Alleviating oxidative damage-induced telomere attrition: a potential mechanism for inhibition by folic acid of apoptosis in neural stem cells

DNA oxidative damage can cause telomere attrition or dysfunction that triggers cell senescence and apoptosis. The hypothesis of this study is that folic acid decreases apoptosis in neural stem cells (NSCs) by preventing oxidative stress-induced telomere attrition. Primary cultures of NSCs were incubated for 9 days with various concentrations of folic acid (0 - 40 µM ) and then incubated for 24 h with a combination of folic acid and an oxidant (100 µM hydrogen peroxide, H 2 O 2 ), antioxidant (10 mM N-acetyl-L-cysteine, NAC) or vehicle. Intracellular folate concentration, apoptosis rate, cell proliferative capacity, telomere length, telomeric DNA oxidative damage, telomerase activity, intracellular reactive oxygen species (ROS) levels, cellular oxidative damage, and intracellular antioxidant enzyme activities were determined. The results showed that folic acid deciency in NSCs decreased intracellular folate concentration, cell proliferation, telomere length and telomerase activity, but increased apoptosis, telomeric DNA oxidative damage and intracellular ROS levels. In contrast, folic acid supplementation dose-dependently increased intracellular folate concentration, cell proliferative capacity, telomere length and telomerase activity but decreased apoptosis, telomeric DNA oxidative damage and intracellular ROS levels. Exposure to H 2 O 2 aggravated telomere attrition and oxidative damage whereas NAC alleviated the latter. High doses of folic acid prevented telomere attrition and telomeric DNA oxidative damage by H 2 O 2 . In conclusion, inhibition of telomeric DNA oxidative damage and telomere attrition in NSCs maybe potential mechanisms of inhibiting NSCs apoptosis by folic acid.


Introduction
Oxidative damage contributes to neuronal cell death and therefore to the important public health problems of aberrant aging and neurodegenerative disease [1,2]. Neural stem cells (NSCs) are the main cell types that produce central nervous system, which are essential for the study of neural development, neurodegeneration and nervous system diseases [3]. NSC homeostasis is in uenced by oxidative damage, when the balance between proliferation and differentiation are destroyed, adult neurogenesis disturbed. NSC oxidative damage, remains in a tight connection with the occurrence of central nervous system diseases [2]. However, the mechanism of oxidative damage the causes neural cell apoptosis remains to be clari ed.
Telomeres are repeating hexameric DNA sequences that protect chromosomes from degradation and end-to-end fusion. Epidemiological studies have revealed that loss of telomere integrity is an important factor in the decay of physiological function associated with ageing and several chronic illnesses [4,5].
Damage to telomeric sequences in stem cells can be repaired by a specialized reverse transcriptase called telomerase [6]. Telomere state depends not only on telomere length but also on the repair that counteracts telomere loss by adding de novo repeats to the 3' ends [7]. Oxidative stress has been observed to accelerate telomere attrition in primary astrocytes [8] and to be associated with apoptosis in endothelial cells [9]. Nevertheless, the role of oxidative stress, telomere attrition and telomerase activity in neurodegeneration is still unclear.
Telomere attrition is associated with ageing, genetic and environmental factors such as nutrients [10,11].
Folate (vitamin B 9 ) is an essential nutrient that acts as a coenzyme to transfer the one-carbon units that are necessary for deoxythymidylate synthesis, purine synthesis, and many methylation reactions [12].
Folic acid has been observed to stimulate the proliferation and neuronal differentiation of NSCs, as well as to decrease their apoptosis [13,14]. Folic acid de ciency has been found to increase reactive oxygen species (ROS) levels and DNA damage in the context of cancer [15,16], while folic acid supplementation inhibits oxidative stress-induced damage, telomere attrition and apoptosis both in primary astrocytes and a murine model of neurodegenerative disease [8,17]. However, the relationship between folate, telomerase activity and telomere attrition in NSCs is still unknown. The hypothesis of this study is that folic acid decreases apoptosis in NSCs by preventing oxidative stress-induced telomere attrition.

