Silencing PP2A Inhibitor by Lenti-shRNA Interference Ameliorates Neuropathologies and Memory Deficits in tg2576 Mice

Transcription

1 originl rticle Silencing Inhibitor by Lenti-shRNA Interference Ameliortes Neuropthologies nd Memory Deficits in tg57 Mice Gong-Ping Liu, Wei Wei,, Xin Zhou, Hi-Rong Shi, Xing-Hu Liu, Go-Shng Chi, Xiu-Qing Yo, Ji-Yu Zhng, Ci-Xi Peng, Jun Hu, Xi-Chun Li, Qun Wng, nd Jin-Zhi Wng Deprtment of Pthophysiology, Key Lbortory of the Ministry Eduction of Chin for Neurologicl Disese, Tongji Medicl College, Huzhong University of Science nd Technology, Wuhn, Chin; Present ddress: Deprtment of Pthophysiology, Institute of Brin Reserch, Key Lbortory of Stte Administrtion of Trditionl Chinese Medicine of the People s Republic of Chin, School of Medicine, Jinn University, Gungzhou, Gungdong, Chin Deficits of protein phosphtse-a () ply crucil role in tu hyperphosphoryltion, myloid overproduction, nd synptic suppression of Alzheimer s disese (AD), in which is inctivted by the endogenously incresed inhibitory protein, nmely inhibitor- of ( ). Therefore, in vivo silencing my rescue nd mitigte AD neurodegenertion. By infusion of lentivirus-shrna trgeting (LV-si ) into hippocmpus nd frontl cortex of -month-old tg57 mice, we demonstrted tht expression of LV-si decresed remrkbly the elevted in both mrna nd protein levels. Simultneously, the ctivity ws restored with the mechnisms involving reduction of the inhibitory binding of to ctlytic subunit ( C ), repression of the inhibitory Leu9-demethyltion nd elevtion of C. Silencing induced long-lsting ttenution of myloidogenesis in tg57 mice with inhibition of myloid precursor protein hyperphosphoryltion nd β-secretse ctivity, wheres simultneous inhibition of bolished the ntimyloidogenic effects of silencing. Finlly, silencing could improve lerning nd memory of tg57 mice with preservtion of severl memory-ssocited components. Our dt revel tht trgeting cn efficiently rescue Aβ toxicities nd improve the memory deficits in tg57 mice, suggesting tht could be promising trget for potentil AD therpies. Received 5 April ; ccepted July ; dvnce online publiction September. doi:.8/mt..89 INTRODUCTION Alzheimer s disese (AD) is the most common neurodegenertive disorder, chrcterized pthologiclly with brin ccumultion of numerous neurofibrillry tngles nd senile plques. Since β-myloid (Aβ) nd hyperphosphorylted tu (p-tu) re respectively the core elements of the plques nd tngles, suppressing Aβ nd p-tu ccumultion could be logic strtegy for rresting AD pthologies. Deficit of severl protein phosphtses (PP), such s, PPB, PP, nd PP5, hs been identified in AD neurodegenertion, mong them, is the most implicted: is the most ctive phosphtse in dephosphorylting the hyperphosphorylted tu proteins isolted from the AD brins nd dephosphoryltion restores the biologicl ctivity of tu proteins in promoting microtubule ssembly nd stbilizing microtubules; ccounts for ~7% of the totl tu phosphtse ctivity in humn brin, nd the ctivity of is significntly decresed in the brin of AD ptients. 4 Studies lso show tht inhibition of cuses tu hyperphosphoryltion nd memory deficits in rts. 5, In ddition to tu proteins, n in vitro study showed tht could lso dephosphorylte myloid precursor protein (APP) nd therefore ttenute Aβ production. 7 Furthermore, ctivtion of promotes xonogenesis nd fcilittes synptic plsticity. 8 These dt suggest tht restortion of ctivity my rescue AD pthologies nd the memory-ssocited synptic deficits. To dte, the ctivtor is very limited. It hs been reported tht cermide cn ctivte ; however, the neurotoxicity of cermide hs gretly restricted its clinicl ppliction. 9 Therefore, restitution of my represent new strtegy in rresting AD neurodegenertion. In the brin, the ctivity of is downregulted by its constitutive inhibitory proteins, nmely inhibitor (I ) nd inhibitor of ( ),, both of which inhibit noncompetitively, including C monomer, dimeric, nd trimeric complexes, with the Ki vlues in low nnomolr rnge. is homologue of SET, which ws first identified in ptient with cute leukemi. Lter studies demonstrte tht SET is multifunctionl protein widely expressed in humn nd mouse. SET cn protect histones from cetyltion, regulte G/M trnsition, modulte HuR mrna binding, or ct s trnscription fctor for P45c7 ctivtion. 4 7 In AD brins, the level of trnscripts ws upregulted nd the elevted protein ws coccumulted with neurofibrillry tngles in the cytoplsm. 8,9 A recent study The first three uthors contributed eqully to this work. Correspondence: Jin-Zhi Wng, Deprtment of Pthophysiology, Key Lbortory of Neurologicl Disese of Eduction Ministry of Chin, Tongji Medicl College, Huzhong University of Science nd Technology, Wuhn 4, P. R. Chin. E-mil: wngjz@mils.tjmu.edu.cn Moleculr Therpy vol. no., dec. 47

2 demonstrte tht upregultion of cn reproduce AD-like histopthologies nd memory deficit in rts. These dt suggest tht the elevted plys crucil role in AD neurodegenertion with mechnisms involving inhibition, therefore, downregultion of my rescue nd therefore mitigte AD neurodegenertion. We tested this ide in the present study. We found tht silencing by in vivo lentivirl vector delivery of shrna trgeting remrkbly ttenuted myloidogenesis by ctivtion of in tg57 mice, recognized mouse model of AD. We lso observed tht silencing improved lerning nd memory of the mice with preservtion of severl memory-ssocited components. We propose tht in vivo trgeting is promising strtegy in mitigting AD pthologies nd memory deficits. RESULTS Level of increses ge dependently in hippocmpus nd frontl cortex of tg57 mice with correlted inhibition of Elevtion of with the inctivtion of hs been detected in the AD brins. 8 To testify whether tg57 is suitble model for the current study, we first mesured the ltertions of nd in the mice. An ge-ssocited elevtion of (Supplementry Figure S c) with inctivtion of (Supplementry Figure Sd) ws observed from to months in both hippocmpus nd frontl cortex of the tg57 mice. By Person nlysis, negtive correltion between level nd ctivity ws shown (Supplementry Figure Se,f). We lso nlyzed the protein level of in hippocmpus of the gemtched wild-type littermtes, but the increse ws only detected t months old (Supplementry Figure S,b). Normlly, is minly locted in the nuclei, but it is lrgely trnslocted into the cytoplsm in AD brins. 8 We therefore mesured the cellulr locliztion of. Compred with the ge-mtched control mice, cytoplsmic trnsloction of ws shown in the hippocmpus of tg57 mice mesured t ~.5 months (Supplementry Figure S). These dt indicte tht tg57 cn serve s perfect model for nd studies, nd the elevted/cytoplsmic trnslocted my underlie the inctivtion of in the mice. Expression of LV-si downregultes in hippocmpus nd the frontl cortex of tg57 mice with stimultion of ctivity To silence in vivo, we infused the virus expressing n egfplbeled LV-si or the scrmbled control (LV-ssi ) into the hippocmpus (CA nd CA) nd frontl cortex of tg57 mice t ~ months old nd nlyzed the mice t 4 or weeks fter injection. Robust expression of the LV-si with significnt reduction of protein nd the trnscript ws detected in hippocmpus nd frontl cortex by direct fluorescence imging (Figure ; for lower mgnifiction, see Supplementry Figure S), immunohistochemistry (Figure b), Western blot (Figure c), nd RT-PCR ssys (Figure d). Expression of LV-si restored or further stimulted ctivity in cortex nd hippocmpus of tg57 mice (Figure ) with increse of totl level of C, decrese of binding of to C (Figure b,c), nd decrese of the inhibitory demethyltion of C (Figure d,e), ll of which my underlie the ctivtion of. We lso mesured the level of A nd B, b GFP Merge Figure Expression of LV-si downregultes (). Virus expressing egfp-lbeled LV- (lentivirus) shrna (si) or the scrmbled control (ssi) ( 9 TU/ml) were injected into the hippocmpl CA nd CA nd the frontl cortex of tg57 mice (tg-ssi nd tg-si) or the wild type littermtes (wt-ssi) t ~ months old under stereotxic pprtus ( μl per site). At ~.5 months, the mice were killed for the following mesurements. () The representtive imges show expression of LV-si (green, direct fluorescent imge) nd the silencing efficcy of (red, with ntibody) t hippocmpl dentte gyrus (DG) of ~.5-month mice (n = ). Scle brs: μm. (b) Level of protein incresed in cortex nd hippocmpus of tg57 mice compred with the wt littermtes, nd expression of si reduced the signl mesured by immunohistochemistry stining using ntibody (the inserts show enlrged imges, n = ). (c) Level of protein in hippocmpus of tg57 mice incresed ~.5-fold of the control level nd expression of LV-si reduced protein lmost to the norml level mesured by Western blotting (n = 5). (d) Level of mrna in hippocmpus of tg57 mice incresed ~4.5-fold of the control level nd expression of LV-si prtilly reduced mrna level mesured by RT-PCR (n = 5). P <. versus wt-ssi; P <. versus tg-ssi (men ± SD). c Hippocmpus DMA Reltive intensity d GADPH Reltive intensity 48 vol. no. dec.

