Mechanism Of Opa1 Hydrolytic Cleavage Induced By Mfn2 Mutation Through Apoptosis And Oxidative Phosphorylation Damage

Charcot-Marie-Tooth type 2A（CMT2A）is a motor and sensory neurological disease caused by Mfn2 mutation,and the inheritance model is autosomal single gene dominant inheritance.Mitofusin 2 encoded by Mfn2 is a highly conserved GTPase,which plays a central role in regulating mitochondrial outer membrane fusion and cell metabolism.Early studies believe that the balance between mitochondrial fusion and fission is critical to the morphology and function of mitochondria,and that impaired mitochondrial fusion can lead to CMT2A.However,current studies also believe that the excessive fusion of mitochondria mediated by Mfn2 mutations is also the cause of the CMT2A pathology.The causes of CMT2A disease based on different allelic mutations of Mfn2 are more complicated.Therefore,it is necessary to construct CMT2A disease models with Mfn2 mutations based on different pathogenic sites to analyze the complex mechanism.In recent years,the CRISPR/Cas9 single-base editing system has been widely used to construct animal models of human genetic diseases,because it can specifically realize the conversion of A·T to G·C by adenine base editor（ABEs）or C·G to T·A bases by cytosine base editor（CBEs）,and will not induce DNA double-strand break（DSB）and homologous directed recombination（HDR）caused by DSB.CMT2A disease is caused by single nucleotide mutation,thus the CRISPR/Cas9 single base editing system is a promising tool for constructing CMT2A disease models.Moreover,the sequence similarity of amino acids encoded by Mfn2 between mouse and human is not less than 95%,so the mouse is an ideal model animal simulating human CMT2A disease.To improve the understanding of the pathogenesis of CMT2A,this study constructed mouse disease models by simulating the known pathogenic site of human Mfn2 gene,providing good gene-edited animal models for the study of the CMT2A disease.Studie on the pathogenesis of CMT2A combined with in vivo and in vitro also provides a theoretical research basis for the treatment of the disease.The research content is as follows:First of all,in this study,the mutations of interest,including heterozygous(Mfn2^R364W/+,Mfn2^G176S/+,Mfn2^H165R/+)and homozygous(Mfn2^R364W/R364W,Mfn2^G176S/G176S,Mfn2^H165R/H165R)mutations,were successfully introduced into C2C12myoblast cells by using ABEmax-NG or Anc BE4max-NG base editor;and the phenotypes of cell models in vitro of Mfn2^G176S,Mfn2^H165R（located in the GTPase domain）and Mfn2^R364W（located in the bundle 1 domain）mutations were characterized.The results showed that there are obvious mitochondrial aggregation and focal distribution of Mfn2 in Mfn2^H165R/H165R mutant cell,while the Mfn2^R364W,Mfn2^G176S and Mfn2^H165R/+mutant cells do not show this feature;the three heterozygous and homozygous mutants all show significant mitochondrial loss and obvious mitochondrial fragmentation.Secondly,we conducted an analysis of the causes of mitochondrial fragmentation and found that Mfn2 mutations do not simply lead to the loss of Mfn2 involved in OMM fusion,but induce the hydrolytic cleavage of optic atrophin 1（Opa1）involved in IMM fusion.Through in-depth studies on the process of apoptosis and mitochondrial oxidative phosphorylation damage,we found that in Mfn2^R364W,Mfn2^G176S and Mfn2^H165R/+,which retain Mfn2 expression,the hydrolysis process is associated with Bax-induced apoptosis and uncoupling of respiration and ATP production due to reduce efficiency of mitochondrial quality control.While in Mfn2^H165R/H165R with Mfn2 loss,this hydrolysis process is associated with Mfn2 loss-induced dissipation of mitochondrial membrane potential（MMP）,which triggers apoptosis and uncoupling of oxidative phosphorylation process.Finally,we used ABEmax-NG or Anc BE4max-NG base editing system and ET technology to construct Mfn2^H165R/H165R,Mfn2^G176S/+,and Mfn2^R364W/+mutant model mice,and described the phenotype and pathophysiological symptoms of neuropathy through behavioral analysis,histopathological analysis and axonal nerve injury research.Mfn2^H165R/H165R mouse shows obvious high arched morphology,slow action,thin body,and died at the age of 1.5 months;Mfn2^R364W/+and Mfn2^G176S/+mice show motor dysfunction and neuromuscular damage;Mfn2^G176S/G176S mice shows obvious swelling and curvature of the toes and died at the age of 2 months.On the basis,the pathogenesis of CMT2A was verified again by exploring the molecular mechanism of apoptosis and mitochondrial oxidative phosphorylation damage in vivo,that is,hydrolytic cleavage of Opa1 in genotypes that retain Mfn2 expression is associated with recruitment of Bax-induced apoptosis and uncoupling of oxidative phosphorylation.The loss of overall fusion activity affects the stability of mitochondrial DNA（mt DNA）,causes mitochondrial dysfunction,and ultimately leads to the occurrence of CMT2A.This study analyzed the molecular mechanism of mitochondrial fusion damage caused by Mfn2 mutations for the first time,and speculated that the differences in pathogenesis of CMT2A caused by Mfn2^R364W,Mfn2^G176S,Mfn2^H165R/+,and Mfn2^H165R/H165R mutations,the changes including the distribution of mitochondria,expression of mitochondrial outer membrane-associated proteins,and enzyme activity of mitochondrial complex I,may be related to the expression of Mfn2.The analysis of the mechanism of mitochondrial fusion damage provides new ideas for the treatment of CMT2A disease,and preclinical diagnostic research for different sites is indispensable.