity among the undifferentiated basal epidermal (stem) cells is supported by single cell transcriptomics (Joost et al. 2016). Novel technologies for tracing and molecularly dissecting single cells over time and in various body sites will further elucidate cell heterogeneity and the influence of the niche on epidermal cell identity in aging and senescence. When differentiating keratinocytes leave the basal layer of the epidermis they do not divide any further. Apart from this growth arrest, differentiating keratinocytes share features of senescent cells like changes in metabolism and chromatin rearrangements. The current consensus view of the International Cell Senescence Association (ICSA) (Gorgoulis et al. 2019) states that terminal differentiation of cells does not qualify for the definition of cellular senescence, as the shared features are not result of severe stress or damage to which senescence would act as a damage response. Also, the differentiating keratinocytes lack several of the molecular key features (macromolecular damage, protein oxidation, telomere shortening, a secretory phenotype and survival within the tissue) of senescence. An interesting question in that regard is, whether terminal differentiation is activated as a consequence of cellular senescence in basal cells (which will be discussed below), or whether senescence can be activated in cells that have already initiated the terminal differentiation program. Recent findings indicate that at least the loss of the nuclear matrix protein Lamin B1, one robust marker of senescence in skin (Dreesen et al. 2013), is inducible in suprabasal keratinocytes by UVB radiation (Wang et al. 2017). In monolayer culture, epidermal keratinocytes develop a senescent phenotype while lacking markers of terminal differentiation (Norsgaard et al. 1996), usually after far less population doublings than fibroblasts. The type of persistent DNA damage signaling that reinforces a senescent phenotype

Journal Pre-proof differs in epithelial keratinocytes from fibroblasts. The first observed growth arrest in course of epithelial cell senescence can be followed by a second phase of replication of cell clones that escape the proliferation stop. Whereas the first arrest is independent of telomere shortening, DDR and ATM/ATR signaling, the second plateau level of proliferation is accompanied by high p21 expression but nevertheless appears somewhat reversible (Abbadie et al. 2017). While in vitro studies reveal mechanistic insights on cell type-specific damage and repair mechanisms, investigating the agerelated phenotype of keratinocytes within the epidermis is complex. In contrast to a dramatic agedependent decline in melanocyte stem cells in the skin, the keratinocyte stem cell numbers (in hair follicles) remain relatively stable (Giangreco et al. 2008) except in areas of age-related hair loss (Matsumura et al. 2016). Heterochronic transplantation does not significantly restore the proliferative capacity (i.e. number of colony forming cells), again hinting to an involvement of niche factors in epidermal aging.

