Selective degradation of damaged mitochondria by autophagy (mitophagy) is proposed to play an important role in cellular homeostasis. However, the molecular mechanisms and the requirement of mitochondrial quality control by mitophagy for cellular physiology are poorly understood. Here, we demonstrated that primary human cells maintain highly active basal mitophagy initiated by mitochondrial superoxide signaling. Mitophagy was found to be mediated by PINK1/Parkin-dependent pathway involving p62 as a selective autophagy receptor (SAR). Importantly, this pathway was suppressed upon the induction of cellular senescence and in naturally aged cells, leading to a robust shutdown of mitophagy. Inhibition of mitophagy in proliferating cells was sufficient to trigger the senescence program, while reactivation of mitophagy was necessary for the anti-senescence effects of NAD precursors or rapamycin. Furthermore, reactivation of mitophagy by a p62-targeting small molecule rescued markers of cellular aging, which establishes mitochondrial quality control as a promising target for anti-aging interventions.

Imbalance of collagen I expression results in severe pathologies. Apart from activation by the TGFβ-receptor/Smad pathway, control of collagen I expression remains poorly understood. Here, we used human dermal fibroblasts expressing a mCherry fluorescent protein driven by endogenous COL1A1 promoter to functionally screen the kinome and phosphatome. We identify 8 negative regulators, revealing that collagen is under tonic repression. The cell surface receptor BDKRB2 represses collagen I and other pro-fibrotic genes. Interestingly, it also promotes other basal membrane ECM genes. This function is independent of the natural ligand, bradykinin, and of SMAD2/3 factors, instead requiring constant ERK1/2 repression. TGFβ stimulation induces rapid BDKRB2 transcriptional downregulation. Human fibrotic fibroblasts have reduced BDKRB2 levels and enhancing its expression in keloid fibroblasts represses COL1A1. We propose that tonic signalling by BDKRB2 prevents collagen overproduction in skin fibroblasts.

Skin ageing is an intricate physiological process affected by intrinsic and extrinsic factors. There is a demand to understand how the skin changes with age and photoexposure in individuals with Fitzpatrick skin types I‐III due to accelerated photoageing and the risk of cutaneous malignancies. To assess the structural impact of intrinsic and extrinsic ageing, we analysed 14 skin parameters from the photoprotected buttock and photoexposed dorsal forearm of young and ageing females with Fitzpatrick skin types II‐III (n = 20) using histomorphic techniques. Whilst the minimum viable epidermis (Emin) remained constant (Q > 0.05), the maximum viable epidermis (Emax) was decreased by both age and photoexposure (Q ≤ 0.05), which suggests that differences in epidermal thickness are attributed to changes in the dermal‐epidermal junction (DEJ). Changes in Emax were not affected by epidermal cell proliferation. For the first time, we investigated the basal keratinocyte morphology with age and photoexposure. Basal keratinocytes had an increased cell size, cellular height and a more columnar phenotype in photoexposed sites of young and ageing individuals (Q ≤ 0.05), however no significant differences were observed with age. Some of the most striking changes were observed in the DEJ, and a decrease in the interdigitation index was observed with both age and photoexposure (Q ≤ 0.001), accompanied by a decreased height of rête ridges and dermal papilla. Interestingly, young photoexposed skin was comparable to ageing skin across many parameters, and we hypothesise that this is due to accelerated photoageing. This study highlights the importance of skin care education and photoprotection from an early age.

Increasingly unparalleled changed in global demographics mean that there is now an even greater need for research focused on ageing; it is estimated that 1 in 6 people will be over 65 in the year 2050. The health and integrity of skin is imperative to general health and wellbeing. However, the unique experience of skin as the barrier to the external environment leads to constant exposure to exogenous stressors, extrinsic ageing, as well as the usual impacts of chronological or intrinsic ageing. In turn, these sustained assaults induce pathological changes in skin structure and function, yet there are few in vitro human skin equivalents (HSEs) which can accurately recapitulate the complex nature of ageing skin. Utilizing a previously established HSE, we have bioengineered full thickness skin tissues which encapsulate various aspects of ageing skin through including senescent cells and fibroblasts/keratinocytes from aged donors. We have found that increasing numbers of senescent cells can be used to successfully generate HSEs with senescent fibroblast burdens similar to the dermis, and that senescent cells can be combined with cells from aged donors to produce more complex and representative models of ageing human skin. In depth characterization shows these HSEs display hallmarks of skin ageing, including changes in epidermal structure and cellular morphology, proinflammatory cytokines and proteases. Recently, we have applied known and novel anti-ageing compounds to attempt to alleviate the ageing phenotype and restore some skin health. This in vitro system provides a reproducible platform technology which can be used to answer fundamental questions about ageing skin biology and identify compounds which can ameliorate the impacts of the ageing process.

