Thiamine deficiency increases beta-secretase activity

Neurobiology of Aging32(2011)42–53Thiamine deficiency increases␤-secretase activity andaccumulation of␤-amyloid peptidesQipeng Zhang a,c,Guang Yang a,Wenxia Li a,Zhiqin Fan a,Anyang Sun c,Jia Luo a,b,∗,Zun-Ji Ke a,∗∗a Key Laboratory of Nutrition and Metabolism,Institute for Nutritional Sciences,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai,200031,PR Chinab Department of Internal Medicine,University of Kentucky College of Medicine,124C Combs Research Building,800Rose Street,Lexington,KY40536,USAc Institute of Neurobiology,Fudan University,138Yixueyuan Road,Shanghai,200032,PR ChinaReceived12August2008;received in revised form28December2008;accepted7January2009Available online23February2009AbstractThiamine pyrophosphate(TPP)and the activities of thiamine-dependent enzymes are reduced in Alzheimer’s disease(AD)patients. In this study,we analyzed the relationship between thiamine deficiency(TD)and amyloid precursor protein(APP)processing in both cellular and animal models of TD.In SH-SY5Y neuroblastoma cells overexpressing APP,TD promoted maturation of␤-site APP cleaving enzyme1(BACE1)and increased␤-secretase activity which resulted in elevated levels of␤-amyloid(A␤)as well as␤-secretase cleaved C-terminal fragment(␤-CTF).An inhibitor of␤-secretase efficiently reduced TD-induced up-regulation of A␤and␤-CTF.Importantly, thiamine supplementation reversed the TD-induced alterations.Furthermore,TD treatment caused a significant accumulation of reactive oxygen species(ROS);antioxidants suppressed ROS production and maturation of BACE1,as well as TD-induced A␤accumulation.On the other hand,exogenous A␤1–40enhanced TD-induced production of ROS.A study on mice indicated that TD also caused A␤accumulation in the brain,which was reversed by thiamine supplementation.Taken together,our study suggests that TD could enhance A␤generation by promoting␤-secretase activity,and the accumulation of A␤subsequently exacerbated TD-induced oxidative stress.©2009Elsevier Inc.All rights reserved.Keywords:␤-Amyloid peptide;␤-Secretase;Thiamine deficiency;Oxidative stress;Alzheimer’s diseaseAbbreviations:A␤,␤-amyloid peptides;AD,Alzheimer’s disease; APP,amyloid precursor protein;APPsw,Swedish mutant APP;BACE1,␤-site APP cleaving enzyme1;CM-H2DCFDA,5-(and-6-)-chloromethyl-2 ,7 -dichlorodi-hydrofluorescein diacetate acetyl ester;␤-CTF,␤-secretase cleaved C-terminal fragment;DCF,dichlorofluorescein;KGDHC,␣-ketoglutarate dehydrogenase complex;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide;PDHC,pyruvate dehydrogenase complex;PI,propidium iodide;ROS,reactive oxygen species;TD,thiamine deficiency;TD5-R,reversal of TD;TPBS,PBS with0.05%Tween-20;WKS, Wernicke–Korsakoff’s syndrome.∗Corresponding author at:Institute for Nutritional Sciences,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai, 200031,PR China.Tel.:+862154920926;fax:+862154920291.∗∗Corresponding author.Tel.:+862154920926;fax:+862154920291.E-mail addresses:jluo@(J.Luo),zjke@(Z.-J.Ke).1.IntroductionAlzheimer’s disease(AD)is the most common cause of dementia in the aged population.The major neuropatho-logical hallmarks of AD are selective neuronal loss,senile plaques,and neurofibrillary tangles.The senile plaques are composed of␤-amyloid peptides(A␤),which are derived from a two-step proteolytic processing of the amyloid pre-cursor protein(APP)by␤-secretase and␥-secretase.This processing is also called the amyloidogenic pathway(Selkoe, 2001).The BACE1(␤-site APP cleaving enzyme1),the protein of␤-secretase,cleaves APP,which produces the C99,or C-termanal fragment(␤-CTF)and releases soluble N-terminal fragment APPbeta(sAPPbeta).␤-CTF is then fur-0197-4580/$–see front matter©2009Elsevier Inc.All rights reserved. doi:10.1016/j.neurobiolaging.2009.01.005Q.Zhang et al./Neurobiology of Aging32(2011)42–5343ther cleaved by␥-secretase complex leading to A␤peptides, mainly of40or42amino acids in length,denoted as A␤40 and A␤42.Increased␤-secretase activity and elevated lev-els of insoluble A␤peptide have been noted in the brain tissue of patients with sporadic AD(Li et al.,2004).Gene mutations in APP,Presenilin1,or Presenilin2can lead to an inherited early onset form of AD,familiar AD(Citron et al.,1992;Russo et al.,2000);however,the molecular physiopathological procedure for sporadic AD is still under investigation.Oxidative stress is proposed to be involved in AD pathogenesis and/or progression(Reddy,2006;Shi and Gibson,2007).Furthermore,oxidative damage is common in many neurodegenerative diseases such as Parkinson’s disease (Onyango,2008),progressive