PGC-1s shape epidermal physiology by modulating keratinocyte proliferation and terminal differentiation.

Skin plays central roles in systemic physiology, and it undergoes significant functional changes during aging. Members of the peroxisome proliferator-activated receptor-gamma coactivator (PGC-1) family (PGC-1s) are key regulators of the biology of numerous tissues, yet we know very little about their impact on skin functions. Global gene expression profiling and gene silencing in keratinocytes uncovered that PGC-1s control the expression of metabolic genes as well as that of terminal differentiation programs. Glutamine emerged as a key substrate promoting mitochondrial respiration, keratinocyte proliferation, and the expression of PGC-1s and terminal differentiation programs. Importantly, gene silencing of PGC-1s reduced the thickness of a reconstructed living human epidermal equivalent. Exposure of keratinocytes to a salicylic acid derivative potentiated the expression of PGC-1s and terminal differentiation genes and increased mitochondrial respiration. Overall, our results show that the PGC-1s are essential effectors of epidermal physiology, revealing an axis that could be targeted in skin conditions and aging.

NRF2 Shortage in Human Skin Fibroblasts Dysregulates Matrisome Gene Expression and Affects Collagen Fibrillogenesis.

NRF2 is a master regulator of the antioxidative response that was recently proposed as a potential regulator of extracellular matrix (ECM) gene expression. Fibroblasts are major ECM producers in all connective tissues, including the dermis. A better understanding of NRF2-mediated ECM regulation in skin fibroblasts is thus of great interest for skin homeostasis maintenance and aging protection. In this study, we investigate the impact of NRF2 downregulation on matrisome gene expression and ECM deposits in human primary dermal fibroblasts. RNA-sequencing‒based transcriptome analysis of NRF2 silenced dermal fibroblasts shows that ECM genes are the most regulated gene sets, highlighting the relevance of the NRF2-mediated matrisome program in these cells. Using complementary light and electron microscopy methods, we show that NRF2 deprivation in dermal fibroblasts results in reduced collagen I biosynthesis and impacts collagen fibril deposition. Moreover, we identify ZNF469, a putative transcriptional regulator of collagen biosynthesis, as a target of NRF2. Both ZNF469 silenced fibroblasts and fibroblasts derived from Brittle Corneal Syndrome patients carrying variants in ZNF469 gene show reduced collagen I gene expression. Our study shows that NRF2 orchestrates matrisome expression in human skin fibroblasts through direct or indirect transcriptional mechanisms that could be prioritized to target dermal ECM homeostasis in health and disease.

Oxidative damage prevention in human skin and sensory neurons by a salicylic acid derivative.

BACKGROUND: Increased protein carbonylation is a hallmark of oxidative stress, protein homeostasis dysregulation and aging in the nervous system and skin. Sensory neurons interact with skin cells and are involved in skin homeostasis. We have previously reported that the 5-octanoyl salicylic acid (C8-SA), a salicylic acid derivative, increased C. elegans lifespan and delayed the accumulation of carbonylated proteins, through the stimulation of autophagy. OBJECTIVES: In this study we aimed to investigate if C8-SA protects human sensory neurons and human skin from extrinsic oxidative stressors as an approach to delay skin aging. METHODS: In vitro reconstituted human epidermis innervated with hiPSc-derived human sensory neurons, as well as ex vivo human organotypic full skin models were used. The fully differentiated sensory neurons were pretreated with C8-SA before oxidative stress induction. Skin explants were maintained in culture and treated topically with C8-SA before the application of urban pollutants. Carbonylated proteins were detected using amino-oxy functionalized fluorophores and quantified. Chaperone mediated autophagy was monitored with LAMP2A immunofluorescence. Inflammation, ROS detoxification and autophagy were assessed by RT-PCR. RESULTS: C8-SA prevented the accumulation of carbonylated proteins, both in human sensory neurons and skin explants. C8-SA stimulated chaperone-mediated autophagy and modulated NRF2 antioxidant response genes, as well as catalase enzymatic activity. CONCLUSIONS: C8-SA acts at two levels to protect skin against oxidative stress: 1) it prevents protein oxidation by stimulating endogenous antioxidant defense and 2) it increases the clearance of oxidized proteins by stimulating chaperone-mediated autophagy. These results suggest that C8-SA maintains skin health in urban polluted environments.