Introduction: The field of skin ageing is important due to the world ageing population. Due to its interface with the external environment, skin ageing is attributed to both intrinsic and extrinsic factors. The aim of this study was to define histomorphic disparities between intrinsically and extrinsically aged skin, and increase understanding of how skin structures change with age and photoexposure. Methods: Full-thickness skin biopsies were obtained from the photoexposed dorsal forearm and photoprotected buttock of young (21-24 years, n=10 females) and aged Caucasian volunteers (61-65 years, n=10 females). Biometrics analysis of epidermal thickness (Emin), epidermal proliferation, basal keratinocyte morphology, interdigitation index and morphology of rête ridges and dermal papilla was performed on blinded histological and immunofluorescence images (n=40 biopsies; n=160-240 images). Results: Our study found that Emin and epidermal proliferation do not change with age or photoexposure (P>0.05). The size and height of basal keratinocytes is increased by photoexposure in young and ageing individuals, but no differences were identified with age (P<0.0001). The dermal-epidermal junction is affected by both age and photoexposure with regards to decreased interdigitation and decreased height of rête ridges and dermal papilla (P<0.0001). Discussion: Our data suggest that basal keratinocyte morphology is affected by photoexposure, and dermal-epidermal junction characteristics are altered by age and photoexposure. Interestingly, flattening of the dermal-epidermal junction was most apparent in photoexposed areas, and we propose that there is an accelerated ageing phenotype due to the contribution of extrinsic factors. These data are important for both academic and industrial scientists to validate in vitro findings, and identify targets for cosmetic interventions.

The ageing global demographic constantly reinforces the need for more age focussed research, in order to better support the population to live healthier for longer. As the interface between the human body and the external environment, the experience of skin is unique, being subjected to both internal and external ageing influences, leading to pathological changes in structure/function. Currently, there are few in vitro ageing skin equivalents to undertake this research, and even fewer which account for both ageing factors. The presence of senescent cells within the dermis is well established, but what is their role in influencing epidermal behavior and morphology and how do they interact with intrinsically aged cells? Modifying an established in vitro human skin model which utilizes scaffold technology, we have combined senescent cells with various primary skin cell populations to recapitulate the structure and function of aged human skin and better understand the role of senescent cells. Adding senescent cells at varying frequencies to young models results in altered cytokine, MMP and TIMP production as expected, indicating a senescence associated secretory phenotype, and a decrease in epidermal markers such as lamin B1 and p63. More recent work has involved adding senescent cells to models created entirely from populations from aged donors, in order to more accurately recapitulate the complexity of aged human skin. These models represent a more physiologically relevant platform for use in a range of age-related applications, such as exploring the mechanisms involved during skin ageing and assessing compounds for senotherapeutic activity in skin, with the aim of alleviating some effects of the ageing process and restoring some skin health.

Understanding the changes in the skin microbiome and their relationship to host skin factors during aging remains largely unknown. To better understand this phenomenon, we collected samples for metagenomic and host skin factor analyses from the forearm, buttock, and facial skin from 158 Caucasian females aged 20-24, 3034, 40-44, 50-54, 60-64, and 70-74 years. Metagenomics analysis was performed using 16S ribosomal RNA gene sequencing, whereas host sebocyte gland area, skin lipids, natural moisturizing factors, and antimicrobial peptides measurements were also performed. These analyses showed that skin bacterial diversity increased at all the skin sites with increasing age. Of the bacterial genera with an average relative abundance >1%, only Lactobacillus and Cutibacterium demonstrated a significant change (decrease) in abundance at all sampled skin sites with increasing age. Additional bacterial genera demonstrated significant age- and site-specific changes in abundance. Analysis of sebocyte area, natural moisturizing factors, lipids, and antimicrobial peptides showed an age-related decrease in sebocyte area and increases in natural moisturizing factors/antimicrobial peptides/ skin lipids, all of which correlated with changes in specific bacterial genera. In conclusion, the human skin microbiome undergoes age-associated alterations that may reflect underlying age-related changes in cutaneous biology.