3 ctivity.5.5 Hip Cortex WB: IP: WB: IP: C 4 C C Input Input d A B DM- PME C C PPMT DMA DMA b c e g Reltive intensity Reltive intensity.. DM- C C A f B PME PPMT Figure Silencing ctivtes by multiple mechnisms. The mice were treted s described in Figure. () The ctivity of in hippocmpus nd cortex extrcts of ~.5-month tg57 mice decresed nd silencing () by expression of LV-si restored or further stimulted ctivity, mesured by using Serine/Threonine Phosphtse Assy Kit (n = 5). (b c) Silencing reduced the inhibitory binding of with C in hippocmpus of tg57 mice, mesured by immunoprecipittion (IP) nd Western blot (WB) using nti- or nti- C ntibodies s indicted (n = ). (d e) Level of Leu9-demethylted C (DM- C ) incresed in hippocmpus of tg57 mice, nd silencing reduced the inhibitory demethyltion of C with significntly incresed totl level of C, wheres the protein levels A nd B were not chnged, mesured by Western blotting (n = ~5). (f g) Silencing incresed protein level of methyltrnsferse (PPMT) with no chnge of methylesterse (PME) in hippocmpus of tg57 mice mesured by Western blotting (n = 5). P <. versus wt-ssi; P <.5; P <. versus tg-ssi (men ± SD). the regultory subunits, but no significnt chnge ws detected (Figure d). To further verify the fctors leding to the reduced C demethyltion, we mesured methyltrnsferse (PPMT) nd methylesterse (PME) tht regulte the methyltion level of. We found tht protein level of PPMT significntly incresed while the PME ws not chnged by silencing (Figure f,g), suggesting n enhnced methyltion of by silencing. Silencing induces long-lsting ttenution of Aβ toxicities in tg57 mice By Western blot nd ELISA ssy, we observed tht levels of Aβ 4 (7788 ± 788 pg/mg protein) nd Aβ 4 (495 ± 4 pg/mg protein) incresed in ~.5-month tg57 mice compred with the ge-mtched controls (Aβ 4, ± pg/mg protein; Aβ 4, 5. ± pg/mg protein), while silencing reduced the Aβ level (Aβ 4, 4945 ± 7 pg/mg protein; Aβ 4, 47 ± 97 pg/mg protein) (Figure,b). An incresed tu phosphoryltion t multiple AD-relted sites ws lso detected in ~.5-month tg57 mice, nd silencing ttenuted tu hyperphosphoryltion t ps4, ps9 nd tu- epitope (Figure c,d). The modified Bielschowsky s silver stining dt showed tht the expression of LV-si significntly decresed tu nd Aβ ggregtion (Figure e). We detected n ge-dependent Aβ ccumultion mesured t,, 8, nd months in tg57 mice (Supplementry Figure S4). Previous study demonstrted tht the lentivirl shrnamedited gene silencing could be long lsting or even be trnsferred to the next genertion, therefore, we further mesured the long-term effects of silencing on Aβ ccumultion. To this end, the tg57 mice were infused with si t ~ months nd exmined t 8 months. As shown in Figure f h, both totl nd insoluble Aβ were still lower in si -infused tg-mice (totl Aβ 4, 888 ± 7 pg/mg protein, Aβ 4, 594 ± 4 pg/mg protein; insoluble Aβ 4, 79 ± 4 pg/mg protein, Aβ 4, 58 ± pg/mg protein) thn in the tg-ssi mice (totl Aβ 4, ± pg/mg protein, Aβ 4, 857 ± 9 pg/ mg protein; insoluble Aβ 4, ± 78 pg/mg protein, Aβ 4, 8 ± pg/mg protein). At months, both density nd the intensity of Aβ-positive plques were lower in si -expressing mice thn in the ge-mtched ssi -expressing control mice (Figure i k). Of note, the impct of silencing on Aβ ccumultion seems to be correlted with the expression of, s the expression of si ws widely spred in the hippocmpus, leding to the ttenution of Aβ ccumultion in whole hippocmpus (CA, CA, nd DG) (Figure i), wheres the expression of si ws only loclized in the injected re of the cortex, resulting in no chnges of the Aβ pthology in the djcent regions (Supplementry Figure S5). Given the estblished role of neuroinflmmtory processes in AD nd the role of in stimulting strocytes, we detected the inflmmtory response to the LV-siRNA tretment in.5- month tg57 mice by mesuring the expression levels of glil fibrillry cidic protein (GFAP, mrker of strocytes), nd Ib- ( mrker of microgli). No significnt difference of GFAP nd Ib- ws observed between si -infused nd the ssi - infused control mice (Supplementry Figure S). These dt together indicte tht in vivo trgeting by lentivirl vector delivery of si not only mitigtes tu pthologies but lso induces long-lsting ttenution of myloidogenesis. Moleculr Therpy vol. no. dec. 49

4 e Aβ DMA 4G8.5 m b c.5 m d, tu- ps4 Level (pg/mg protein) 5, 5, Silver stining (.5 m) Aβ 4 Aβ 4.5 m pt ps9 tu-5 DMA f Reltive intensity 4 tu- g ps4 pt ps9 h i Aβ ( m) j Insoluble level (pg/mg protein),,, Aβ 4 Aβ 4 8 m Figure Silencing induces long-lsting ttenution of myloidogenesis in tg57 mice. The mice were treted s described in Figure. () The representtive Western blot show n incresed Aβ level in hippocmpl extrcts of ~.5-month tg57 mice infused with the scrmbled si (tg-ssi) compred with the ge-mtched wild-type littermtes (wt-ssi), nd silencing by LV-si (tg-si) reduced Aβ level compred with the tg-ssi controls (n = ). (b) ELISA dt show incresed levels of Aβ 4 nd Aβ 4 in hippocmpl extrcts of tg57 mice nd the reduction of Aβ by silencing. The experiments were repeted for t lest three times with triplictes. (c d) An incresed tu phosphoryltion t multiple AD-relted sites in tg57 mice nd the ttenution by knockdown detected by Western blotting (note tht tu- rects with the unphosphorylted tu, therefore, n incresed immunorection indictes reduced tu phosphoryltion) (n = 5). (e) The representtive silver stining imges show n enhnced intrcellulr ccumultion of the rgyrophilic substnces in hippocmpus nd the cortex of tg57 mice, nd the reduced ccumultion by LV-si. Scle brs: μm (n = ). (f h) At 8 months, LV-si infused (t ~ months) tg57 mice still show lower levels of totl nd insoluble Aβ in hippocmpus thn the LV-ssi-infused control mice. The ELISA ssy ws repeted t lest three times in triplictes. (i k) The representtive imges nd the quntittive nlyses show increse of plque lod in hippocmpus nd cortex of ~-month tg57 mice immunostined with Aβ ntibody, nd ttenution of by knockdown mesured t the virus injection sites. Scle brs: μm (n = ). P <. versus wt-ssi; P <.5; P <. versus tg-ssi (men ± SD).,, Aβ 4 8 m Cortex Hippocmpus CA DG Totl level (pg/mg protein), Aβ 4 Soluble level (pg/mg protein) k % Are covered in cortex % Are covered in hippocmpus..8 Aβ m Aβ 4 Activtion of medites the si -induced ttenution of Aβ ccumultion with mechnisms involving APP dephosphoryltion nd β-secretse inhibition To verify the role of in si -induced ttenution of myloidogenesis, we infused OA into the hippocmpl CA nd CA regions of tg57 mice for 48 hours to inhibit fter expression of si for weeks. Compred with the tg-mice injected with si lone (Aβ 4, 49 ± 45 pg/mg protein, Aβ 4, 47 ± pg/mg protein), simultneous inhibition of by OA bolished the si -induced reduction of Aβ 4 (8 ± pg/mg protein) nd Aβ 4 (48 ± 8 pg/mg protein) in hippocmpus (Figure 4,b). We further confirmed the role of in ttenuting myloidogenesis in N/APP cells. As shown in Figure 4c,d, inhibition of by OA incresed Aβ level, wheres ctivtion of with C-cermide (DES) ttenuted 5 vol. no. dec.

