The authors have declared that no competing interests exist.
Conceived and designed the experiments: EEP AR DS. Performed the experiments: AR DS AUB AS KH JG. Analyzed the data: EEP AR AUB DS AS. Contributed reagents/materials/analysis tools: EEP AUB AEMS RM. Wrote the paper: EEP AR.
Current address: Novartis Pharma Stein AG, Stein, Switzerland
Apart from the first family member, uncoupling protein 1 (UCP1), the functions of other UCPs (UCP2-UCP5) are still unknown. In analyzing our own results and those previously published by others, we have assumed that UCP's cellular expression pattern coincides with a specific cell metabolism and changes if the latter is altered. To verify this hypothesis, we analyzed the expression of UCP1-5 in mouse embryonic stem cells before and after their differentiation to neurons. We have shown that only UCP2 is present in undifferentiated stem cells and it disappears simultaneously with the initiation of neuronal differentiation. In contrast, UCP4 is simultaneously up-regulated together with typical neuronal marker proteins TUJ-1 and NeuN during mESC differentiation in vitro as well as during murine brain development
The subfamily of uncoupling proteins (UCP1-UCP5) belongs to the superfamily of mitochondrial carriers that are alleged to shuttle metabolic substrates across the mitochondrial inner membrane
In 1997, UCP2 was discovered and its ubiquitous expression was mainly postulated on the basis of mRNA distribution
Since knowledge about the correct protein localization seems to be a crucial prerequisite for the recognition of UCP function(s), we have now applied a dynamic approach which allows us to follow the protein expression under changing developmental and metabolic conditions. We initiated the differentiation of murine embryonic stem cells into neurons and analyzed the expression of UCP subfamily members. Our results strengthen the hypothesis that the expression of UCP2 is tightly connected to the cell metabolic state and thereby changes simultaneously upon its alteration.
Undifferentiated murine embryonic stem cells (mESCs, clone D3,
Initiation of neural differentiation and maintenance of differentiated cells were performed as previously described with minor modifications
N18TG2 cells (Deutsche Sammlung von Mikroorganismen & Zellkultur GmbH (DSMZ), Braunschweig, Germany) and BV-2 cells (Banca Biologica e cell Factory, Genova, Italy) were kept in 25 cm2 cell culture flasks with 5 ml medium in an incubator at 37°C, 5% CO2 and 100% humidity and split when reaching confluence. Cell culture media contained DMEM (4.5 mg/ml glucose) supplemented with either 9.7% fetal bovine serum, 3.85 mM glutamine and 1.94 mM sodium pyruvate (N18TG2 cells) or 9.8% fetal bovine serum and 3.92 mM glutamine (BV-2 cells) (all obtained from Sigma-Aldrich). Prior to experiments, cells were collected by centrifugation for 10 min at 178 g and re-suspended in serum-free media with afore mentioned concentrations of glutamine and sodium pyruvate. In addition, 2% B27® without antioxidants (Invitrogen) was added to the medium. Cells were cultivated in 6 well plates with 2 ml medium per well for another 48 h before the experiments.
This study was carried out in strict accordance with the recommendations specified in the European guidelines (2010/63/EU) for the use of laboratory animals. The protocol was approved by the Committee on the Ethics of Animal Experiments (Landesamt für Gesundheit und Soziales, Berlin (LAGeSo); permit number: T0108/11). Pregnant, postnatal and adult C57BL/6 mice obtained from the central animal facility at Charité – Universitätsmedizin's Research Institutes for Experimental Medicine, were kept under standard laboratory conditions (12 hour light/dark cycle; (55±15)% humidity; (24±2)°C room temperature and water ad libitum, enriched and grouped). Pregnant and postnatal animals were sacrificed by decapitation; all efforts were made to minimize suffering. For one sample, six whole embryos (E8–E9), embryonic heads (E10–E12) or isolated organs from embryonic/young mice were collected. Samples were frozen in liquid nitrogen and stored at −80°C until protein or RNA isolation.
