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DNA methylation manipulation of memory genes is involved in sevoflurane induced cognitive impairments in aged rats

Citation

Ni, Cheng et al. (2020), DNA methylation manipulation of memory genes is involved in sevoflurane induced cognitive impairments in aged rats, Dryad, Dataset, https://doi.org/10.5061/dryad.69p8cz8xw

Abstract

DNA methylation is an essential epigenetic mechanism involving in gene transcription modulation. An age-related increase in promoter methylation has been observed for neuronal activity and memory genes, and participates in neurological disorders. However, the position and precise mechanism of DNA methylation for memory gene modulation in anesthesia related cognitive impairment remained to be determined. Here, we tested whether sevoflurane anesthesia would suppress the transcription of memory genes in aged rat hippocampus. Then, we investigated changes in DNA methylation of involved genes and verify whether dysregulated DNA methylation would contribute to anesthesia induced cognitive impairment. The results indicated that sevoflurane anesthesia down-regulated the mRNA and protein levels of three memory genes, Arc, Bdnf and Reln, which were accompanied with promoter hypermethylation and increased Dnmt1, Dnmt3a and Mecp2 expression, and finally impaired hippocampus dependent memory. Furthermore, inhibition of DNA hypermethylation by 5-Aza rescued sevoflurane induced memory gene expression decrease and cognitive impairment. These findings provides an epigenetic understanding for the pathophysiology of cognitive impairment induced by general anesthesia in aged brain.

Methods

MassARRAY EpiTYPER Assay

In brief, DNA was extracted from C6 glioma cells and rat Hippocampi using Cell/Tissue DNA Extraction Kit (BioTeke, Beijing, China), then was bisulfite converted using Zymo Research EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA). Primers were designed using the EpiDesigner Software (http://www.epidesigner.com/index.html), and their sequences are listed in table 2. PCR amplification was carried out in a 8 μl reaction volume containing 0.8 μl of 10×PCR Buffer, 0.8 μl of dNTPs, 0.1 μl PCR enzyme, 0.2 μl of each primer, 1.0 μl of bisulfite-converted DNA and H2O, and the amplification was carried out with an initial denaturation step at 94 oC for 4 min followed by 45 cycles of 94 oC for 20 s, 56 oC for 30 s and 72 oC for 1 min, then final extension at 72 oC for 3 min. After PCR and Shrimp Alkaline Phosphatase treatment, fragments were ligated to a T7 promoter segment, and then transcribed into RNA. The synthesized RNA was cleaved with RNase A and all cleavage products were analyzed on a mass spectrometer, according to the manufacture’s protocol. The generated mass signal patterns were translated into quantitative DNA methylation levels of different CpG sites of the selected genes by MassARRAY EpiTYPER Analyzer software. The locations of promoter regions encompassing the transcription start and the number of CpG sites assessed in the promoter regions are listed in table 2. The results were processed and analyzed by the MassARRAY Workstation software. All measurements were performed in triplicate and the average was used for statistical analysis.

 Table 2 The primer sequences and assessed locations for Bisulfite sequencing PCR

Genes

Orientation

Sequence (5’ to 3’)

Location

CpG sites

Arc

Forward

aggaagagagGGTAGAGGAGAGTGT

TTTTGGTTTT

-274 to +318

42

Reverse

cagtaatacgactcactatagggagaaggctACA

CTTACCAATCTACAAAATCACATT

Bdnf

Primer I

Forward

aggaagagagATTGTGATTTTTTTGGT

AAAAAGGA

-644 to -99

16

Reverse

cagtaatacgactcactatagggagaaggctCCA

AAACCCACCTTCTAAAACTTAT

Bdnf

Primer II

Forward

aggaagagagTTATTTTTTAGTATTTG

TTGGGGAGA

-634 to -36

6

Reverse

cagtaatacgactcactatagggagaaggctCCTT

TCCATATATAAAAACATTACCCA

Bdnf

Primer III

Forward

aggaagagagTGTTTATTTATAATGAA

ATGGGTAATGT

-1216 to -730

18

Reverse

cagtaatacgactcactatagggagaaggctACC

AAAAATCTATTCCAACCTACAC

Bdnf

Primer IV

Forward

aggaagagagTTGTTGTTGTTTAGATG

ATGAAAGG

-245 to +346

18

Reverse

cagtaatacgactcactatagggagaaggctACC

CACCTTTTTCAATCACTACTTA

Bdnf

Primer VI

Forward

aggaagagagGAGTTTTGGGGTTAAG

TAGTTGGTT

-192 to +349

35

Reverse

cagtaatacgactcactatagggagaaggctCCT

CAAAATCCACACAAAACTCTC

Reln

Forward

aggaagagagGTAGTTAGGTTGAAA

GGGAGATTGG

-1383 to -834

17

Reverse

cagtaatacgactcactatagggagaaggctTAA

TACCCTTTTCCCAAACTCAAAC

           

Funding

National Natural Science Foundation of China, Award: No. 81771146

National Natural Science Foundation of China, Award: No. 81970994

National Natural Science Foundation of China, Award: No. 81400869