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中华妇幼临床医学杂志(电子版)

Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition) ›› 2022, Vol. 18 ›› Issue (05): 528 -539. doi: 10.3877/cma.j.issn.1673-5250.2022.05.006

Original Article

Mechanism of MMACHC gene mutation of cblC-type methylmalonic acidemia on apoptosis and Wnt/β-catenin signaling pathway in mouse neural cells

Wei Zhou1, Heng Cai2, Haidi Fan3, Huizhong Li1, Chuanxia Wang1, Maosheng Gu1,†()   

  1. 1Center of Genetic Medicine, Xuzhou Maternity and Child Health Care Hospital, Xuzhou 221009, Jiangsu Province, China
    2Xuzhou Medical University, Xuzhou 221004, Jiangsu Province, China
    3Clinical Laboratory, The First Branch of Huai′an First Poeple′s Hospital, Huai′an 223300, Jiangsu Province, China
  • Received:2021-09-24 Revised:2022-04-29 Published:2022-10-01
  • Corresponding author: Maosheng Gu
  • Supported by:
    Maternal and Child Health Research Project of Jiangsu Province(F201912); Xuzhou Science and Technology Program(KC19028, KC20074)
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Objective

To investigate the mechanism of novel variants in MMACHC gene resulting in cblC-type methylmalonic acidemia (MMA) on neural cell apoptosis and Wnt/β-catenin signaling pathway in mice.

Methods

Two novel missense variants of c. 626_627del and C. 228_231del of MMACHC gene which were detected in blood samples of two infants with cblC-type MMA screening and diagnosed by Xuzhou Branch Center of Jiangsu Newborn Screening Center in May and July 2017, were selected as research materials. After analyzing the conservation of amino acid residues involved in the two novel variants and predicting the pathogenicity, two new recombinant plasmids of MMACHC gene were constructed, and with the recombinant plasmids of wild-type MMACHC gene, they were transiently transfected into mouse nerve cells (neuron cell line HT22 and nerve stem cell line C17.2) for in vitro experiments. Mice HT22 and C17.2 cells transfected with MMACHC gene c.626_627del plasmid were included into neuron observation group 1 and nerve stem observation group 1, respectively. Mice HT22 and C17.2 cells transfected with MMACHC gene C. 228_231del plasmid were included into neuron observation group 2 and nerve stem observation group 2, respectively. Mice HT22 and C17.2 cells transfected with wild-type MMACHC gene recombinant plasmid were included into neuron control group and nerve stem control group, respectively. Immunofluorescence staining, TUNEL fluorescence detection and Western blotting were used to analyze the apoptosis of mouse nerve cells and the expression of Wnt/β-catenin signaling pathway related proteins. One-way ANOVA and Dunnett t test were used to statistically compare the apoptosis rate, apoptosis-related proteins and the expression levels of Wnt/β-catenin signaling pathway related proteins between two observation groups and control group, respectively. This study was approved by the Ethics Committee of Xuzhou Maternity and Child Health Care Hospital [Approval No.2019(09)].

Results

① The amino acid sites of two novel variants in MMACHC gene (positions 77 and 209) were highly conserved among H. sapiens, M. musculus, R. norvegicus, D. rerioand and M. mulatta. Bioinformatics analysis indicated that both variants may be pathogenic. ② Compared to control group, the DAPI staining of nuclei of nerve cells in corresponding two observation groups were darker (bright blue), and the number of cells with positive TUNEL fluorescence (green) were more. The apoptosis rate (DAPI staining) and apoptosis rate (TUNEL staining) of neuron observation group 1 were (25.8±1.1)% and (23.1±1.4)%, respectively, which were significantly higher than those of (4.9±0.9)% and (3.2±0.4)% in neuron control group, and the differences were statistically significant (t=25.95, 24.41; all P<0.001). With similar results to this were, neuron observation group 2 compared with neuron control group, also nerve stem observation group 1 and group 2 compared with nerve stem control group, respectively, and the differences were statistically significant (all P<0.05). ③ The expression levels of apoptosis-related proteins of caspase-3 and cleaved caspase-3, and intracytoplasmic β-catenin in HT22 cells in neuron observation groups 1 and 2 were higher than those in neuron control group, respectively; while the expression levels of Wnt/β-catenin signaling pathway related proteins of postsynaptic density(PSD)-95, myc-MMACHC, phosphorylation-glycogen synthase kinase(p-GSK)-3β(Ser) and nuclear nucleus β-catenin were lower than those of neuron control group, respectively. And the above differences were statistically significant (P<0.05). ④ The expression levels of caspase-3, PSD-95, myc-MMACHC, p-GSK-3β(Ser) and nuclear nucleus β-catenin in C17.2 cells in nerve stem observation group 1 were lower than those in nerve stem control group; while the expression levels of cleaved caspase-3, GSK-3β and intracytoplasmic β-catenin were significantly higher than those in nerve stem control group. And the above differences were statistically significant (P<0.05). The expression levels of caspase-3, cleaved caspase-3, GSK-3β and intracytoplasmic β-catenin of C17.2 cells in nerve stem observation group 2 were higher than those in nerve stem control group, while the expression level of nuclear nucleus β-catenin was lower than that in nerve stem control group. And the above differences were statistically significant (P<0.05).