5 b c ctivity Reltive intensity of Aβ Ctr + OA OA OA + DES Level in hippocmpus (pg/mg protein) 5, 5, + OA d e f 5, Aβ DMA Aβ 4 Aβ 4 C DMA ssil sil sil + si C Reltive level in medium.. WB(E) Ctr OA OA + DES ssil sil sil + si C Aβ 4 Aβ 4 Figure 4 Simultneous inhibition of bolishes the si -induced ttenution of Aβ ccumultion. () The mice were first injected s described in Figure. After ~.5 months, okdic cid (OA) ws infused into the hippocmpl CA nd CA (. μmol/l, μl per site), nd inhibition of ctivity by OA ws detected in hippocmpl extrcts by using Serine/Threonine Phosphtse Assy Kit (n = 5 in triplictes). (b) The mice were treted s described in pnel. Levels of Aβ were reduced by expression of LV-si in.5-month tg57 mice, while simultneous inhibition of by OA reversed prtilly the si-induced reduction of Aβ levels mesured by ELISA ssy (n = 5 in triplictes). (c d) N/APP cells treted with 5 nmol/l OA for 4 hours incresed Aβ levels, wheres ctivtor C-cermide (DES) (7 nmol/l) tretment ttenuted the OA-induced Aβ production. The proteins were isolted by ntive Tris-tricine grdient gel electrophoresis (8 %) nd probed by ntibody E. The experiment ws repeted for three times. (e) Silencing incresed C in N/APP cells, wheres simultneous expression of si C for 4 hours downregulted the si-induced overexpression of C mesured by Western blotting. The experiment ws repeted t lest three times. (f) Silencing reduced Aβ levels, wheres simultneous downregultion of C by si C prtilly reversed the si-induced Aβ reduction in the culture medium of N/ APP cells mesured by ELISA. The experiment ws repeted t lest three times with triplictes. P <.5 versus ssi; P <. versus tg-ssi or ssi or Ctr; P <.5; P <. versus tg-si or si or OA (men ± SD). the OA-induced Aβ overproduction. Furthermore, simultneous inhibition of by expression of si C bolished the si -induced reduction of Aβ (Figure 4e,f). These dt strongly suggest tht the ctivtion of medites the si -induced ttenution of Aβ ccumultion. It hs been well chrcterized tht is the most implicted phosphtse in dephosphorylting tu proteins; however, the role of in Aβ production is not fully illustrted. We therefore studied how ctivtion my ttenute Aβ production. First, we mesured the level of APP phosphoryltion t Thr8 (), becuse the phosphoryltion of APP t this site hs been reported to fcilitte Aβ production.,4 Compred with the ge-mtched wild-type littermte, significnt increse of APP phosphoryltion ws observed in tg57 mice (Figure 5). Expression of si -inhibited APP phosphoryltion (Figure 5), which ws bolished by simultneous inhibition of through hippocmpl infusion of OA (Figure 5b). To further verify the role of in APP phosphoryltion, we treted N/APP cells with OA lone or together with DES. As shown in Figure 5c, ctivtion of by DES not only decresed the endogenous, but lso ttenuted the OA-induced APP phosphoryltion. Furthermore, overexpression of wild-type C (wt C ) decresed, wheres downregultion of by si C prtilly reversed the si -induced reduction of APP phosphoryltion (Figure 5d,e). These dt suggest tht restitution of my ttenute Aβ genertion through dephosphorylting APP. Aβ production is lso regulted by α-, β- nd γ-secretses; therefore, we mesured the ltertions of the secretses. The ctivity ssy showed tht only β-secretse but not α- nd γ-secretse ws significntly ctivted in hippocmpus nd the cortex of ~.5-month tg57 mice, nd silencing completely reversed the ctivity of β-secretse (Figure,b). In ddition, upregultion of β-secretse nd the ttenution by si in hippocmpus of.5-month tg57 mice were lso detected by Western blot (Figure c,d) nd RT-PCR nlyses (Figure e,f), wheres inhibition of by brin injection of OA bolished the si -induced inhibition of β-secretse in the mice (Figure g). In N/APP cells, inhibition of by OA ctivted β-secretse, wheres stimultion of by DES or overexpression of wtp- PA C inhibited β-secretse (Figure h,i). These dt suggest tht β-secretse inhibition is lso involved in the si -induced ttenution of Aβ genertion. Silencing improves lerning nd memory with preservtion of severl memory-ssocited components Tg57 mice show lerning nd memory deficits t.5 months compred with the ge-mtched wild-type controls, nd downregultion of remrkbly improved the cognitive functions of these mice s mesured by Morris wter mze test t ~ months (Figure 7,b) nd step-down voidnce test t ~.5 months (Figure 7c,d). The improved cognitive function ws still significnt t 8 months mesured by step-down voidnce test (Figure 7e,f). The role of ctivtion in the improved memory ws evluted by hippocmpl infusion of OA for 48 hours fter expression of si for weeks. We observed unexpectedly tht simultneous inhibition of by OA did not induce Moleculr Therpy vol. no. dec. 5

6 DMA b Reltive intensity 4 8 DMA + OA c DMA d Reltive intensity 9 Ctr OA OA + DES DES C DMA pcdna wt C Ctr OA OA + DES DES e Reltive intensity DMA.. ssil sil sil + sic ssil sil sil + si C Reltive intensity + OA Reltive intensity.8.9 pcdna wt C Figure 5 Activtion of medites the si -induced APP dephosphoryltion t Thr8. () The mice were treted s described in Figure. The levels of totl APP () nd the Thr8-phosphorylted APP () were incresed in hippocmpl extrcts of ~.5 months tg57 mice (tg-ssi) compred with the ge-mtched wild-type littermtes (wt-ssi), both mice were trnsfected with the scrmbled si ; expression of si (tg-si) decresed the phosphoryltion level of APP compred with tg-ssi, mesured by Western blotting (n = ). (b) In tg57 mice, simultneous inhibition of by hippocmpl (CA nd CA) infusion of okdic cid ( μl ech) reversed the si -suppressed APP phosphoryltion (n = ). (c) In N/APP cells, inhibition of by OA incresed APP phosphoryltion compred with the control (Ctr), wheres simultneous ctivtion of by DES ttenuted the OA-induced APP phosphoryltion. The experiments were repeted two times in triplictes. (d) In N/APP cells, upregultion of by overexpression of wild type C (wt C ) reduced the phosphoryltion