Selective degradation of damaged mitochondria by autophagy (mitophagy) is proposed to play an important role in cellular homeostasis. However, the molecular mechanisms and the requirement of mitochondrial quality control by mitophagy for cellular physiology are poorly understood. Here we demonstrated that primary human skin fibroblasts in cell culture maintain highly active basal mitophagy driven by mitochondrial ROS signalling. Mitophagy was found to be mediated by PINK1/Parkin-dependent pathway involving p62 as a selective autophagy receptor. Importantly, this pathway was disturbed upon the induction of cellular senescence leading to a robust shutdown of mitophagy. Suppression of mitophagy was sufficient to trigger the senescence programme, whilst re-activation of mitophagy was necessary for anti-senescence effects of NAD precursors or rapamycin. Furthermore, activation of mitophagy by a p62-targeting small molecule suppressed markers of senescence establishing mitochondrial quality control as a promising target for the development of novel anti-ageing interventions.Funding Information: This study was supported by Fellowships from Uehara Memorial Foundation and the International Medical Research Foundation to T.K.; RESETageing H2020 grant (952266) to L.F. and V.I.K.; BBSRC DTP PhD studentship, BBSRC (BB/M023389/1) and BBSRC (BB/R008167/2) grants to V.I.K.Declaration of Interests: J.E.O. and C.C.B. are employees of The Procter & Gamble Company, USA. All other authors declare they have no competing interests.Ethical Approval Statement: The REC reference 19/NE/004_Lovat quoted within the study by Hill et al relates to the full ethical permission granted to the Newcastle University Dermatology Biobank that I am the principle investigator of, granted for the isolation of primary cutaneous cells (fibroblasts, melanocytes and keratinocytes) and their use in cutaneous research and which includes the use of cells in the present study. I therefore confirm The REC reference 19/NE/004 (valid until March 2024) covers these follow on studies.

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction.

Skin ageing is defined in part by collagen depletion and fragmentation that leads to a loss of mechanical tension. This is currently believed to reflect, in part, the accumulation of senescent cells. We compared the expression of genes and proteins for components of the extracellular matrix (ECM) as well as their regulators and found that senescent cells produced more matrix metalloproteinases (MMPs) than proliferating cells from adult and neonatal donors. This was consistent with senescent cells contributing to increased matrix degradation with age; however, cells from adult donors proved significantly less capable of producing new collagen than neonatal or senescent cells, and they showed significantly lower myofibroblast activation as determined by the marker α-SMA. Functionally, adult cells also showed slower migration than neonatal cells. We concluded that while increased collagen degradation with age might reflect senescent cell accumulation, the reduced collagen production that prevents the skin from maintaining homeostasis must reflect senescence-independent processes.

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration by a poorly understood mechanism. Here we identified an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring cells with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarisation and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient cells and organisms. Our study provides a mechanistic link between autophagy and NAD metabolism, and identifies novel targets for interventions in human diseases associated with autophagic, lysosomal and mitochondrial dysfunction.

Mechanotransduction is defined as the ability of cells to sense mechanical stimuli from their surroundings and translate them into biochemical signals. Epidermal keratinocytes respond to mechanical cues by altering their proliferation, migration, and differentiation. In vitro cell culture, however, utilises tissue culture plastic, which is significantly stiffer than the in vivo environment. Current epidermal models fail to consider the effects of culturing keratinocytes on plastic prior to setting up three-dimensional cultures, so the impact of this non-physiological exposure on epidermal assembly is largely overlooked. In this study, primary keratinocytes cultured on plastic were compared with those grown on 4, 8, and 50 kPa stiff biomimetic hydrogels that have similar mechanical properties to skin. Our data show that keratinocytes cultured on biomimetic hydrogels exhibited major changes in cellular architecture, cell density, nuclear biomechanics, and mechanoprotein expression, such as specific Linker of Nucleoskeleton and Cytoskeleton (LINC) complex constituents. Mechanical conditioning of keratinocytes on 50 kPa biomimetic hydrogels improved the thickness and organisation of 3D epidermal models. In summary, the current study demonstrates that the effects of extracellular mechanics on keratinocyte cell biology are significant and therefore should be harnessed in skin research to ensure the successful production of physiologically relevant skin models.

