Tet1-mediated DNA demethylation regulates neuronal cell death induced by oxidative stress

Abstract

Epigenetic regulations including DNA methylation and demethylation play critical roles in neural development. However, whether DNA methylation and demethylation may play a role in neuronal cell death remains largely unclear. Here we report that the blockade of DNA methyltransferase inhibits apoptosis of cerebellar granule cells and cortical neurons in response to oxidative stress. We found that knockdown of ten-eleven translocation methylcytosine dioxygenase (Tet1), a critical enzyme for DNA demethylation, significantly increase apoptosis of cerebellar granule cells induced by hydrogen peroxide. Moreover, cerebellar granule cells from tet1(null) mice appeared to be more sensitive to oxidative stress, suggesting the critical role of Tet1 in neuronal cell death. We further showed that the expression of Klotho, an antiaging protein, in cerebellar granule cells is tightly regulated by DNA methylation. Finally, we found that knockdown of Klotho diminished the rescue effects of DNA methyltransferase inhibitors and Tet1 on neuronal cell death induced by oxidative stress. Our work revealed the role of Tet1-mediated DNA demethylation on neuronal protection against oxidative stress and provided the molecular mechanisms underlying the epigenetic regulation of neuronal cell death, suggesting the role of Klotho in regulating neuronal cell death in response to oxidative stress.

Figure 1. DNA demethylation inhibits neuronal cell death induced by oxidative stress.

(a) CGNs transfected with GFP vector with or without pretreated with 5-aza-2-dC, a Dnmt inhibitor, and treated with H₂O₂. Yellow arrowhead stands for the healthy cells and red arrowhead indicates apoptotic cells. Chromatin condensation was monitored by Hoechst staining. Scale bar = 50 μm. (b) Quantitation of (a). (ANOVA followed by Tukey's test, ***p < 0.001, **p < 0.01, *p < 0.05, n = 4). Error bar represents s.e.m. Each bar represents at least 600 cells of each condition. (c) Validation for Tet1 shRNA efficacy. Cortical neurons were electroporated with control and Tet1 shRNA expressing plasmids and lysated for Western blot analysis. (d) CGNs transfected with GFP-expressing plasmid with or without Tet1 shRNA plasmid, were treated with H₂O₂. Yellow arrowhead stands for the healthy cells and red arrowhead indicates apoptotic cells. Scale bar = 50 μm. (e) Quantitation of (d). (ANOVA followed by Tukey's test, ***p < 0.001, n = 4). Error bar represents s.e.m. ANOVA, analysis of variance; CGNs, cerebellar granule neurons; GFP, green fluorescent protein; shRNA, short hairpin RNA.

Figure 2. Tet1 is involved in neuronal cell death induced by oxidative stress.

(a) Tet1-null neurons showed higher sensitivity to oxidative stress. CGNs were cultured from Tet1 KO mice and control littermates, treated with H₂O₂ of a gradient 0, 100 μM, 200 μM. Cell condensation was monitored by Hoechst staining. Yellow arrowhead stands for the healthy cells and red arrowhead indicates apoptotic cells. (b) Quantitation of (a). (student t test, **p < 0.01, *p < 0.05, n = 3. Error bar represents s.e.m. Scale bar = 20 μm. WT, wild type; KO, Tet1 knockout). (c) CGNs transfected with HA-tagged Tet1CD and Tet1CDm together with GFP were treated with H₂O₂. Yellow arrowhead stands for the healthy cells and red arrowhead indicates apoptotic cells. (d) Quantitation of (c). (ANOVA followed by Tukey's test, ***p < 0.001, n = 4). Error bar represents s.e.m. Scale bar = 50 μm.

Figure 3. Expression of Klotho is regulated by oxidative stress and DNA methylation.

(a–b) Expression of Klotho in cortical neurons is regulated by DNA methylation. Cortical neurons were treated with 5-aza-2-dC for 6–12 h and collected for analyzing mRNA levels of Klotho and BDNF. Quantitative PCR were performed. All data points are normalized to untreated samples. (c) Klotho protein expression was up-regulated with 5-aza-2-dC treatment. CGNs were treated with 5-aza-2-dC for 12–48 h and collected for examination protein level of Klotho. Lower panel: Quantification of western blot results. (d) Klotho protein expression was down-regulated with H₂O₂ treatment. CGNs were treated with various concentrations of H₂O₂ for 12 h and collected for examination protein level of Klotho. Antibodies against Klotho and Tubulin were used. Lower panel: Quantification of western blot results. (e) H₂O₂-induced Klotho downregulation is rescued by 5-aza-2-dC treatment. CGNs were treated with 5-aza-2-dC 6 h prior to H₂O₂ stimulation for 12 h and collected for examination protein level of Klotho. Lower panel: Quantification of western blot results. (f) Klotho protein level was down-regulated by knockdown of Tet1. Lentivirus harboring Tet1 shRNA were seed to mouse cortical neurons and cell samples were harvested 4–5 days after viral infection. Western blots with specific antibody were performed to examine protein level of Klotho. Right panel: quantitation of (f). (Student t test, n = 4, *P < 0.05, Error bar represents s.e.m.) (g) Klotho in the neurons was down-regulated in the MCAO model. Mouse focal cerebral ischemia (I) was induced for 2 h, and after reperfusion (R) for different periods 0 h, 6 h and 12 h, immunoblots were done of the extracts from the contralateral (c) or ipsilateral (i) cortex. Tubulin served as a loading control. Lower panel: quantification of the normalized klotho protein levels (Student t test, n = 3–4 mice per time point, *P < 0.05 versus contralateral, Error bar represents s.e.m. I, ischemia; R, reperfusion). All data were collected from 3–6 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001; one-way ANOVA with Tukey's test.

Figure 4. Klotho is involved in neuronal protection in response to oxidative stress.

(a) Validation of Klotho shRNA. CGNs were electroporated with plasmids containing control and Klotho shRNA and collected for Western blot analysis. (b) CGNs transfected with GFP-expressing plasmids along with control and Klotho shRNA plasmids, treated with H₂O₂ and immunostained for GFP and Hoechst for measure neuronal cell death. Yellow arrowhead stands for the healthy cells and red arrowhead indicates apoptotic cells. (c) Quantitation of (b). (ANOVA followed by Tukey's test, ***p < 0.001, n = 4). Error bar represents s.e.m. Scale bar = 50 μm.

Figure 5. Klotho is critical for DNA demethylation-mediated neuronal protection in response to oxidative stress.

(a) CGNs were transfected with shKlotho, pretreated with 5-aza-2-dC 6 h prior to H₂O₂ stimulation for 12 h. Cell condensation was monitored by Hoechst staining. Yellow arrowhead stands for the healthy cells and red arrowhead indicates apoptotic cells. (b) Quantitation of (a). (ANOVA followed by Tukey's test, ***p < 0.001, **p < 0.01, n = 4). Error bar represents s.e.m. Scale bar = 50 μm.

Figure 6. Klotho is critical for Tet1-mediated neuronal protection in response to oxidative stress.

(a) CGNs were transfected with either Klotho shRNA plasmid or HA-tagged Tet1-CD and both plasmids, treated with H₂O₂ and immunostained for HA, GFP and Hoechst for examining neuronal cell death. Yellow arrowhead stands for the healthy cells and red arrowhead indicates apoptotic cells. (b) Quantitation of (a). (ANOVA followed by Tukey's test, ***p < 0.001, **p < 0.01, n = 4). Error bar represents s.e.m. Scale bar = 50 μm.