level of APP. The experiments were repeted two times in triplictes. (e) In N/APP cells, knockdown by expression of si (si) reduced APP phosphoryltion, while simultneous inhibition of by expression of si C reversed the si-suppressed APP phosphoryltion. The experiments were repeted t lest for three times. P <.5; P <. versus wt-ssi or Ctr or ssi; P <.5 versus pcdna; P <. vs tg-ssi or tg-si or si or OA (men ± SD). ny obvious difference when compred with the control group (Figure 7g,h). These dt suggest tht silencing improves the lerning nd memory of tg57 mice nd this effect seems independent of ctivtion. To further explore the mechnisms tht my underlie the improved lerning nd memory of the mice, we mesured the levels of severl memory-relted proteins. Levels of NRA/B, c-fos, totl camp response element-binding protein (CREB), nd the phosphorylted form t Ser (pcreb) were decresed in ~.5- month tg57 mice compred with the wild-type littermtes, nd silencing incresed the levels of NRA/B, synptophysin (Syt), c-fos, tcreb, nd pcreb (Figure 8,b). The level of synpsin- (Syn-) incresed in ~.5-month tg57 mice, nd silencing did not obviously ffect the levels, nd no difference ws observed for PSD95 (Figure 8,b). We lso found tht the dendrite brnches, density of dendritic spine nd the mushroom-like spines decresed significntly in ~.5-month tg57 mice, which were prtilly restored by silencing (Figure 8c h). These dt together indicte tht downregultion of my improve lerning nd memory by preservtion of severl memory-ssocited components in tg57 mice. Since the ttenution effect of si in pthologies nd memory deficits ws still significnt t 8 months of the mice, we detected level nd ctivity t this ge. We found tht decrese of level (Supplementry Figure S7) nd preservtion of ctivity (Supplementry Figure S8) were still significnt t 8 months. These dt further confirm the role of nd in the observed ttenution effects. DISCUSSION is the most ctive phosphtse in dephosphorylting the hyperphosphorylted tu isolted from the AD brins, in which ctivity is significntly inhibited. 4 lso plys role in Aβ production 7 nd synptic functions. 8,5 is constitutive protein inhibitor of., Its level is elevted in the AD brins, 8 nd upregultion of reproduces AD-like histopthology nd memory deficit in rts. These studies indicte tht silencing my ctivte nd therefore improve the pthologies nd cognitive functions of AD. In the present study, we found n ge-dependent elevtion of with subsequent inctivtion of in tg57 mice, in which tu hyperphosphoryltion, Aβ ccumultion nd memory deficit were reported,7 nd confirmed in the current study. Expression of LV-si downregulted nd ctivted with simultneous ttenution of tu hyperphosphoryltion, Aβ ccumultion nd memory deficits. We lso demonstrte tht ctivtion is essentil in ttenuting myloidosis by silencing, wheres the improved cognitive function seems independent of ctivtion but 5 vol. no. dec.

7 Activity (Hip) Reltive intensity..8.8 BACE PS BACE g h i β-secretse ctivity (Hip) + OA b Activity (Cortex) β-secretse ctivity Ctr OA DES PS BACE DMA α β γ α β γ d e f. GADPH..8 c β-secretse ctivity Reltive intensity 4 pcdna wt C Figure Activtion of medites the si -induced inhibition of β-secretse. ( b) The mice were treted s described in Figure, nd then the ctivity of α-, β-, nd γ-secretses in hippocmpus nd cortex extrcts of ~.5 months tg57 mice nd the wild-type littermtes (wt) ws mesured. β-secretse ws ctivted in tg57 mice compred with the wt, nd expression of LV-si (si) inhibited β-secretse. The experiments were repeted t lest two times in triplictes. (c d) The protein level of β-site APP-cleving enzyme (BACE) ws incresed in hippocmpl extrcts of ~.5 months tg57 mice mesured by Western blotting, nd expression of LV-si reduced the protein level of BACE (n = 5). (e f) The mrna level of BACE ws incresed in hippocmpl extrcts of ~.5 months tg57 mice mesured by RT-PCR, nd expression of LV-si reduced the mrna level of BACE (n = 5). (g) Simultneous inhibition of by hippocmpl infusion of OA ( μl ech t CA nd CA) for 48 hours bolished the LV-si-induced inhibition of β-secretse in ~.5 months tg57 mice fter injection of LV-si for weeks (n = ). (h) In N/ APP cells, inhibition of by OA (5 nmol/l) for 4 hours ctivted β-secretse, wheres ctivtion of by DES (7 nmol/l) for 4 hours inhibited β-secretse. The experiments were repeted t lest three times in triplictes. (i) In N/APP cells, overexpression of wild-type C (wt C ) for 4 hours inhibited β-secretse when compred with pcdna vector trnsfected cells. The experiments were repeted t lest three times in duplictes. P <.5; P <. versus wt-ssi or Ctr or pcdna; P <.5; P <. versus tg-ssi or tg-si or OA (men ± SD). b c d Ltency (s) (d) Opp AL Trg AR STM LTM STM LTM months months.5 months.5 months Qudrnt time (s) e f g h Error/ min 9 STM LTM 8 months Ltency (s) STM LTM 8 months + OA + OA Figure 7 Silencing improves lerning nd memory independent of ctivtion in tg57 mice. () Tg57 mice (tg) t ~ months hd sptil lerning deficit compred with the wild-type littermtes (wt), nd silencing () improved the lerning bility mesured by the ltency to find the hidden pltform during 7 dys wter mze trining (n = 5 ech group). (b) At ~ months, the tg-mice showed sptil memory deficits nd silencing improved the memory bility mesured by the time spent in ech qudrnt fter removed pltform t dy 9 (OPP, opposite qudrnt; AL, djcent left qudrnt; Trg, trget qudrnt; AR, djcent right qudrnt; n = 5 ech group). (c d) At ~.5 months, the tg-mice showed deficits of short-term memory (STM) (mesured t 5 minutes fter trining) nd long-term memory (LTM) (mesured t 4 hours fter trining) by step-down voidnce test, nd knockdown improved the memory (n = 5 for ech group). (e f) At ~8 months, the tg-mice showed deficits of STM nd LTM mesured by step-down voidnce test, nd knockdown improved the memory (n = 8 ech group). (g h) Simultneous inhibition of by hippocmpl (CA nd CA) infusion of OA fter expression of LV-si for weeks did not significntly ffect the si-induced improvement of STM nd LTM mesured t ~.5 months (n = 7 ech group). P <.5; P <. versus wt-ssi; P <.5; P <. versus tg-ssi (men ± SD). Error/ min Error/ min 9 STM LTM.5 months Ltency (s) Ltency (s) STM LTM.5 months Moleculr Therpy vol. no. dec. 5

