| BACKGROUND AND AIMFragile X syndrome(FXS)is one of the most common genetic mental disorders,the incidence rate of male is about 1/1200 ~ 1/2500,and female is about 1/1650 ~ 1/5000.Fragile X syndrome is due to over repeated CGG trinucleotide sequence of Fmr1 gene on the patient’s X chromosome,leading to gene inactivation and its encoded FMRP deletion.The m Glu R theory of FXS suggests that the absence of FMRP increases the synthesis of related proteins,thereby leading to the over-internalization of AMPA receptors,making the m Glu R-mediated LTD overactive.Caveolin,a membrane endocytosis molecule,may mediate the AMPAR internalization in the process of m Glu R-LTD,and thereby be involved in the pathogenesis of FXS.In this study,we used multiple experimental methods to explore the mechanism of caveolin-1 mediated AMPA receptor over-internalization of abnormal synaptic plasticity in the FXS,and to further enrich the m Glu R theory and lay the foundation for treatment of FXS.METHODS 1.The wild-type(WT)and Fmr1 knock-out(KO)mouse neurons were cultured,and RT-PCR and western blot were used to observe the expression of caveolin after m Glu R activation,and compare CAV1 expression in WT and KO mice.2.In normal mouse neuronal cells,we detected the effect of m Glu R downstream src kinase on CAV1 expression by administering src kinase inhibitor.3.By using CAV1 overexpression and sh RNA lentivirus in WT and KO neurons to control CAV1 expression,or using dynamin inhibitor to block the internalization process,and isolating the membrane protein,we investigated the AMPA receptor subtype Glu R1 and Glu R2 internalization after activation of m Glu R pathway under different conditions,thus distinguishing the different mechanism of caveolin and clathrin mediated AMPA receptor internalization.4.The CAV1 overexpression virus was injected into the WT mice to enhance the expression of CAV1 in KO mice,and the performance in open field test,water maze test and fear memory test was detected.5.The src kinase inhibitor was injected into KO mice to reduce the expression of CAV1,and observed KO mice performance in the open field,water maze and fear memory tests.RESULTS 1.In WT mice,the gene and protein levels of caveolin were significantly increased by activating m Glu R in vivo and in vitro,and the growth trend decreased with the activation time prolonged,suggesting that CAV1 expression was increased under the activation of m Glu R pathway.2.The increase of CAV1 expression induced by DHPG was significantly inhibited by addition of m Glu R downstream src kinase inhibitor A-419259,and high concentration of A-419259 could inhibit the expression of CAV1 to the level lower than control.It was confirmed that m Glu R1/5 control CAV1 expression through downstream src kinase.3.The expression of CAV1 in KO mice brain was significantly higher than that in WT mice.It suggested that CAV1,which was abnormally overexpressed in KO mice,could be involved in the regulation of abnormal synaptic plasticity.4.In animal behavioral experiments,CAV1 overexpression virus was injected into WT mice to increase CAV1 expression in WT mice and affected the activity of WT mice in open field experiments and to influence their learning and memory in water maze and fear memory experiments.5.In animal behavioral experiments,it was found that treatment of src kinase inhibitor in KO mice could ameliorate the hyperactivity of KO mice and improve the learning and memory of KO mice in water maze and fear memory experimentsCONCLUSIONS The results showed that m Glu R1/5 activation through the downstream src kinase can increase CAV1 expression,and internalize AMPA receptor;however,in the fragile X syndrome,due to loss of FMRP translation inhibition,CAV1 expression was high and overexpressed after m Glu R1/5 activation,which led to excessive internalization of AMPA receptors.This caused abnormalities of m Glu R-LTD,which affected the learning and memory ability of patients with fragile X syndrome. |
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