The Role Of LSD1 In Regulating Chloride Homeostasis And Neurotransmitter Excitation-inhibition Imbalance In Rat Autism

Objective: Autism spectrum disorders are a group of neurodevelopmental disorders that begin in early childhood and are characterized by social deficits,repetitive stereotypic behaviors,and anxious behaviors.Its incidence is increasing year by year,but the pathogenesis remains unknown.Aluminum is widely distributed and is closely related to the occurrence of autism.Aluminum is the most abundant metal element in the earth’s crust and widely exists in daily life.Infants are exposed to a large amount of aluminum in different ways after birth,such as infant formula,vaccination,and parenteral nutrition.Early neonatal exposure to toxic metals is strongly associated with ASD-related abnormal developmental outcomes.Studies have found that an E/I imbalance in brain neurotransmission may be the pathological basis of behavioral deficits in autism,but the specific regulatory mechanism is still unknown.In addition,it has been confirmed that the LSD1 inhibitor TAK-418 promotes Ucp2 mRNA expression by inhibiting LSD1 enzyme activity to improve autism behavior deficits,but the exact mechanism remains unclear.In order to better simulate the occurrence of early postnatal autism,this study intends to use aluminum combined with VPA and LPS to construct a new autism rat model and to detect the changes of LSD1 in the regulation of chloride homeostasis and neurotransmitter excitatory and inhibitory imbalance in autism rats.To further improve the regulatory relationship between E/I imbalance and ASD,to clarify the role and possible mechanism of LSD1 in the pathogenesis of ASD,and to provide the important experimental basis and clues for the study of the pathological mechanism of ASD.Methods: A new autism model was established to observe the growth and development,general behavior,and neurobehavioral performance of pups.HE staining was used to evaluate the pathological damage to brain tissue.The level of NSE in the pups was measured by immunofluorescence to assess neuronal damage.LSD1 enzyme activity,GABA,and Glu levels were measured by ELISA.The activity of SOD,level of GSH and level of ATP were detected by kits to evaluate the oxidative damage of pups.The protein levels of GABRG2,KCC2,NKCC1,and UCP2 were detected by Western blotting.The level of Ucp2 mRNA was detected by real-time quantitative PCR.The expression of the GABRG2 protein was detected by immunofluorescence and the co-localization of GABRG2 and Gephyrin was analyzed.MQAE imaging was used to assess changes in chloride ion concentration and Image J was used for quantitative analysis.LSD1 inhibitor TAK-418 was used to inhibit LSD1 enzyme activity,and behavioral changes and physiologically biochemical changes in pups were observed.Results: Model establishment.1.Growth and development.The autistic animal model constructed by aluminum combined with sodium valproate or lipopolysaccharide showed more pronounced growth and developmental malformations,such as tail bending and short tail than sodium valproate or lipopolysaccharide alone.2.General behavior development.The animal model constructed by aluminum combined with sodium valproate or lipopolysaccharide showed a significant increase in the achievement time in the air righting reflex,swimming test,auditory startle,and antennae positioning reflex.The time spent on the surface righting reflex,negative geotaxis reflex,and cliff aversion experiment increased significantly.In the forelimb suspension,the suspension time decreased significantly.3.Neuroethology.The combined model group showed more obvious social defects,repetitive stereotyped behavior,and anxiety behavior.Discussion on mechanism.1.Autistic rats in the model group showed impaired morphological structure in PFC;LSD1 activity increased;the levels of UCP2 protein and Ucp2 mRNA decreased.SOD activity,GSH,and ATP levels decreased significantly.KCC2down-regulation and NKCC1 up-regulation,MQAE fluorescence intensity decreased significantly,and intracellular chloride concentration increased.GABA level decreased,Glu and Glu/GABA ratio increased.The expression of GABRG2 protein decreased,and the co-localization coefficient of GABRG2 and Gephyrin decreased significantly.The intervention of LSD1 inhibitor TAK-418 can reduce ASD-like behavior,which is manifested as improving the pathological tissue damage of prefrontal cortex in autism rat model;reducing LSD1 activity;promoting the expression of UCP2 protein and Ucp2 mRNA;SOD activity,GSH,and ATP levels were increased;promote KCC2 up-regulation and NKCC1 down-regulation,reduce intracellular chloride ion concentration;elevate GABA levels,reduced Glu and Glu/GABA ratio levels;promote the express of GABRG2 protein and colocation of GABRG2 and Gephyrin.Conclusions: We successfully constructed a novel autistic rat model and found that LSD1 induced oxidative damage via the down-regulating UCP2 to induce dysregulation of chloride transport proteins,resulting in an imbalance of chloride homeostasis and GABA excitatory inhibition.TAK-418 ameliorated autism-like behavior in ASD rats by inhibiting LSD1 activity,rescuing abnormal chloride ion transport,and the imbalance of neurotransmitter excitatory inhibition.