Role of DNA Methylation in Hypobaric Hypoxia-Induced Neurodegeneration and Spatial Memory Impairment

Abstract

Hypobaric hypoxia (HH) is a major stress factor that is associated with physiological, biochemical, molecular and genomic alterations. Brain is the organ that reacts sensitively to oxygen deprivation, which leads to oxidative stress and cognitive function impairment. Our previous studies have reported that downregulation of brain derived neurotrophic factor (BDNF) leads to neurodegeneration and memory impairment. The aim of the present study was to investigate the effect of HH exposure on DNA methylation and its regulation in BDNF expression, neurodegeneration and spatial memory impairment. For this purpose, Sprague Dawley rats were exposed to HH at a simulated altitude of 25,000 feet for 14 days. Real-time polymerase chain reaction was used for transcriptional expression of DNA Methyltransferases (DNMTs) including DNMT1, DNMT3a and -DNMT3b, and immunoblotting was used for the translational expression of DNMT1, DNMT3a, DNMT3b, Methyl CpG binding protein 2 (MeCP2), pMeCP2 and BDNF in rat hippocampus. Additionally, neuronal morphology alteration and neurodegeneration in CA1 region of hippocampus were investigated though Cresyl violet (CV) staining and Fluoro-Jade C staining respectively. Results obtained suggested that HH exposure increased the expression of DNMT1 DNMT3b at the mRNA as well as protein level, whereas no significant change was observed in the level of DNMT3a. Furthermore, the level of pMeCP2 and BDNF were significantly decreased; however, the expression level of MeCP2 was significantly increased. The CV and Fluoro-Jade C-positive cells were significantly enhanced in the CA1 region of hippocampus in the HH exposed group as compared to unexposed rats. Thus, the present study concluded that HH decreases neuronal activation by the upregulation of DNA methylation and MeCP2 and decreased the expression of pMeCP2, which result in the downregulation of BDNF. The decreased BDNF expression is associated with neuronal loss and spatial memory impairment. This study highlights that DNMT inhibition could be an important therapeutic target for neurodegenerative diseases.

Keywords: Brain-derived neurotrophic factor; DNA methyltransferase; Hypobaric hypoxia; Methyl CpG binding protein 2; Neurodegeneration.

Fig. 1.

Effect of HH exposure on spatial reference memory impairment. HH-exposed spatial memory impairment was estimated through Morris water maze (MWM). A, B Representative traces plot of MWM for probe trial and memory test respectively. The bar diagram (Aa, Ab) represent a significant increase in path-length (* p < 0.05) and latency (*** p < 0.01) in HH-exposed rats as compared to normoxia rats (control) respectively. The bar diagram of (Ba, Bb) represent a significant decrease in the time spent (* p < 0.05) and the number of crossing (** p < 0.01) in the targeted zone as compared to Normoxia rats (control) respectively. Data represents mean ± SEM of 4 independent experiments. Student t test was used to calculate significance.

Fig. 2.

The effect of HH on the mRNA level of different DNA Methyltransferases (DNMTs) in a rat's hippocampus. The fold change of different DNMTs as DNMT1, DNMT3a and DNMT3b was quantified through real-time PCR in both normoxia and HH-exposed rat's hippocampus. Fold changes of DNMT1, DNMT3a and ­DNMT3B have been shown through bar diagram as (a–c) respectively. Fold change in DNMT1 and DNMT3b was increased significantly (p < 0.01) in HH-exposed rats as compared to normoxia rats (Control). Data represents mean ± SEM of 4 independent experiments. Student t test was used to calculate significance; ** p value < 0.01.

Fig. 3.

HH exposure increased the protein level of DNA Methyltransferases (DNMTs) in hippocampus of rats. The levels of ­DNMTs including DNMT1, ­DNMT3a and DNMT3b were estimated through western blotting shown in (a). The alterations in the level of DNMT1 (b) and ­DNMT3b (d) were significantly increased (* p < 0.05, *** p < 0.001) in HH-exposed rats as compared to normoxia rats (Control), although no significant change was observed in DNMT3a (c). Data represents mean ± SEM of 4 independent experiments. Student t test was used to calculate significance.

Fig. 4.

HH exposure alters the level of MeCP2 and MeCP2 phosphorylation in hippocampus. Alteration in the level of MeCP2 and MeCP2 phosphorylation at position Ser. 421 was analysed through western blotting in both HH-exposed and normal rats. An equal amount of proteins (in both control and HH exposed rats) was loaded for both MeCP2 and pMeCP2 (a). A significant decrease in MeCP2 phosphorylation (b; * p < 0.05) and increase in the level of MeCP2 (c; ** p < 0.01) were observed in the HH-exposed group as compared to control rats. Data represents mean ± SEM of 4 independent experiments. Student t test was used to calculate significance. MeCP2, methyl CpG binding protein 2.

Fig. 5.

HH exposure leads to alteration in the BDNF level in hippocampus. Change in the level of BDNF expression was quantified through western blotting in both normoxia (control) and HH-exposed rats. The immunoblotting of BDNF obtained on Nitrocellulose membrane (a) at corresponding molecular weight revealed a significant decrease in the level of BDNF expression in the HH-exposed group of rats (b; * p < 0.05) as compared to normoxia rats (Control). Data represents mean ± SEM of 4 independent experiments. Student t test was used to calculate significance. BDNF, brain derived neurotrophic factor.

Fig. 6.

HH induced neuronal morphology and apoptosis in the CA1 region of hippocampus. Neuronal morphology alteration and apoptotic neurons were examined through CV staining and Fluoro-Jade C staining, respectively, as shown in (a). A significant increase in the number of CV positive neurons (b; * p < 0.05) and Fluoro-Jade C positive neurons (c; ** p < 0.01) were observed in the HH-exposed group as compared to Normoxia rats (Control). Data represents mean ± SEM of 6 independent experiments. ­Student t test was used to calculate significance. HH, hypobaric hypoxia; CV, crystal violet.