Apelin-13Improve Cognition And Memory Function Through ABCA1in APP/PS1Mice

Alzheimer disease (AD) is a kind of higher incidence and more destructivedegenerative disease in central nervous system with clinical features of progressivecognition functional disturbance and the decrease of memory ability. Senile plaque (SP)which generated from production and deposition of β-Amyloid protein（Aβ）in the brainplays an important role in the pathological process of AD. Production increase,insufficient degradation mediated by enzyme and clearance reduction from the brain ofAβ is the important reasons causing gather of Aβ. Too much Aβ gather and form intoamyloid deposition and SP, which produces neurotoxicity and induces impairment ofcognitive and memory.ATP binding cassette from the A1(ABCA1) is closely linked with Aβ metabolism.Lack of ABCA1protein can reduce lipid-loaded level of apolipoprotein E (apoE) andincrease of Aβ level in the brain. However, overexpression of ABCA1protein canincrease lipid-loaded level of apoE and reduce the level of Aβ, so that influence theprogress of AD. High expression of ABCA1promotes decrease of Aβ production andincrease of degradation and clearance. ABCA1plays a very important role in Aβmetabolism and gather, so ABCA1can be as an important target for the treatment of AD.Therefore, it is crucial to looking for a kind of material to effectively increase ABCA1expression.Apelin is a kind of peptide hormone with biological activity secreted in adipose cellsand has an extensive histologic distribution in the body. Recent studies have shown thatapelin plays an important role in the protection process of hippocampal neuron. Based onthe important function of hippocampal neurons in the learning and memory, it suggestsapelin can adjust learning and memory. Our previous studies have shown that apelin-13increased ABCA1level in THP-1derived macrophages, but its effect on Aβ metabolismwas unclear. Given the close relationship between the ABCA1protein and Aβmetabolism, we put forward the following hypothesis: apelin-13decreases Aβ deposition through increasing ABCA1protein level and improve cognition and memory function inAD model rat.This research was divided into three parts to verify the hypothesis. In part I we usedapelin-13to treat rat pheochromocytoma cell line (PC-12cell) with the feature ofneuroendocrine cell and further observed whether apelin-13adjusted Aβ metabolism inPC-12cells is by influencing the ABCA1protein level, and then clarified its mechanisms.In part Ⅱ we used apelin-13to treat APP/PS1transgenic mice and further observedwhether apelin-13improved learning and memory function of AD model rats and reducedits Aβ deposition. In part Ⅲwe used ABCA1SiRNA to silence ABCA1expression inbrain tissue of APP/PS1transgenic mouse and observed whether apelin-13improvedlearning and memory function of AD model rats is by influencing ABCA1protein leveland reducing its Aβ deposition. This study provides an experimental basis for theprevention and treatment of AD.Part I Apelin-13increases ABCA1protein level toadjust Aβ metabolism in PC-12cellsObjective: To observe whether adjustment of apelin-13to Aβ metabolism is byinfluencing the ABCA1protein level and clarify its mechanisms.Methods:①Putative receptor protein related to the angiotensin receptor AT1(APJ) was detected by Western blot PC-12cells to determine the receptor of apelin-13existed in PC-12cells.②PC-12cells were treated with different concentrations ofapelin-13(1,10,100nmol/L) for24h and treated with100nmol/L apelin-13withdifferent time (12h,24h,48h), the method of enzyme-linked immunosorbent assay(ELISA) was used to detect Aβ1-40and Aβ1-42, Western blot was used to detect APP,BACE1and NEP protein levels, the kits was used to detect enzyme activity of BACE1and NEP, RT-PCR was used to detect ABCA1mRNA level, Western blot was used todetect ABCA1protein levels, the method of liquid scintillation counting was used todetect intracellular cholesterol efflux, Oil red O staining was used to detect the contents of lipids in the cells.③ABCA1siRNA was used to inhibit ABCA1protein expression inPC-12cells, then PC-12cells were treated with100nmol/L apelin-13for24h, ELISAwas used to detect Aβ1-40and Aβ1-42levels, the kits were used to detect enzyme activity ofBACE1and NEP. Treatment of PC-12cells with ABCA1siRNA is to study the role ofABCA1in the effects of apelin-13on Aβ metabolism in PC-12cells.④PKCαsiRNAwas used to inhibit expression of PKCα, and then100nmol/L apelin was used to treatPC-12cells for24h, the affects of apelin on ABCA1protein levels were detected. PC-12cells were treated with different concentrations (1,10,100nmol/L) of apelin-13for24h,calpain activity was test.⑤PC-12cells were treated with different concentrations (1,10,100nmol/L) of apelin-13for24h and Western blot was used to detect apoE protein levels.ApoE siRNA was used to inhibit expression of apoE and PC-12cells were treated with100nmol/L apelin for24h. Then the impact of apelin on BACE1and the enzyme activityof NEP were detected.Results:①APJ existed in PC-12cells.