8 PSD95 NRA/B pcreb c d f Syn- Syt c-fos CREB DMA Reltive intensity b Figure 8 Silencing preserves severl memory-ssocited components. ( b) Levels of NRA/B, c-fos, CREB, nd pcreb were reduced in ~.5 months tg57 mice, wheres knockdown () restored the levels of the memory-ssocited proteins nlyzed by Western blot (n = 5). (c e) The representtive imges showing decresed dendrite brnch numbers in hippocmpl DG region of ~.5-month tg57 mice, nd preservtion of the dendrite complexity by knockdown. (c) Dendritic rbors shown by Golgi stin. (d) Reconstruction of the dendritic rbors. (e) Quntittive nlysis of dendrite brnch numbers. (f h) The totl spine number nd the mushroom-shped spines were decresed in ~.5-month tg57 mice, nd knockdown preserved spine plsticity nlyzed by Golgi stin nd NeuronStudio system. Scle brs: μm. P <.5; P <. versus wt-ssi; P <.5; P <. versus tg-ssi (men ± SD). involves preservtion of severl synpse/memory-ssocited components. These findings indicte tht trgeting by LV-shRNA could be promising strtegy in rescuing AD, nd the mechnistic studies provide new insights on how regultes, nd how medites the effects of on myloid metbolisms nd the cognition-relted mchineries. Though it is currently not fully understood how my interct with nd therefore inhibit its ctivity, it is known tht the biologicl ctivity of is regulted by the totl level of ctlytic subunit ( C ) (the ctivity-executing subunit), nd its inhibitory phosphoryltion t Tyr7 nd demethyltion t Leu9. 8 Our dt showed tht silencing stimulted ctivity through mechnisms involving increse of protein level of C nd decrese of Leu9-demethyltion, nd reduction 4 PSD95 Stubby Thin Syn- NRA/B e g h Mushroom shped No. of dendritic brnches Spine density/μm Mushroom spine % Syt c-fos pcreb CREB of the binding level of C with. In line with our results, recent study lso reported tht silencing results in ctivtion of due to reduced binding of C with. Our results show tht regultes ctivity by multiple pthwys, involving gene expression, direct interction nd modulting the upstrem regultory fctors, such s methyltrnsferse. Of note, we lso noticed tht downregultion of t hippocmpus not only restored but even further stimulted ctivity. Given tht the ctivity of is significntly decresed in the AD brins, silencing could be plusible strtegy to rescue. It is well documented tht is the most implicted phosphtse in dephosphorylting the hyperphosphorylted tu, wheres much less is known regrding the role of in Aβ-relted myloidosis. Aβ is produced from APP by β- nd γ-secretse clevge pthwy. Under physiologicl conditions, APP is lrgely metbolized through α-secretse pthwy, by which only low level of Aβ is produced. Mny fctors, such s ctivtion of β- or/nd γ-secretse, cn stimulte Aβ production.,4 In vivo studies strongly suggest tht APP phosphoryltion fcilittes Aβ genertion through β-secretse pthwy, s in the AD brins nd its trnsgenic mouse models, ll Aβ plques contin pt8-app or β-secretse, nd the production of Aβ is significntly reduced when Thr8-phosphoryltion is either bolished by muttion or inhibited with kinse inhibitors.,4 On the other hnd, recent in vitro study indictes tht Thr8-phosphoryltion of APP does not ffect the C-terminl γ-secretse clevge. 5 Our current dt show tht the ctivtion of by silencing decreses APP phosphoryltion with remrkble reduction of Aβ, wheres simultneous inhibition of increses APP phosphoryltion with ccumultion of Aβ; we lso demonstrte tht silencing suppresses β-secretse without ffecting γ-secretse. These dt together indicte tht phosphoryltion of APP t Thr8 my fcilitte the β-secretse but not γ-secretse clevge of APP to increse Aβ genertion. Since OA my lso inhibit PP, PP4 nd PP5, we used different pproches, including expression of wt C or si C plsmids nd specific ctivity ssy to confirm the role of in mediting the effects of silencing. These dt strongly suggest tht stimultion of by silencing cn efficiently inhibit myloidogenesis through inhibiting APP phosphoryltion nd β-secretse ctivity. We lso noticed tht the effects of inhibition on tu dephosphoryltion were not s prominent s expected in tg57 mice. It could be becuse inhibition ffects other protein phosphtses (such s PPB, PP, nd PP5), nd s well s tu kinses, which lso ffect tu phosphoryltion. Given the more pronounced effects of si on Aβ deposition nd memory impirment thn tu phosphoryltion, it implies tht Aβ my ply more criticl role thn tu hyperphosphoryltion in tg57 mice. We lso observed tht the effects of inhibition were much more drstic on Aβ 4 thn Aβ 4 levels. Of note, silencing did not ffect γ-secretse, wheres it reduced phosphoryltion of APP t Thr8. Consistent with our results, previous studies lso observed tht phosphoryltion of APP led to more prominent elevtion of Aβ 4 thn Aβ 4. 7,8 On the bsis of these dt, we propose tht the phosphoryltion sttus of APP t Thr vol. no. dec.

9 my chnge the conformtion of APP nd therefore differentilly ffect its clevge t the C-terminl. The tg57 mice used in the present study show memory deficits t ~ months old onwrd, which is consistent with the previous reports.,7 We therefore injected the mice t months nd did the behviorl tests t.5 months. We found tht downregultion of remrkbly improved the lerning nd memory with ttenution of tu nd Aβ pthologies, nd more impressively, the improvement ws still significnt when the mice were rered to old ges. The long-term efficiency of lentivirl vector-medited gene trgeting ws lso observed in previous studies. Unexpectedly, we observed tht the cognitive improvement by silencing seemed independent of ctivtion. We found tht knockdown incresed the levels of NRA/B, c-fos, synptophysin, totl, nd the phosphorylted CREB. Since cn lso regulte gene repliction, 9 core histone cetyltion, 7,4 nd protein trnscription; 4 these functions of my contribute to the ltertions of the observed synpse- or memory-ssocited proteins, which deserves further studies. In ddition, silencing lso incresed the dendritic complexity with incresed spine density nd mushroom-like spines. The role of neuronl dendrites is to receive nd process synptic inputs; recent study demonstrtes tht rpid spine stbiliztion nd synptic enhncement ply n importnt role t the onset of behviorl lerning. 