| Part One: CUMS-induced LRP1 protein change and depressive-like behaviorObjective: To explore the relationship between LRP1 and depressive-like behavior.Methods: A total of 30 rats were separated into control(CTRL,n = 15),CUMS(CUMS,n = 15).After 1-week adaption,CUMS group were exposed to CUMS for 4weeks,CTRL group were housed normally.The body weight was determined once a week,and behavior tests,including sucrose preference test,forced swimming test and open field test were performed before and after 4-week CUMS.Western blot and immunohistochemical staining analysis was conducted to determine the protein levels of LRP1 in all experimental rats after behavior performance,Pearson correlation analysis was performed for the correlation of LRP1 level and sucrose consumption.Results: Compared with the rats in CTRL group,the rats in CUMS group presented decreased body weight,lower sucrose consumption,less moving distance,lower velocity,decreased frequency of rearing,more immobility time,significantly.Western blot results showed more significantly increased LRP1 levels in CUMS group rats.Pearson correlation analysis showed LRP1 level was negative with sucrose consumption.Immunohistochemical staining analysis showed elevated LRP1 level in hippocampal DG sub-region significantly.Conclusion: CUMS could induce higher LRP1 level in hippocampal DG sub-region and the hippocampal LRP1 level correlates positively with depressive-like behavior.Part Two: Fluoxetine reverses the increased LRP1 levels mediating microtubule instability and depressive-like behaviorObjective: To explore the impacts of fluoxetine on LRP1,mediating microtubule instability and depressive-like behavior.Methods: A total of 50 rats were separated into control(CTRL,n = 12),CUMS(CUMS,n = 10),CUMS + saline(CUMS + S,n = 14),or CUMS+ fluoxetine(CUMS+ F,n = 14)groups.After 1-week adaption,CUMS,CUMS + S and CUMS + F groups were exposed to CUMS for 4 weeks,CUMS + S and CUMS + F groups were treated with saline or fluoxetine for another 4 weeks.CTRL group were housed normally.The body weight was determined once a week,and behavior tests,including sucrose preference test,forced swimming test and open field test were performed before,after 4-week CUMS and after 4-week fluoxetine or saline treatment.Western blot,RT-PCR and immunohistochemical staining analysis were conducted to determine the protein levels of LRP1 in all experimental rats after behavior performance.Western blot were performed to determine the protein levels of tubulin,Acet-tub,Tyr-tub,Tau and p-Tau(262,404).Finally,double-immunofluorescence staining were applied for the co-location of p-tau(404,262)and Acet-tub.Results: Compared with the rats in CTRL group,the rats in CUMS group presented decreased body weight and fluoxetine could not reverse the decrement.Behavior performance presented lower sucrose consumption,less moving distance,lower velocity,decreased frequency of rearing and more immobility time significantly.After fluoxetine treatment,the rats in CUMS group became less anhedonia,increased space exploration,and decreased desperate behavior.More significantly increased LRP1 levels in CUMS group rats,fluoxetine treatment could decrease LRP1 level,especially in hippocampal DG sub-region.The levels of Acet-tub increased following CUMS,accompanied by elevated levels of p-tau(404,262),and fluoxetine treatment could reverse the increments.Tyr-tub levels were not decreased in CUMS group rats significantly.Double-immunofluorescence staining results showed the decreased co-localization of Acet-tub and p-Tau(262,404)and fluoxetine could reverse the decrements.Conclusion: Fluoxetine could reverse the increased LRP1 level in hippocampal DG sub-region induced by CUMS,increase microtubule dynamic and improve the depressive-like behavior.Part Three: LRP1 regulates microtubule instability and depressive-like behavior through PI3K/Akt/GSK-3?Objective: To explore the regulation of LRP1 in depressive-like model rats on microtubule instability and depressive-like behavior via PI3K/Akt/GSK-3?.Methods: A total of 52 rats were separated into control AAV-CON(n = 13),control AAV-sh LRP1(n = 14),CUMS AAV-CON(n = 13),CUMS AAV-sh LRP1(n =14)groups.After 1-week adaption,the rats in AAV-CON group were infused AAV-CON and separated into control and CUMS,the rats in AAV-sh LRP1 group were infused AAV-sh LRP1 and separated into control and CUMS.Two weeks after AAV infusion,CUMS groups were exposed to CUMS for 4 weeks,CTRL group were housed normally.The body weight was determined once a week,and behavior tests,including sucrose preference test,forced swimming test and open field test were performed before,after 4-week CUMS.Western blot analysis was conducted to determine the protein levels of LRP1,tub,Acet-tub,Tyr-tub,Tau and p-Tau(262,404)and PI3K/Akt/GSK-3?.Results: The basal behavior performance in each group did not present difference.The rats in CTRL groups with AAV-sh LRP1 presented decreased LRP1 level,compared with the rats with AAV-CON and rats in CUMS group with AAV-sh LRP1 showed lower LRP1 level.Compared with the rats in CUMS group with AAV-CON,the body weight change in the fourth week in the rats with AAV-sh LRP1 did not present recovery significantly.After 4-week CUMS,the rats in CTRL groups presented no difference in behavior performance,but the rats in CUMS group with AAV-sh LRP1 showed higher sucrose consumption,increased moving distance,higher velocity,increased frequency of rearing and less immobility time significantly,compared with rats with AAV-CON.The levels of Acet-tub and p-Tau(262,404)had no significance in rats in CTRL groups.The rats in CUMS group with AAV-sh LRP1 presented decreased significantly,compared with AAV-CON.To determine the mechanism of the role of LRP1 on microtubule dynamic,our results found that in CTRL groups the levels of PI3K/Akt/GSK-3? had no difference.However,the rats in CUMS group with AAV-sh LRP1 presented higher levels of p-PI3 K and p-Akt and lower levels of p-GSK-3?,compared with the rats with AAV-CON.Conclusion: Low LRP1 levels could regulate microtubule instability and improve depressive-like behavior through PI3K/Akt/GSK-3? axis. |
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