Conclusions

Two novel variants in MMACHC gene might inhibit GSK-3β-Wnt/β-catenin signaling pathway to promote nerve cell apoptosis in mice.

Key words: Methylmalonic academia, MMACHC gene, Neurons, Neural stem cells, Apoptosis, Glycogen synthase kinase 3, Beta catenin, Mice
图1 对人源、家鼠、褐鼠、斑马鱼及猕猴5个不同物种MMACHC基因2种新突变的氨基酸位点同源性分析(图1A表示c.228_231del突变的MMACHC蛋白氨基酸位点同源性分析；图1B表示c.626_627del突变的MMACHC蛋白氨基酸位点同源性分析)注：Homo sapiens为人类、Mus musculus为家鼠、Rattus norvegicus为褐鼠、Danio rerio为斑马鱼、Macaca mulatta为猕猴
图2 对人源MMACHC蛋白氨基酸残基空间位阻示意图[图2A：MMACHC蛋白野生型与p.D77Qfs*22突变型组合的三维结构示意图；图2B：MMACHC蛋白野生型第77位天冬氨酸残基空间结构(绿色)示意图；图2C：MMACHC蛋白突变型第77位谷氨酸残基空间结构(红色)示意图；图2D：MMACHC蛋白野生型与突变型第77位氨基酸残基空间结构组合示意图；图2E：MMACHC蛋白野生型与p.V209Dfs*35突变型组合的三维结构示意图；图2F：MMACHC蛋白野生型第209位缬氨酸残基空间结构(绿色)示意图；图2G：MMACHC蛋白突变型第209位天冬氨酸残基空间结构(红色)示意图；图2H：MMACHC蛋白野生型与突变型第209位氨基酸残基空间结构组合示意图]注：Asp为天冬氨酸，Gln为谷氨酰胺，Val为缬氨酸
图3 人源MMACHC蛋白三维结构及其表面电荷分布示意图(图3A：MMACHC蛋白野生型及2种突变型组合的三维结构示意图，绿色为野生型、红色为p.D77Qfs*22突变型、灰蓝色为p.V209Dfs*35突变型；图3B：MMACHC蛋白结构域简图，并标注2种突变的氨基酸位置；图3C~3E：MMACHC蛋白3种型表面电荷分布示意图，蓝色为负电荷聚集区、红色为正电荷聚集区)
表1 转染MMACHC重组质粒后，3组小鼠神经元HT22细胞凋亡率及凋亡相关蛋白表达水平比较(±s)
组别 例数 细胞凋亡率(%)(DAPI染色) caspase-3相对表达量 cleaved caspase-3相对表达量 细胞凋亡率(%)(TUNEL染色)
①神经元观察1组 3 25.8±1.1 0.86±0.01 0.80±0.00 23.1±1.4
②神经元观察2组 3 12.5±1.2 1.10±0.03 1.13±0.02 32.5±1.2
③神经元对照组 3 4.9±0.9 0.65±0.00 0.69±0.02 3.2±0.4
总体比较(F值/P值)   289.40/<0.001 394.30/<0.001 720.70/<0.001 572.50/<0.001
两两比较(t值/P值)          
  ① vs ②   14.04/0.001 12.06/<0.001 35.87/<0.001 8.91/0.001
  ① vs ③   25.95/<0.001 32.40/<0.001 9.60/0.001 24.41/<0.001
  ② vs ③   8.66/0.001 24.31/<0.001 29.85/<0.001 38.86/<0.001
表2 转染MMACHC重组质粒后，3组小鼠神经干C17.2细胞凋亡率及凋亡相关蛋白表达水平比较(±s)
组别 例数 凋亡率(%)(DAPI染色) caspase-3相对表达量 cleaved caspase-3相对表达量 凋亡率(%)(TUNEL染色)
①神经干观察1组 3 45.80±1.07 1.19±0.01 1.45±0.12 18.9±1.0
②神经干观察2组 3 35.53±3.11 1.40±0.05 1.40±0.08 22.1±1.0
③神经干对照组 3 5.70±0.26 1.23±0.01 0.74±0.02 2.2±0.5
总体比较(F值/P值)   358.00/<0.001 41.30/<0.001 71.40/<0.001 436.40/<0.001
两两比较(t值/P值)          
  ① vs ②   5.40/0.006 7.32/0.002 0.60/0.584 3.72/0.204
  ① vs ③   63.02/<0.001 4.25/0.013 10.32/<0.001 30.60/<0.001