②Apelin-13significantly reduced Aβ1-40and Aβ1-42levels in PC-12cells, had no significant effect on the APP, BACE1and NEPprotein expression, reduced the enzyme activity of BACE1, increased the enzyme activityof NEP, had no effect on ABCA1mRNA level, increased ABCA1protein levels,promoted cholesterol efflux and reduced the lipid accumulation in the cells.③Afterusing ABCA1siRNA to inhibit ABCA1protein expression in PC-12cells, the loweringaffects of apelin-13on Aβ1-40and Aβ1-42levels significantly reduced, the lowering affectsof apelin-13on the enzyme activity of BACE1and the increasing affects of apelin-13onthe enzyme activity of NEP were significant inhibited.④After PKCαsiRNA was used toinhibit PKCαexpression in PC-12cells, the effects of apelin increasing ABCA1proteinlevels weakened. Apelin-13obviously inhibited calpain activity in PC-12cells. Apelin-13had no significant effect on expression of apoE protein in the PC-12cells.⑤After usingapoE siRNA to suppress apoE expression in PC-12cells, the lowering affects ofapelin-13on the enzyme activity of BACE1and the increasing affects of apelin-13on theenzyme activity of NEP significantly decreased.Conclusion:①Apelin-13increases ABCA1protein level through PKCα pathway in the PC-12 cells.②Apelin-13increases ABCA1protein levels, decreases Aβ production, andincreases Aβ degradation in the PC-12cells.Part Ⅱ Apelin-13improves cognition and memoryfunction in APP/PS1miceObjective: To observe whether apelin-13impact cognition and memory functionand Aβ metabolism in APP/PS1mice.Methods: Lateral ventricle injection with different doses (1,2,4μg) ofapelin-13were used to APP/PS1mice after30days (1time/day), Y maze experiment,new object recognition and Morris water maze experiments were used to test cognitionand memory function in mice.Then mice were killed, ELISA was used to test the Aβ1-40and Aβ1-42levels in hippocampus and prefrontal tissues, Congo red staining was used todetect Aβ deposition in hippocampal tissue, Western blot was used to detect APP, BACE1and NEP protein levels in hippocampus and prefrontal tissues, the kits were used to testenzyme activity of BACE1and NEP in hippocampus and prefrontal tissues, ELISA testwas used to test Aβ1-40and Aβ1-42levels in peripheral blood, Western blot was used todetect ABCA1protein levels of hippocampus and prefrontal tissues.Results: Apelin-13significantly improved accuracy in the Y maze test inAPP/PS1mice, significant increased the cognition to the new object in the new objectrecognition experiments (whether short-term or long-term test) in the APP/PS1mice, andsignificant reduced the average swimming distance and the average latency period inMorris water maze experiment in APP/PS1mice. Apelin-13significantly reduced Aβ1-40and Aβ1-42levels in hippocampus and prefrontal tissues in APP/PS1mice, had nosignificant effect on the APP, BACE1and NEP protein levels, reduced the enzymeactivity of BACE1in hippocampus and prefrontal tissues, increased the enzyme activityof NEP, significantly increased Aβ1-40and Aβ1-42levels in the peripheral blood, andimproved ABCA1protein levels in the hippocampus and prefrontal tissues. Conclusion:①Apelin-13improves cognition and memory function inAPP/PS1mice.②Apelin-13reduces Aβ production, increases Aβ degradation and Aβ clearance inhippocampus and prefrontal tissues in APP/PS1mice.③Apelin-13increases ABCA1protein levels in hippocampus and prefrontaltissues in APP/PS1mice.Part Ⅲ Apelin-13improves memory and cognition functionin APP/PS1mice through ABCA1Objective: To observe whether the impact of apelin-13on the cognition andmemory function and Aβ metabolism in APP/PS1mice is by ABCA1protein.Methods: Lateral ventricle injection with ABCA1siRNA was used to silenceABCA1protein expression in brain in APP/PS1mice, the affects of apelin-13oncognition and memory function and Aβ metabolism in brain in AD model mice wereobserved. Y maze experiment, new object recognition experiment and Morris water mazeexperiment were used to test cognition and memory function in mice. After mice werekilled, ELISA was used to test Aβ1-40and Aβ1-42levels in hippocampus and prefrontaltissues in mice, Congo red staining was used to detect Aβ deposition in hippocampaltissues, the kits were used to test enzyme activity of BACE1and NEP in hippocampusand prefrontal tissues, ELISA was used to test Aβ1-40and Aβ1-42levels in peripheral bloodof mice.Results: After ABCA1expression was silent in the brain tissue, the improvingaffects of apelin-13on cognition and memory function and Aβ deposition in brain inAPP/PS1mice were reduced, inhibition of apelin-13on BACE1activity and promotionof apelin-13on NEP activity decreased in hippocampus and prefrontal tissues in APP/PS1mice. Increase effects of apelin-13on Aβ1-40and Aβ1-42levels in peripheral blood of micedecreased. Conclusion:①Apelin-13improves cognition and memory function by increasing ABCA1protein levels in the APP/PS1mice.②Apelin-13reduces Aβ production, increases Aβ degradation and clearance inhippocampus and prefrontal tissue in APP/PS1mice by increasing ABCA1protein levels.