Date Published: June 23, 2017
Publisher: Public Library of Science
Author(s): Daniel Mellem, Martin Sattler, Sonja Pagel-Wolff, Sören Jaspers, Horst Wenck, Michael Alexander Rübhausen, Frank Fischer, Irene Georgakoudi.
Mitochondria form dynamic networks which adapt to the environmental requirements of the cell. We investigated the aging process of these networks in human skin cells in vivo by multiphoton microscopy. A study on the age-dependency of the mitochondrial network in young and old volunteers revealed that keratinocytes in old skin establish a significantly more fragmented network with smaller and more compact mitochondrial clusters than keratinocytes in young skin. Furthermore, we investigated the mitochondrial network during differentiation processes of keratinocytes within the epidermis of volunteers. We observe a fragmentation similar to the age-dependent study in almost all parameters. These parallels raise questions about the dynamics of biophysical network structures during aging processes.
Cells in human tissue produce their energy in form of adenosine triphosphate (ATP) basically by oxidative phosphorylation. This process takes place in mitochondria, that form highly dynamic networks adapting fast to the cells’ environment and their metabolic requirements.  In recent years, numerous experiments have been performed to investigate correlations between mitochondrial network states and corresponding metabolic processes within cells. Cancer cells lacking of a glycolytic medium establish fusion states of the mitochondrial network correlating with a change in energy production going from glycolysis to oxidative phosphorylation.  In contrast, a transfer from respiratory to glycolytic conditions leads to a fragmentation of the mitochondrial network.  During moderate stress mitochondrial networks form hyperfusion states which coincide with increased ATP production.  On the contrary, high stress levels induce to a fragmentation of networks.  Mitochondrial fission and fusion states are considered to be quality saving mechanisms of the cell. Fission states support repelling of heavily damaged mitochondria from the network, e.g. during autophagy and mitophagy. [6, 7] In contrast, fusion states help to compensate for defect protein complexes or rare metabolites among mitochondria. [8, 9] Recently, a fragmentation of the mitochondrial network with age was observed in vitro.  The interplay of mitochondrial dynamics was simulated biophysically in a probabilistic quality model which also revealed a significant fragmentation of the mitochondrial network during aging. 
Tissue can be investigated non-invasively in vivo by imaging endogenous fluorophores using multiphoton microscopy. [12–14] An important metabolic fluorophore in the epidermis is nicotinamide adenine dinucleotide (NADH) [15, 16] which serves as an electron carrier from the Krebs cycle to respiratory chain. Thus, NADH is almost exclusively located in mitochondria. We tested this premise by in vitro investigations using a confocal scanning microscope (Leica TCS SP5, Leica Microsystems, Mannheim, Germany). Neonatal human epidermal keratinocytes were purchased from Lonza Group AG (Basel, Switzerland) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Carlsbad, USA), supplemented with 10% fetal calf serum (PAA Laboratories, Pasching, Austria), L-glutamine and penicillin/streptomycin (both: Life Technologies, Carlsbad, USA). During culturing cells were maintained in a humidifying incubator with a 5% CO2 atmosphere at 37°C. Mitochondria in the cells were marked with MitoTracker Red CMXRos (Life Technologies, Carlsbad, USA). We correlated the fluorescence of the dye from 570nm to 650nm to the autofluorescence of keratinocytes in the NADH emission spectrum from 410 nm to 540 nm (Fig 1). The measurements were performed with a 63x objective (HCX PL APO lambda blue 63.0×1.20 WATER UV, Leica). Single images of 2048*2048 pixels were generated with a bidirectional scan either at zoom 3.0 (82μm x 82 μm, step size: 0.04 μm) with scanning rate of 100Hz or at zoom 7.9 (31.2μm x 31.2μm, step size: 0.04μm) with scanning rate of 200Hz.
In the first study the mitochondrial morphology in epidermal keratinocytes of twelve young (mean ± SD: 23.75 ± 1.67 years) and twelve old (72.17 ± 4.15 years) volunteers was analysed by statistically comparing 48 areas in each age group. We analysed the number, area and circularity of the mitochondrial clusters per keratinocyte in the stratum granulosum in a depth of about 15 μm. The results of all examined parameters were averaged in each area before statistical comparison. Mitochondrial clusters in the stratum granulosum of young volunteers are significantly larger (p = 0.029) and have a significantly higher circularity (p = 0.014) (Fig 2). In contrast, the number of mitochondrial clusters normalized to the size of each keratinocyte is significantly higher (p = 0.006) in old skin (S2 Fig).
Our study of the mitochondrial network during aging reveals, that granular epidermal keratinocytes in young skin have less clusters in total than keratinocytes in old skin. Additionally, clusters in granular keratinocytes of young volunteers are bigger in size and less circular in shape. These findings reveal a high connectivity among mitochondria in the stratum granulosum in young skin and a fragmented mitochondrial network in the stratum granulosum of old skin. Granular keratinocytes of young volunteers seem to prefer a fusion state of the mitochondrial network which is not maintained during aging.
In conclusion, we have measured and analyzed the morphology of mitochondrial networks in human skin in vivo for the first time. We found a significant fragmentation of mitochondrial morphologies in granular keratinocytes during aging. Our results are qualitatively in good agreement with age-dependent investigations in vitro and with simulations of biophysical models. Moreover, we observed a fragmentation of the network during the differentiation process of keratinocytes in the epidermis. The parallels of both studies raise questions about the linkage of aging and differentiation concerning mitochondrial morphologies.