Amyloid beta-peptide worsens cognitive impairment following cerebral ischemia-reperfusion injury*****

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1 NEURL REGENERTION RESERCH Volume 8, Issue 26, September doi:1.3969/j.issn [ Song, o Q, Niu Y, Shen Q, Zuo HC, Zhng XF, Gong YD. myloid bet-peptide worsens cognitive impirment following cerebrl ischemi-reperfusion injury. Neurl Regen Res. 213;8(26): myloid bet-peptide worsens cognitive impirment following cerebrl ischemi-reperfusion injury***** o Song 1, 2, Qing o 1, 3, Ying Niu 2, Qin Shen 1, Huncong Zuo 3, Xiufng Zhng 2, Yndo Gong 2 1 Reserch Center of Stem Cells nd Regenertive Medicine, School of Medicine, Tsinghu University, eijing 184, Chin 2 Stte Key Lortory of iomembrne nd Membrne iotechnology, School of Life Sciences, Tsinghu University, eijing 184, Chin 3 Institute of Neurology Disorders, Yuqun Hospitl, Tsinghu University, eijing 149, Chin Reserch Highlights (1) This study combined intrcerebroventriculr injection of myloid β-peptide with cerebrl ischemi nd reperfusion to estlish new niml model of lzheimer s disese. We found tht single injection of myloid β-peptide lone could not impir lerning nd memory ilities in rts or cuse deth of hippocmpl neurons. However, intrcerebroventriculr injection of myloid β-peptide following cerebrl ischemi nd reperfusion could ggrvte lerning nd memory in rts nd cuse nerve cell deth. (2) In this study, the synergistic effect of myloid β-peptide nd cerebrl ischemi-reperfusion injury excerbted nerve dmge by inducing glycogen synthse kinse 3β nd protein phosphtse 2 ctivity, which resulted in the phosphoryltion of tu protein. bstrct myloid β-peptide, mjor component of senile plques in lzheimer s disese, hs been implicted in neuronl cell deth nd cognitive impirment. Recently, studies hve shown tht the pthogenesis of cerebrl ischemi is closely linked with lzheimer s disese. In this study, rt model of globl cerebrl ischemi-reperfusion injury ws estlished vi occlusion of four rteries; menwhile, fibrillr myloid β-peptide ws injected into the rt lterl ventricle. The Morris wter mze test nd histologicl stining reveled tht dministrtion of myloid β-peptide could further ggrvte impirments to lerning nd memory nd neuronl cell deth in the hippocmpus of rts subjected to cerebrl ischemi-reperfusion injury. Western blot showed tht phosphoryltion of tu protein nd the ctivity of glycogen synthse kinse 3β were significntly stronger in cerebrl ischemi-reperfusion injury rts subjected to myloid β-peptide dministrtion thn those undergoing cerebrl ischemi-reperfusion or myloid β-peptide dministrtion lone. Conversely, the ctivity of protein phosphtse 2 ws remrkly reduced in rts with cerebrl ischemi-reperfusion injury following myloid β-peptide dministrtion. These findings suggest tht myloid β-peptide cn potentite tu phosphoryltion induced by cerebrl ischemi-reperfusion nd thereby ggrvte cognitive impirment. o Song, M.D. Corresponding uthor: Yndo Gong, Mster, Stte Key Lortory of iomembrne nd Membrne iotechnology, School of Life Sciences, Tsinghu University, eijing 184, Chin, gongyd@ tsinghu.edu.cn. Qing o, M.D., Reserch Center of Stem Cells nd Regenertive Medicine, School of Medicine, Tsinghu University, eijing 184, Chin; Institute of Neurology Disorders, Yuqun Hospitl, Tsinghu University, eijing 149, Chin, oqing@ tsinghu.edu.cn. Received: ccepted: (N ) cknowledgments: We would like to thnk Gong K from the Stte Key Lortory of iomembrne nd Membrne iotechnology, School of Life Sciences, Tsinghu University, eijing, Chin for providing technicl ssistnce. Key Words neurl regenertion; brin injury; cerebrl ischemi-reperfusion; lzheimer s disese; myloid β-peptides; tu proteins; glycogen synthse kinse 3β; protein phosphtse 2; phosphoryltion; grnts-supported pper; neuroregenertion 2449