Cell Culture
The Tianjin Medical University Animal Ethics Committee approved all experimental protocols in this study. Hippocampus and striatum were dissected from neonatal (postnatal less than 24 hours) Sprague-Dawley rats (Charles River Laboratories, Beijing, China) and NSCs were prepared as described previously [12]. Hippocampus and striatum tissues were cut into 1 mm 3 pieces and digested with 0.25% trypsin at 37 ℃ for 15 min. This step was followed by agitation, centrifugation and resuspension of the cells in serum free Dulbecco's modi ed Eagle's medium (DMEM) and nutrient mixture F-12 Ham (F12) (1:1) (Corning, NY, USA), supplemented with 2% B27 supplement (Gibco, USA), 20 ng/mL epidermal growth factor (EGF; PeproTech, USA), 20 ng/mL basic broblast growth factor (bFGF; PeproTech), 100 U/mL penicillin and phytomycin (Slarbio) and 2 mmol/L L-glutamine (Sigma, USA). The resulting cell suspension was plated at 1 × 10 6 cells/mL in T25 culture asks (Corning, NY, USA), then cultured in 95% air and 5% CO2 at 37 ℃, and the culture medium was changed every 2 or 3 days. After puri cation (7 days), NSCs were incubated for 9 days with various concentrations of folic acid (0-40 µM). Then the cells were exposed for 24 h to medium containing a combination of folic acid and either oxidant (100 µM hydrogen peroxide, H 2 O 2 ) or antioxidant (10 mM N-acetyl-L-cysteine, NAC) or vehicle. The dosage ranges of H 2 O 2 and NAC were selected according to data from the literature [18,19] and the results of our preliminary experiments. After subjected to the different study conditions for 10 days, cells were harvested to detect intracellular folate concentration, apoptosis, cell viability, ROS and oxidative damage, antioxidant activities and telomere attrition.

Cell Identi cation
By the end of 7 days in culture, NSC neurospheres were gently mechanically dissociated and plated on laminin (100 µg/mL) coated coverslips, incubated with 1mM 5-bromo-20-deoxyuridine (BrdU, Sigma, USA) in proliferative medium for 24 h, then xed with 4% paraformaldehyde for 20 min at room temperature. The cells were washed with phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 for 15 min at room temperature, blocked with 10% goat serum for 1 h at 37 °C, and incubated with primary antibodies (mouse anti-BrdU antibody [1:50, Sigma, USA]; rabbit anti-SOX2 antibody [1:200, Abcam, UK] overnight at 4 °C. After another washing with PBS, the coverslips were incubated with secondary antibodies (tetramethyl rhodamine isothiocyanate [TRITC]-conjugated antimouse antibody, 1:100; uorescein isothiocyanate-conjugated antirabbit antibody, 1:100, Jackson, USA) for 1 h at room temperature and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) contained Vectashield (H1200, Vector). The potential capacity of cultured NSC was assessed by immunocytochemistry. NSC neurospheres were gently mechanically dissociated and the resulting cells were plated on laminin (100 µg/mL) coated coverslips at a density of 2 × 10 4 cells/mL in DMEM/F12 medium supplemented with 5% fetal bovine serum (FBS; Gibco), 2% N2 (Gibco) and 100 U/mL penicillin and phytomycin (Gibco), but without B27, EGF, or bFGF [12]. After 6 days of differentiation, the cells were xed with 4% paraformaldehyde for 20 min at room temperature. After washing with PBS, the cells were blocked with 10% goat serum for 1 h at 37 °C, and incubated with primary antibodies (mouse anti-β-III- This terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine monophosphate (dUTP) nick end labeling (TUNEL) assay detects nuclear DNA fragmentation, which is a hallmark of apoptosis. Brie y, NSCs on coverslips were xed in 4% paraformaldehyde for 25 min, washed twice with PBS, permeabilized with 0.2% Triton X-100 for 5 min and then incubated in the dark with Equilibration Buffer for 10 min at room temperature. rTdT incubation buffer was added for 60 min at 37 ℃. Then terminate the reactions by 2× SSC for 15 min at room temperature. Finally, the cells were incubated with DAPI, washed twice with PBS, and mounted with a uorescent mounting medium. We detected localized green uorescence of apoptotic cells ( uorescein-12-dUTP) in a blue background (DAPI) by a uorescence microscope (Olympus, Tokyo, Japan). Positive cells were counted using Image Pro Plus 6.0 software.

