J. Cosmet. Sci., 67, 13–20 (January/February 2016) 13 Autophagy in human skin fi broblasts: Comparison between young and aged cells and evaluation of its cellular rhythm and response to Ultraviolet A radiation NADINE PERNODET, KELLY DONG, and EDWARD PELLE, Estee Lauder Research Laboratories, Melville, NY (N.P., K.D., E.P.), and Environmental Medicine, New York University School of Medicine, New York, NY (E.P.). Accepted for publication November 3, 2015. Synopsis Autophagic mechanisms play critical roles in cell maintenance. Damaged organelles that are not removed by autophagosomes, which act by engulfi ng and degrading these cellular components, have been linked to various pathologies. Recently, the progression of aging has also been correlated to a compromised autophagic response. Here, we report for the fi rst time a signifi cant reduction in autophagic levels in synchronized aged normal human skin fi broblasts as compared to young fi broblasts. We measured a 77.9% reduction in autophagy as determined by reverse transcription-polymerase chain reaction for LC3B expression, a microtubule- associated protein correlated to late stage autophagosome formation. In addition, we visualized these same changes by immunocytofl uorescence with antibodies directed against LC3B. By harvesting synchronized, as well as unsynchronized cells over time, we were also able to measure for the fi rst time a nighttime peak in autophagy that was present in young but absent in aged fi broblasts. Finally, since human skin is constantly subjected to environmentally induced oxidative stress from sunlight, we exposed fi broblasts to 10 J/cm2 ultraviolet A and found, in good agreement with current literature, not only that irradiation could partially reactivate autophagy in the aged cells, but also that this increase was phase shifted earlier from its endogenous temporal pattern because of its loss of synchronization with circadian rhythm. INTRODUCTION Autophagy, a major cellular degradative and recycling pathway, has now been shown to be essential for health and longevity, as well as a critical player in the aging process (1). As a highly conserved mechanism, it is responsible for the continuous recycling and renewal of intracellular organelles, lipids, and proteins and is a vital secondary source of energy for cells. It is a well-calibrated pathway that supports cellular homeostasis and responds to stress. By ensuring that malfunctioning or damaged intracellular components are removed and recycled, further intracellular damage can be minimized. The autophagic process removes cellular debris by fi rst sequestering material in an autophagosome, followed by Address all correspondence to Nadine Pernodet at npernode@estee.com.

JOURNAL OF COSMETIC SCIENCE 14 fusion with a lysosome leading to the formation of an autolysosome, which then degrades and recycles cellular components. Autophagy can be considered to be a very effi cient quality control mechanism that maintains cell effi ciency and health (2). Decreases in autophagic activity have recently been associated with age. For example, hepatocytes were found to accumulate intracellular damage over time (3) and the loss of activity as described by Cuervo (4) has now been recognized as a major aging pathway. Furthermore, the link between impaired autophagy and age-associated illness has now been established for cancer, diabetes, and Alzheimer’s and Parkinson’s diseases and is cur- rently an active area of investigation. Aging tissues show an expansion of lysosomal com- partments, accumulation of autophagic vacuoles, and deposition of undigested materials inside the cells. These changes result in a decrease of energy supply and an increase in intracellular damages and oxidative stress, all of which have been shown to accelerate aging. Since metabolic intermediates are generated as a consequence of autophagy, caloric restric- tion has been shown to play a role in the life span extension associated with autophagy and this has been genetically confi rmed in Caenorhabditis elegans (5). In addition, circadian rhythms, which also coordinate metabolic activity, have been observed in yeast to temporally affect autophagy (6). To understand how these processes may infl uence cutaneous health and aging, we compared autophagy levels by measuring LC3B, a microtubule-associated protein correlated to late stage autophagosome formation (7) between young and aged skin fi broblasts, and then also analyzed these levels as a function of time. Furthermore, since human skin is constantly exposed to sunlight, we determined their response to environmental stress induced by ultraviolet A (UVA 320–400 nm). MATERIALS AND METHODS TISSUE CULTURE Normal human dermal skin fi broblasts from 2 day and 67-year-old donors were obtained from The Coriell Institute (Camden, NJ) and grown on 4-well chamber slides (2 × 104 cells/well) for immunocytofl uorescence or on 60-mm dishes for RNA extraction (5 × 104 cells/dish). They were cultured in Dulbecco’s Modifi ed Eagle’s Media (DMEM) media with 5% bo- vine calf serum and 1% penicillin/streptomycin at 37°C in a 5% CO2 humidifi ed incuba- tor. Cells were cultured for 72 h before the start of the experiment. CELL SYNCHRONIZATION Cells were synchronized via serum starvation. Cells to be synchronized were washed once with Dulbecco’s Phosphate Buffered Saline (D-PBS) and then treated with medium without serum. Medium with serum was placed on cells that were not synchronized. Cells were incubated for 24 h. After synchronization, the media were removed and medium with serum was added to both unsynchronized and synchronized cells. RNA was extracted at 0, 1, 2, 4, and 8 h after release from starvation. In a second experiment, synchronized cells were irradiated just before release from starvation and RNA extracted at the same time intervals as before. IMMUNOCYTOFLUORESCENCE Cells were fi xed with 4% paraformaldehyde and then permeabilized with methanol. Cells were blocked with 5% goat serum and 0.3% Triton X-100 (Sigma-Aldrich, St. Louis,