  ② vs ③   16.54/<0.001 5.69/0.005 14.92/<0.001 25.51/<0.001
图4 转染MMACHC突变质粒后小鼠神经细胞凋亡变化[图4A：3组HT22细胞的免疫荧光图(细胞核DAPI荧光染色呈蓝色，高倍镜)；图4B：Western blotting检测3组HT22细胞凋亡相关蛋白相对表达量；图4C: 3组HT22细胞的免疫荧光图(TUNEL荧光染色的凋亡细胞呈绿色，高倍镜)；图4D：3组C17.2细胞的免疫荧光图(细胞核DAPI荧光染色呈蓝色，高倍镜)图4E：Western blotting检测3组C17.2细胞凋亡相关蛋白相对表达量；图4F: 3组C17.2细胞的免疫荧光图(TUNEL荧光染色的凋亡细胞呈绿色，高倍镜)]注：1表示MMACHC基因c.626_627del质粒转染神经细胞，2表示MMACHC基因c.228_231del质粒转染神经细胞，3表示野生型MMACHC基因重组质粒转染神经细胞。α-tubulin为α-微管蛋白(内参蛋白)，caspase为半胱氨酸天冬氨酸蛋白酶，cleaved caspase为活化的半胱氨酸天冬氨酸蛋白酶。Western blotting为蛋白质印迹法
表3 3组小鼠神经元H2TT细胞Wnt/β-catenin信号通路相关蛋白表达水平比较(±s)
组别 例数 PSD-95相对表达量 myc-MMACHC相对表达量 p-GSK-3β(Ser9)相对表达量 GSK-3β相对表达量 β-catenin相对表达量
细胞质内 细胞核内
①神经元观察1组 3 0.85±0.00 0.78±0.00 0.89±0.00 0.87±0.01 1.36±0.02 0.49±0.02
②神经元观察2组 3 0.96±0.02 0.88±0.01 0.95±0.02 0.87±0.02 1.12±0.01 0.68±0.01
③神经元对照组 3 1.02±0.01 1.24±0.01 1.04±0.02 0.90±0.03 1.10±0.00 0.76±0.00
总体比较(F值/P值)   119.70/<0.001 1 211.00/<0.001 72.29/<0.001 1.33/0.033 357.20/<0.001 537.10/<0.001
两两比较(t值/P值)              
  ① vs ②   8.33/0.001 11.68/<0.001 4.51/0.011 0.17/0.871 18.45/<0.001 19.15/<0.001
  ① vs ③   44.73/<0.001 53.16/<0.001 15.91/<0.001 1.59/0.188 20.82/<0.001 27.19/<0.001
  ② vs ③   4.41/0.012 30.25/<0.001 6.16/0.004 1.53/0.199 4.02/0.016 19.05/<0.001
图6 MMACHC基因2种突变质粒转染小鼠神经干细胞C17.2后，Wnt/β-catenin信号通路相关蛋白Western blotting电泳图
表4 3组小鼠神经干C17.2细胞Wnt/β-catenin信号通路相关蛋白表达水平比较(±s)
组别 例数 PSD-95相对表达量 myc-MMACHC相对表达量 p-GSK-3β(Ser9)相对表达量 GSK-3β相对表达量 β-catenin相对表达量
细胞质内 细胞核内
①神经干观察1组 3 1.17±0.02 0.92±0.01 1.76±0.01 1.82±0.00 0.92±0.02 0.91±0.01
②神经干观察2组 3 1.23±0.01 1.24±0.01 1.83±0.00 1.84±0.00 0.86±0.01 0.96±0.01
③神经干对照组 3 1.29±0.04 1.25±0.02 1.83±0.01 1.68±0.02 0.66±0.01 1.01±0.01
总体比较(F值/P值)   15.53/0.004 573.90/<0.001 138.50/<0.001 172.80/<0.001 370.50/<0.001 99.80/<0.001
两两比较(t值/P值)              
  ① vs ②   5.53/0.005 32.50/<0.001 15.91/<0.001 5.41/0.006 5.38/0.006 6.48/0.003
  ① vs ③   4.66/0.010 27.40/<0.001 12.80/<0.001 12.55/<0.001 23.34/<0.001 21.13/<0.001
  ② vs ③   2.21/0.091 1.08/0.339 1.02/0.365 14.28/<0.001 25.19/<0.001 6.02/0.004
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