Cell Proliferation Assay
NSC proliferation was measured by BrdU incorporation [20]. Brie y, NSCs on coverslips were incubated with 10 µM BrdU (Sigma, USA) in proliferative medium for 24 h, then xed in 4% paraformaldehyde for 20 min at room temperature. The cells were washed twice with PBS, permeabilized with 0.1% Triton X-100 for 15 min at room temperature, blocked with 10% goat serum for 1 h at 37 °C, and incubated with primary mouse anti-BrdU antibody (1:100, Sigma) overnight at 4 °C. After another washing with PBS, the coverslips were incubated with secondary TRITC-conjugated goat anti-mouse antibody (1:100, Proteintech) for 1 h at room temperature and counterstained with DAPI contained Vectashield (H1200, Vector). Immuno uorescence signals were captured using Olympus IX81 microscope (Olympus) and analyzed by Image-Pro Plus 6.0 software.

Cell Viability Assay
Cell viability was measured with the CellTiter 96® AQueous One Solution Cell Proliferation Assay . Cells were seeded into a 96-well plate at a density of 1 × 10 4 cells per well (200 µL/well). Then 20 µL of MTS (5 mg/mL) was added to each well, and the mixture was incubated at 37 °C for an additional 3 h in a humidi ed, 5% CO 2 atmosphere. The absorbance at 490 nm was recorded using a microplate reader (ELX800uv™; BioTek Instruments Inc).

Measurement of Telomere Length by Southern Blot
The telomere restriction fragment (TRF) analysis was performed using a commercial kit (TeloTAGGG Telomere Length Assay, Roche Life Science, Mannheim, Switzerland), based on the instruction. Brie y, 150ng genomic DNA sample was digested with Hinf I and Rsa I for 2 h at 37°C. Digested DNA was then electrophoresed on 1% agarose pulsed eld gels at 6 V/cm for 11 h. The blotting membrane was washed, blocked, incubated with anti-DIG-alkaline phosphatase (1:4000 dilution, Roche, Mannheim, Switzerland) for 4 h, washed, and exposed with CDP-star (Roche, Mannheim, Switzerland) for 15 min. After exposure of the blot to an X-ray lm, an estimate of the mean TRF length can be obtained by comparing the mean size of the smear to the molecular weight marker with Telo Tool.

Measurement of Telomerase Activity
Telomerase is a ribonucleic acid-protein complex composed of a single long non-coding RNA, called telomerase RNA, and associated proteins. We used a telomerase activity quanti cation qPCR assay kit (ScienCell, Carlsbad, CA, USA #8928) to measure the products of telomerase activity that are ampli ed by qPCR. The cell lysis buffer enables the release of telomerase in the native state and the telomere primer set (TPS) recognizes and ampli es newly synthesized telomere sequences in the assay. Cell proteins were extracted by cell lysis buffer supplemented with PMSF 0.1 M in isopropanol and β-mercaptoethanol. After cell protein extraction telomerase reaction was performed as described in the product protocol.
Cellular T-AOC, SOD, CAT, GSH-PX activities were normalized to the cellular protein levels, determined using a BCA protein assay kit (BosterBio).

Statistical Analysis
Data are expressed as mean ± SEM values based on three independent experiments. Two-way analysis of variance was used to evaluate differences between the treatment groups, and the Student-Newman-Keuls test was used for multiple comparisons to determine signi cant differences among the experimental groups. Correlations were assessed using the Pearson's method. The statistical software package SPSS 24.0 was used to evaluate the differences within groups, and p value less than 0.05 was considered statistically signi cant.

Cell Identi cation
By the end of 7 days in culture, almost all the NSCs in neurospheres had the potential to proliferate as indicated by incorporation of Brdu (Additional gure 1a) and they also were SOX2-positive (Additional gure 1b). Then the neurospheres were mechanically dissociated and the resulting cells were cultured in DMEM/F12 medium supplemented with 5% FBS, 2% N2, but without B27, EGF, or bFGF. After 6 days culture in this differentiation medium, most of the NSCs had differentiated into neurons or astrocytes, which were identi ed as β-III-tubulin-positive and GFAP-positive, respectively (Additional gure 1e-h). These results demonstrate that the cultured neurospheres were comprised of NSCs with the capacity for self-renewal as well as for neuronal and astrocytic differentiation.