Multi-dimensional computational pipeline for large-scale deep screening of compound effect assessment: an in silico case study on ageing-related compounds.

Designing alternative approaches to efficiently screen chemicals on the efficacy landscape is a challenging yet indispensable task in the current compound profiling methods. Particularly, increasing regulatory restrictions underscore the need to develop advanced computational pipelines for efficacy assessment of chemical compounds as alternative means to reduce and/or replace in vivo experiments. Here, we present an innovative computational pipeline for large-scale assessment of chemical compounds by analysing and clustering chemical compounds on the basis of multiple dimensions-structural similarity, binding profiles and their network effects across pathways and molecular interaction maps-to generate testable hypotheses on the pharmacological landscapes as well as identify potential mechanisms of efficacy on phenomenological processes. Further, we elucidate the application of the pipeline on a screen of anti-ageing-related compounds to cluster the candidates based on their structure, docking profile and network effects on fundamental metabolic/molecular pathways associated with the cell vitality, highlighting emergent insights on compounds activities based on the multi-dimensional deep screen pipeline.

A salicylic acid derivative extends the lifespan of Caenorhabditis elegans by activating autophagy and the mitochondrial unfolded protein response.

Plant extracts containing salicylates are probably the most ancient remedies to reduce fever and ease aches of all kind. Recently, it has been shown that salicylates activate adenosine monophosphate-activated kinase (AMPK), which is now considered as a promising target to slow down aging and prevent age-related diseases in humans. Beneficial effects of AMPK activation on lifespan have been discovered in the model organism Caenorhabditis elegans (C. elegans). Indeed, salicylic acid and acetylsalicylic acid extend lifespan in worms by activating AMPK and the forkhead transcription factor DAF-16/FOXO. Here, we investigated whether another salicylic acid derivative 5-octanoyl salicylic acid (C8-SA), developed as a controlled skin exfoliating ingredient, had similar properties using C. elegans as a model. We show that C8-SA increases lifespan of C. elegans and that a variety of pathways and genes are required for C8-SA-mediated lifespan extension. C8-SA activates AMPK and inhibits TOR both in nematodes and in primary human keratinocytes. We also show that C8-SA can induce both autophagy and the mitochondrial unfolded protein response (UPRmit ) in nematodes. This induction of both processes is fully required for lifespan extension in the worm. In addition, we found that the activation of autophagy by C8-SA fails to occur in worms with compromised UPRmit , suggesting a mechanistic link between these two processes. Mutants that are defective in the mitochondrial unfolded protein response exhibit constitutive high autophagy levels. Taken together, these data therefore suggest that C8-SA positively impacts longevity in worms through induction of autophagy and the UPRmit .

Longevity effect of a polysaccharide from Chlorophytum borivilianum on Caenorhabditis elegans and Saccharomyces cerevisiae.

The traditional Indian medicine, Ayurveda, provides insights and practical solutions towards a healthy life style. Rasayana is a branch of Ayurveda known for preserving and promoting health, enhancing the quality of life and delaying the aging process. In the traditional knowledge, the Rasayana herb, Chlorophytum borivilianum (C. borivilanum) is regarded as a general health promoting tonic that delays aging and increases lifespan, cognitive function and physical strength. Aging is a complex and multifactorial physiological phenomenon that manifests itself over a wide range of biological systems, tissues, and functions. Longevity is an obvious marker of physiological aging. Simple model systems such as the single-cell budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the nematode, Caenorhabditis elegans (C. elegans) are widely used to study the aging process and longevity. Here, we show that a polysaccharide fraction obtained from C. borivilianum increases the lifespan of S. cerevisiae and C. elegans, using an automated screening platform (ChronoscreenTM). Chemical analysis of this extract revealed a low molecular weight polysaccharide of 1000 Da, predominantly comprising Glu1→6Glu linkage. This polysaccharide showed significant dose-dependent extension of the median lifespan of S. cerevisiae by up to 41% and of the median lifespan of C. elegans by up to 10%. Taking cue from these results and the traditionally described benefits of Rasayanas on skin rejuvenation, we tested in vitro the polysaccharide for potential skin benefits. In a keratinocyte culture, we observed that this polysaccharide increased cell proliferation significantly, and induced synthesis of hyaluronic acid (HA), a well-known extracellular matrix component. Furthermore, when added to culture medium of human reconstructed epidermis, we observed an enhanced production of epidermal markers, e.g. CD44 and HA that are otherwise diminished in aged skin. Together, these results suggest that in addition to life-span extension of S. cerevisiae and C. elegans, a polysaccharide from the Rasayana herb, C. borivilianum may have beneficial effects on skin aging parameters.