supranuclear palsy(Albers et al.,1999),Huntington’s disease(Trushina and McMurray, 2007)and Wernicke–Korsakoff’s syndrome(WKS)(Victor et al.,1989).Thiamine(Vitamin B1)deficiency(TD)is a classic model for mild impairment of oxidative metabolism,which produces WKS in humans.Regionally selective neuronal death was found in the brains of humans and animals with TD(Ke et al.,2003;Ke and Gibson,2004;Victor et al.,1989).TD impairs the activity of thiamine-dependent enzymes,including transketolase(EC 2.2.1.1),pyruvate dehydrogenase complex(PDHC,EC1.2.4.1,EC2.3.1.12 and EC1.6.4.3),and␣-ketoglutarate dehydrogenase com-plex(KGDHC,EC1.2.4.2,EC2.3.1.61,EC1.6.4.3)that play important roles in glucose metabolism(Ke and Gibson, 2004).Subsequently,TD causes mitochondrial dysfunction, energy shortage and chronic oxidative stress.Since TD causes selective neuronal death,inflammation,glial activation and abnormalities that are common features of many aging-related neurodegenerative diseases,TD has been used to model some neurodegenerative diseases(Ke and Gibson, 2004).The concentrations of thiamine pyrophosphate and the activity of thiamine-dependent enzymes are decreased in AD brains(Heroux et al.,1996;Mastrogiacoma et al.,1996a,b). Furthermore,the phosphorylated tau level in CSF is elevated in patients with TD(Matsushita et al.,2008).Sub-clinical thiamine deficiency is frequently observed in the elderly pop-ulation(O’Keeffe,2000).However,the relationship between TD and AD remains to be elucidated.In this study,we used both cellular and animal models of TD to determine the effect of TD on APP processing. We demonstrated that TD treatment promoted the matu-ration of BACE1,as well as the accumulation of both secreted and intracellular A␤.On the other hand,exogenous A␤could exacerbate TD-induced oxidative stress.Impor-tantly,re-administration of thiamine or antioxidant treatment reversed the TD-induced maturation of BACE1and accu-mulation of A␤.Taken together,these results imply that TD-induced oxidative stress promotes the amyloidogenic APP processing and A␤accumulation through regulating BACE1maturation,and elevated A␤may further enhance oxidative stress.2.Materials and methods2.1.Chemical and reagentsAll chemical reagents were purchased from Sigma Chem-ical Co.(St.Louis,MO,USA)unless otherwise mentioned. Annexin-V FITC Apoptosis Detection Kit and␤-Secretase Activity Assay Kit was purchased from Biovision Inc. (Mountain View,CA,USA).␥-Secretase Activity Assay Kit was purchased from R&D Systems Inc.(R&D Systems, Minneapolis,MN,USA).Human or mouse amyloid1–40and A␤1–42Colorimetric Immunoassay Kits were ordered from Biosource International Inc.(Camarillo CA,USA).5-(and-6-)-chloromethyl-2 ,7 -dichlorodi-hydrofluorescein diacetate acetyl ester(CM-H2DCFDA)was obtained from Molecu-lar Probes(Eugene,OR,USA).Trolox,␤-secretase inhibitor IV and␥-secretase inhibitor IX(DAPT)were purchased from Calbiochem(San Diego,CA,USA).Anti-Alzheimer precursor protein A4monoclonal antibody(APP22c11)was purchased from Chemicon International Inc.(Temecula, CA,USA),anti-BACE1antibody was purchased from Cal-biochem Inc.(San Diego,CA,USA),anti-A␤monoclonal antibody(6E10)was purchased from Signet Laboratories Inc. (Signet Labs,Dedham,MA,USA)and anti-␤-actin antibody was purchased from Sigma Chemical Co.(St.Louis,MO, USA).Synthetic A␤1–40and A␤40–1were purchased from Sigma Chemical Co.(St.Louis,MO,USA).2.2.Cell culturesHuman neuroblastoma SH-SY5Y cells stably expressing Swedish mutant APP(APPsw)gene were a generous gift from Dr.Weihong Song(University of British Columbia, Canada).Cells were maintained in Dulbecco’s modified Eagle’s medium(DMEM,Invitrogen)supplemented with 10%fetal bovine serum(Hyclone),100␮g/mL streptomycin sulphates and100U/mL penicillin G(Gibco).To ensure APPsw gene expression,zeocin TM(Invitrogen)was added to the culture medium at afinal concentration of100␮g/mL. The thiamine deficiency DMEM medium was obtained from JRH Biosciences Inc.(Lenexa,Kansas,USA).2.3.Induction of TD and drug treatmentWe used both the thiamine deficiency medium and a thi-amine antagonist pyrithiamine to induce thiamine deficiency in vitro as previously described(Wang et al.,2000).Pyrithi-amine is an analog of thiamine,which inhibits thiamine metabolism and induces neurological symptoms similar to that of WKS in animals(Liu et al.,2006).Three hundred nanomoles per liter of pyrithiamine was added to the thiamine deficiency medium.To measure␤-CTF levels,␥-secretase inhibitor IX(DAPT,final2␮mol/L)was added to the culture medium24h prior to harvesting cells.The animal TD model has been previously described(Ke et al.,2003).Briefly,adult male C57BL/6J mice(20–25g)44Q.Zhang et al./Neurobiology of Aging 32(2011)42–53Fig.1.Effect of thiamine deficiency on cell viability.