Identification of telomere dysfunction in Friedreich ataxia

The telomere length in FRDA human and transgenic mouse fibroblasts was measured by a Q-FISH protocol adapted for interphase cells. A total of 100–150 interphase nuclei per cell line were captured and the mean telomere fluorescence intensity per cell was used to determine the mean difference between FRDA fibroblasts and controls. Initially, telomere fluorescence intensity was analysed in mouse FRDA (YG8R and YG22R) and control (Y47R and B6) fibroblasts at passage 7. To quantify the results, two mouse lymphoma cell lines, LY-R and LY-S, with known telomere lengths of 49 kb and 7 kb, respectively, were used as calibration standards [12]. The results revealed that the mouse FRDA cell lines, YG8R and YG22R, have significantly increased telomeric repeat fluorescence (P < 0.001) compared to the controls, Y47R and B6 (Fig. 1a). LY-R control cells were shown to have approximately 6 times greater telomere fluorescence than LY-S control, which confirmed the accuracy of the technique. Subsequently, telomere fluorescence intensities of PNA probes were analysed in human FRDA and control fibroblasts that had each undergone 7 passages subsequent to acquisition from the Coriell Cell Repository (details are shown in Table 1). The results showed that the mean telomere fluorescence intensity of the PNA probes, and hence telomere length, in all FRDA fibroblast cells was significantly greater (P < 0.001) than those of controls (Fig. 1b). The values for human FRDA cells also appeared to be higher than those of the FRDA mouse cells, suggesting potential differences due to factors such as GAA repeat size. However, a direct comparison between these human and mouse results cannot effectively be made due to the relative rather than absolute nature of the two experiments. To further confirm the Q-FISH results, qPCR was performed to evaluate the telomere length in the human FRDA and control cell lines. Three epithelial cell samples with known telomere length, previously measured by telomere repeat amplification (TRF), were used as the controls to examine the accuracy of the technique. The results obtained by qPCR were in good agreement with those previously acquired by Q-FISH and TRF methods. Overall, the results demonstrated that FRDA fibroblasts have significantly higher average telomere length of 19 kb compared to normal fibroblasts (14 kb, P < 0.001) and normal human breast epithelial cells (15 kb, P < 0.05) (Fig. 1c).

Subsequently, peripheral blood leukocyte genomic DNA samples from 18 FRDA patients and 12 healthy controls (details shown in Table 2) were used to measure the telomere length by qPCR. The results indicated that, although there was a considerable variation in the telomere length of the patients (Additional file 1), the mean telomere length was significantly (P < 0.05) shortened in FRDA leukocyte cells compared to controls (Fig. 1d). These results are in good agreement with those previously reported by Castaldo and colleagues [5]. Although we were not able to investigate any correlation between telomere length and age, as this data was not available to us, we did investigate the correlations between telomere length and gender or GAA repeat sizes, but no significant correlations were observed (Additional file 2).

Next, we measured the telomere lengths of DNA samples from cerebellum autopsy tissues from FRDA patients compared with age-matched unaffected individuals to provide in vivo validation of our observation made from fibroblast cells in vitro. Human DNA sample details are shown in Additional file 3. The results showed that telomere lengths in FRDA cerebellar tissues were significantly reduced (P < 0.05) compared to those of unaffected age-matched controls. However, there were no correlations between telomere length and age of onset or GAA allele sizes (Additional file 4).

