Study On The Effect And Mechanism Of GTPase MMAA On The Nervous System

BackgroundMethylmalonic acidemia(MMA)is the most common autosomal recessive organic acid metabolism disease in China.The age of onset of methylmalonic acidemia is different,the clinical manifestations are not specific,and all organs and tissues can be affected,often manifested as feeding difficulties,vomiting,shortness of breath,dystonia,convulsions,lethargy,and neurological symptoms,such as cognitive impairment,psychomotor disorders,epilepsy,developmental delay,cerebral hemorrhage,cerebral edema,etc.MMA has a variety of pathogenic genes,MMAA(metabolism of cobalamin associated A)is one of them,its coding protein is a GTPase in the methylmalonic acid metabolic pathway,localized to mitochondria,involved in the transport of vitamin B12 from the cytoplasm to the mitochondria.Defects in the Mmaa gene can lead to abnormal methylmalonic acid metabolism.Clinical cases of Mmaa gene mutations show that neurological symptoms are common,and the function and mechanism of MMAA in the nervous system are unknown.PurposeMmaa gene knockout mice were used to construct methylmalonic acidemia model to study the effect of Mmaa gene on nervous system,and further explore the changes of nervous system related metabolic molecules and the mechanism of action.Methods1.Mmaa-/-mice were constructed using CRISPRJ/Cas9 gene editing technique.Changes of metabolites associated with methylmalonic acidemia in Mmaa-/-mice were detected by tandem mass spectrometry.The basal metabolic rate and exercise of Mmaa/-mice were observed in metabolic cage.The histopathologic conditions of brain,heart,liver,kidney and testis of Mmaa-/-mice were observed by H&E staining.2.Mmaa-/-mice were observed for behavioral abnormalities through open field experiments and Barnes mazes.Nissl staining and immunofluorescence were used to visualize the morpHology and number of neurons and glial cells.Western blotting detects mitochondria-associated protein expression in mouse cerebral cortex tissue.3.The activities of superoxide dismutase(SOD),glutathione peroxidase(GSH/Px),peroxidase(POD),NADH enzyme and malondialdehyde(MDA)content were detected by biochemical detection kit.The structure and morpHology of mitochondria and synapses were observed by transmission electron microscopy.mRNA expression levels of inhibitory neurotransmitters and vesicle transport-related molecules were detected by qPCR.Results1.Tandem mass spectrometry showed an increase in propionylcarnitine(C3)and C3/C2 ratio,and a decrease in arginine,ornithine and glycine.2.The oxygen consumption rate and carbon dioxide production rate of Mmaa-/mice were significantly lower than that of the control group,and the diet and water consumption were also greatly reduced.Serious growth and metabolism problems appeared in Mmaa-/-mice,but the motor behavior was abnormally increased.3.Open field experiments and Barnes maze experiments showed that Mmaa-/-mice developed neurological symptoms of anxiety and memory loss.The results of Nissl staining showed that the Mmaa-/-mouse cerebral cortex neurons were wrinkled and decreased in number.Immunofluorescence staining showed neuronal skeleton rupture in the hippocampal region and activation of glial cells.4.Transmission electron microscopy(TEM)and biochemical tests showed mitochondrial damage,structural cavitation,reduced or even disappeared ridge structure,and oxidative stress in Mmaa-/-mice.The mRNA expression of inhibitory neurotransmitter related molecules in Mmaa-/-mice was decreased by qPCR.Conclusion1.The A mouse model of methylmalonic acidemia caused by Mmaa gene knockout was successfully constructed and showed serious growth and energy metabolism problems.2.Mmaa-/-Mice exhibit anxious behaviors and memory loss,which may be associated with cerebral cortical damage and reduced transcription levels of inhibitory neurotransmitter receptors.3.Mmaa-/-Mice cerebral cortical injury is associated with mitochondrial damage to the nervous system and decreased arginine and ornithine.