Total RNA from murine tissue samples and cultivated cells was extracted using TRIzol® reagent (Invitrogen). Production of cDNA was completed using the “High Capacity cDNA reverse Transcription kit” (Applied Biosystems, Foster City, CA, USA). Reverse transcriptase qRT PCR was performed with the following gene expression assays (Applied Biosystems): Mm00627598_m1, Mm01277266_m1, Mm00488302_m1, 4352932E and ID 4352933E for UCP2, UCP4, UCP5, GAPDH and β-actin respectively. For HPRT, separate primer and probe were used (Primer Mix: for
The collection of total cellular protein from tissue and cell culture samples and Western blot (WB) analysis for UCP4 and UCP2 was performed as described previously
Cells plated on coverslips were fixed in ice cold 4% PFA for 25 min, washed three times in 0.1 M PBS and incubated with blocking solution containing 10% fetal calf serum (Biochrom, Berlin, Germany) and 0.05% Triton X-100 (Sigma-Aldrich) for 1 h at RT. Thereafter, cells were labeled overnight at 4°C using antibodies described above. The dilutions of antibodies for immunocytochemistry were: 1∶400-1∶1000 for anti-UCP4 and 1∶1000 for MAP2 (Sigma-Aldrich). After washing the cells with PBS, samples were incubated for 1 h at RT with the appropriate goat anti-mouse IgG Alexa-488 and goat anti-rabbit IgG Alexa-568 (Invitrogen) secondary antibodies, diluted 1∶1000 in blocking solution for 1 h at RT. After rinsing in PBS, coverslips were embedded in mounting medium containing DAPI (Vectashield; Vector Laboratories, Burlingame, CA, USA), dried and stored at 4°C.
Naïve C57BL/6 mouse was deeply anaesthetized with a mixture containing ketamine (Pfizer, Karlsruhe, Germany) and xylazine (
For light microscopy, brain sections were prepared in the same manner. Then the sections were incubated with 3% H2O2 for blocking of endogenous peroxidase, washed three times with 0.1 M PBS, and soaked for 1 hour in 10% normal goat or donkey serum to block non-specific binding. Thereafter, the free floating sections were incubated overnight at 4°C with primary antibodies diluted as described above in 1% serum and 0.5% Triton X-100. As secondary antibodies, biotinylated anti-rabbit IgG, anti-mouse IgG and anti-goat IgG (Vector Laboratories, Burlingame, CA, USA) were incubated in dilution 1∶1000 for 2 h at RT. Next, sections were pre-incubated with ABC-Elite (Vector Laboratories) and developed with 0.03% H2O2 and 1% 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich). After rinsing in PBS, tissues were mounted on slides, dehydrated through a graded series of ethanol, cleared in xylene, and coverslipped with Entellan® (Merck, Darmstadt, Germany).
Confocal microscopy was performed using an inverse confocal microscope (TCS SP5 Leica Microsystems) equipped with argon and helium-neon lasers with excitation wavelength 488 nm, 543 nm and 633, respectively. Image processing was performed with Leica Confocal Software and Image J. Light microscopy was performed as described in Smorodchenko et al., 2009
Previously, we and other research groups have reported that UCP2 is expressed in haematopoietic and human pluripotent stem cells
A–E. Representative Western blot images showing UCP expression in mESCs using antibodies against UCP1 (A), UCP2 (B), UCP3 (C), UCP4 (D) and UCP5 (E). Brown adipose tissue (BAT), activated T-cells, brain from adult mice and recombinant mUCP5 were used as positive controls for the respective protein antibodies. Gels were loaded with 20 µg protein per lane. Antibodies directed against VDAC, GAPDH and Hsp 60 were used to visualize the respective proteins as loading controls. mESCs from at least 3 different passages were analyzed in each experiment.
Because both UCP2 and UCP4 were reported to be abundant in neurons, we analyzed their expression at mRNA and protein levels in mESC cultures at day 0 (cells before the initiation of differentiation) and day 7, 9, 12, 14, 21 and 28 of neuronal differentiation (
A. Representative Western blots show the time-dependent expression of neuronal (TUJ-1 for young and NF for adult neurons) and astrocyte (GFAP) markers during mESCs differentiation in culture. Gels were loaded with 20 µg protein per lane. B. Real-time PCR analysis of mESCs shows the amount of UCP2, UCP4 and UCP5 mRNA relative to mRNA amounts of the housekeeping gene GAPDH at different time points during neuronal differentiation. Each data point represents the mean value and SD of 3 independent differentiation experiments.
