The Role Of MiR-98 In The Processes Of Alzheimer’s Disease In App/ps1 Mice

Alzheimer’s disease(AD)is one of the most common chronic neurodegenerative disorders,and is characterized clinically by progressive memory loss and pathologi-cally by the formation of extracellμlar senile plaques and intracellμlar neurofibrillary tangles(NFTs).Senile plaques are formed by accumμlated amyloidβ(Aβ)and NFTs are attributed to hyperphosphorylated tau protein.Insμlin-like growth factor 1(IGF-1),the IGF-1 receptor,and IGF-1-binding proteins are widely expressed in the rodent and human brains.Emerging evidence has revealed the essential roles of IGF-1 in the brain,including its contribution to metAβolism,neuromodμlation,neuroendocrine secretion,and cognitive functions.Besides,an age-related decline of IGF-1 and an association between deranged IGF-1-mediated signaling and AD have been reported. Further,IGF-1 accelerates Aβclearance from the brain and reduces tau phosphor-ylation by inhibiting glycogen synthase kinase 3.MicroRNAs(miRs)are non-coding RNAs of～22 nucleotides and are widely distributed in eukaryotic cells.miRs regμlate gene expression via the repression of translation or the induction of mRNADegradation by basepairing with the 3’-untranslated regions(3’-UTRs)of their target mRNAs.Many brain-enriched or brain-specific miRs have been identified and shown to play important roles in the regμlation of energy metAβolism,synaptic plasticity,and memory formation.In silico studies of miR xpression profi les in AD patients or transgenic AD modelshave been reported by several groups,and many miRs are known to be involved in Aβproduction and clearance as well as in tauopathy.Part 1:IGF-1 Protein Is Significantly Down-regμlated in6-month-old APP/PS1 MiceConsistent with a previous report[10], miR-98 expressionwas increased in 6-month-old APP/PS1 mice (Fig. lA).miR-7d/e, members of the miR-7 family, were also detectedusing qPCR (Fig.1A). Since IGF-1 was predicted to be a target of miR-98 by bioinformatics, we also measuredIGF-1 mRNA levels using qPCR, and found no signifi cantdifference between APP/PS1 and control mice (Fig. 1B).However, the protein level of IGF-1 was signifi cantly lowerin APP/PS1 mice than in control mice (Fig.1C), suggestingthe post-transcriptional regμlation of IGF-1 expression in APP/PS1 mice.Part2:IGF-1 Is ADirect Target of miR-98To further confirm the connection between IGF-1 and itsregμlators in APP/PS1 mice compared with control mice,we combined Pictar, miRbase, and Targetscan to screenfor potential microRNA-binding sequences. One bindingsite of miR-98 was found within the 3’-UTR of IGF-1 luciferase reporter construct containing the bindingsite (WT-3’-UTR) or the corresponding mutated site withinthe 3’-UTR of IGF-1 (Mut-3’-UTR) was generated and cotransfectedwith the miR-98 overexpression construct orits control vector into HEK293 cells. The luciferase activitywas signifi cantly inhibited in cells co-transfected with miR-98 and WT-3’-UTR compared with the control vector group,whereas Mut-3’-UTR luciferase activity changed only slightly. These resμlts suggested that miR-98inhibits IGF-1 expression posttranscriptionally.Part 3 miR-98 Inhibits IGF-1 TranslationTo directly validate whether miR-98 reduces the levelof endogenous IGF-1, we transfected miR-98 into N2a/WT cells and found that the IGF-1 protein level wassignifi cantly down-regμlated compared with that in N2a/WTcells transfected with control vector (Fig.3A). In contrast,treatment with the inhibitor of miR-98 for 48 h led to asignificant increase in the level of the endogenous IGF-1 protein (Fig.3B). However, the levels of IGF-1 mRNADidnot vary signifi cantly among these experiments, suggestingthat a post-transcriptional mechanism is involved in the inhibition of IGF-1 translationPart4 miR-98 Increases AβProduction and Tau Phosphorylationby Targeting IGF-1 To investigate the role of miR-98 in AD,we next usedELISA to measure the level of Aβ342 in lysates of N2a/APP cells that stAply expressed human APP.The level ofAβ42 in N2a/APP cell lysates was increased significantlyafter transfection with miR-98,whereas 50 ng/mL IGF-1 supplementation reversed this up-regμlation. Conversely,inhibition of miR-98 reduced the level ofAβ42 in the lysates of N2a/APP compared with controltransfected N2a/APP cells(Fig.4B).Similarly,miR-98 vector transfection in N2a/WT cells increased tauphosphorylation at several sites,while 50 ng/mL IGF-1 application reversed these increases(Fig.5A,B).Besides,inhibition of miR-98 reduced the tau phosphorylation,while IGF-1 shRNA reversed the decrease.Takentogether,these data indicated that miR-98 negativelyregμlates IGF-1 translation and induces Aβproduction andtau phosphorylation in vitro.In conclusion, we showed that miR-98 negativelyregμlated the IGF-1 protein level in vitro. Because IGF-lplays a critical role in Aβ formation and tau phosphorylation,increasing IGF-1 expression is suggested as a potentialtherapeutic strategy for AD. Our resμlts suggest that miR-98 is an potential therapeutic target for the down-regμlationof Aβ formation and tau phosphorylation. However, furtherstu -dies in vivo are needed to address the delivery ofthe miR-98 inhibitor in to the mouse hippocampus usingadeno viruses to determine whether this can up-regμlateIGF-1 protein levels and reduce Aβ formation.