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3.1 Novel findings damage, dysfunction and repair of epidermal keratinocytes in aging (Fig. 1 VI-XI) The role of proteostasis in aged keratinocytes is less well studied than in fibroblasts and somewhat controversial. An age-related decline of the proteasomal subunit PSMD8 was found in epidermal keratinocytes, and RNA-interference of this gene simulated the phenotype of aged epidermis in a three-dimensional model (Ishii et al. 2018). Autophagy is not essential for the formation of the epidermis and barrier function in homeostasis, but keratinocytes lacking autophagy develop a senescent phenotype and DNA damage in culture more easily (Eckhart et al. 2019;Song et al. 2017). However, other studies have shown that autophagy is required for induction of senescence in other cell types, e.g. hepatocytes, thus further investigations, especially on autophagy in epidermal stem cells and epidermal metabolism are required. A recent study uncovered that the high levels of NAD+ promote a proliferative undifferentiated state in keratinocytes while depletion results in premature differentiation and elevated senescence markers (Tan et al. 2019). This finding further links the metabolic state to DNA damage repair, keratinocyte differentiation and senescence. Senescent cultured keratinocytes accumulate redox stress-induced single strand breaks (Nassour et al. 2016). These breaks remained unrepaired due to a decrease in PARP1 activity and led to accumulation of large XCRR1 foci which ultimately promote p16 dependent cell cycle arrest. Mutations in the Cockayne syndrome a (CSA) gene impair nucleotide excision repair of DNA and cause a premature aging phenotype and photosensitivity of the skin, besides other premature aging defects. Gene correction of CSA restored NER and prevented premature in vitro senescence of keratinocytes from affected individuals (Cordisco et al. 2019). As epidermal NER capacity declines with age in humans in vivo (Yamada et al. 2006), this type of DNA damage repair appears to be a major factor in keratinocyte-specific intrinsic and extrinsic aging. In vivo, aged, quiescent keratinocyte stem cells of the murine hair follicle display permanent DDR signaling, and recently Matsumura and colleagues reported that sustained gamma-H2AX and p53BP1 foci in keratinocyte stem cells of the hair follicle induced a proteolytic signal, that in turn depletes these cells of the hemi-desmosomal structural protein collagen 17A1 (Matsumura et al. 2016). The loss of collagen 17A1 ultimately promotes the clearance of the aged cells via terminal differentiation and ultimately shedding as corneocytes. As we will discuss below, collagen 17A1 appears to have a central role in epidermal stem cell aging in vivo. 3.2 Communication and plasticity of keratinocytes in aging (Fig. 1 1-3; 10-12) Transient amplifying and differentiating epidermal keratinocytes do not reside for a prolonged time at fixed positions. The findings on Col17A1 proteolysis in aging keratinocyte stem cells (see above) were extended by Liu and colleagues who found that PDGFRa+ fibroblasts form junctional structures that anchor the epidermal basal keratinocytes as long as they express Col17A1 (Liu et al. 2019). These structures were decreased in aging, and the authors suggest that the ensuing reduction of physical interaction between fibroblasts and keratinocytes could impair the epidermal stem cell

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maintenance. This also affected melanocytes that were attached to the Col17a1 positive stem cells and, upon UVB photodamage to the keratinocytes (and their hemi-desomosomes) co-delaminated and thereby caused dyspigmentation. Keratinocytes of aged epidermis display a lower activation state of IGF-1R which causes defects of DNA damage responses and increases the susceptibility to accumulating mutations. This is not a cell intrinsic phenomenon, but results from reduced IGF-1 supplementation through the (aged) dermal fibroblasts (Kemp et al. 2017). As to signals emanating from aged or senescent keratinocytes, they have mostly been described in vitro; while in vivo findings are mainly limited to oncogene induced senescence. One example of the latter was recently reported in a mouse model in which the SASP from oncogene induced senescent keratinocytes displayed a dual role on non-senescent keratinocytes (Ritschka et al. 2017). In the short term, the SASP increased locally the plasticity and stemness of neighboring epithelial cells which apparently cleared and replaced senescent cells. A prolonged SASP however led to accumulation of stem cells without a tissue regenerative effect and to bystander senescence of neighboring cells possibly through paracrine mechanisms. It has to be noted that only parts of this concept were modeled in vivo in the skin, and that the authors merely refer to papillomas as an (oncogene induced) senescent model but did not directly identify and trace the senescent keratinocytes in situ. The keratinocyte SASP is not only under transcriptional control of the canonical transcription factors like NFkB, but additionally under translational control of the RNA binding protein YBX1 which is high in progenitor cells but its decrease in senescent cells promotes translation of SASP cytokines (Kwon et al. 2018).

4 Conclusion

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Wiley et al. reported that in another mouse model induced mitochondrial dysfunction also elicited an untypical SASP lacking the major inflammatory component which drives senescence in human and murine keratinocytes and accelerated KC differentiation (Wiley et al. 2016). Yet, also this study lacks a definite attribution of senescence markers to keratinocytes in vivo. The mice do however display a skin aging phenotype and the data are compatible with the concept that senescent keratinocytes are effectively removed by differentiation (Velarde et al. 2015). Also this study corroborates an association of low NAD+ levels with cellular senescence in keratinocytes. Of note, a recent study (Hu et al. 2017) suggested that the epidermis of aged mice, especially in response to barrier disruption, is a source not only for elevated cutaneous but also for elevated serum inflammatory cytokine levels. Thus, the keratinocyte SASP (or more generally age related secretome) composition may adapt to stress.

