Role of mitochondrial DNA 6mA in the hippocampal neurons in vascular cognitive impairment
10.3760/cma.j.cn115354-20240618-00360
- VernacularTitle:海马神经元线粒体DNA 6mA在血管性认知障碍中的作用研究
- Author:
Ziyi CHEN
1
;
Lingfei YANG
;
Kaixin WANG
;
Qingsheng LI
;
Yanjie JIA
;
Zhe GONG
Author Information
1. 郑州大学第一附属医院神经内科，郑州 450000
- Keywords:
Vascular cognitive impairment;
Chronic cerebral hypoperfusion;
Mitochondria DNA 6mA;
METTL4
- From:
Chinese Journal of Neuromedicine
2024;23(8):757-768
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the role and mechanism of mitochondrial DNA N6-methyladenine (6mA) in the hippocampal neurons in vascular cognitive impairment.Methods:(1) In vivo experiments: SPF male rats were randomly divided into sham-operated group and chronic cerebral hypoperfusion (CCH) group ( n=12). CCH models in the CCH group were established by ligating bilateral carotid arteries, while rats in the sham-operated group were only bilaterally dissected without ligation. Exploratory ability was detected by open field test 50 d after modeling, cognitive function was evaluated by novel object recognition test 51-53 d after modeling, and learning and memory abilities were tested by Morris water maze 54-59 d after modeling. And then, rats were sacrificed; ATP concentration and reactive oxygen species (ROS) level in the hippocampal tissues were detected, and neuron apoptosis in the hippocampal CA1 area was detected by TUNEL. (2) In vitro experiments: HT-22 cells were divided into normal control (NC) group, oxygen-glucose deprivation (OGD) group, OGD+siControl group, and OGD+siMETTL4 group. Cells in the NC group were cultured routinely, cells in the OGD group were subjected to low sugar and low oxygen for 12 h, and cells in the OGD+siControl group and OGD+siMETTL4 group were, respectively, transfected with NC-siRNA or METTL4-siRNA after being subjected to low sugar and low oxygen for 12 h. Mitochondria morphology was observed by transmission electron microscopy, ROS was detected by flow cytometry, mitochondria membrane potential was detected by JC-1 fluorescent staining, and mitochondrial complex I and III activity was detected by kit. (3) In vivo and in vitro experiments: METTL4 and DNA 6mA expressions in neuronal mitochondria of rat hippocampal tissues and mitochondria of HT-22 cells were detected by immunofluorescent staining and Western blotting. Results:(1) CCH rats had cognitive impairment: compared with the sham-operated group, CCH group had significantly increased frequency of entering the central area and reduced time in exploring new objects in open field experiment,and significantly decreased frequency of crossing the platform and prolonged escape latency in water maze experiment ( P<0.05). Compared with rats in the sham-operated group, rats in the CCH group had significantly decreased hippocampal ATP content ([18.820±1.177] nmol/L vs. [10.190±0.519] nmol/L) and increased ROS content ([4 488.00±255.70] AU vs. [11 644.00±530.20] AU, P<0.05). TUNEL results showed that the number of apoptotic neurons in the hippocampal CA1 area of CCH group was obviously increased than that in sham-operated group. Immunofluorescent staining results showed that 6mA and METTL4 mainly distributed in the mitochondria of hippocampal neurons in CCH group, and the 6mA and METTL4 expressions were obviously increased compared with those in the sham-operated group. Western blotting results showed that METTL4 expression in the hippocampal mitochondria of CCH group was significantly higher than that in the sham-operated group (1.729±0.168 vs. 1.000±0.000). (2) In vitro experiment: under transmission electron microscope, compared with the NC group, HT-22 cells in the OGD group showed obvious mitochondrial ridge disappearance, membrane rupture and vacuolation. Compared with the OGD group, the OGD+siMETTL4 group had significantly increased ATP production, decreased mtROS production, increased mitochondrial membrane potential, and increased mitochondrial complex I and III activities ( P<0.05). Immunofluorescent staining results showed that the mtDNA 6mA and METTL4 expressions in the OGD group were obviously higher than those in the NC group, and both mainly expressed in the mitochondria; mtDNA 6mA expression in the OGD+siMETTL4 group was obviously lower than that in OGD group. Western blotting results showed that METTL4 expression in the OGD+siMETTL4 group was significantly higher than that in the OGD group (1.578±0.261 vs. 2.970±0.280). Conclusion:Specific high expression of methylase METTL4 in hippocampal neurons of rats with cognitive impairment after CCH promotes the increased mtDNA 6mA expression and leads to mitochondrial energy metabolism disorders and increased ROS, which is speculated to be one of the mechanisms causing vascular cognitive impairment.