2 Song, et l. / Neurl Regenertion Reserch. 213;8(26): Funding: This work ws supported by the Ntionl High Technology Reserch nd Development Progrm of Chin ("863"Progrm), No ; the Ntionl Nturl Science Foundtion of Chin, No nd ; Ntionl Nturl Science Foundtion of Chin (NSFC)/Reserch Grnts Council (RGC) Joint Reserch Scheme, No ; nd Tsinghu- Yue-Yuen Medicl Sciences Fund. uthor contributions: Song completed the experiments nd wrote the mnuscript. o Q nd Gong YD provided reserch ides nd reserch design. Niu Y ws responsible for dt nlysis, nd prticipted in the study. Shen Q nd Zuo HC guided the study. Zhng XF provided reserch ides nd guided the study. ll uthors pproved the finl version of the pper. Conflicts of interest: None declred. Ethicl pprovl: The study ws pproved by the niml Ethics Committee of Tsinghu University, Chin. uthor sttements: The mnuscript is originl, hs not been submitted to or is not under considertion by nother publiction, hs not been previously published in ny lnguge or ny form, including electronic, nd contins no disclosure of confidentil informtion or uthorship/ptent ppliction/funding source disputtions. 245 INTRODUCTION lzheimer s disese is chrcterized by two hllmrk brin lesions extrcellulr deposition of myloid β-peptide (β) nd intrcellulr neurofibrillry tngles [1]. β, the mjor constituent of senile plques in the brin of lzheimer s disese ptients, hs been implicted in the pthogenesis of the disese. Ynkner et l [2] reported tht β ws neurotoxic to cultured hippocmpus neurons, while Iwski et l [3-4] found tht fibrillr β could induce prominent neuronl cell deth nd the expression of cell deth-relted genes in primry corticl neuronl cultures. These dying neurons exhibited morphologicl fetures of poptosis [5]. Neurofibrillry tngles re composed of bundles of pired helicl filments nd stright filments, the mjor protein component of which is normlly hyperphosphorylted tu protein [1]. The biologicl function of tu is regulted by its degree of phosphoryltion. Hyperphosphoryltion of tu depresses its microtubule ssembly ctivity nd its binding to microtubules, thus leding to microtubule destiliztion, the ppernce of neurofibrillry tngles nd neurodegenertion in the brin of lzheimer s disese ptients [6]. Phosphoryltion of tu cn be regulted by mny protein kinses nd phosphtses. Glycogen synthse kinse 3 is serine/ threonine kinse tht ws first isolted nd purified s n enzyme cple of phosphorylting nd inctivting glycogen synthse [7-8]. Glycogen synthse kinse 3 is inhibited when phosphorylted t serine residue (Ser 9 for glycogen synthse kinse 3β nd Ser 21 for glycogen synthse kinse 3α) locted in the N-terminl domin [7-8]. Glycogen synthse kinse 3β is shown to phosphorylte tu protein both in vitro nd in vivo t multiple sites, some of which re normlly hyperphosphorylted in the brins of lzheimer s disese ptients [9]. Currently, glycogen synthse kinse 3β, is recognized s multifunctionl kinse involved in mny kinds of biologicl processes, such s embryonic development, tumorigeness, neurodegenertion nd cell deth [9]. growing number of studies hve shown tht glycogen synthse kinse 3β is the most likely cndidte for the protein kinse responsible for the norml phosphoryltion of tu in the brin of lzheimer s disese ptients [1-11]. Immunohistochemicl studies hve found tht glycogen synthse kinse 3β is locted in neurofibrillry tngles [1]. Moreover, protein levels of glycogen synthse kinse 3β re elevted in the brin of lzheimer s disese ptients [11]. Protein phosphtse 2 is member of the fmily of serine/threonine phosphtses nd is ubiquitously expressed in most tissues nd cells. Protein phosphtse 2 ccounts for s much s 1% of totl cellulr protein nd is responsible for the mjor portion of serine/threonine phosphtse ctivity [12-13]. The ctivity of protein phosphtse 2 is decresed in the brins of lzheimer s disese ptients [14], but its function remins uncler. Ischemi-induced cell deth is one of the most serious effects cused by brin ischemi [15-16]. The precise pthogenic mechnism of brin ischemi is not cler. Despite numerous gents tht cn prevent the cscde of events leding to ischemic neuronl deth in niml models, humn clinicl trils with these gents hve proven disppointing [15-16]. In the lst ten yers, incresing evidence hs indicted tht cerebrovsculr diseses hve importnt roles in the pthogenesis nd evolution of lzheimer s disese. Stroke hs been shown to be closely ssocited with lzheimer s disese in mny studies [17-21]. Wen et l [22] found tht tu hyperphosphoryltion my contribute to the brin dmge induced by trnsient cerebrl ischemi. Mny studies hve found tht brin ischemi cn result in lzheimer s disese-like neuropthology [23-24], such s incresed production of β, norml phosphoryltion of tu protein nd overexpression of presenilin nd polipoprotein E. Clinicl studies hve shown tht lzheimer s disese ptients with history of cerebrovsculr disese hve more rpid development of dementi [25]. Other studies hve found tht hypoxi

3 Pltform-pssing times Escpe ltency (second) Song, et l. / Neurl Regenertion Reserch. 213;8(26): cn promote β-induced poptosis of hippocmpl neurons cultured in vitro [26]. Epidemiologicl studies suggest tht elderly people with silent stroke hve n incresed risk of lzheimer s disese [27]. Despite mny studies reporting close ssocition between brin ischemi nd lzheimer s disese, the underlying mechnisms remin uncler. To further disclose the possible ssocition between lzheimer s disese nd brin ischemi, we developed n in vivo rt model of dementi using cerebrl ischemi-reperfusion combined with intrcerebroventriculr dministrtion of β. s β is metolized very rpidly in the brin, we dministered β dily. Sptil lerning nd memory of rts ws monitored, nd the survivl of hippocmpl pyrmidl cells, phosphoryltion of tu protein, nd the ctivity of glycogen synthse kinse 3β nd protein phosphtse 2 were exmined. The pthogenesis of lzheimer s disese is very complicted. Mny environmentl fctors nd genetic fctors my jointly contribute to the formtion of lzheimer s disese. To dte, there re no idel in vivo niml models tht possess ll pthologicl fetures, which gretly restrict the development of therpeutic drugs for lzheimer s disese. lthough mny niml models of lzheimer s disese re ville, these models only contin one or severl pthologicl fetures of lzheimer s disese. In this study, we developed n in vivo rt model of dementi by injecting β following cerebrl ischemi-reperfusion; two importnt fctors for inducing lzheimer s disese. Our results showed tht neuronl dmge ws ggrvted in this model. Therefore, this niml model of dementi my be used for studying the pthogenic mechnism of lzheimer s disese nd for therpeutic drug development. RESULTS Quntittive nlysis of experimentl nimls Eighty dult mle Sprgue-Dwley rts were rndomly divided into shm surgery, cerebrl ischemi-reperfusion, β dministrtion without brin ischemi (β), nd cerebrl ischemi-reperfusion plus β dministrtion (combintion) groups. The numbers of rts in ech group were 2. ll 8 rts were included in the finl results nlysis. Effects of cerebrl ischemi-reperfusion nd β dministrtion on sptil memory The Morris wter mze test ws conducted on rts fter 15 minutes brin ischemi followed by 3 dys of reperfusion. In the plce nvigtion test, the escpe ltency in the cerebrl ischemi-reperfusion group ws significntly longer thn tht in the shm opertion group (Figure 1). The escpe ltency in the combintion group ws significntly longer thn tht in the cerebrl ischemi-reperfusion group (P <.5). However, the escpe ltency in the β dministrtion group ws not significntly different from tht of the shm opertion group Time of reperfusion (dy) Shm Ischemi Ischemi+β β Shm Ischemi Ischemi+β Figure 1 Morris wter mze test for sptil lerning nd memory ility of rts subjected to myloid β-peptide (β) dministrtion. () The plce nvigtion test: Time to find the hidden pltform. The escpe ltency in the cerebrl ischemireperfusion group ws significntly longer thn tht in the shm group. P <.5, vs. shm group; b P <.5, vs. cerebrl ischemi-reperfusion group. () Pltform-pssing times in the probe tril test. P <.5, vs. shm group; b P <.5, vs. cerebrl ischemireperfusion group. Dt re expressed s men ± SD, n = 1. Sttisticl nlysis ws crried out by one-wy nlysis of vrince followed by the lest significnt difference test or Student Newmn-Keuls test. Shm: Shm opertion group; ischemi: cerebrl ischemi-reperfusion group; ischemi + β: cerebrl ischemi-reperfusion + β group. In the probe tril test, the pltform-pssing times in the cerebrl ischemi-reperfusion group were significntly fewer thn those in the shm opertion group (P <.5; Figure 1). The pltform-pssing times in the combintion group were significntly fewer thn those in the β 2451