As FRDA fibroblasts were found to have chromosomes with relatively longer telomeric repeats, it was hypothesized that these cells might have overcome telomere shortening through activation of telomerase. To investigate this hypothesis, human FRDA fibroblast cells were screened for expression of telomerase activity using the telomeric repeat amplification protocol (TRAP) assay. In this method, telomerase adds telomeric repeats to the forward primer, provided that it is active in these cells. The extended primer would then be amplified by qPCR with a reverse primer complementary to the telomeric repeat. The results revealed that telomerase activity was not present in FRDA fibroblasts (Additional file 5). Consequently, the FRDA cells were considered to be negative for telomerase activity. Since the TRAP assay is a semi-quantitative method and there exists a strong correlation between human telomerase reverse transcriptase (hTERT) mRNA expression levels and telomerase activity [13], hTERT mRNA expression was measured in FRDA cells. The quantitative assay for hTERT mRNA expression was conducted using TaqMan qRT-PCR. The results indicated that FRDA cells do not express the hTERT gene, further confirming lack of telomerase activity in these cells (Additional file 5). Since FRDA fibroblasts exhibited longer telomeres with no detectable telomerase activity, we investigated another mechanism of telomere length maintenance, commonly known as ALT, by measuring ALT-associated PML bodies (APBs). The enhanced formation of APBs in cells is an indicator of ALT activity. FRDA cells were first examined for the presence of PML bodies using immunofluorescence. No difference was detected in the number and distribution of PML nuclear foci in FRDA cells compared to controls. Subsequently, co-localisation of PML bodies with telomeres was investigated in these cells using combined immunofluorescence to PML followed by Q-FISH for telomere detection (Fig. 2a,b). The results indicated that in 50 randomly analysed FRDA cells, approximately 35 % of the PML foci co-localised with telomeric DNA. By comparison, the mean co-localisation values of PML and telomeres in ALT positive U2OS cells, normal human fibroblasts and HeLa negative controls were 99 %, 11 % and 8 % respectively (Fig. 2a,b). These results suggested that an ALT-like mechanism is involved in FRDA cells but it is not as prominent as in ALT positive cells. Previous results have shown that cellular stress may promote aggregation of PML bodies or conversely disperse them into microspeckles [14]. Therefore, the size of co-localised PML bodies with telomeres was examined. However, no significant difference was detected in the size of telomere-associated PML bodies compared with non-telomere-associated PML bodies in FRDA cells (Additional file 6). Consequently, these results did not provide any further information regarding the assembly or disassembly of PML bodies in FRDA cells due to cellular stress. Subsequently, telomere recombination frequencies between sister telomeres were analysed in human FRDA and control primary fibroblasts, using the chromosome orientation FISH (CO-FISH) method. This method is used to measure levels of T-SCE – another marker of ALT activity in cells. The results indicated a significant increase (P < 0.001) in T-SCE levels in FRDA cells compared to controls (Fig. 2c,d). These results suggested that T-SCEs occur much more frequently in FRDA cells than normal cells, further confirming the existence of an ALT-like mechanism in these cells.

To investigate whether FRDA fibroblasts can activate an ALT-like mechanism during culturing in vitro to become immortalised, growth curves and cumulative population doubling time analyses were performed. Growth curve analysis of human FRDA and control fibroblasts was carried out from the earliest passage number available for each cell line. FRDA fibroblasts underwent growth arrest after 124 to 175 days with average cumulative population doubling of 47. In contrast, normal fibroblasts exhibited earlier growth arrest, after 124 to 138 days, with an average of 36 cumulative population doubling (Fig. 3a). These results indicated that FRDA cells were not immortalised by the ALT-like mechanism alone, suggesting that the frequency of the PML bodies may not have been sufficient to prevent senescence. However, FRDA cells senesced on average approximately 11 population doublings later than the controls (P < 0.001). The presence of senescent cells was confirmed by β-galactosidase (β-Gal) assay (Additional file 7). This delay in transition from proliferation to senescence in FRDA cells could in part be due to the difference in the original telomere lengths of these cell lines. Subsequently, the effect of replicative aging of culture cells at the 7th, 15th and 26th passages following acquisition from the Coriell Cell Repository (Table 1) was studied by qPCR telomere length assay to determine telomeric DNA loss due to cell divisions. The results of this analysis revealed an accelerated telomere shortening in FRDA fibroblasts, taking initial differences in passage numbers into consideration (Fig. 3b). Although initial results indicated that FRDA fibroblasts contained relatively longer telomeres compared to controls after 7 passages, further analysis after 15 and 26 passages showed that these cells reduced telomere length more rapidly throughout culture than normal cells (Fig. 3b). FRDA fibroblasts showed an average telomere loss of 186 bp per population doubling (PD), which was four times greater than the 47 bp average value of normal fibroblasts (Fig. 3c). The accelerated telomere shortening observed in FRDA fibroblasts suggested that the telomeres may be dysfunctional in these cells.

To confirm the presence of a telomere dysfunction phenotype in FRDA fibroblasts, the telomere dysfunction-induced foci (TIF) assay was performed using an antibody against DNA damage marker γ-H2AX together with the synthetic PNA telomere probe. The presence of TIFs in a cell is indicative of DNA damage at telomeres [15] and is used to quantitatively measure levels of telomeric dysfunction. As evident in Figs. 4 a and c, the frequency of γ-H2AX foci was significantly higher in FRDA cells compared to controls (P < 0.001). Similarly, FRDA cells had greater TIF frequencies in comparison to controls (P < 0.001) (Fig. 4b,c), indicating that FRDA fibroblasts have greater induced telomere dysfunction.

