Regulation Of TSG Et Al On APP Splicing And The Intervention On AD Pathology

Part I: Role of TSG in the alternative splicing of APP exon 7Objectives: A characteristic hallmark of Alzheimer’s diseases(AD)is the presence of extracellular senile plaques composed of amyloid beta(Ab),whichis derived from the membrane protein,amyloid precursor protein(APP).In the brain,3 APP isoforms(APP770,APP751 and APP695)are produced by the alternative splicing of APP.In the healthy brain,APP695 is the main form of APP m RNA in neurons.Inclusion of APP exon 7 generates the isoforms containing a Kunitz protease inhibitor(KPI)domain.APP-KPI level in brain is correlated with Ab production.Glycogen synthase kinase 3β(GSK3β)is a proline-directed serine/threonine protein kinase,and is emerging as a key kinase that interacts with many proteins involved in the etiology of AD.We reported recently that truncation of GSK3β markedly increases its activity in AD brain.Here,we studied whether the activation of AKT-GSK3β by tetrahydroxystilbene glucoside(TSG),apart from affecting protein phosphorylation,also affects the regulation of alternative splicing of APP.Methods:(1)APP mini-gene comprising of exon 7 and its flanking exons and introns was successfully constructed by PCR and subcloned into PCI-neo vector.HEK-293 FTand N2a cell lines was transfected with APP mini-gene.The splicing products of APP mini-gene were detected by RT-PCR.(2)We designed primers specific for detecting endogenous and exogenous APP splicing products generated from RT-PCR and real-time PCR.The endogenous APP splicing products from human neuroblastoma SH-SY5 Y cells was examined by RT-PCR.Mouse neuroblastoma N2 a cellswere transfected together with APP mini-gene and GSK3β plasmid to determine the role of GSK3β on APP splicing.(3)In addtion,the role of TSG on AKT-GSK3β pathway was determined by Western blot and PCR.Co-immunoprecipitation and co-localization was applied to detect interaction between GSK3β and ASF.We injected insulin into the cerebral ventricles of 5 months old rats to activate AKT-GSK3β pathway.We then measured the levels of AKT and GSK3β in the rat brain at various time points after injection.(4)TSG was intragastricly adiministrated at a dose of 50mg/kg/day into 5 months’ old APP/PS1 mice for 12 months.The brain tissue was subjected to RT-PCR and Western blots.Results:(1)We observed that the 3 APP isoforms were produced from the minigene in HEK-293 FT and N2 a cells,of which the APP-KPI+ isoforms were at higher levels.Overexpression of GSK3β promoted APP-KPI inclusion,whereas the si RNA of GSK3β had the opposite effect.The suppression of GSK3β by its specific inhibitor Li Cl reduced the APP-KPI+ levels in SH-SY5 Y cells in a dose-dependent manner.(2)APP minigene together with several known splicing factors into HEK-293 FT cells and we observed that ASF promoted APP-KPI inclusion effectively.In addition,We found that GSK3β co-immunoprecipitated with ASF by anti-HA,confirming the interaction between ASF and GSK3β(13)Moreover,we observed that a single transfection of ASF or GSK3β resulted in their localization in the nucleus or soma,respectively.In contrast,the transfection of GSK3β and ASF revealed their co-localization in both the soma and the nucleus.GSK3β overexpression significantly increased ASF’s serine sites’ phosphorylation level,confirming that GSK3β phosphorylates ASF mostly at the serine sites.Moreover,treatment with TSG drastically suppressed ASF phosphorylation by GSK3β(13)(3)TSG slightly decreased the expression of APP-KPI+ in HEK-293 FT cells.Additionally,The incubation of TSG(50,100 and 200 mM)significantly increased the phosphorylation of AKT at serine 473(p S473-AKT)and GSK3β at serine 9(p S9-GSK3β)in SH-SY5 Y cells.We found that the levels of p S473-AKT and p S9-GSK3β significantly increased at 6 h post insulin injection into rat the cerebral ventricles,while level of APP-KPI inclusion was suppressed at 6 h after insulin injection.