4 Neuronl density Song, et l. / Neurl Regenertion Reserch. 213;8(26): cerebrl ischemi-reperfusion group (P <.5). However, the pltform-pssing times in the β dministrtion group were not significntly different when compred to the shm opertion group. Effects of cerebrl ischemi-reperfusion nd β dministrtion on hippocmpl neurl cells The effects of cerebrl ischemi-reperfusion nd β dministrtion on the survivl of hippocmpl neurl cells were exmined using cresyl violet stining of rt brin sections. s shown in Figure 2, the number of surviving hippocmpl C1 neurons in the cerebrl ischemi-reperfusion group ws significntly lower thn tht in the shm opertion group. between the cerebrl ischemi-reperfusion group nd shm opertion group (Figure 4). C D The number of surviving hippocmpl C1 neurons in the combintion group ws lso significntly lower thn tht in the shm opertion group (P <.5). There ws no sttisticl difference in the number of surviving hippocmpl C1 neurons between the cerebrl ischemireperfusion nd combintion groups (P <.5). However, in the cerebrl ischemi-reperfusion group, the loss of neurons ws minly observed in the hippocmpl C1 region (P <.5). dditionlly, in the combintion group, the loss of neurons ppered in both the C1 nd C2 regions. E G F H The phosphoryltion of tu in rts with cerebrl ischemi-reperfusion injury subjected to β dministrtion Next, the level of phosphorylted tu ws exmined by western blot. s shown in Figure 3, the level of tu phosphorylted t pired helicl filment-1 (Ser 396/44 ) ws not significntly different between the cerebrl ischemi-reperfusion group nd shm opertion group. The level of phosphorylted tu in the combintion group ws significntly higher thn tht in the shm opertion group (P <.5). Moreover, the level of phosphorylted tu in the β dministrtion group ws significntly higher thn tht in the shm opertion group, but lower thn the combintion group (P <.5). Protein levels of phosphorylted glycogen synthse kinse 3β nd protein phosphtse 2 in rts subjected to cerebrl ischemi-reperfusion nd β dministrtion Considering tht glycogen synthse kinse 3β is the mjor tu kinse, the ctivity of glycogen synthse kinse 3β during cerebrl ischemi-reperfusion combined with β dministrtion ws determined by western blot nlysis. Protein levels of glycogen synthse kinse 3β when phosphorylted t Ser 9 showed no significnt difference I Shm Ischemi Ischemi+β β Figure 2 Effect of myloid β-peptide (β) dministrtion on the number of nerve cells in the rt hippocmpus following cerebrl ischemi-reperfusion (cresyl violet stining, light microscope). oxed res in the left column re shown t higher mgnifiction thn tht in the right column. Scle br: 5 μm (left column), 8 μm (right column). (, ) Shm group. (C, D) Cerebrl ischemi-reperfusion group. (E, F) Cerebrl ischemi-reperfusion + β dministrtion group. (G, H) β dministrtion (without brin ischemi) group. (I) Quntittive representtions of the of surviving hippocmpl C1 neurons per 1 mm length in ll groups. The number of surviving hippocmpl C1 pyrmidl cells per 1 mm length ws counted s the density of neurons. P <.5, vs. shm group. Dt re expressed s men ± SD, n = 8. Sttisticl nlysis ws crried out by one-wy nlysis of vrince followed by the lest significnt difference test or Student-Newmn-Keuls test. Shm: Shm opertion; ischemi: cerebrl ischemi-reperfusion group; ischemi + β: cerebrl ischemi-reperfusion + β group. 2452

