| Experimental Study on the Effect and Mechanism of PPARγAgonist on AβDegrading Enzyme: Insulin-Degrading EnzymeAlzheimer's disease(AD)is the most common cause of dementia in the elderly. Acetylcholinesterase inhibitors(AchEI) have been the main therapeutic approach for years. However, this symptomatic treatment can only benefit patients with mild-moderate efficacy. The abnormal accumulation of Aβin the brain is an early and invariant feature in AD and is believed to play a pivotal role in the etiology and pathogenesis of the disease. Recently, Aβimbalance, thus the overproduction or the dysfunction of degradation or clearance for Aβhas been considered as the underlying cause of Aβaccumulation. As such, reducing Aβgeneration&aggregation, promoting clearance°radation will surely be the promising strategies of AD treatment in the near future. Altering catabolism of Aβis an novel and inexpensive way to reduce Aβburden in the brain. Many proteases or peptidases have been reported with the capability of cleaving Aβ. Insulin-degrading enzyme(IDE)is one of them. Therefore, a possible therapeutic approach for treatment of AD might be a chronic upregulation of IDE. Zhao and his colleague's study indicated that IDE could be a downstream target of insulin signaling pathway. By enhancing CNS insulin signaling, we can rectify IDE deficit or dysfunction. Moreover, converging evidence also showed that insulin resistance or insulin signaling dysfunction itself might progress during ageing and might play a role in AD pathogenesis. Therefore, we hypothesize that: by enhancing insulin signaling pathway, we can not only rectify underlying age-related insulin resistance, but upregulate IDE thus increase Aβmetabolism. To test this hypothesis, we used PPARγagonists as an intervention both in vitro and in vivo, to determine the possibility of upregulating IDE, accelerating Aβdegradation by enhancing insulin signaling pathway.Part 1 PPARγAgonists Upregulate IDE in Primary Cultured Hippocampal Neurons Objectives To investigate the effect of PPARγagonists on IDE expression in vitro, comparing them with insulin. Further, to find out the best intervention drug, optimal dosage and time.Materials and Methods Primary cultured hippocampal neurons, 7-10 days old, were identified with MAP-2 immunofluorescence staining to determine the purity. Then they were treated separately with insulin(INS)2,20,200 nmol/L, pioglitazone(PGZ)0.01,0.1,1μmol/L, rosiglitazone(RSG)0.01,0.1,1μmol/L and PBS 0.01 mol/L for 6h, 12h,24h, respectively. Cell survival rate were assessed by MTT assay. IDE mRNA expression and protein level were determined by RT-PCR and Western blot.Results By MAP-2 identification, the purity of cultural hippocampal neuron had reached 95ï¼…. In comparison with PBS, insulin, PGZ and RSG could all increase the expression of IDE in a dose-dependent manner, both on mRNA and protein level. When treated for 6h, the mRNA expression of IDE was not significantly increased, while the protein level of IDE slightly rose. By 12h, both RT-PCR and Western blot results showed that, all the three interventions could raise IDE expression, also in a dose-dependent manner. Results of 24h were similar with 12h. Among all the groups, RSG 1μmol/L was the most effective.The results of part 1 experiment indicated that PPARγagonists could upregulate IDE expression, which was comparable with or even more potent than insulin. There existed a drug-, dose- and time-dependent manner of the upregulation effect. RSG 1μmol/L, treating 12h was the most optimal intervention.Part 2 Neuroprotection against AβToxicity by Rosiglitazone: Correlation with IDE Objectives To evaluate the protective effect of RSG against Aβtoxicity in primary cultured neurons. And to elucidate the mechanism of the neuroprotection effect.Materials and methods primary cultured hippocampal neuron was treated with Aβ40 5μM, Aβ40 5μM+RSG 1μM, Aβ42 1μM, Aβ42 1μM+RSG1μM, Aβ421μM+RSG1μM+Wortmannin 50nM, Aβ42 1μM+RSG1μM+1, 10phenanthroline10μM, separately for 12h. Morphology changes were recorded. Apoptosis and cell survival rate were determined by Hoechst33258 staining, flow cytometry (FCM) and MTT assay. Protein expression of IDE, pAKT/tAKT, pGSK3β/tGSK3βwere assayed. Aβ40/42 level in cultured medium was obtained by ELISA.Results As shown by Hoechst33258 staining, the apoptosis proportion rose strikingly after Aβ40,Aβ42 treatment whereas RSG reduced them by 31.91ï¼…and 30.79ï¼…(p＜0.05). FCM results showed that apoptotic cell proportion were reduced by 