PTPα/NMDA-R Signal Toxicity In Hippocampus And Its Mechanism In Cuprizone Induced Model

Objectives:Multiple sclerosis(MS)is a neurodegenerative disease caused by the damage of myelin cells in the brain and spinal cord,which leads to the blockage of nerve conduction and further affects neurological function.The impairment of learning and memory function in patients with multiple sclerosis may be related to the damage of hippocampus.Neuroimaging showed that the hippocampus of patients was significantly smaller than that of normal people of the same age.The pathological results of the patient’s autopsy showed that there were extensive demyelinating lesions in the hippocampus.Previous studies in our laboratory have shown that Protein-tyrosine Phosphatase Alpha(PTPα)can regulate the formation of myelin sheath.Our research group has accumulated a large amount of experience in PTPα and multiple sclerosis,mainly focusing on the spinal cord.However,we found that cuprizone-induced demyelination in MS mice significantly reduced the hippocampal volume,enlarged the cerebral ventricle,and reduced the mass of the cerebral ventricle,and PTPα deficiency aggravated hippocampal atrophy.Hippocampus is the core area of the brain to regulate learning and memory,and the damage of hippocampus is directly related to the impairment of learning and memory function.How PTPα is involved in the regulation of hippocampal demyelination,remyelination and its regulatory mechanism are still unknown.Abnormal glutamatergic neurotransmission has been found in the pathological process of multiple sclerosis.N-methyl-d-aspartate receptor(NMDAR)is one of the most important excitatory glutamate ion receptors in the central nervous system.The ion channel protein TRPM4 can endow NMDA receptors with toxicity and plays a central role in neurodegenerative diseases [3].In this study,the effect of PTPα/NMDAR on hippocampus was investigated to explore the molecular mechanism of PTPαsignaling pathway regulating hippocampal demyelination and remyelination.Methods:Healthy SPF-grade male PTPα wild-type and knockout mice were randomly divided into control group,Cuprizone model group and MK-801 treatment group.Experiments were carried out in batches.1.The behavioral effects of PTPα knockout and MK-801 treatment on cuprizoneinduced emotional states were evaluated by open field test and black and white box test(5 mice/group).After the mice completed the behavioral test,specimens were randomly selected from each group for collection.2.PTPα wild type and gene knockout mice in control group,Cuprizone model groups for 1,2,3,and 6 weeks,and 9-week groups(0w,1w,2w,3w,6w,and 9w),which were fed for 6 weeks and then resumed normal diet for 3 weeks.Real-time fluorescent quantitative polymerase chain reaction(q RT-PCR)was used to detect and analyze the m RNA expression of myelin-related proteins,oligodendrocyte progenitor cells,neural stem cells,astrocytes,inflammatory factors,NMDAR2 A and NMDAR2 B in the hippocampus during demyelination and remyelination.The expression of Fyn,NMDAR1,NMDAR2 A and NMDAR2 B m RNA in the hippocampus of PTPα wild type and gene knockout mice in the control group was detected.3.PTPα wild-type and knockout mice in the control group(0w)and Cuprizone model group at 1,2 and 3 weeks(1w,2w and 3w)were selected to detect ATP content in the hippocampus of mice.4.PTPα wild type and knockout mice were selected from the control group(0w)and Cuprizone group at 2 and 3 weeks(2w and 3w).NMDA receptor subunits NR2 A,NR2B and TRPM4 were double stained by immunofluorescence,and their protein expression in the hippocampus was observed by fluorescence section.5.PTPα of Cuprizone model group(1w,3w)and MK-801 treatment group(1w+MK-801,3w+MK-801)were selected the effect of MK-801 on the expression of NMDAR1,NMDAR2 A and NMDAR2 B m RNA in hippocampus of wild-type mice during demyelination was detected by real-time fluorescent quantitative polymerase chain reaction(q RT-PCR).PTPα of control group(0w),Cuprizone model group(1w,3w)and MK-801 treatment group(1w+MK-801,3w+MK-801)were selected the m RNA expressions of myelin-related proteins,oligodendrocyte progenitor cells,neural stem cells,neurons,astrocytes and inflammatory factors in hippocampus were detected in wild-type mice after MK-801 treatment.Results:1.In cuprizone-induced mice,mice treated for the first three weeks showed increased excitability and anxiety compared with control untreated mice,and treated mice showed increased mortality,which were alleviated by PTPα deficiency.Especially at the second week of treatment,PTPα wild-type mice in the Cuprizone model group showed significantly increased mortality compared with PTPα deficient mice.2.In the open field test,compared with the control group,Cuprizone-induced PTPα wild type and knockout PTPα mice increased the total distance and time in the corner area,and decreased the distance and time in the central area of the open field.Compared with PTPα KO mice,PTPα WT mice had longer total regional movement distance,longer central movement distance and longer movement time in open field,but shorter activity time in corner area.In the black and white box experiment,compared with the control group,the mice in the Cuprizone model group spent more time in the dark box,and spent less time in the light box and the number of shuttletimes.3.Cuprizone toxicity induced demyelination in mice,and the demyelination in mouse hippocampus could be regenerated when the mice returned to normal diet.Depletion of PTPα promoted the proliferation of neural stem cells,oligodendrocyte progenitor cells,and astrocytes,as shown by q RT-PCR.4.The expression of NMDAR2A/TRPM4 and NMDAR2B/TRPM4 protein in PTPα wild-type and knockout mice fed with Cuprizone mixed diet for 3 weeks was significantly higher than that in control group.Moreover,the expression of complexes in most hippocampal regions of PTPα wild-type mice was relatively higher than that of PTPα knockout mice.In the ATP detection experiment,the ATP content of PTPα wild type and PTPα knockout mice in Cuprizone model group was significantly lower than that of the control group.And its trend has a certain correlation with the m RNA expression level of inflammatory factors.5.Compared with the Cuprizone model group,MK-801 treatment group did not affect the total distance in the open field,but reduced the activity time and distance in the central area and the activity time in the corner area.Compared with the Cuprizone model group,MK-801 treatment reduced the time spent in the dark box and increased the time spent in the light box.MK-801 at an appropriate concentration can slow down the demyelination induced by Cuprizone in mice.There is a significant correlation between inflammatory factors and the early onset of multiple sclerosis in mouse model.Appropriate concentration of MK-801 can reduce the expression of TNF-α,IL-1β and IL-6.Conclusions:1.During the first three weeks of Cuprizone induction,the mortality of mice was high,and the mortality of PTPα wild-type mice was significantly higher than that of PTPα knockout mice.2.Cuprizone toxicity can affect excitability and anxiety in mice.PTPα knockdown slows excitability.3.The loss of PTPα can reduce the expression of NMDAR/TRPM4.Mitochondrial dysfunction and inflammatory response may contribute to demyelination and even death.4.Administration of the antagonist MK-801 attenuated cuprizone-induced demyelination and alleviated the excitability and anxiety,but reduced the exploratory behavior of the mice.