5 bsorbnce (folds vs. shm) bsorbnce (folds vs. shm) bsorbnce (folds vs. shm) Song, et l. / Neurl Regenertion Reserch. 213;8(26): Shm Ischemi Ischemi+β β p-tu (PHF-1) Shm Ischemi Ischemi+β β p-gsk3β(ser 9 ) GSK3β tu p-gsk3β 1.2 GSK3β shm ischemi ischemi+β β Figure 3 Effect of myloid β-peptide (β) dministrtion on phosphorylted tu protein (p-tu) levels in the rt hippocmpus following cerebrl ischemi-reperfusion. () Western blot ssy of tu phosphoryltion in the shm group, cerebrl ischemi-reperfusion group, cerebrl ischemi-reperfusion + β group nd β dministrtion group. () Semiquntittive representtions of p-tu. nds were scnned nd the intensities were determined by sorbnce (). P <.5, vs. shm group; b P <.5, vs. cerebrl ischemi-reperfusion group. Dt re expressed s men ± SD, n = 8. Sttisticl nlysis ws crried out by one-wy nlysis of vrince followed by the lest significnt difference test or Student-Newmn-Keuls test. Shm: Shm opertion group; ischemi: cerebrl ischemi-reperfusion group; ischemi + β: cerebrl ischemi-reperfusion + β group. PHF-1 tu Shm Ischemi Ischemi+β β Figure 4 Effect of myloid β-peptide (β) dministrtion on phosphorylted glycogen synthse kinse 3 (p-gsk-3β) protein levels in the rt hippocmpus following cerebrl ischemi-reperfusion. () Protein levels of p-gsk-3β in the shm, cerebrl ischemi-reperfusion, cerebrl ischemi-reperfusion + β, nd β dministrtion groups were determined using western blot. () Semiquntittive representtions of protein levels of p-gsk-3β. nds were scnned nd the intensities were determined by sorbnce (). P <.5, vs. shm group; b P <.5, vs. cerebrl ischemi-reperfusion group. Dt re expressed s men ± SD, n = 8. Sttisticl nlysis ws crried out by one-wy nlysis of vrince followed by the lest significnt difference test or Student-Newmn-Keuls test. Shm: Shm opertion group; ischemi: cerebrl ischemi-reperfusion group; ischemi + β: cerebrl ischemi-reperfusion + β group. Shm Ischemi Ischemi+β β p-pp2 PP2 p-pp2 (Tyr 37 ) PP Shm Ischemi Ischemi+β β Figure 5 Effect of myloid β-peptide (β) dministrtion on phosphorylted protein phosphtse 2 (p-pp2) protein levels in the rt hippocmpus following cerebrl ischemi-reperfusion. () Protein levels of p-pp2 in the shm, cerebrl ischemi-reperfusion, cerebrl ischemi-reperfusion + β, nd β dministrtion groups were determined using western blot. () Semiquntittive representtions of p-pp2 protein levels. nds were scnned nd the intensities were determined by sorbnce. P <.5, vs. shm group; b P <.5, vs. cerebrl ischemi-reperfusion group. Dt re expressed s men ± SD, n = 8. Sttisticl nlysis of the results ws crried out by one-wy nlysis of vrince followed by the lest significnt difference test or Student-Newmn-Keuls test. Shm: Shm opertion group; ischemi: cerebrl ischemi-reperfusion group; ischemi + β: cerebrl ischemi-reperfusion + β group. 2453

6 Song, et l. / Neurl Regenertion Reserch. 213;8(26): The level of phosphorylted glycogen synthse kinse 3β in the combintion group ws significntly lower thn tht in the shm opertion group (P <.5), which suggested the ctivity of glycogen synthse kinse 3β ws incresed in the combintion group. The level of phosphorylted glycogen synthse kinse 3β in the β dministrtion group ws lso significntly lower thn tht in the shm opertion group, but higher thn tht in the combintion group (P <.5). In ddition, the ctivity of protein phosphtse 2, the mjor tu phosphtse, ws detected using western blot. The level of protein phosphtse 2 phosphorylted t Tyr 37 ppered to hve no significnt difference between the cerebrl ischemi-reperfusion group nd shm opertion group (Figure 5). The level of phosphorylted protein phosphtse 2 in the combintion group ws significntly higher thn tht in the shm opertion group (P <.5), which mens the ctivity of protein phosphtse 2 ws reduced in the combintion group. The level of phosphorylted protein phosphtse 2 in the β dministrtion group ws lso not significntly different from tht of the shm opertion group. DISCUSSION β is produced by the proteolytic processing of the myloid precursor protein. Under norml physiologicl conditions, β is enzymticlly degrded nd metolized. The two specific forms