Mouse fibroblast cell lines were established from the kidney tissues of C57BL/6 J (B6) mice and previously reported FXN YAC transgenic mouse models: Y47R (9 GAA repeats), YG8R (90 and 190 GAA repeats) and YG22R (190 GAA repeats) [42, 43]. All human fibroblasts derived from FRDA patients and unaffected controls were obtained from the Coriell Cell Repository (Table 1). Cells were cultured in DMEM culture medium (Gibco) supplemented with 10-15 % fetal bovine serum (Gibco) and 2 % penicillin-streptomycin (Gibco) at 37 °C with 5 % CO₂. Mouse lymphoma LY-R (radio-resistant) and LY-S (radio-sensitive) cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10 % FBS and 2 % Pen-Strep at 37 °C with 5 % CO2. U2OS (human osteosarcoma) and HeLa (human cervical carcinoma) cell lines were purchased from ECCC (European Collection of Cell Culture) and ATCC (American Tissue Culture Collection), respectively. The U2OS cell line was grown in McCoy’s culture medium (Sigma-Aldrich) supplemented with 10 % FBS, 2 % Pen-Strep and 2 mM glutamine (Gibco) at 37 °C with 10 % CO₂. The HeLa cell line was grown in DMEM culture medium supplemented with 10 % FBS and 2 % Pen-Strep at 37 °C with 10 % CO₂. For growth curve analysis, FRDA and control fibroblast cell lines were serially passaged by re-seeding at 100,000 cells/25 cm² flasks (Fisher Scientific) and harvesting at 80 % confluence from earliest passage until they entered replicative senescence. For each harvest, cells were counted with a haemocytometer (Invitrogen) and transferred every 6–7 days. Cell population doublings per passage (PDP) and cumulative population doublings (CPD) were calculated from the first passage onward by the following formula [44]: PD = (Log N₁ /Log2)-(Log N_o / Log2); CPD_n = ΣPD_{(1, n)}, where N_o is the number of cells at the beginning; N₁ is the number of cells at the end of each cell culture period and n is the number of passages. β-galactosidase activity was determined as described [45].

Genomic DNA was extracted from FRDA and control human and mouse fibroblasts, blood samples and cerebellum autopsy tissues by standard phenol/chloroform extraction and ethanol precipitation. Telomere length was determined by a previously described quantitative polymerase chain reaction (qPCR) method [46]. In brief, the relative telomere length was calculated from the ratio of the telomere (T) repeat copy number to a single gene (36B4, which encodes the acid ribosomal phosphoprotein PO) (S) copy number (T/S ratio) for each sample using standard curves. For quality control, all qPCR telomere length measurements were performed in triplicate. To quantify telomere shortening per PD, the following formula was used: (N₁-N_o) / CPD, where N_o is the telomere length at the initial passage and N₁ is the telomere length at the later passage.

Interphase quantitative fluorescent in situ hybridisation (IQ-FISH) was performed using the FITC-conjugated peptide nucleic acid (PNA) telomeric oligonucleotide (CCCTAA)₃ probe as previously described [47]. Images of interphase cells were acquired on a digital fluorescence microscope (Zeiss Axioskop 2) equipped with a CCD camera (Photometrics) and Smart Capture software (Digital Scientific) using fixed exposure time of 0.5 second and magnification of 63×. Telomere fluorescence intensity per cell was analysed using IP Lab software (Digital Scientific) and the average signal was evaluated by subtracting the background signal from the total telomeric signal intensity.

Chromosome orientation fluorescence in situ hybridisation (CO-FISH) was performed as previously described [48]. Briefly, cells were grown in the presence of BrdU/BrdC (3:1) (Sigma Aldrich) at a concentration of 1 × 10⁻⁵ for 24 h. Slides containing chromosome preparation were stained with DNA-binding fluorescent dye Hoechst 33258 (0.5 μg/ml; Sigma Aldrich) for 15 min at room temperature and were exposed to 365 nm UV light (Stratelinker 1800 UV irradiator) for 30 min. The nicked BrdU-substituted DNA strands were then digested by 3U/ml of Exonuclease III (Promega) in the buffer supplied by the manufacturer at room temperature for 10 min. In situ hybridisation was performed using the Cy3-conjugated PNA telomeric oligonucleotide (CCCTAA)₃ probe as previously reported [49]. Images of metaphase spreads were acquired using a Zeiss fluorescence microscope equipped with a CCD camera (Photometrics) and ISIS Capture software (in situ imaging system “ISIS”, Meta Systems, Altlussheim, Germany).