A–B. Representative Western blots of UCP2 (A) and UCP4 (B) expression during the differentiation of mESCs in culture. Activated T-cells and primary neuronal cultures (13 days) were used as positive controls. Gels were loaded with 20 µg protein per lane. Cells were collected at different time points from at least three independent differentiation experiments. C. Representative fluorescent images showing the time course of UCP4 and MAP2 expression during differentiation of mESCs to neurons. Primary antibodies were visualized by Alexa-488 (MAP2, green) and Alexa-567 (UCP4, red) respectively. Cell nuclei were counterstained with DAPI (blue).
In contrast to its nearly constant mRNA levels, UCP2 promptly disappeared on the protein level with the initiation of neuronal differentiation (
Immunocytochemical staining of cells at days 7, 14 and 21 of neuronal differentiation showed the results which confirm our WB data. UCP4 and the neuronal marker MAP2 are only slightly expressed at day 7 (
To support the data revealed in an
A. UCP2, UCP4 and UCP5 mRNA levels during neuronal development analyzed by quantitative PCR. UCP mRNA levels in mouse head are shown as a ratio to GAPDH at embryonic day 12 (E12; inset) and as a ratio (UCP mRNA)/(GAPDH mRNA) at different days to (UCP mRNA)/(GAPDH mRNA) at E8. B. Representative Western blot indicates the simultaneous start of UCP4 protein expression with the expression of the neuronal marker TUJ-1. C-D. Representative Western blots demonstrate that UCP2 is not present at the protein level in the tested embryonic tissue (C) as well as in young postnatal neocortical brain tissue (NC) (D). Gels were loaded with 20 µg protein per lane. GAPDH, β-actin and VDAC were used as loading controls. At least 3 samples of pooled embryonic and postnatal tissue from at least 6 mice were analyzed at each condition (Experiments A–D).
It is known that adult brain neurogenesis occurs in restricted regions such as in the subventricular zone of the lateral ventricle (SVZ) and the subgranular zone of gyrus dentatus (SGZ)
A. Schematic drawing illustrates the localization of the SVZ of the lateral ventricle in adult mouse brain. B–C. Light microscopy analysis of the representative immunohistostained sample shows the distribution of UCP4- and Dcx-positive cells within the SVZ in 50 µm thick coronal sections of adult mouse brain. D. Representative CLSM images of UCP4 (green), Dcx (red) and NeuN (blue) stained with respective antibodies and visualized using Alexa 488, Alexa 594 and Alexa 633 fluorescent dyes.
Unfortunately, there is no appropriate antibody against UCP2. Our antibody is only reliable when used specifically in WB. Therefore, we were not able to test the expression of UCP2 in this region.
The function of neuronal UCPs and other proteins are often analyzed in immortalized cell lines. The data presented in
A. Representative Western blot analysis of UCP4 expression in the murine neuroblastoma cell line N18TG-2 and murine microglial cell line BV-2. Mouse brain tissue was used as a positive control for the antibody against UCP4. B. Representative Western blot analysis of UCP2 expression in the murine neuroblastoma cell line N18TG-2 and murine microglial cell line BV-2. Thymus of UCP2 knockout (KO) and wild type (wt) mice were used as negative and positive controls for the antibody directed against UCP4. Gels were loaded with 20 µg protein per lane. Cells from at least three different passages were analyzed in each experiment.
In this work, we, for the first time, performed the comprehensive analysis of UCP2 and UCP4 expression in mouse embryonic stem cells (mESC) during their differentiation into neural cells. We revealed that only undifferentiated highly proliferative stem cells express UCP2. After the initiation of neuronal differentiation, UCP2 protein levels dropped abruptly and did not appear at later time points of differentiation, whereas UCP2 mRNA remained nearly constant throughout the differentiation period. Moreover, we could not detect UCP2 in murine embryonic brain tissue after the start of neurogenesis.