Intracellular Folate Level
After 10 days of intervention, folic acid increased the intracellular folate concentration in a dosedependent manner (F=233.601, p<0.001) (Figure 1). In contrast, NAC had no effect on intracellular folate. H 2 O 2 also had no effect on the intracellular folate when medium contain high level of folic acid. However, These results showed that folic acid de ciency decreased NSC proliferation and cell viability, and folic acid supplementation could increase cell proliferative capacity. The expression of telomerase RNA was too low to detect (de ned by the kit manufacturer's instructions as a Cq-value higher than 33) in 3 treatment groups, namely, 0 µM folic acid with or without H 2 O 2 , and 10 µM folic acid with H 2 O 2 ( Table 1). Folic acid (10-40 µM) increased the telomerase activity dosedependently (F=35.746, p<0.001).
The oxidant H 2 O 2 decreased the telomerase activity in the NSCs that received 0 and 10 µM folic acid (Table 1), whereas the antioxidant NAC increased the telomerase activity in folic acid de cient NSCs (0 µM folic acid, Table 1).

Discussion
The results of the present study showed that folic acid de ciency in NSCs decreased intracellular folate concentration, cell proliferative capacity, telomere length and telomerase activity, but increased apoptosis, telomeric DNA oxidative damage and intracellular ROS levels. In contrast, folic acid supplementation dose-dependently increased intracellular folate concentration, cell proliferative capacity, telomere length and telomerase activity but decreased apoptosis, telomeric DNA oxidative damage and intracellular ROS levels. Exposure to H 2 O 2 aggravated telomere attrition and oxidative damage whereas NAC alleviated the latter. High doses of folic acid prevented telomere attrition and telomeric DNA oxidative damage by H 2 O 2 .
Neurodegenerative diseases are characterized by a chronic and selective process of neuronal cell death. Folic acid is an essential nutrient that is vitally important to neural cell survival [14]. Folic acid de ciency has obvious impact on neural cell proliferation and could induce cell apoptosis [26]. In vitro studies have found that folic acid supplementation decreases apoptosis in astrocytes [8] and stimulates cell proliferation in embryonic NSCs [27]. In vivo studies have discovered that folic acid supplementation delays age-related neurodegeneration in the cerebral cortex and hippocampal CA1 region of senescence-accelerated mouse prone 8 (SAMP8) mice [17], whereas folic acid de ciency worsens neural cell injury in hippocampus following ischaemia/reperfusion injury [28]. Furthermore, in a recent study of Chinese patients with Parkinson's disease, relatively higher homocysteine and lower folate levels correlated with white matter hyperintensities [29]. The present study provided further insight by showing in primary NSCs that folic acid de ciency increased apoptosis and decreased cell proliferative capacity and whereas folic acid supplementation did the opposite.
Oxidative stress, resulting from an imbalance between ROS production and antioxidant defenses, contributes to aging and the pathogenesis of numerous diseases including neurodegenerative diseases [30][31][32][33]. SOD, CAT and GSH-PX are antioxidant enzymes that contribute importantly to cells' capacity for surviving oxidative stress [34,35]. The severity of oxidative stress in cultured cells can be assessed by measuring intracellular ROS, LPO and MDA, as well as by measuring the release of LDH from cells to the medium. In the present study, folic acid decreased medium LDH concentrations, lowered intracellular LPO and MDA levels, and increased the intracellular SOD, CAT, GSH-PX and T-AOC activities of NSCs. Taken together, these results indicated that su cient amounts of folic acid could enhance antioxidant capacity and protected cells from oxidative damage.
Telomeres are dynamic nucleoprotein-DNA structures that cap and protect linear chromosome ends [36]. Critically short telomeres trigger cellular senescence in mature cells, which contributes to agingrelated degenerative diseases [37,38]. While numerous genetic and environmental factors are associated with telomere attrition, oxidative stress has been identi ed as an underlying mechanism [39]. Telomeric DNA is particularly vulnerable to oxidative damage because of its high level of guanine in the 5'-TTAGGG-3' repeat sequence [40,41]. Guanine is the most susceptible of the natural bases to oxidation, commonly generating 8-OxoG, which is the most widely recognized biomarker for detecting oxidative damage in DNA [42,43]. A link between telomerase and oxidative stress is evident since it has been shown in human endothelial cells that chronic oxidative stress rapidly downregulates telomerase activity and accelerates telomere attrition [44].
Folate is essential for nucleotide synthesis and maintaining redox status [45]. A recent study showed that folic acid de ciency increases intracellular ROS levels, and aggravates telomere attrition in primary cultures of astrocytes [8]. There is evidence that a similar mechanism may occur in vivo since folic acid supplementation decreases ROS levels and alleviates both telomere attrition and telomeric DNA damage in SAMP8 mice [17]. The present study suggested that folic acid's actioned of suppressing ROS levels, telomere attrition and telomeric DNA oxidation, while increasing telomerase activity, might account for the vitamin's protection against NSC apoptosis.
Our previous study found that folic acid de ciency worsened neural cell injury in hippocampus following ischaemia/reperfusion injury [28]. Folic acid supplementation stimulates NSCs proliferation [12,27] and neuronal differentiation [13]. We also found folic acid inhibited apoptosis in astrocytes in vitro and endothelial cells in vivo and in vitro. The protective effects may be due to folic acid decreased oxidative stress [8,9]. Moreover, Folic acid supplementation delayed age-related neurodegeneration and cognitive decline in SAMP8 mice, and alleviating telomere attrition could serve as one in uential factor in the process [17]. For the depth of previous study, the effect of folic acid on apoptosis and telomeric attrition in primary NSCs were discussed in this study.