of β, β1 4 nd β1 42, re prone to dopt β-sheet conformtion nd ggregte s myloid fibrils in the brin. Incresing evidence suggests tht the β-sheet conformtion of β hs neurotoxic effects. Ynkner et l [2] reported tht β could induce neuronl cell deth in cultured neurons, nd My et l [28] suggested tht the toxicity of β is ssocited with β-sheet conformtion. In ddition, Pike et l [29] found tht β solution incubted for 24 hours t 37 C hs much stronger neurotoxicity thn newly dissolved β. Owing to the hydrophobic nture of β, β monomers ggregte into polymers in queous solution to form insoluble ggregtes, which is probly responsible for the β-sheet structure. Mny studies hve found tht β fibrils cn induce neuronl deth in vitro. Here, we conducted n in vivo study nd found tht fibrillr β lone filed to impir sptil lerning ility nd induce neuronl loss. However, combintion of brin ischemi nd β dministrtion could further ggrvte neuronl injury. Using western blot nlysis, we found tht the ctivity of glycogen synthse kinse 3β ws remrkly incresed in the combintion group compred with the cerebrl ischemireperfusion group or β tretment group. Moreover, the ctivity of protein phosphtse 2 ws significntly reduced in the combintion group compred with the cerebrl ischemi-reperfusion group or β tretment group. The level of phosphorylted tu protein ws rnked s follows: the combintion group > β dministrtion group > shm opertion group. s glycogen synthse kinse 3β is the mjor tu kinse nd protein phosphtse 2 is the mjor tu phosphtse, the incresed ctivity of glycogen synthse kinse 3β nd reduced ctivity of protein phosphtse 2 jointly contributed to n increse in tu phosphoryltion in the combintion group. These findings suggest tht β dministrtion synergisticlly improves trnsient ischemic brin injury. ccording to the β theory of lzheimer s disese pthogenesis, β lies t the top of the signl pthwy. β cn ctivte glycogen synthse kinse 3β nd induce incresed phosphoryltion of tu, which my result in the formtion of neurofibrillry tngles. However, the underlying mechnism is not cler. lzheimer s disese is chrcterized by two hllmrk brin lesions, extrcellulr β deposits nd intrcellulr neurofibrillry tngles, but β deposits lso exist in the brin of some helthy elderly people, suggesting β is not necessry for dementi. This is consistent with the results of our present study. The ility of lerning nd memory nd the neuronl survivl of rts in the β dministrtion group showed no significnt difference when compred with the shm opertion group. Previous studies hve found tht neurofibrillry tngles re more relevnt to lzheimer s disese thn β deposits [3-31]. Neurofibrillry tngles re composed of normlly hyperphosphorylted tu proteins. Hyperphosphoryltion of tu depresses its microtubule ssembly ctivity nd its binding to microtubules [32-33], thus cusing neurodegenertion in the brin of lzheimer s disese ptients. In word, tu my be the min executor for neuronl cell deth. The synergistic ctions of brin ischemi nd β my induce tu hyperphosphoryltion, but the underlying mechnism needs further study. In recent yers, mny reserchers hve found tht there is close ssocition between brin ischemi nd lzheimer s disese. rin ischemi cn result in lz- 2454

7 Song, et l. / Neurl Regenertion Reserch. 213;8(26): heimer s disese-like brin injuries, such s incresed production of β nd hyperphosphoryltion of tu protein. Some studies propose n ischemi-reperfusion theory of lzheimer s disese pthogenesis. The results from this study lso support this new theory. Recent progress in lzheimer s disese reserch hs begun to unrvel the pthogenesis of the disese, but few in vivo niml models hve been shown to be suitle for therpeutic drug studies. In this study, we creted rt model of dementi by combining intrcerebroventriculr dministrtion of β nd brin ischemi. This model could ggrvte sptil memory impirment nd neuronl cell deth. Therefore, this model my be useful for studying the pthogenesis of lzheimer s disese nd ischemic stroke, nd be useful for the