A jasmonic acid derivative improves skin healing and induces changes in proteoglycan expression and glycosaminoglycan structure.

BACKGROUND: Jasmonates are plant hormones that exhibit anti-cancer and anti-inflammatory properties and have therefore raised interest for human health applications. The molecular basis of these activities remains poorly understood, although increasing evidence suggests that a variety of mechanisms may be involved. Recently, we have reported that a jasmonate derivative (JAD) displayed anti-aging effects on human skin by inducing extracellular matrix (ECM) remodeling. Based on this observation, we have investigated here the effects of JAD on proteoglycans and glycosaminoglycan (GAG) polysaccharides, which are major cell-surface/ECM components and are involved in a multitude of biological processes. In parallel, we have examined the ability of JAD to promote growth factor activities and improve skin wound healing. METHODS: Proteoglycan expression was analyzed on epidermal primary keratinocytes and reconstituted skin epidermis, using electron/immunofluorescence microscopy, western blotting and flow cytometry. GAG composition was determined by disaccharide analysis. Finally, biological activities of JAD were assessed in cellulo, in FGF-7 induced migration/proliferation assays, as well as in vivo, using a suction blister model performed on 24 healthy volunteers. RESULTS: JAD was found to induce expression of major skin proteoglycans and to induce subtle changes in GAG structure. In parallel, we showed that JAD promoted FGF-7 and improved skin healing by accelerating epithelial repair in vivo. CONCLUSION: This study highlights JAD as a promising compound for investigating GAG structure-function relationships and for applications in skin cosmetic /corrective strategies. GENERAL SIGNIFICANCE: We propose here a novel mechanism, by which jasmonate derivatives may elicit biological activities in mammals.

AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha.

Activation of AMP-activated kinase (AMPK) in skeletal muscle increases glucose uptake, fatty acid oxidation, and mitochondrial biogenesis by increasing gene expression in these pathways. However, the transcriptional components that are directly targeted by AMPK are still elusive. The peroxisome-proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) has emerged as a master regulator of mitochondrial biogenesis; furthermore, it has been shown that PGC-1alpha gene expression is induced by exercise and by chemical activation of AMPK in skeletal muscle. Using primary muscle cells and mice deficient in PGC-1alpha, we found that the effects of AMPK on gene expression of glucose transporter 4, mitochondrial genes, and PGC-1alpha itself are almost entirely dependent on the function of PGC-1alpha protein. Furthermore, AMPK phosphorylates PGC-1alpha directly both in vitro and in cells. These direct phosphorylations of the PGC-1alpha protein at threonine-177 and serine-538 are required for the PGC-1alpha-dependent induction of the PGC-1alpha promoter. These data indicate that AMPK phosphorylation of PGC-1alpha initiates many of the important gene regulatory functions of AMPK in skeletal muscle.

A fundamental system of cellular energy homeostasis regulated by PGC-1alpha.

