A Study Of The Effects Of MsrB1 Knockout On Mice Central Neuronal System By Electrophysiology And Calcium Fluorescence Imaging

In the process of biological metabolism,methionine residues of proteins can be easily oxidized into methionine sulfoxide,leading to the change of spatial structure,and even function change.However,this post-translation modification can be reversed by methionine sulfoxide reductase in organisms.In mammals,methionine sulfoxide reductase family consists of four members.Among of them,as including selenocysteine,Msr B1 is a kind of selenium protein with high reducibility(high oxidize resistance).Msr B1 was highly expressed in liver,kidney and other organs,and in knockout model mice,the levels of liver and kidney oxidation were significantly increased.In addition,some studies have shown that Msr B1 can interact with actin,TRPM6 and amino beta in vitro.One of them,Actin is one of the main members of cytoskeleton,and regulation of cytoskeleton play an important role in the process of muscle contraction/relaxation,cell movement,cytokine and cytoplasmic flow,cell migration,cell polarity,axon growth,synapsis and signal transmission between synapses.It can be seen that Msr B1 may play an important role in the development and function of the central nervous system,but whether the knockout of Msr B1 affects the normal function of the central nervous system in mice has not been studied and reported.Therefore,in this study,we took the calcium fluorescence imaging system and electrophysiological system to study the function of Msr B1 in mouse brain.Firstly,from the morphological point of view,the effects of Msr B1 knockout on brain tissue structure and distribution of different types of brain cells were observed.HE staining results showed that there was no obvious change in brain structure of KO mice.But immunofluorescence staining results showed that astrocytes proliferated in the hippocampus of KO mice.It can be easily seen that loss of Msr B1 have a great impact on hippocampus.Therefore,we tested the spatial learning and memory ability of mice through water maze and open field behavior test.The results showed that,compared with wild-type mice,the ability of autonomous exploration and spatial learning of the Msr B1 KO mice with deficiency decreased significantly,but there is no difference on anxiety.In order to further confirm the results,we also used electrophysiological technology to detect LTP and LTD in CA1 area of hippocampus,and found that LTP / LTD of KO mice decreased significantly.It can be seen that knockout of Msr B1 has obvious damage to the signal transmission function of neurons.The signal transmission of neurons depends on the plasticity of synaptic structure,and there are abundant actin microfilaments in the dendritic spines on which synapses are formed.The dynamic aggregation and depolymerization of microfilaments are very important to the morphology,function and transmitter transport of dendritic spines,and the change of actin microfilaments is affected by their own redox state.In vitro studies showed that msrb1 could restore the aggregation ability of actin by reducing the oxidized 44 and 47 methionine of actin.In addition,the results of this study showed that the level of methionine oxidation at the position 44 of actin in the brain of Msr B1 deficient mice was significantly increased.It can be seen that the increased level of methionine oxidation of actin is related to the spatial learning disorder of Msr B1 deficient mice,and the results of proteomic analysis also show that after the knockout of Msr B1,part of proteins related to physiological and pathological processes have obvious changed,including complement system,actin skeleton regulation,Rap1 signaling pathway,AD,neuron signaling pathway,LTP,calcium signaling regulation,MAPK signaling pathway and endoplasmic reticulum protein synthesis.In addition,a large number of studies have confirmed that NMDAR molecules play an important role in the process of LTP and LTD.Therefore,we detected the expression of Glu N2 A and Glu N2 B,which are mainly expressed in the neurons of vertebrae,and the expression of synaptophysin and PSD-95,which are located in presynaptic and postsynaptic respectively.The results show that loss of Msr B1 leads to the decrease of NMDARs expression and damage of synaptic structure.At the same time,it is reported that the activity of neurons can lead to a significant increase of intracellular calcium concentration,and then,as a second messenger,calcium can mediate a variety of cell responses.Calcium combines with calmodulin to stimulate the activation of a variety of enzymes,including calmodulin kinase and calcium sensitive adenylate cyclase,which can help the transmission of calcium signals and affect short-term biological responses,as well as long-term neuronal responses that need to change gene expression.And Ca MKII is phosphorylated under the action of Ca2+-CAM complex,and retains its catalytic activity under the initial stimulation condition,forming a memory molecular device.This process has long been considered to play an important role in LTP and learning and memory.The WB results also show that the phosphorylation level of Ca MKII in KO mice is significantly lower than that in WT mice.In order to prove the above results in vivo,intracellular calcium fluorescence imaging was used to detect the calcium level in primary neurons of WT and KO mice.The results show that under normal conditions,the calcium level in neurons of KO mice was significantly lower than that of WT mice,and even higher than that of KO mice after stimulated by glutamate.It can be seen that the knockout of Msr B1 lead to the normal calcium level of neurons and calcium uptake under glutamate stimulation.To sum up,adopting Msr B1 KO mouse as the model,this study first reveals the role of Msr B1 in the brain: firstly,the loss of Msr B1 will not affect the normal shape of the mouse brain,but will lead to the proliferation of astrocytes in the mouse brain,especially in hippocampus;secondly,the loss of Msr B1 will affect the expression of synaptic signaling function protein and the activation of calcium signaling function protein,and then lead to the impairment of signal transmission function among neurons Finally,the memory ability of mice was weakened.This study not only expands the research field of Msr B1 function,but also provides new evidence for the role of Msr B1 family members in CNS.In addition,the relationship between Msr B1 and neurodegenerative behavior provides a new way to diagnose and treat neurodegenerative diseases.