development of new therpeutic drugs. In conclusion, the combintion of brin ischemi nd β dministrtion ggrvtes sptil memory impirment nd neuronl cell deth. These findings suggest tht brin ischemi is n importnt fctor tht fcilittes the development of lzheimer s disese. This in vivo model my be useful for therpeutic drug development in the tretment of lzheimer s disese nd other neurodegenertive diseses. MTERILS ND METHODS Design prllel controlled, comprtive, in vivo experiment. Time nd setting Experiments were performed from My 28 to July 21 t the School of Life Sciences, Tsinghu University, Chin. Mterils totl of 8 helthy, clen mle Sprgue-Dwley rts, 7 weeks of ge, were purchsed from the Vitl River Corportion, eijing, Chin. Experimentl procedures for nimls were in strict ccordnce with the Guidnce Suggestions for the Cre nd Use of Lortory nimls, issued by the Ministry of Science nd Technology of Chin [34]. Methods Estlishment of rt model of cerebrl ischemireperfusion Trnsient brin ischemi followed by reperfusion ws induced by the four-vessel occlusion method, s described by Pulsinelli et l [35]. riefly, rts were nesthetized intrperitonelly (i.p.) with 3 mg/kg chlorl hydrte (Sigm-ldrich, St. Louis, MO, US) nd immobilized in stereotxic pprtus (RWD Life Science Co., Ltd., Shenzhen, Gungdong Province, Chin). The bilterl vertebrl rteries were electrocuterized with bipolr cogultor. On the next dy, the bilterl common crotid rteries were occluded with neurysm clips to induce brin ischemi. fter 15 minutes of occlusion, the neurysm clips were removed followed by 7 dys of reperfusion. Rts in the shm opertion group received the sme surgicl procedures except the crotid rteries were occluded. The body temperture of rts ws mintined t 37 C using heting pd. Rts tht did not exhibit loss of their righting reflex during brin ischemi were excluded from the subsequent experiment. Intrcerebroventriculr injection of β β 1 42 ws purchsed from NSPEC, Sn Jose, C, US. β 1 42 (1 μg) ws dissolved in 1 μl sline nd incubted for 72 hours t 37 C. Twenty-four hours fter cerebrl ischemi-reperfusion, the rts were nesthetized with chlorl hydrte (3 mg/kg, i.p.; Sigm) nd plced in stereotxic pprtus (RWD Life Science Co., Ltd.). β 1 42 (1 μl; 1 μg/μl) ws dministrted unilterlly to rts every 24 hours for 6 dys through the left ventricle of the brin (nteroposterior,.8 mm; lterl, 1.5 mm; depth, 3.5 mm from the bregm). Rts in the β dministrtion group received the sme surgicl procedures except for cerebrl ischemi-reperfusion. Western blot nlysis of protein smples Rts were decpitted immeditely fter 15 minutes brin ischemi followed by 7 dys reperfusion. The hippocmpi were quickly seprted nd dipped into liquid nitrogen, nd then stored t 8 C. Hippocmpi were homogenized in ice-cold homogeniztion buffer. The homogentes were centrifuged t 8 g for 15 minutes t 4 C to collect the superntnt, nd the protein concentrtions were determined by the method of Lowry. For western blot nlysis, proteins were seprted by 1% (w/v) sodium dodecyl sulfte polycrylmide gel electrophoresis (SDS-PGE) nd then electrotrnsfered onto nitrocellulose membrne (pore size,.45 μm). The membrne ws probed with rbit nti-phospho- GSK3β (Ser 9 ) monoclonl ntibody (1:1, Cell Signling Technology, Inc., M, US, rbit nti-gsk3β monoclonl ntibody (1:1, Cell Signling), mouse nti-tu monoclonl ntibody (1:1, Cell Signling), mouse nti-phf-1 monoclonl ntibody (1:1, provided by Dr. Peter Dvies, lbert Einstein College of Medicine, ronx, NY, US), rbit nti-phospho-protein phos- 2455