4 We believe tht the influence of on the memory-ssocited proteins nd/or dendritic morphology t lest contribute to the improved synptic plsticity nd cognition. Tken together, we demonstrte here tht silencing the elevted by in vivo lentivirl vector delivery ttenutes tu pthologies nd myloidogenesis by restortion of ctivity in tg57 mice; it lso improves lerning nd memory with the mechnisms involving preservtion of severl memory-ssocited components. Therefore, could be promising trget for AD. MATERIALS AND METHODS Production nd brin infusion of lentivirl constructs. The -shrna sequence ws selected by referring previous study, 4 nd it ws cloned into LentiLox.7. The egfp sequence ws driven by cytomeglovirus promoter nd terminted using polydenyltion signl in the long terminl repet. The third genertion pckging systems ws used for lentivirl production. These vectors include: pmdlg/prre (gg/pol elements), prsv-rev, nd pmd.g (env elements). The egfp-lbeled lentivirl - shrna (LV-si ) nd the scrmbled control (LV-ssi ) were constructed ccording to the stndrd procedure. The recombinnt lentivirus ws produced by trnsient trnsfection of HEK9T cells using the clcium phosphte method; the virus ws hrvested t 48 nd 7 hours posttrnsfection nd purified by centrifugtion t 4 C. The titer of the virus ws 9 TU/ml. The in vivo knock-down efficiency ws mesured by immunofluorescence stining, Western blotting, nd RT-PCR fter injection of LV-si into the mouse brins. For brin injections, ~-month-old tg57 mice hrboring the humn myloid precursor protein 95 with Swedish double muttion (APP95; K7N/M7L, Jckson Lb) were positioned respectively in stereotxic instrument, then μl LV-si (tg-si ) or LV-ssi (tg-ssi ) were unilterlly injected into the hippocmpus CA (AP -., ML -.5, DV -.), CA (AP -., ML -.8, DV -.) nd the frontl cortex (AP -., ML -., DV -.5) t rte of.5 μl/min. To receive comprble result, the ge-mtched wild-type littermtes lso received infusion of ssi (wt-ssi ). The syringe ws left in plce for ~ minutes before being withdrwn. The genotype of the mice ws ssessed by PCR from til clip digestions. All mice were kept t 4 ± C on dily hours light-drk cycles with d libitum ccess to food nd wter. The triple sites injection did not significntly increse the deth rte or chnge the norml ctivity of the mice compred with the noninjected controls. The expression of the lentivirl vectors in hippocmpus ws detected in CA, CA, nd DG regions, wheres the expression in the frontl cortex ws only detected in the injected site. Therefore, we used the whole hippocmpus nd the injected site of the cortex, respectively, for the biochemicl mesurements. The niml experiments were crried out ccording to the Policies on the Use of Animls nd Humns in Neuroscience Reserch pproved by the Society for Neuroscience in 995; nd supervised by Animl Administrtion nd Ethics Committee of Tongji Medicl College, Huzhong University of Science nd Technology. Behviorl procedures. Four weeks fter brin infusion of the lentivirl vectors, the sptil lerning nd memory were ssessed by Morris wter mze test. 44 For sptil lerning, mice were trined in wter mze to find hidden pltform for 7 consecutive dys, three trils per dy with -second intervl from 4: to : pm. On ech tril, the mice strted from one of the four qudrnts fcing the wll of the pool nd ended when the niml climbed on the pltform. If the mice did not locte the pltform in seconds, they were guided to the pltform. The swimming pth nd the time used to find the pltform (ltency) or to pss through the previous pltform qudrnt were recorded ech dy by video cmer fixed to the ceiling,.5 meters from the wter surfce. The cmer ws connected to digitl-trcking device ttched to n IBM computer. The sptil memory ws tested dys (dy 9) fter the lst trining. The longer mouse styed in the previous pltform-locted qudrnt, the better it scored the sptil memory. The step-down voidnce test ws performed 7 dys fter the Morris wter mze test t ~.5 months or when the mice were rered to ~8 months by following previous procedure. 45 Briefly, the mouse ws kept in the cge for minutes to dpt to the environment before experiments, then the mice received trining by delivering electricl shock ( v) for minutes (the mice will step up onto the pltform). The short-term memory (STM) nd long-term memory (LTM) were tested respectively t 5 minutes nd 4 hours fter the trining by mesuring the step-down ltency nd the errors mde in minutes. Cell culture nd trnsfection. N cells with stble expression of APP95 (N/APP) (kind gift of Dr. Xu, Burnhm Institute, SD) were cultured in : mixture of DMEM nd OPTI-MEM supplemented with 5% FBS nd U/ml penicillin/ mg/ml streptomycin in humidified tmosphere of 5% CO in ir t 7 C. The cells were plted onto six-well pltes overnight nd the plsmids were trnsfected the next dy using Lipofectmine ccording to the mnufcturer s instruction (Invitrogen, CA). Rel time PCR. Totl RNA ( μg in 5 μl), isolted using Trizol (Invitrogen, CA), ws reversely trnscribed nd the produced cdna ( μl) ws mesured by rel time PCR. The primers for : 5 -CTTCAACTCTGGTCAAATAATGCA-, 5 -GAACAAAAATATAAC AAACTCCGC- ; for β-site APP cleving enzyme (BACE): 5 -CGG-GA GTGGTATTATGAAGTG-, 5 -AGGATGGTGATGCGGAAG- ; nd for glycerldehyde phosphte dehydrogense (GAPDH): 5 -GAAGGTGAA GGTCGGAGTC-, 5 -GAAGATGGTGATGGGATTTC-. Western blot. The hippocmpi were rpidly removed nd homogenized t 4 C using Teflon glss homogenizer in 5 mmol/l Tris-HCl, ph 7.4, 5 mmol/l NCl, mmol/l NF, mmol/l N VO 4, 5 mmol/l EDTA, mmol/l benzmidine, nd mmol/l phenylmethylsulfonyl fluoride. The extrct ws mixed with smple buffer (:, v/v) contining mmol/l Tris-HCl, ph 7., 8% SDS, 4% glycerol, 4 mmol/l dithiothreitol, boiled for minutes, nd then centrifuged t,g for minutes t 5 C. The superntnt ws stored t 8 C for Western blot nlysis. The protein concentrtion in the superntnt ws estimted by bicinchoninic cid Moleculr Therpy vol. no. dec. 55

10 (BCA) kit ccording to mnufcturer s instructions. For generl protein nlyses, % SDS-polycrylmide gel electrophoresis ws used to isolte the protein nd nitrocellulose membrne ws used for trnsfer ( ma for hours followed by 7 ma for hour). For nlyzing Aβ, the proteins were seprted on 8 % Tris-tricine grdient gel nd trnsferred to. μm PVDF membrnes (5 ma for 4 hours followed by ma for hour). The membrnes were blocked with 5% nonft milk dissolved in TBS Tween- (5 mmol/l Tris HCl, ph 7., 5 mmol/l NCl,.