Ablative fractional laser treatment is considered the gold standard for skin rejuvenation. In order to understand how fractional laser works to rejuvenate skin, we performed microarray profiling on skin biopsies to identify temporal and dose-response changes in gene expression following fractional laser treatment. The backs of 14 women were treated with ablative fractional laser (Fraxel®) and 4 mm punch biopsies were collected from an untreated site and at the treated sites 1, 3, 7, 14, 21 and 28 days after the single treatment. In addition, in order to understand the effect that multiple fractional laser treatments have on skin rejuvenation, several sites were treated sequentially with either 1, 2, 3, or 4 treatments (with 28 days between treatments) followed by the collection of 4 mm punch biopsies. RNA was extracted from the biopsies, analyzed using Affymetrix U219 chips and gene expression was compared between untreated and treated sites. We observed dramatic changes in gene expression as early as 1 day after fractional laser treatment with changes remaining elevated even after 1 month. Analysis of individual genes demonstrated significant and time related changes in inflammatory, epidermal, and dermal genes, with dermal genes linked to extracellular matrix formation changing at later time points following fractional laser treatment. When comparing the age-related changes in skin gene expression to those induced by fractional laser, it was observed that fractional laser treatment reverses many of the changes in the aging gene expression. Finally, multiple fractional laser treatments, which cover different regions of a treatment area, resulted in a sustained or increased dermal remodeling response, with many genes either differentially regulated or continuously upregulated, supporting previous observations that maximal skin rejuvenation requires multiple fractional laser treatments. In conclusion, fractional laser treatment of human skin activates a number of biological processes involved in wound healing and tissue regeneration.

Human C2orf69 is an evolutionary-conserved gene whose function is unknown. Here, we report 9 children from 5 unrelated families with a fatal syndrome consisting of severe auto-inflammation, progredient leukoencephalopathy with recurrent seizures that segregate homozygous loss-of-function C2orf69 variants. C2ORF69 orthologues, which can be found in most eukaryotic genomes including that of unicellular phytoplanktons, bear homology to esterase enzymes. We find that human C2ORF69 is loosely bound to the mitochondrion and its depletion affects mitochondrial membrane potential in human fibroblasts and neurons. Moreover, we show that CRISPR/Cas9-inactivation of zebrafish C2orf69 results in lethality by 8 months of age due to spontaneous epileptic seizures which is accompanied by persistent brain inflammation. Collectively, our results delineate a novel auto-inflammatory Mendelian disorder of C2orf69 deficiency that disrupts the development/homeostasis of the immune and central nervous systems as demonstrated in patients and in a zebrafish model of the disease.

At birth, human infants are poised to survive in harsh, hostile conditions. An understanding of the state of newborn skin development and maturation is key to the maintenance of health, optimum response to injury, healing and disease. The observational study collected full-thickness newborn skin samples from 27 infants at surgery and compared them to skin samples from 43 adult sites protected from ultraviolet radiation exposure, as the standard for stable, mature skin. Transcriptomics profiling and gene set enrichment analysis were performed. Statistical analysis established over 25,000 differentially regulated probe sets, representing 10,647 distinct genes, in infant skin compared to adult skin. Gene set enrichment analysis showed a significant increase in 143 biological processes (adjusted p < 0.01) in infant skin, versus adult skin samples, including extracellular matrix (ECM) organization, cell adhesion, collagen fibril organization and fatty acid metabolic process. ECM organization and ECM structure organization were the biological processes in infant skin with the lowest adjusted P-value. Genes involving epidermal development, immune function, cell differentiation, and hair cycle were overexpressed in adults, representing 101 significantly enriched biological processes (adjusted p < 0.01). The processes with the highest significant difference were skin and epidermal development, e.g., keratinocyte differentiation, keratinization and cornification intermediate filament cytoskeleton organization and hair cycle. Enriched Gene Ontology (GO) biological processes also involved immune function, including antigen processing and presentation. When compared to ultraviolet radiation-protected adult skin, our results provide essential insight into infant skin and its ability to support the newborn’s preparedness to survive and flourish, despite the infant’s new environment laden with microbes, high oxygen tension and potential irritants. This fundamental knowledge is expected to guide strategies to protect and preserve the features of unperturbed, young skin.

