The Effects Of Xestospongin C, A Reversible InsP3 Receptor Antagonist, Alleviates The Cognitive And Pathological Impairments In Alzheimer’s Disease APP/PS1 Mice

Objective:Alzheimer’s disease（AD）,is a kind of primary,progressive and irreversible neurodegenerative disease,which primarily occurs in the elderly.However,there is no cure against AD at present.Exaggerated calcium signaling is a robust proximal phenotype cause of neural dysfunction in AD.And the perturbation of calcium homeostasis are associated with endoplasmic reticulum（ER）stress,a key process during the adaptation,damage and apoptosis of stressed cells,thereby generating amyloid-β（Aβ）and other pathology of AD.The present study showed for the first time the effects of Xestospongin C（XeC）,a reversible IP₃ receptor antagonist,on the cognitive behavior and pathological features of AD mice.We also tested endoplasmic reticulum stress-related proteins including GRP78,caspase-12,and CHOP in the hippocampus with wstern blotting technique.By using primary cell cutrue,non-invasive micro-test technology（NMT）and calcium imaging,we further investigated the neuroprotective effects of XeC against Aβ-induced neurotoxicity,intracellular calcium overloading and its possible molecular mechanisms.Methods:1.In vivo transgenic mice experimentThe 7-month-old APP/PS1 transgenic mice and wild type C57BL/6 mice were randomly divided into four groups:WT+PBS,APP/PS1+PBS,WT+PBS and APP/PS1+XeC.Then XeC（268 ng/200 ul）or PBS were continuously injected by Alzet osmotic pump for four weeks.Four weeks later,behavioral tests were performed.On the first day,the open field test was used to eliminate the motor exploration ability disorder in mice.On the second day,Y-maze was used to test the short-term working memory of mice by recording the percentage of spontaneous alteration.Finally Morris water maze（MWM）test was performed,lasting for six days.Druing the first five days of place navigation tests,escape latency was recorded to evaluate special learning ability of mice;on the sixth day,probe test was performed to observe the long-term spatial memory ability by calculating the number of platform crossing and the swimming time percentage in the target quadrant;in the end,the visible platform test was carried out to avoid the influence of visual and motor ability on the cognitive function.After behavioral test,mice were sacrificed.Half of them was used in immunohistochemistry（IHC）for testing the distribution and amount of amyloid-βand evaluating the effect of XeC on the pathological impairments of AD.Another half of mice was used in western blotting for determining the expression levels of GRP-78,caspase-12and CHOP in the hippocampi and to clarifying the mechanism of neuroprotection of XeC.2.In vitro primary cell experimentCultured primary hippocampal neurons were divided into groups as follow:Vehicle+PBS,Vehicle+Aβ_1-42,XeC+PBS,XeC+Aβ_1-42,Ni+NP+D-APV+Aβ,EGTA+Aβ,XeC+Dantrolene+Aβ,YM-58483+Aβ,XeC+YM-58483+Aβ.Primary hippocampal neurons were prepared from 1-3 days SD rats,and experiments were performed on 8-d-old cultures.Cells cultured in 6-well plates were used for apoptosis examination with Annexin V-FITC/PI by flow cytometry.Cells cultured in 35 mm dishes were incubated with XeC and other antagonists for observing the changes of intracellular calcium concentration after applying Aβ_1-42-42 using calcium imaging.In addition,the transmembrane calcium fluxs in the cells pretreated with XeC and YM-58483 were also tested by using NMT.Results:1.In open field test,there was no difference in the total distance during 5 min of running among four groups（P>0.05）.As for the percentage of time spent in the central area,mice in the APP/PS1+PBS group were higher than that in the WT+PBS control group（P<0.01）,while XeC treated transgenic mice had short percentage compared with transgenic mice treated with PBS（P<0.05）.2.In Y maze,there was no difference in the number of total arm entries among the four experimental groups（P>0.05）.Compared with the WT+PBS control group,the percentage of spontaneous alteration in the APP/PS1+PBS group was lower（P<0.001）,which was elevated by XeC treatment（P<0.001）.3.In the place navigation tests of MWM,the escape latency of four groups had a on the decline with the increase of the training as the day went on.There were was no significant difference between WT+PBS group and APP/PS1+PBS group in the first training day.However,APP/PS1+PBS group displayed a longer escape latency compared with the WT+PBS groupcontrol group from the second training day（P<0.01 or P<0.001）,while transgenic mice pretreated with XeC manifested a shorter escape latency than APP/PS1mice+PBS from the third training day（P<0.01 or P<0.001）.In the probe test,both the number of platform crossing and the percentage in the target quadrant decreased in the APP/PS1+PBS group compared with WT+PBS group（P<0.001）.Importantly,those two index increased with XeC treated compared to the APP/PS1 mice without XeC.The visible platform test showed that there was no significant difference in escape latency and swimming speed among four groups（P>0.05）.4.In IHC,we found that the distribution of Aβburden in hippocampi of APP/PS1+PBS group was significantly more than those in WT+PBS group（P<0.001）,which was decreased after XeC treatment（P<0.01）.5.In western blotting,compared with WT+PBS group,APP/PS1+PBS group manifested a higher level in the expression of GRP-78、caspase-12 and CHOP（P<0.05 or P<0.01）.Importantly,these increase was suppressed by XeC treatment in APP/PS1 mice（P<0.001 or P<0.01）.6.In flow cytometry,the rate of early apoptosis of neurons was much more high in Aβmodel group than control group（P<0.001）.However,groups pretreated with XeC manifested a lower early apoptosis rate than Aβmodel group（P<0.001）.7.In calcium imaging,XeC pretreatment significantly suppressed Aβ-induced intracellular calcium([Ca²⁺]_i)elevation,either in the speed or amount（P<0.001）.Moreover,XeC,the antagonist of intracellular calcium release channel,showed greater inhibitory effects on the Aβ-induced[Ca²⁺]_i increase compared with co-application of Nifedipine,NP118809 and D-APV（P<0.001）.Furthermore,there was no significant difference between the groups incubated with XeC alone and co-incubated with XeC and Dantrolene（P>0.05）.8.In NMT,we found that XeC pretreatment apparently decreased the speed and amount of Aβ_1-42-induced calcium influx（P<0.01 or P<0.001）.Similarly,pretreatment with YM-58483 also suppressed the Aβ_1-42-induced calcium influx（P<0.01 or P<0.001）,but showed no significant difference compared with the co-pretreated group with XeC and YM-58483.Conclusion:XeC ameliorated the disinhibitory behavior,short-term working memory and long-term spatial learning and memory;XeC also reduced the deposition of Aβin the hippocampus,reduced the level of ER stress associated proteins.The pretreatment of XeC also protected cultured hippocampal neurons against Aβ-induced apoptosis and inhibited Aβ-induced intracellular calcium overload and extracellular calcium influx.We also found that intracellular calcium release had greater contribution in Aβ-induced[Ca²⁺]_i elevation than extracellular calcium entry.The increased calcium entry and the following intracellular calcium release may further exaggerated calcium overload by stimulating the that the IP₃R in the endoplasmic reticulum may be a novel target of AD,and suppression of IP₃R like using XeC could be potentially beneficial in the prevention and treatment of early AD.