% Tween-) for hour nd probed with primry ntibody t 4 C for overnight. Then, the blots were incubted with ntimouse or nti-rbbit IgG conjugted to horserdish peroxidse (:5,) for hour t 7 C, nd visulized with enhnced chemiluminescence. The blots were quntittively nlyzed by Kodk Digitl Science D softwre (Estmn Kodk, New Hven, CT). The ntibody used in the current study is listed in Supplementry Tble S. ELISA, immunocytochemistry nd quntittive nlysis. The Aβ levels were mesured using Signl Select β-amyloid ELISA Kits (Biosource, NY). Immunohistochemicl studies were performed s described previously, 4 nd immunofluorescence imging ws crried out using lser scnning confocl microscope (Olympus FV5, Tokyo, Jpn). The quntittive nlysis of Aβ deposits ws performed s described by Oddo et l. 47 Briefly, brin sections were stined with monoclonl nti-β-myloid ntibody, which detects Aβ 4 nd Aβ 4. The imges of stined sections were cptured using n Olympus Provis AX8 microscope (B&B Microscopes, Phildelphi, PA) nd imported into Imge-Pro Plus. softwre (Medi Cybernetics, Bethesd, MD). The re covered with Aβ deposits in the virus-injected regions of the hippocmpus or cortex ws mesured. Three to five coronl sections were nlyzed per mouse, nd the verge rtios were used to clculte group mens. nd secretse ctivity ssy. ctivity ws mesured using Serine/Threonine Phosphtse Assy Kit by following the mnufcturer s instructions, which cn specificlly mesure but not the other protein phosphtses through selection of specific substrte nd lterntive buffer systems (Promeg, MA). The bsorbnce ws red t nm (BioTek Instruments, VT). The ctivity of α-, β- or γ-secretse ws estimted using α-, β- or γ-secretse Activity Kits by following the mnufcturer s instructions (R&D Systems, MN) nd the bsorbnce ws red t excittion 55 nm nd emission 5 nm (BioTek Instruments, VT). Golgi impregntion nd dendritic morphology nlysis. For Golgi stin, the mice were nesthetized nd then perfused trnscrdilly with 4% prformldehyde, nd brin tissue ws processed s described. 48 Individul sections were incubted overnight t room temperture in wter solution of.5% K Cr O 7 nd.4% OsO 4. The sections were then sndwiched in two glss slides nd incubted in % AgNO (q) for 5 hours t room temperture in drk. Then the slide ssemblies were dismntled in wter nd the sections were mounted on gel-coted slides (.5% porcine geltin), dehydrted in series of grded ethnol rinses, clered with Histocler, nd cover slipped with cytosel. The imges were tken using Olympus BX (Tokyo, Jpn). To nlyze the dendritic morphology, Z-stcks (step size μm) from 5 to 7 neurons were generted using confocl microscope (LSM5, Zeiss) in bright-field mode ( objective) nd reconstructed in ImgePro in combintion with the NeuroDrw toolbox for ech niml. Totl number of brnch points ws nlyzed for every neuron. In ddition, the spine density ws determined in two segments of dendrites t distnce of 9 μm (proximl) nd 9 μm (distl) from the som. To cquire imges for spine nlysis, the dendritic segments were imged under bright-field illumintion on Zeiss Axioimger microscope with oil immersion objective, nd spine morphology ws nlyzed ccording to previously reported method, 49 which does not ssess spine density in three dimensionl mnner but focuses on spines prlleled to the plne of section. Although the method my underestimte the totl number of spines, it fcilittes direct comprison of tretment groups when they re nlyzed in n identicl mnner. Imge J softwre ws used to clculte liner spine density, 5 which ws presented s the number of spines per mm of dendrite length. On the bsis of morphology, spines were clssified into the following ctegories: (i) thin: spines with long neck nd visible smll hed; (ii) mushroom: big spines with well-defined neck nd very voluminous hed; nd (iii) stubby: very short spines without distinguishble neck nd stubby ppernce. The rtio of mushroom spine ws clculted. Dt from 5 to 7 neurons were verged per niml nd used in further sttisticl nlysis. Sttisticl nlysis. Dt were nlyzed with SPSS. sttisticl softwre. The one-wy nlysis of vrince procedure followed by lest significnt difference post hoc tests ws used to determine the sttisticl significnce of the mens. SUPPLEMENTARY MATERIAL Figure S. The ge-dependent ltertions of nd ctivity in tg57 mice nd the wild-type littermtes. Figure S. Altertion of the cytoplsmic trnsloction of in the hippocmpus of.5-month tg57 mice. Figure S. The direct fluorescence imges fter hippocmpl (CA nd CA) nd frontl cortex infusion of LV-si for weeks. Figure S4. An ge-dependent ltertion of Aβ ccumultion in tg57 mice. Figure S5. The representtive imges show plque lod in the cortex of ~ months tg57 mice djcent to the injection site. Figure S. Mesurement of glil fibrillry cidic protein (GFAP, mrker of strocytes) nd Ib- ( mrker of microgli) by Western blotting. Figure S7. Altertions of in hippocmpus of tg57 mice mesured t 8 months fter infused with LV-si or the scrmbled control t months. Figure S8. Altertion of ctivity in hippocmpus of tg57 mice mesured t 8 months fter infused with LV-si or the scrmbled control t months. Tble S. Antibodies employed in the study. ACKNOWLEDGMENTS This work ws supported by grnts from the Ntionl Nturl Science Foundtion of Chin (8748 nd 95), Alzheimer s Assocition (IIRG-9-4), nd FIRCA grnt (R TW8744- A). The uthors declre no conflict of interest. REFERENCES. Brk, H nd Brk, E (99). Neuropthologicl stgeing of Alzheimer-relted chnges. Act Neuropthol 8: Wng, JZ, Gong, CX, Zidi, T, Grundke-Iqbl, I nd Iqbl, K (995). Dephosphoryltion of Alzheimer pired helicl filments by protein phosphtse-a nd -B. J Biol Chem 7: Liu, F, Grundke-Iqbl, I, Iqbl, K nd Gong, CX (5). Contributions of protein phosphtses PP,, PPB nd PP5 to the regultion of tu phosphoryltion. Eur J Neurosci : Gong, CX, Shikh, S, Wng, JZ, Zidi, T, Grundke-Iqbl, I nd Iqbl, K (995). Phosphtse ctivity towrd bnormlly phosphorylted tu: decrese in Alzheimer disese brin. J Neurochem 5: Sun, L, Liu, SY, Zhou, XW, Wng, XC, Liu, R, Wng, Q et l. (). Inhibition of protein phosphtse A- nd protein phosphtse -induced tu hyperphosphoryltion nd impirment of sptil memory retention in rts. Neuroscience 8: Tin, Q, Lin, ZQ, Wng, XC, Chen, J, Wng, Q, Gong, CX et l. (4). Injection of okdic cid into the meynert nucleus bslis of rt brin induces decresed cetylcholine level nd sptil memory deficit. Neuroscience : Sontg, E, Nunbhkdi-Crig, V, Sontg, JM, Diz-Arrsti, R, Ogris, E, Dyl, S et l. (7). Protein phosphtse A methyltrnsferse links homocysteine metbolism with tu nd myloid precursor protein regultion. J Neurosci 7: Zhu, LQ, Zheng, HY, Peng, CX, Liu, D, Li, HL, Wng, Q et l. (). Protein phosphtse A fcilittes xonogenesis by dephosphorylting CRMP. J Neurosci : Sentelle, RD, Senkl, CE, Jing, W, Ponnusmy, S, Gencer, S, Selvm, SP et l. (). Cermide trgets utophgosomes to mitochondri nd induces lethl mitophgy. Nt Chem Biol 8: Li, M, Guo, H nd Dmuni, Z (995). Purifiction nd chrcteriztion of two potent het-stble protein inhibitors of protein phosphtse A from bovine kidney. Biochemistry 4: vol. no. dec.

