Figure 1.
Age-related DNA damage accumulation in HFSCs.
(A) IFM micrograph of CD34+ HFSCs (green) in the bulge region, in comparison to the schematic representation of the hair follicle. (B) Quantification of 53BP1-foci in HFSCs and epidermal cells analyzed 0.5 h after irradiation with different doses. IFM micrograph of radiation-induced 53BP1-foci in HFSCs, consistently colocalizing with γH2AX (0.5 h after 100 mGy). (C) Quantification of HFSCs and epidermal cells with spontaneous 53BP1-foci in repair-proficient C57BL/6 mice aged 0.5, 3, 12, 18, 24 months compared to repair-deficient SCID mice aged 3 and 12 months. Data are presented as means from three different experiments ±SE. * significant difference to 0.5-month-old mice; ** significant difference to the younger age-group. (Note: The hair shaft itself and hair-shaft cells display autofluorescence due to the keratin and melanin.).
Figure 2.
Colocalization of 53BP1-foci with telomeric DNA.
(A) IFM micrograph of FISH combined with immunofluorescence to visualize telomeric DNA and 53BP1-foci in skin sections of 24-month-old mice. High numbers of telomeric spots of similar size and intensity (green), but only single, larger 53BP1-foci (red, arrows) were randomly distributed throughout the nuclei. (B) Serial optical section show that only a minority of age-related 53BP1-foci overlaps with telomeric DNA (arrow). (C) Quantitative analysis of the colocalization of 53BP1-foci and telomeric spots, compared to the total number of spontaneous 53BP1-foci, in hair follicle cells and epidermal cells of 3- and 24-month-old mice. For every data point, at least 50 foci were analyzed for colocalization in tissue sections obtained from three different mice, and data are presented as means ±SE. * significant difference to 3-month-old mice.
Figure 3.
DNA damage response after exposure to ionizing radiation.
(A, B) Age-related decrease in DNA repair capacity: DSB repair capacity of HFSCs and epidermal cells was analyzed in skin sections of whole-body irradiated mice by counting cells with 53BP1-foci at defined time-points after 2 Gy irradiation (A). Additionally, the number of 53BP1-foci per cell was counted at defined time-points after 2 Gy irradiation. To analyze the repair capacity independent of the endogenous effect, controls were subtracted from the quantified 53BP1-foci (B). The number of 53BP1-foci is depicted separately for repair-proficient C57BL/6 mice compared to repair-deficient SCID mice. At late repair-times (compare inset for 48 h post-irradiation), HFSCs reveal age-related differences in the number of cells with 53BP1-foci. Data are presented as means from three different experiments ±SE. * significant difference to 0.5-month-old mice; ** significant difference to the younger age-group. (C) DNA damage accumulation during fractionated low-dose radiation: To evaluate the impact of fractionated low-dose radiation (10×, 20×, 30×, and 40× 10 mGy) on cumulative DNA damage, HFSCs and epidermal cells with residual 53BP1-foci were quantified at 24 h and 72 h after the last exposure. Persisting 53BP1-foci levels increase with cumulative radiation doses. Data are presented as means from three different experiments ±SE. * significant difference to unirradiated mice; ** significant difference to the lower dose-group.
Figure 4.
Characterization of radiation-induced DNA lesions by TEM.
(A) TEM micrographs of double-labeling of 53BP1 (6-nm) and pKu70 (10-nm) in HFSCs analyzed 0.5 h after 2 Gy irradiation. pKu70 consistently colocalized with 53BP1 in heterochromatic regions, but only pKu70 clusters (without 53BP1) were detected in euchromatic regions. (B) TEM micrographs of double-labeling of 53BP1 (10-nm) and H3K9me3 (6-nm) in HFSCs analyzed 72 h after protracted low-dose radiation (40× 10 mGy). Persistent 53BP1 clusters (green circles at higher magnification) colocalized with the heterochromatin marker H3K9me3 and were localized predominantly in tightly packed heterochromatin (dark grey regions in TEM; compare inset with the overview of the whole nucleus).
Figure 5.
Characterization of age-related DNA lesions by TEM.
(A) TEM micrographs of double-labeling of 53BP1 (6-nm) and pKu70 (10-nm) in HFSCs of 3- and 24-month-old mice. In skin samples of aged mice, but not young mice, multiple 53BP1 clusters of 2–6 gold-beads are scattered throughout the highly condensed heterochromatin of HFSCs. These persistent 53BP1 clusters did not colocalize with pKu70. (B) The area highlighted by a circle in the TEM micrograph indicates the region studied. Quantitative analysis of the 53BP1 and pKu70 cluster distribution in eu- and heterochromatin of HFSCs in skin sections of 3- and 24-month-old animals. Data are presented as means from three different experiments ±SE. * significant difference to 3-month-old mice.
Figure 6.
Biological outcome of HFSCs in aged and irradiated skin.
(A) IFM micrographs of Caspase-3 staining of skin sections derived from aged (24-months-old) and low-dose irradiated animals (40× 10 mGy). Aged skin revealed slightly increased levels of apoptotic cells (red), but not in the CD34+ HFSCs (green) of the bulge region. After protracted low-dose radiation, slightly higher levels of apoptotic cells were observed, and even some CD34+ HFSCs undergo apoptosis (arrow in the enlarged image). (B) Micrographs of the histochemical detection of SA-β-gal activity in skin sections derived from aged (24-months-old) and low-dose irradiated animals (40× 10 mGy). SA-β-gal staining of aged skin revealed an increase of the blue-dyed precipitate in the upper isthmus and infundibulum of the hair follicle, but not in CD34+ HFSCs of the bulge region (brown). In irradiated skin, some CD34+ HFSCs stained positive for SA-β-gal (two arrows in the enlarged lower image). Sporadic staining of the lumen of sebaceous ducts was also seen (enlarged upper image). (C) Analysis of the DNA damage response by flow-assisted cytometry. Relative fluorescence intensity of repair proteins, histone modifications, apoptosis and senescence markers in CD34+ and CD34– cells derived from aged (24-month-old) and irradiated (40× 10 mGy; 72 h) skin normalized to unirradiated young skin (3-month-old). Data are presented as means from three different experiments ±SE. * significant difference compared with unirradiated young skin.