Conclusion
In conclusion, inhibition of oxidative damage-induced telomere attrition in NSCs may be a potential mechanism of inhibiting NSCs apoptosis by folic acid. The results suggested that folic acid supplementation might be a therapeutic strategy to people with neurodegenerative disease by preventing NSC apoptosis.

Declarations
Funding This research was supported by a grant from the National Natural Science Foundation of China (No. 81730091), Natural Science Foundation of Tianjin (No. 19JCQNJC11700).
Data Availability All data generated or analyzed during this study are available from the corresponding author on reasonable request.

Compliance with Ethical Standards
Con icts of interest The authors state that they have nothing to disclose and declare no con ict of interest.
Ethics Approval Not applicable to this study.   Cell apoptosis rates. NSCs were assessed to treatment groups as described in Figure 1, but were in adherence to a laminin substrate. (a-d) Cell apoptosis detected by TUNEL assay. Representative images of NSCs were stained with markers of uorescein-12-dUTP (green), cell nuclei were stained with DAPI (blue), Double staining for uorescein-12-dUTP and DAPI were indicated with arrows, the scale bar is 50 µm. (e) Quanti cation of apoptotic-positive cells/total number of DAPI-stained nuclei. The plotted values represent the mean ± SEM values of three independent experiments. Statistical analysis was performed using two-way ANOVA. *, p<0.05 compared with 0 µM folic acid. #, p<0.05 compared with 10 µM folic acid. &, p<0.05 compared with 20 µM folic acid. $, p<0.05 compared with the group without H2O2 or NAC at the same folic acid level.

Figure 3
Cell proliferative capacity and cell viability. NSCs were incubated as described in Figure 1.  Telomere length of cells. NSCs were incubated as described in Figure 1. p<0.05 compared with 0 µM folic acid. #, p<0.05 compared with 10 µM folic acid. &, p<0.05 compared with 20 µM folic acid. $, p<0.05 compared with the group without H2O2 or NAC at the same folic acid level.

Figure 5
Telomeric DNA oxidative damage of cells. NSCs used into qPCR were incubated as described in Figure 1 and cells used into IF-FISH were in adherence to a laminin substrate. (a) Representative images of telomere IF-FISH (red, telomeric probe; green, -H2AX probe; blue, DAPI-stained nuclei). Scale bar=10 µm. with 0 µM folic acid. #, p<0.05 compared with 10 µM folic acid. &, p<0.05 compared with 20 µM folic acid. $, p<0.05 compared with the group without H2O2 or NAC at the same folic acid level.

Figure 6
Oxidative injury and antioxidant activities. NSCs were incubated as described in Figure 1.