Human C2orf69 is an evolutionarily conserved gene whose function is unknown. Here, we report eight unrelated families from which 20 children presented with a fatal syndrome consisting of severe autoinflammation and progredient leukoencephalopathy with recurrent seizures; 12 of these subjects, whose DNA was available, segregated homozygous loss-of-function C2orf69 variants. C2ORF69 bears homology to esterase enzymes, and orthologs can be found in most eukaryotic genomes, including that of unicellular phytoplankton. We found that endogenous C2ORF69 (1) is loosely bound to mitochondria, (2) affects mitochondrial membrane potential and oxidative respiration in cultured neurons, and (3) controls the levels of the glycogen branching enzyme 1 (GBE1) consistent with a glycogen-storage-associated mitochondriopathy. We show that CRISPR-Cas9-mediated inactivation of zebrafish C2orf69 results in lethality by 8 months of age due to spontaneous epileptic seizures, which is preceded by persistent brain inflammation. Collectively, our results delineate an autoinflammatory Mendelian disorder of C2orf69 deficiency that disrupts the development/homeostasis of the immune and central nervous systems.

Bioengineered skin constructs provide a physiologically relevant platform for fundamental and translational research. We have developed a complex, robust and reproducible full thickness skin equivalent, which has been validated against in vivo skin using in-depth analyses. To mimic the dermis in vitro, primary fibroblasts were cultured within the Alvetex® Scaffold to produce endogenous extracellular matrix proteins. The robust dermal compartment supports the generation of an organised, stratified and keratinised epidermis. Additional cell types have also been incorporated, such as melanocytes and immune cells, to increase the skin equivalent complexity. The human skin equivalent recapitulates the organised structure of human skin in vitro. Skin constructs that represent different ages have been developed using neonatal, young or ageing cells. The use of ageing and senescent cells recreates the ageing skin phenotype in vitro, with regards to epidermal thinning, decreased keratinocyte proliferation and reduced extracellular matrix synthesis. Pigmented skin equivalents include melanocytes, which localise to the basal layer and transfer melanin to neighbouring keratinocytes to form a protective supranuclear cap. The pigmented skin equivalents are functional, and exposure to ultraviolet radiation induces a tanning effect. Langerhans cells have also been incorporated into the epidermis to produce an immune-competent skin equivalent, which is responsive to topical allergens and irritants. We have developed advanced, robust and reproducible skin constructs that resemble the structure of human skin. These skin equivalents have multifaceted research applications such as investigating the underpinning mechanisms of skin ageing, assessing the effect of exogenous stressors and evaluating cosmetic formulations.

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

Type I collagen is a key protein of most connective tissue and its up-regulation is required for wound healing but is also involved in fibrosis. Control of expression of this collagen remains poorly understood apart from Transforming Growth Factor beta (TGF-β1)-mediated induction. To generate a sensitive, practical, robust, image-based high-throughput-compatible reporter system, we genetically inserted a short-lived fluorescence reporter downstream of the endogenous type I collagen (COL1A1) promoter in skin fibroblasts. Using a variety of controls, we demonstrate that the cell line faithfully reports changes in type I collagen expression with at least threefold enhanced sensitivity compared to endogenous collagen monitoring. We use this assay to test the potency of anti-fibrotic compounds and screen siRNAs for regulators of TGF-β1-induced type I collagen expression. We propose our reporter cell line, Red-COLA1, as a new efficient tool to study type I collagen transcriptional regulation.