Basal Level of Autophagy Is Increased in Aging Human Skin Fibroblasts In Vitro, but Not in Old Skin

May Basal Level of Autophagy Is Increased in Aging Human Skin Fibroblasts In Vitro, but Not in Old Skin Data Availability Statement: All relevant data are within the paper. 0 1 2 Dino Demirovic 0 1 2 Carine Nizard 0 1 2 Suresh I. S. Rattan 0 1 2 0 1 Laboratory of Cellular Ageing, Department of Molecular Biology and Genetics, Aarhus University , Aarhus , Denmark , 2 LVMH Research, St. Jean de Braye , France 1 Funding: The Laboratory of Cellular Ageing received a PhD fellowship grant from LVMH Research, France. The funder provided support in the form of salary for author CN, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section 2 Academic Editor: Dimitris Kletsas, National Centre for Scientific Research , 'Demokritos', GREECE Intracellular autophagy (AP) is a stress response that is enhanced under conditions of limitation of amino acids, growth factors and other nutrients, and also when macromolecules become damaged, aggregated and fibrillated. Aging is generally accompanied by an increase in intracellular stress due to all the above factors. Therefore, we have compared the basal levels of AP in serially passaged human facial skin fibroblasts undergoing aging and replicative senescence in vitro, and ex vivo in the skin biopsies from the photo-protected and photo-exposed area of the arms of 20 healthy persons of young and old ages. Immunofluorescence microscopy, employing antibodies against a specific intracellular microtubuleassociated protein-1 light chain-3 (LC3) as a well established marker of AP, showed a 5fold increase in the basal level of LC3 in near senescent human skin fibroblasts. However, no such age-related increase in LC3 fluorescence and AP could be detected in full thickness skin sections from the biopsies obtained from 10 healthy young (age 25 to 30 yr) and 10 old (age 60 to 65 yr) donors. Furthermore, there was no difference in the basal level of LC3 in the skin sections from photo-protected and photo-exposed areas of the arm. Thus, in normal conditions, the aging phenotype of the skin cells in culture and in the body appears to be different in the case of AP. - Competing Interests: CN is a senior scientist and an employee of LVMH Research, France. DD and Optimal stress response (SR) is an essential aspect of the biological property of homeostasis/ homeodynamics [1,2]. Among the major intracellular SR pathways, autophagy (AP) is a response to insufficiency of nutrients; and is being increasingly recognized for its role in survival, aging, longevity and age-related pathology, including cancer [3,4]. AP is a regulatory self-eating process, which involves cytoplasmic degradation of macromolecules and other large compartments of the cell, such as the mitochondria. Under normal conditions, AP operates constitutively at a basal level concurrent with lysosomes and proteasomes [5]. However, AP is enhanced under conditions of limitation or deprivation of amino acids, growth factors and other nutrients, or when macromolecules become damaged, aggregated, fibrillated or in some SISR have declared that no competing interests exist. This does not alter their adherence to PLOS ONE policies on sharing data and materials. other way modified and are not used by the cells. Thus, AP is one of the survival mechanisms for cells during extrinsic and intrinsic stress, whose biological consequences depend on the level of the stress. For example, whereas too much AP can lead to cell death, moderately enhanced AP during dietary restriction slows down aging and increases the lifespan of cells and organisms [6]. Since the process of aging is characterized by a progressive occurrence and accumulation of damage, including protein aggregation, fibrillation, mitochondrial damage and reduced efficiency of energy production [7,8], it is relevant to find out if the basal level of AP is altered during aging. One of the widely used approaches to study AP is the appearance and disappearance of a specific intracellular microtubule-associated protein 1 light chain 3 (LC3), which can be used as a marker for the autophagic flux [9]. LC3 is a cytoplasmic and constitutively expressed protein, which is cleaved at its C-terminus by Atg4 protease generating the cytosolic form LC3-I. It is then conjugated to phosphatidylethanolamine (PE) in a ubiquitin-like reaction and the lipidated form of LC3, known as LC3-II, is attached to the autophagosome membrane. The formation of a cytoplasmic double membrane, the so-called phagophore, probably originating from the endoplasmatic reticulum, then expands and forms a closed sphere called the mature autophagosome, which is recognized by the lysosomal associated membrane proteins (LAMPs), and the contents of the autophagosome, including LC3-II, are degraded by the lysosomal enzymes [10]. We have studied and compared the basal levels of LC3-II as an indicator of autophagic flux in serially passaged human facial skin fibroblasts undergoing aging and replicative senescence in vitro, and ex vivo in the skin biopsies from healthy persons of different ages. The cell culture model system of serial passaging and cellular aging in vitro, also known as the Hayflick system, is a widely used system for understanding the phenomenological and mechanistic aspects of aging [11,12]. However, not all aspects of aging are reflected in this model system in an exact manner, and some changes observed in vitro may be exaggerated or diminished in the normal situation in vivo. Therefore, comparative studies are important to understand and resolve the significance of age-related changes in different model systems and conditions. Facial adult skin fibroblast cell strain designated as FSF-1, was established from a healthy 40-yr old womans eye-lid, at LVMH-Research, St. Jean de Braye, France, and stored frozen in liquid nitrogen at passage 2, as described previously [13]. FSF-1 cultures were grown and maintained in plastic tissue culture flasks (NUNC, Roskilde, Denmark), in an incubator at 37C, 95% relative humidity and 5% CO2, using Dulbeccos Modified Eagles Medium, (DMEM; Biowhittaker, Viviers, Belgium), supplemented with 10% bovine Fetal Calf Serum (FCS; Biological Industries, Beit, Haemek, Israel), and 100 U/mL penicillin/streptomycin (Biowhittaker, Viviers, Belgium). For sub-culturing, monolayer cultures near confluency (9095% of the growth surface covered by cells) were trypsinized with 0.25% trypsin-EDTA, and distributed into new cell culture flasks. Cells were serially sub-cultured or passaged at 1:2 or 1:4 split ratio until they stopped dividing, became irreversibly growth arrested and entered a state of senescence. In order to estimate the proliferative lifespan of FSF-1 cells, 1 or 2 passages (P) were added to the age of the cultures at each sub-culturing at 1:2 or 1:4 split ratio, respectively. H (...truncated)