(4)Intragastric administration of TSG(50 mg/kg)for 12 months increased the phosphorylation level of GSK3β at Serine 9 in APP/PS1 mouse brain.We also determined the APP-KPI+ expression level by PCR and Western blot in APP/PS1 mice and found that both m RNA and protein levels of APP-KPI inclusion were decreased after TSG treatmentConclusion: In this study,we successfully constructed the APP mini-gene.Our data demonstrates the neuroprotective effect of TSG on APP processing.TSG suppressed APP-KPI+ inclusion in vitro and in vivo.The neuroprotective effect of TSG might be through the activation of AKT-GSK3β signaling pathway,followed by attenuation of the splicing activity of ASF,and finally the decrease in APP-KPI+ level.Thus,TSG might be beneficial for AD prevention and treatment.Part II: Regulation of APP exon 7 alternative splicing by Dyrk1AObjectives: The deposition of amyloid β(Aβ)in brains is a common feature in Alzheimer’sdisease(AD).In our preliminary study,we found the kinase activityof dual-specificity tyrosine-phosphorylated and regulated kinase 1A(Dyrk1A)was elevated in AD brains.Dyrk1 A is a SR protein kinase and was alsoreported to regulate tau exon 10 alternative splicing.However,the exact role of Dyrk1 A in amyloid precursor protein(APP)processing remain unclear.Aβ peptide is a cleaved product of APP,whereas the function of APP holoprotein is not yet established.In adult humanbrain,APP gene undergoes an alternative splicing regulation,which leads to thegeneration of 3 isoforms.Among all these isoforms,APP-KPI+ isoforms are reported to be correlated tightly to the Aβ production.Thus,we investigated the role of Dyrk1 A on endogeneous and exogeneous APP-KPI+ expression.Together,we explored the alterations of APP-KPI+ level and learning memory behavior changes of Ts65 Dn mice after inhibition of Dyrk1 A.Methods:(1)In this study,we will use APPmini-gene to mimic APP exon 7 alternative splicing in vivo and detect the changesof splicing pattern when overexpression of Dyrk1 Aor kinase dead form Dyrk1 ADNin N2acell lines by real-time PCR.(2)To evaluate the endogenous APP-KPI+ levels after inhibition of Dyrk1 A,we treated the SH-SY5 Y cells with various concentration of Harmine(2.5/5/10/20/40 mM)and followed by RNA extraction and RT-PCR analysis.Next,we will construct APP770 plasmid and transfected it into HEK-293 FT cells.Together,we will treat the cells with Dyrk1 A inhibitors Harmine or Green tea flavonol epigallocatechin-gallate(EGCG).After 24 h,we will detect the APP-KPI levels by Western blots.(3)The brain tissue of Ts65 Dn is subjected for APP-KPI+ m RNA and protein levels evaluation.Next,to learn whether the inhibition of Dyrk1 A could attenuate the dysregulated APPexon 7 splicing in Ts65 Dn,we adminstrated green tea extracted EGCG to mice in drinking water from gestation(started during initial mating period)to ~5 months old,and then analyzed the levels of APP-KPI+ by Western blots and PCR.We will also detect the behaviorchanges between EGCG feeding group and none feeding group.Results:(1)We found the overexpression of Dyrk1 A promotes APP-KPI+ inclusion in N2 a cells,whereas the kinase dead form Dyrk1 ADN shows no impact on APP splicing.(2)Inhibition of Dyrk1 A with harmine significantly suppressed APP-KPI+ level and promoted APP-KPI-expression,resulting in increases of APP-KPI+/KPI-ratio at m RNA levels in a dose-dependent manner.Both Harmine and EGCG decreased the overexpressed APP770 level in HEK-293 FT cell lines in a dose-dependent manner.(3)Series of Dyrk1 A deletion mutants promotes APP-KPI+ inclusion,especially the Dyrk1 A C-terminal deletions,suggesting the truncation forms of Dyrk1 A displayed higher kinase activities.RT-PCR results revealed the truncations of Dykr1 A impacted the APP splicing.(4)In Ts65 Dn mice brains,we observed impaired spatial learning memory and abnormal general behaviors.After a period of EGCG treatment,we found that the level of APP-KPI+ decreased in EGCG treated Ts65 Dn brain compared with that of EGCG non-feeding groups.In addition,we found that EGCG recued the learning ability of Ts65 Dn.EGCG treated mice spent more time in target quadrant and crossed the target quadrant more times than control littermates,suggesting EGCG attenuates the memory deficit in Ts65 Dn.Moreover,EGCG treated Ts65 Dn stayed less time in open arms than control treated Ts65 Dn,suggesting EGCG has an impact on mice general behavior.Conclusions: In this study,we demonstrated the role of Dyrk1 A in regulating APP splicing.Dyrk1 A promotes APP-KPI+ expression in different cell lines.Inhibiton of Dyrk1 A by its spesific inhibitor Harmine or EGCG suppressed APP-KPI+ level in vitro and in vivo.Administration of EGCG in Ts65 Dn rescued the impaired anxiety and spatial learning memory deficts.This study will help us to better understand themolecular mechanisms of AD early pathogenesis and provide some new evidence forsearching effective intervention strategy.