11 . Li, M nd Dmuni, Z (998). I nd. Two potent protein phosphtse A-specific inhibitor proteins. Methods Mol Biol 9: 59.. Li, M, Mkkinje, A nd Dmuni, Z (99). The myeloid leukemi-ssocited protein SET is potent inhibitor of protein phosphtse A. J Biol Chem 7: 59.. von Lindern, M, vn Bl, S, Wiegnt, J, Rp, A, Hgemeijer, A nd Grosveld, G (99). Cn, puttive oncogene ssocited with myeloid leukemogenesis, my be ctivted by fusion of its hlf to different genes: chrcteriztion of the set gene. Mol Cell Biol : Brennn, CM, Gllouzi, IE nd Steitz, JA (). Protein lignds to HuR modulte its interction with trget mrnas in vivo. J Cell Biol 5: Cnel, N, Rodriguez-Vilrrupl, A, Estnyol, JM, Diz, C, Pujol, MJ, Agell, N et l. (). The SET protein regultes G/M trnsition by modulting cyclin B-cyclindependent kinse ctivity. J Biol Chem 78: Compgnone, NA, Zhng, P, Vigne, JL nd Mellon, SH (). Novel role for the nucler phosphoprotein SET in trnscriptionl ctivtion of P45c7 nd initition of neurosteroidogenesis. Mol Endocrinol 4: Seo, SB, McNmr, P, Heo, S, Turner, A, Lne, WS nd Chkrvrti, D (). Regultion of histone cetyltion nd trnscription by INHAT, humn cellulr complex contining the set oncoprotein. Cell 4: Tnimuki, H, Grundke-Iqbl, I nd Iqbl, K (5). Up-regultion of inhibitors of protein phosphtse-a in Alzheimer s disese. Am J Pthol : Tsujio, I, Zidi, T, Xu, J, Kotul, L, Grundke-Iqbl, I nd Iqbl, K (5). Inhibitors of protein phosphtse-a from humn brin structures, immunocytologicl locliztion nd ctivities towrds dephosphoryltion of the Alzheimer type hyperphosphorylted tu. FEBS Lett 579: 7.. Wng, X, Blnchrd, J, Kohlbrenner, E, Clement, N, Linden, RM, Rdu, A et l. (). The crboxy-terminl frgment of inhibitor- of protein phosphtse-a induces Alzheimer disese pthology nd cognitive impirment. FASEB J 4: Mäkinen, PI, Koponen, JK, Kärkkäinen, AM, Mlm, TM, Pulkkinen, KH, Koistinho, J et l. (). Stble RNA interference: comprison of U nd H promoters in endothelil cells nd in mouse brin. J Gene Med 8: Liu, XP, Zheng, HY, Qu, M, Zhng, Y, Co, FY, Wng, Q et l. (). Upregultion of strocytes protein phosphtse-a stimultes strocytes migrtion vi inhibiting p8 MAPK in tg57 mice. Gli : Shin, RW, Ogino, K, Shimbuku, A, Tki, T, Nkshim, H, Ishihr, T et l. (7). Amyloid precursor protein cytoplsmic domin with phospho-thr8 ccumultes in Alzheimer s disese nd its trnsgenic models: role to medite interction of Abet nd tu. Act Neuropthol : Lee, MS, Ko, SC, Lemere, CA, Xi, W, Tseng, HC, Zhou, Y et l. (). APP processing is regulted by cytoplsmic phosphoryltion. J Cell Biol : Jouvenceu, A, Billrd, JM, Hditsch, U, Mnsuy, IM nd Dutr, P (). Different phosphtse-dependent mechnisms medite long-term depression nd depotentition of long-term potentition in mouse hippocmpl CA re. Eur J Neurosci 8: Hsio, K, Chpmn, P, Nilsen, S, Eckmn, C, Hrigy, Y, Younkin, S et l. (99). Correltive memory deficits, Abet elevtion, nd myloid plques in trnsgenic mice. Science 74: Irizrry, MC, McNmr, M, Fedorchk, K, Hsio, K nd Hymn, BT (997). APPSw trnsgenic mice develop ge-relted A bet deposits nd neuropil bnormlities, but no neuronl loss in CA. J Neuropthol Exp Neurol 5: Chen, J, Mrtin, BL nd Brutign, DL (99). Regultion of protein serine-threonine phosphtse type-a by tyrosine phosphoryltion. Science 57: Sontg, E, Hldik, C, Montgomery, L, Lungpirom, A, Mudrk, I, Ogris, E et l. (4). Downregultion of protein phosphtse A crboxyl methyltion nd methyltrnsferse my contribute to Alzheimer disese pthogenesis. J Neuropthol Exp Neurol : Sontg, JM, Nunbhkdi-Crig, V, Mitterhuber, M, Ogris, E nd Sontg, E (). Regultion of protein phosphtse A methyltion by LCMT nd PME- plys criticl role in differentition of neuroblstom cells. J Neurochem 5: Arnud, L, Chen, S, Liu, F, Li, B, Khtoon, S, Grundke-Iqbl, I et l. (). Mechnism of inhibition of ctivity nd bnorml hyperphosphoryltion of tu by ()/ SET. FEBS Lett 585: Vssr, R, Bennett, BD, Bbu-Khn, S, Khn, S, Mendiz, EA, Denis, P et l. (999). Bet-secretse clevge of Alzheimer s myloid precursor protein by the trnsmembrne sprtic protese BACE. Science 8: Luo, X nd Yn, R (). Inhibition of BACE for therpeutic use in Alzheimer s disese. Int J Clin Exp Pthol : Selkoe, DJ (998). The cell biology of bet-myloid precursor protein nd presenilin in Alzheimer s disese. Trends Cell Biol 8: Mtsushim, T, Sito, Y, Elliott, JI, Iijim-Ando, K, Nishimur, M, Kimur, N et l. (). Membrne-microdomin locliztion of myloid ß-precursor protein (APP) C-terminl frgments is regulted by phosphoryltion of the cytoplsmic Thr8 residue. J Biol Chem 87: Swingle, M, Ni, L nd Honknen, RE (7). Smll-molecule inhibitors of ser/thr protein phosphtses: specificity, use nd common forms of buse. Methods Mol Biol 5: Liu, F, Su, Y, Li, B, Zhou, Y, Ryder, J, Gonzlez-DeWhitt, P et l. (). Regultion of myloid precursor protein (APP) phosphoryltion nd processing by p5/cdk5 nd p5/cdk5. FEBS Lett 547: Ryoo, SR, Cho, HJ, Lee, HW, Jeong, HK, Rdnbzr, C, Kim, YS et l (8) Dulspecificity tyrosine (Y)-phosphoryltion regulted kinse A-medited phosphoryltion of myloid precursor protein: evidence for functionl link between Down syndrome nd Alzheimer s disese. 4: Mtsumoto, K, Ngt, K, Ui, M nd Hnok, F (99). Templte ctivting fctor I, novel host fctor required to stimulte the denovirus core DNA repliction. J Biol Chem 8: Kutney, SN, Hong, R, Mcfrln, T nd Chkrvrti, D (4). A signling role of histone-binding proteins nd INHAT subunits pp nd Set/TAF-Ibet in integrting chromtin hypocetyltion nd trnscriptionl repression. J Biol Chem 79: Okuwki, M nd Ngt, K (998). Templte ctivting fctor-i remodels the chromtin structure nd stimultes trnscription from the chromtin templte. J Biol Chem 7: Roberts, TF, Tschid, KA, Klein, ME nd Mooney, R (). Rpid spine stbiliztion nd synptic enhncement t the onset of behviourl lerning. Nture 4: ten Klooster, JP, Leeuwen, Iv, Scheres, N, Anthony, EC nd Hordijk, PL (7). Rc- induced cell migrtion requires membrne recruitment of the nucler oncogene SET. EMBO J : Morris, RG, Grrud, P, Rwlins, JN nd O Keefe, J (98). Plce nvigtion impired in rts with hippocmpl lesions. Nture 97: Nghr, AH, Merrill, DA, Coppol, G, Tsukd, S, Schroeder, BE, Shked, GM et l. (9). Neuroprotective effects of brin-derived neurotrophic fctor in rodent nd primte models of Alzheimer s disese. Nt Med 5: Liu, GP, Wei, W, Zhou, X, Zhng, Y, Shi, HH, Yin, J et l. (). I()() regultes p5 nd Akt correltively nd leds the neurons to bort poptosis. Neurobiol Aging : Oddo S, Cccmo A, Smith IF, Green KN, LFel FM (). LRP/myloid β-peptide interction medites differentil brin efflux of Aβ isoforms. Neuron 4: Woolley, CS nd McEwen, BS (99). Estrdiol medites fluctution in hippocmpl synpse density during the estrous cycle in the dult rt. J Neurosci : Mgriños, AM, Li, CJ, Gl Toth, J, Bth, KG, Jing, D, Lee, FS et l. (). Effect of brin-derived neurotrophic fctor hploinsufficiency on stress-induced remodeling of hippocmpl neurons. Hippocmpus : Spires-Jones, TL, Ky, K, Mtsouk, R, Rozklne, A, Betensky, RA nd Hymn, BT (). Clcineurin inhibition with systemic FK5 tretment increses dendritic brnching nd dendritic spine density in helthy dult mouse brin. Neurosci Lett 487:. Moleculr Therpy vol. no. dec. 57