Study Of Oxidative Stress And Metabolomics In Dip2a Knock-out Mice

Autism spectrum disorders(ASD)are a kinds of neurodevelopmental disorders.The core symptoms are defects in social interaction,language communication,and repetitive stereotyped behaviors.The etiology of ASD is complicated and the pathogenic mechanism is not clear.Disconnected(disco)-interacting protein 2A(DIP2A)is a susceptibility gene for ASD.In our previous studies,DIP2A,the expression of Dip2a product,regulates endogenous acetyl-coenzyme A(ac-Co A)metabolism,an important intermediate metabolite.Deletion of Dip2a in themice decrease stability of postsynaptic scaffold proteins causing synaptic dysfunction,which mimicked the ASD phenotype.To further understandt the role of DIP2A in metabolism,we performed a series of studies in this thesis.Firstly,we found that DIP2A was abundant in mitochondria.The binding proteins of DIP2A in mouse brain were isolated with N-termianl fusion with GST-DMAP1 functional domain through Pull down assay.The specific bands were collected and identified by LC-MS/MS mass spectrometry.A total of 1512 proteins were identified,of which 91 proteins were localized in mitochondria.Gene ontology analysis of the mitochondria-related proteins revealed that 13 binding proteins were categorized into the oxidoreductase subfamily.We further analyzed the binding activity of the 13 oxidoreductases and found that only SOD1 and SOD2 have binding activity.Since SOD is a major antioxidative enzyme,we speculated that DIP2A was involved in antioxidative reactions.Using co-immunoprecipitation,we found that DIP2A bind with SOD2.In cultured HEK293 cells,DIP2A co-localized with SOD2 partially.We further found that SOD activity was decreased in the cerebral cortex of Dip2a KO mice,but the expression level of SOD2 was no detectable changes.Correspondingly,deletion of Dip2a also increased the level of ROS in the murine brain.Whereas,DIP2A overexpression in cultured cells increased SOD activity and inhibited the generation of ROS under oxidative stress.Because mitochondria were susceptible to be attacked by excess ROS,we further analyzed the morphology of mitochondria of brain in the Dip2a KO mice.Results showed deletion of Dip2a led to decreased mitochondria area and shortened mitochondria length and diameter.Mitochondria are the main site of metabolism in cells.Defects in mitochondria morphology usually associates with serious metabolic disorders.In order to explore the effect of DIP2A deficiency on mice metabolism,serum metabolites were studied by non-targeted-metabonomics.Serum samples of Dip2a^+/+,Dip2a^+/-,and Dip2a^-/-mice were collected at post-natal day 30.The data of endogenous metabolites were preprocessed by LC-MS mass spectrometry.The preprocessed data were analyzed by meta analysis 4.0 software.Compared with control group,the overall changes of serum metabolites were showed that lipids were the major compounds of changed metabolites,in which the fatty acids and phosphatidylglycerides were significantly decreased,whereas the sphingolipids were increased.Besides that,concentrations of carbohydrates and some amino acids were also increased.Interestingly,we found 19 common differential metabolitesby comparing differential metabolites among Dip2a^+/+,Dip2a^+/-,and Dip2a^-/-mice,which included 15 lipids.These results suggested that deletion Dip2a impaired glucose metabolism and increased lipid metabolism as a compensatory pathway for energy supply,which might be the reason for the increase of oxidative stress in Dip2a^-/-mice.To further confirm the association between DIP2A and oxidative stress,we selected the commonly used antioxidants,co-enzyme Q10 and vitamin E,to treat the Dip2a KO mice after weaning.The results indicated that co-enzyme Q10 and vitamin E effectively reduced the ROS accumulation in the brain tissue of Dip2a KO mice,and significantly improved the repetitive stereotype behavior of mice.However,we had no find the effect of improvement on the social deficit of Dip2a^-/-mice.In conclusion,deficiency of Dip2a caused a decrease in SOD activity and an increase in ROS level in the cerebral cortex of mice.Elevated ROS level caused abnormal mitochondrial morphology,damaged mitochondrial glucose metabolism and excessive lipid metabolism.It is speculated that excessive lipid metabolism leads to abnormal synaptic morphology and function.These results revealed that an undiscovered function of DIP2A in antioxidant protection,and provide another possible explanation for the pathophysiology of DIP2A-mediated ASD.