Results presented in this work clearly show that both proteins (but not mRNA of these proteins) do not occur in the same cell type at the same time (
mRNA ratio to GAPDH | ||||||
+++ > 0.1 | protein detection | |||||
0.1 > ++ > 0.005 | in 20 µg total protein | |||||
0.005 > + > 0.001 | ||||||
UCP2 | UCP4 | UCP5 | UCP2 | UCP4 | UCP5 | |
brain | ++ | ++ | ++ | O | X | O |
spinal cord | ++ | ++ | + | O | X | O |
heart | ++ | + | O | O | O | O |
skeletal muscle | + | O | O | O | O | O |
BAT | n.a. | n.a. | n.a. | O | O | O |
WAT | +++ | + | + | O | O | O |
spleen | +++ | + | + | X | O | O |
thymus | +++ | ++ | + | X | O | O |
lungs | +++ | ++ | + | (X) | O | O |
stomach | +++ | + | + | (X) | O | O |
intestine | n.a. | n.a. | n.a. | (X) | O | O |
liver | ++ | O | O | O | O | O |
kidney | ++ | + | + | O | O | O |
mononuclear immune cells | n.a. | n.a. | n.a. | X | O | O |
neurons | O | + | + | O | X | O |
astrocytes | ++ | + | + | (X) | X | O |
microglia | +++ | O | O | X | O | O |
stem cells | +++ | ++ | + | X | O | O |
neuroblastoma cells | n.a. | n.a. | n.a. | X | O | O |
Crosses and circles indicate the positive and negative tested tissues of adult mice and murine cells, respectively. X indicates tissues in which UCP2 was always detected. (X) indicates tissues in which UCP2 was often but not always detected or tissues where the protein abundance variation was very strong. Non-analyzed tissues are marked as n.a. Data were published in
Whereas fast proliferating cells use aerobic glycolysis to promote cell growth, neurons, also known to have high metabolic demands and strong dependence on glucose as an energy source, rely on a permanent ATP supply by oxidative phosphorylation
UCP2's presence in other cell types cannot be excluded either, especially in those which are able to acutely induce aerobic glycolysis from a proliferative and metabolic standstill, e.g. intestinal cells and fibroblasts in the course of logarithmic growth
The detection of UCP2 in cell lines (neuroblastoma, BV-2) shown in this work may indicate that metabolism of neuroblastoma cells is not comparable to that of native neurons. Therefore, caution is required in studies of cell metabolism when using corresponding cell lines that express UCP2 as a characteristic feature. Moreover, the overexpression of UCP2 in cells that normally lack this protein may present inaccurate results because of its intervention in metabolism, which is not typical for the primary cells.
The role of UCP2 in mitochondria of fast proliferating cells is still uncertain. A recent finding showed that by its artificial expression, UCP2 hindered the differentiation of pluripotent stem cells
The importance of UCP2 for proliferating cells may explain the ubiquitous presence of UCP2 mRNA in all tissues, because it ensures the possibility of sudden proliferation as required during development and growth, due to increased activity, uncontrolled proliferation in cancerogenesis and repair after tissue injury. The latter would explain, why UCP2 is often reported to be up-regulated in a range of neurodegenerative and ischemic disease such as multiple sclerosis, seizure, stroke, brain trauma, and ischemia
The presence of UCP2 in the mouse embryonic stem cell clone D3 and neuroblastoma cells evaluated in the present study is in agreement with our formerly proposed distribution pattern of UCP2. Based on the presented and previous results, demonstrating the expression of UCP2 in embryonic cells, cancer cells, (activated) lymphocytes and macrophages, we suggest that the presence of UCP2 may be characteristic for cells with high proliferative and anabolic potential. The expression of UCP2 in adult neurons under physiological conditions seems very unlikely, due to their different metabolic features. Our present results indicate a new pathway for the study of UCP2/UCP4 functions.
(TIFF)
We thank C. Krewenka (University of Veterinary Medicine, Vienna, Austria), B. Brokowski and R. Dannenberg (Charité-Universitätsmedizin, Berlin, Germany) for the excellent technical assistance. We are grateful for providing stimulated T-cells to C. Infante-Duarte (Experimental and Clinical Research Center, a joint cooperation between the Charité-Universitätsmedizin Berlin and the Max-Delbrück Center for Molecular Medicine, Berlin, Germany). We are grateful to Quentina Beatty for the excellent editorial assistance.