39.15ï¼…and 50.48ï¼…(p＜0.05), while necrotic cell percentage dropped by 35.29ï¼…and 20.30ï¼…(p＜0.05), respectively. MTT assay indicated that RSG raised cell survival rate by 32.89% and 43.91% (p＜0.05). According to Western blot results, after treated with RSG, the IDE level of Aβ340, Aβ342 group rose by 168% (p＜0.05) and 150% (p＜0.05). pAKT/tAKT ratio increased by 123.33% (p＜0.01) and 150% (p＜0.01). pGSK3β/tGSK3βratio increased by 66.67% (p＜0.05) and 45.83% (p＜0.05), respectively. Besides, the Aβlevel in cultured medium decreased from 4.56μM to 3.65μM in Aβ40 group, from 0.90μM to 0.67μM in Aβ42 group (p＜0.05). PI3K inhibitor----wortmannin could abolish RSG's effect on IDE by 50% (p＜0.01), reduce its effect on pAKT by 57% (p＜0.01). And accordingly, apoptosis percentage increased significantly (from 22.03% to 42.15%, increased by 99.13%, p＜0.01). Meanwhile, IDE specific inhibitor----1,10-phenanthroline had little impact on IDE and pAKT level whereas RSG's anti-apoptosis effect was partially lost(apoptotic percentage increased by 57.92%, p＜0.05).The results of Part 2 suggested that RSG could protect neurons from neurotoxicity induced by Aβ40 and Aβ42, which was accomplished by enhancement of insulin signaling pathway, upregulation of IDE and increased Aβdegradation.Part 3 Reversion of Age-related IDE Dysfunction by Rosiglitazone & its Effect on an AD-like Rat ModelObjectives To get a better understanding of age-related alteration of IDE, insulin signaling and to explore RSG's effect on them. To investigate the efficacy of RSG on an Aβ-induced AD-like rat model.Materials and methods Sprague Dawley rats were divided into 6 groups according to different ages and treatments: 3 months old, 8; 12 months old, 12; 18 months old, 12; 18 months old, RSG lmg/kg/d, by gavage, q.d. for 4 months, 12; 18 months old, bilateral intrahippocampal infusion of Aβ42, 12; 18 months old, bilateral intrahippocampal infusion of Aβ42, RSG, 1mg/kg/d, by gavage, q.d. for 4 months, 12. Cognitive function was evaluated by Morris Water Maze. IDE and pAKT expression were assayed by Western-blot and immunofluorescenct staining. IDE activity was obtained by RIA. Aβlevel was assayed by ELISA.Results Cognition decreased progressively with age. 3m rats had the shortest escape latency in Morris Maze (9.70s), while 18m rats had the longest (27.80s). 12m rats were in between (15.50s). RSG administration might attenuate this impairment (14.60s) (p＜0.05, vs 18m rats). In space probing test, the counts of crossing the platform decreased by 22.22 %(p＜0.05) for 12m rats and by 27.78% (p＜0.05) for 18m rats when comparing with 3m rats. RSG increased the counts by 23.08% as compared by 18m rats (p＜0.05). Meanwhile, IDE and pAKT expression also declined with age. The difference between 3m and 12m rats was obvious (p＜0.05) while the gap between 3m and 18m was even more significant, as shown by Western blot. Immunofluorescent staining resembled the above tendency. RSG could rectify the IDE & pAKT decrement significantly (p＜0.05, vs 18m rats). There also existed an age-dependent decline of IDE activity, which dropped sharply in 12m rats (p＜0.01). Chronical administration of RSG could moderately upregulate IDE activity in liver (p＜0.05) and slightly in brain. RSG could also modestly improve cognition in Aβ-induced AD-like rat model, shortening its mean escape latency from 79.40s to 58.51s (by 26.31%, p＜0.05 ), reducing intracerebral Aβlevel from 235.53pM to 187.34 pM (by 20.46%, p＜0.05) as well.The results in Part 3 signified that, during normal ageing, both insulin signaling pathway and IDE expression & activity declined in an age-dependent manner. So did cognition. RSG could reverse this deterioration in some extent. RSG could also attenuate cognitive impairment and reduce Aβburden in an Aβ-induced AD-like rat model.Conclusions1. PPARγagonists could exert neuroprotective effect through upregulating IDE expression and activity, enhancing Aβdegradating, both in vitro and in vivo. Rosiglitazone was a typical example.2. The neuroprotection effect was mainly accomplished by enhancing insulin signaling pathway and upregulating IDE.3. There existed age-dependent insulin signaling impairment and IDE downregulation during normal ageing, which might be partially responsible for cognitive decline and played a role in the pathogenesis of AD. Rosiglitazone could reverse insulin signaling dysfunction and upregulate IDE thus might have potential therapeutic value for AD.
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