Telomere dysfunction induced foci (TIF) assays were performed as previously described [50]. Briefly, cells were grown on poly-L-lysine-coated slides (Poly-Prep slides, Sigma-Aldrich) for 24 h prior to immunofluorescence staining. The slides were fixed in 4 % formaldehyde in PBS for 15 min and were permeabilised with 0.2 % Triton X-100 (Sigma-Aldrich) for 10 min at 4 °C. The cells were then blocked with 0.5 % BSA in PBS for 30 min. 100 μl of mouse monoclonal antiphospho-histone H2AX (Ser 139, Millipore) was added to the slides at the desired concentration (1:500 in 0.5 % BSA/PBS) and the slides were incubated for 1 h in a humid container. After three washes in TBST (Tris-Buffered Saline with 0.1 % Tween20) for 5 min, the slides were incubated with 100 μl of secondary FITC-conjugated anti-mouse IgG antibody (Sigma) at the desired concentration (1:1000 in 0.5 % BSA/PBS) for 1 h in a humid container. The slides were washed again as above and hybridised for 2 min at 70-75 °C with Cy3-conjugated PNA (CCCTAA)₃ probe. The slides were analysed using a Zeiss fluorescence Axioplan microscope equipped with a CCD camera and Smart Capture software. The frequency of γH2AX was analysed from the TIF results since the TIF protocol simultaneously detects γH2AX foci and telomeres.

For immuno-FISH, cells were grown on poly-L-lysine-coated slides (Poly-Prep slides, Sigma-Aldrich) for 24 h, then fixed in ice-cold methanol-acetone (1:1) for 10 min and washed three times in PBS. The cells were then blocked with 1 % NCS (Newborn Calf serum) in PBS for 30 min. 75 μl of promyelocytic leukemia (PML) primary antibody (Rabbit polyclonal antibody against PML, ab53773, Abcam) was added to the slides at the desired concentration (1:100 in 1 % NCS/PBS) and the slides were then incubated for 1 h in a humid container. The slides were then washed three times in PBS for 5 min. 75 μl of anti-rabbit secondary antibody labelled with FITC (F1262-1ML, Sigma) was added to the slides at the desired concentration (1:100 in 1 % NCS/PBS) and the slides were subsequently incubated for 1 h in a humid container. After rinsing in PBS, the FISH method was applied with a Cy3-conjugated PNA probe (CCCTAA)₃ followed by standard formamide and SSC washes. Cells were counterstained with DAPI and were examined on a Zeiss Axioplan fluorescence microscope equipped with a CCD camera (Photometrics) and Smart Capture software (Digital Scientific) using fixed exposure time of 1 s and magnification of 1000×. The co-localisation of PML and telomere signals was analysed using ImageJ software (National Institutes of Health, Bethesda, MD).

Cells (10⁵-10⁶) were lysed in 200 μl of CHAPS buffer, containing RNase inhibitor, and were incubated at 4 °C for 30 min. The lysates were then centrifuged at 15700 X g for 20 min at 4 °C. The protein concentration was measured using a Pierce® BCA Protein Assay Kit (Thermo scientific) following the manufacturer’s instructions. In order to obtain the final concentration of 125 ng/μl, the protein concentration of the tested samples was measured from the standard curve and the samples were diluted in CHAPS lysis buffer accordingly. Telomerase activity was measured using TRAPEZE® Telomerase Detection Kit (Chemicon, Millipore) according to the manufacturer’s instructions.

Total RNA was extracted from 10⁶ cells using the Trizol® method (Invitrogen) and reverse transcribed using AMV reverse transcriptase (Invitrogen) with random hexanucleotide primers following the manufacturer’s instructions. hTERT mRNA expression levels were quantified by qRT-PCR using an ABI Prism 7900HT Sequence Detection System and TaqMan® Fast Universal Master Mix (Applied Biosystems) [51]. An hTERT assay (Applied Biosystems), containing hTERT forward (5′-GAGCTGACGTGGAAGATGAGC-3′) and reverse (5′-GGTGAACCTCGTAAGTTTATGCAA-3′) primers and a TaqMan TAMRA™ probe (5′-CACGGTGATCTCTGCCTCTGCTCTCC-3′) giving a 260 bp fragment, and a GAPDH assay (Assay ID: Hs99999905_m1, 124 bp amplicon size, Applied Biosystems), containing GAPDH forward and reverse primers and a TaqMan MGB probe, were used in this experiment. The cycling protocol consisted of 20 s at 95 °C, followed by 50 cycles (95 °C for 1 s and 60 °C for 20 s). Reactions were carried out in triplicate for each sample.

Comparisons between population doublings were assessed by two-way analysis of variance (ANOVA) while relationships between telomere length and other variables were calculated by multiple regression analysis. All other data were analysed by the Student’s t test, with a significance value set at P < 0.05.