8 Song, et l. / Neurl Regenertion Reserch. 213;8(26): phtse 2 (Tyr 37 ) monoclonl ntibody (1:1, Epitomics, Inc., Hngzhou, Zhejing Province, Chin nd rbit nti-protein phosphtse 2 monoclonl ntibody (1:1, Epitomics) overnight t 4 C. Detection ws performed using lkline phosphtse conjugted got-ntirbit IgG (1:1 ) or got nti-mouse IgG(1:1 ). The bnds on the membrne were scnned nd nlyzed by n imge nlyzer (Lworks Softwre, UVP Inc., Uplnd, C, US). The intensities of the bnds were determined by the sorbnce vlues. The results were expressed s fold vs. shm control. Morris wter mze test The Morris wter mze ws used to test sptil lerning nd memory in rts [36]. The wter mze test ws performed in blck circulr pool (dimeter 1.5 m, height 6 cm, filled with wter of C to height of 3 cm). fter 15 minutes brin ischemi followed by 3 dys reperfusion in rts, the test ws conducted. In the hidden-pltform trils, 1-cm-dimeter pltform ws plced 2. cm below the wter line in the southestern qudrnt of the pool. The rt ws plced in the wter fcing the wll t one rndom strt loction of four qudrnts. Ech rt ws llowed to find the submerged pltform within 12 seconds, nd rest on it for 2 seconds. If rts filed to find the hidden pltform within 12 seconds, the rt ws plced on the pltform for 2 seconds. Two sessions of four trils were conducted on the first testing dy, nd the intervl ws 4 hours. The first session ws considered s the trining procedure. Then, one session of four trils ws conducted dily within the next 3 dys. Four hours fter the lst tril, probe tril ws given within 12 seconds in which the pltform ws removed from the pool. The escpe ltency (time to find the pltform) nd pltform-pssing times were monitored by cmer (eijing New World Technology Co., Ltd., eijing, Chin) directly ove the pool. Histologicl stining of rt brin sections Rts were deeply nesthetized with chlorl hydrte nd perfused trnscrdilly with chilled norml sline for 3 minutes followed by tretment with 4% (w/v) prformldehyde. rins were removed nd stored in the sme prformldehyde solution overnight. Frozen sections (1 µm) were cut coronlly nd stined with cresyl violet (Sigm). The sections were exmined under light microscopy (Olympus, Tokyo, Jpn) nd the surviving pyrmidl cells in the hippocmpl C1 per 1 mm length were counted s the density of neurons. Sttisticl nlysis Vlues were expressed s men ± SD from t lest eight independent nimls. Sttisticl nlysis of the results ws conducted using Origin 7. softwre (OriginL Corportion, US), nd one-wy nlysis of vrince followed by the lest significnt difference test or Student-Newmn-Keuls test. P <.5 ws considered significnt. REFERENCES [1] Gndy S, DeKosky ST. Towrd the tretment nd prevention of lzheimer s disese: rtionl strtegies nd recent progress. nnu Rev Med. 213;64: [2] Ynkner, Duffy LK, Kirchner D. Neurotropic nd neurotoxic effects of myloid β protein: reversl by tchykinin neuropeptides. Science. 199;25(4978): [3] Iwski K, Kitmur Y, Ohgmi Y, et l. The disruption of sptil cognition nd chnges in brin mino cid, monomine nd cetylcholine in rts with trnsient cerebrl ischemi. rin Res. 1996;79(2): [4] Iwski K, Sunderlnd T, Kusik JW, et l. Chnges in gene trnscription during bet-medited cell deth. Mol Psychitry. 1996;1(1): [5] Egshir N, Iwski K, Ishibshi M, et l. Hypoxi enhnces bet-myloid-induced poptosis in rt cultured hippocmpl neurons. Jpn J Phrmcol. 22;9(4): [6] lzquez-llorc L, Grci-Mrin V, Merino-Serris P, et l. bnorml tu phosphoryltion in the thorny excrescences of C3 hippocmpl neurons in ptients with lzheimer s disese. J. lzheimers Dis. 211;26(4): [7] Embi N, Ryltt D, Cohen P. Glycogen synthse kinse-3 from rbit skeletl muscle. Seprtion from cyclic- MP-dependent protein kinse nd phosphorylse kinse. Eur J iochem. 198;17(2): [8] Woodgett JR, Cohen P. Multisite phosphoryltion of glycogen synthse. Moleculr bsis for the substrte specificity of glycogen synthse kinse-3 nd csein kinse-ii (glycogen synthse kinse-5). iochim iophys ct. 1984;788(3): [9] Mondrgón-Rodríguez S, Perry G, Zhu X, et l. Glycogen synthse kinse 3: point of integrtion in lzheimer s disese nd therpeutic trget? Int J lzheimers Dis. 212;212:27683 [1] Pei JJ, rk E, rk H, et l. Distribution of ctive glycogen synthse kinse 3β (GSK-3β) in brins stged for lzheimer disese neurofibrillry chnges. J Neuropthol Exp Neurol. 1999;58(9): [11] Ymguchi H, Ishiguro K, Uchid T, et l. Preferentil leling of lzheimer neurofibrillry tngles with ntiser 2456

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