Maintenance of ATP levels is a critical feature of all cells. Mitochondria are responsible for most ATP synthesis in eukaryotes. We show here that mammalian cells respond to a partial chemical uncoupling of mitochondrial oxidative phosphorylation with a decrease in ATP levels, which recovers over several hours to control levels. This recovery occurs through an increased expression of the transcriptional coactivator peroxisome proliferator-activated receptor-coactivator 1alpha (PGC-1alpha) and mitochondrial genes. Cells and animals lacking PGC-1alpha lose this compensatory mechanism and cannot defend their ATP levels or increase mitochondrial gene expression in response to reduced oxidative phosphorylation. The induction of PGC-1alpha and its mitochondrial target genes is triggered by a burst of intracellular calcium, which causes an increase in cAMP-response-element-binding protein and transducer of regulated cAMP-response-element-binding proteins actions on the PGC-1alpha promoter. These data illustrate a fundamental transcriptional cycle that provides homeostatic control of cellular ATP. In light of this compensatory system that limits the toxicity of mild uncoupling, the use of chemical uncoupling of mitochondria as a means of treating obesity should be re-evaluated.

Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators.

PPARgamma coactivator 1alpha (PGC-1alpha) is a potent stimulator of mitochondrial biogenesis and respiration. Since the mitochondrial electron transport chain is the main producer of reactive oxygen species (ROS) in most cells, we examined the effect of PGC-1alpha on the metabolism of ROS. PGC-1alpha is coinduced with several key ROS-detoxifying enzymes upon treatment of cells with an oxidative stressor; studies with RNAi or null cells indicate that PGC-1alpha is required for the induction of many ROS-detoxifying enzymes, including GPx1 and SOD2. PGC-1alpha null mice are much more sensitive to the neurodegenerative effects of MPTP and kainic acid, oxidative stressors affecting the substantia nigra and hippocampus, respectively. Increasing PGC-1alpha levels dramatically protects neural cells in culture from oxidative-stressor-mediated death. These studies reveal that PGC-1alpha is a broad and powerful regulator of ROS metabolism, providing a potential target for the therapeutic manipulation of these important endogenous toxins.

Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1alpha null mice.

PGC-1alpha is a coactivator of nuclear receptors and other transcription factors that regulates several metabolic processes, including mitochondrial biogenesis and respiration, hepatic gluconeogenesis, and muscle fiber-type switching. We show here that, while hepatocytes lacking PGC-1alpha are defective in the program of hormone-stimulated gluconeogenesis, the mice have constitutively activated gluconeogenic gene expression that is completely insensitive to normal feeding controls. C/EBPbeta is elevated in the livers of these mice and activates the gluconeogenic genes in a PGC-1alpha-independent manner. Despite having reduced mitochondrial function, PGC-1alpha null mice are paradoxically lean and resistant to diet-induced obesity. This is largely due to a profound hyperactivity displayed by the null animals and is associated with lesions in the striatal region of the brain that controls movement. These data illustrate a central role for PGC-1alpha in the control of energy metabolism but also reveal novel systemic compensatory mechanisms and pathogenic effects of impaired energy homeostasis.

The Caenorhabditis elegans F-box protein SEL-10 promotes female development and may target FEM-1 and FEM-3 for degradation by the proteasome.

The Caenorhabditis elegans F-box protein SEL-10 and its human homolog have been proposed to regulate LIN-12 Notch signaling by targeting for ubiquitin-mediated proteasomal degradation LIN-12 Notch proteins and SEL-12 PS1 presenilins, the latter of which have been implicated in Alzheimer's disease. We found that sel-10 is the same gene as egl-41, which previously had been defined by gain-of-function mutations that semidominantly cause masculinization of the hermaphrodite soma. Our results demonstrate that mutations causing loss-of-function of sel-10 also have masculinizing activity, indicating that sel-10 functions to promote female development. Genetically, sel-10 acts upstream of the genes fem-1, fem-2, and fem-3 and downstream of her-1 and probably tra-2. When expressed in mammalian cells, SEL-10 protein coimmunoprecipitates with FEM-1, FEM-2, and FEM-3, which are required for masculinization, and FEM-1 and FEM-3 are targeted by SEL-10 for proteasomal degradation. We propose that SEL-10-mediated proteolysis of FEM-1 and FEM-3 is required for normal hermaphrodite development.