The recent years have highlighted an unexpected level of cellular plasticity in the aging skin, both in the epidermis that regenerates fast mainly through keratinocyte stem cells, but also in dermal fibroblasts. The plasticity appears to be elicited by internal, cell-autonomous factors and factors derived from the respective niches, including the secretome of aging cells or senescent cells. Besides cyto- and chemokines, physical interactions between the cells, secreted vesicles, and lipids have been identified to contribute to the senescence secretome. The exact mechanisms of how these signaling methods elicit bystander senescence, evasion of clearance through the immune system and consequently tissue damage or carcinogenesis are yet to be uncovered. Another evolving research field concerns the mechanisms by which targeted interventions into cellular metabolism can crosstalk with damage repair, stemness and senescence mechanisms. The finding that caloric restriction, the most robust health-span prolonging strategy, is able to revert age-related plasticity and senescence of dermal fibroblasts opens new perspectives on targeting negative effects of skin aging.

Journal Pre-proof 5 Funding FG and CK are grateful for support by the Federal Ministry for Digital and Economic Affairs of Austria and the National Foundation for Research, Technology and Development of Austria and CHANEL Fragrance Beauty, Research & Innovation to the Christian Doppler Laboratory for Biotechnology of Skin Aging. ET is grateful for support by CHANEL Fragrance Beauty, Research & Innovation.

Figure Legend Figure 1 - Recent findings on cell aging and senescence in dermal fibroblasts and epidermal keratinocytes. I-XI Damage and functional decline

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I: RRAD, a novel functional pan-senescence marker (Wei et al. 2019) ; II: Impaired proteostasis in senescent FB (Cavinato and Jansen-Durr 2017); III: Impaired immunoproteasome/immune signaling (Pickering et al. 2015); IV: Aberrant ECM production in Nrf2 overexpressing senescent FB (Hiebert et al. 2018); V: NEMO induces SASP (Meyer et al. 2017); VI: DNA SSB induce senescence and lower NER in aging epidermis (Cordisco et al. 2019;Nassour et al. 2016); VII: Aged, quiescent KC SC – permanent DDR (Matsumura et al. 2016); VIII: Aging associated decline in proteasomal PSMD8 (Ishii et al. 2018); IX: NAD+ depletion induces senescence in KC (Tan et al. 2019); X: YBX1 represses SASP in KC SC (Kwon et al. 2018); XI DDR induces premature differentiation (Liu et al. 2019) 1-12 Communication and identity

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1 : Fibroblasts anchor Col17A1 expressing KC SC in junctional structures (Liu et al. 2019); 2: Short term SASP enhances stemness and regeneration (Ritschka et al. 2017); 3,4: Reduced IGF1 synthesis by FB, reduced IGF1R activation and DNA damage repair in KC (Kemp et al. 2017); 5: Bystander senescence in FB (da Silva et al. 2019); 6: Transfer of SASP-EVs from FB to KC (Terlecki-Zaniewicz et al. 2019); 7: Aged FB take on adipocyte identity (Salzer et al. 2018); 8: Static FB extend membranes to compensate for aging dependent cell losses (Marsh et al. 2018); 9: Senescent FB evade clearance by HLA-E expression (Pereira et al. 2019); 10: untypical, non-inflammatory SASP in KC upon MiDAS (Wiley et al. 2016); 11: Barrier disruption in aged epidermis induces systemic cytokine release (Hu et al. 2017); 12: DDR induces COL17A1 proteolysis in HF-KSC (Matsumura et al. 2016)

6 References

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