(A)Images of SH-SY5Y cells over-expressing APPsw in four kinds of culture media.Cells were grown in different culture media for specified times.CM:control medium;CM +Py:control medium +thiamine antagonist pyrithiamine;TDM:thiamine deficient medium;TDM +Py:thiamine deficient medium +thiamine antagonist pyrithiamine.(B)TD-induced apoptosis of SH-SY5Y cells over-expressing APPsw.Cells were grown in different media for 5days.Apoptosis was determined by Annexin-V staining as described under the Section 2.(C)TD-induced reduction of cell viability.Cells were grown in different media for 5days and cell viability was determined by MTT assay as described under the Section 2.The experiments were replicated three times.*P <0.05;**P <0.01,compared with CM group.were housed in a controlled environment (at 23◦C and 53%humidity).The animals were fed with either a control diet or a thiamine deficient diet (ICN Nutrition Biomedicals,Cleve-land,OH,USA)ad libitum .TD animals also received a daily i.p.injection of pyrithiamine hydrobromide (5␮g/10g body weight,Sigma Chemical Co.),while control animals were injected with saline.2.4.Cell growth and death assayIn the initial study,four experimental groups were included:control group (DMEM with thiamine 4mg/L),control +pyrithiamine (Py)group (DMEM with thiamine 4mg/L plus 300nmol/L pyrithiamine),thiamine deficiency medium (TDM)group (thiamine deficiency DMEM medium)and TDM +Py group (thiamine deficiency DMEM medium plus 300nmol/L pyrithiamine).Since TDM +Py group displayed most severe cell damage (Fig.1),this treat-ment was selected as TD for the rest of the studies,and DMEM containing 4mg/L thiamine served as a con-trol.Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)method as previously described (Chen et al.,2004).TD-induced alter-ations in cell viability were expressed as a percentage of the control.Apoptotic cell death was determined using an Annexin-V FITC apoptosis detection kit (BioVision,Moun-tain View,CA)as previously described (Wang et al.,2007).Briefly,2.0×105cells were resuspended in 0.5ml binding buffer and incubated with Annexin-V FITC and propidium iodide (PI)for 5min in the dark at room temperature.FAC-Scan cytometer (BD Biosciences)equipped with a FITC signal detector FL1(excitation 488nm,green)and a phy-coerythrin emission signal detector FL2(excitation 585nm,red)was used to analyze the Annexin-V binding.The results were expressed as the percentage of apoptotic cells calculated from the total (2×104)using the Cellquest pro-gram.Q.Zhang et al./Neurobiology of Aging32(2011)42–53452.5.AβELISAThe levels of both secreted A␤and intracellular A␤were simultaneously quantified in SH-SY5Y cells stably expressing APPsw using a commercial ELISA assay kit (Human Amyloid1–40and A␤1–42Colorimetric Immunoas-say Kit)from Biosource International;the manufacturer’s instructions were followed.The cells and culture mediums were collected for ELISA assay.Briefly,an equal amount of cells was plated into60mm culture dishes;the culture medium was changed after24h and TD was induced as described above.At specified times following TD induc-tion,cell lysate and culture medium were collected to detect intracellular and secreted A␤by ELISA,respectively.To determine the involvement of␤-secretase in TD-induced A␤generation,a␤-secretase inhibitor IV(1␮mol/L)was added to the culture medium for the last24h during TD treatment.To investigate the effect of antioxidants on TD, 200␮mol/L Trolox,a water-soluble derivative of vitamin E,was added to the culture medium at the onset of TD. After5days of treatment,the cell lysate and culture medi-ums were collected for A␤assay.Reversal of TD(TD5-R) was achieved by replacing the TD medium with a con-trol medium after5-day TD treatment.For the TD reversal experiment in the mouse model,the mice were given the control diet instead of the TD diet after8days TD intro-duction.These mice were sacrificed after9days of TD treatment.The procedure for animal surgery was performed in accordance with the Guideline of Animal Care and Use Committee of the Institute for Nutritional Sciences.The mice were deeply anesthetized by i.p.injection of chlo-ral hydrate(350mg/kg),then briefly perfused with saline. Selected regions of the brain were rapidly dissected on ice, weighed,and immersed in liquid nitrogen.Brain samples were stored at−70◦C until use.Tissue samples were homog-enized with0.2%diethylamine in50mM NaCl and then ultracentrifuged at100,000×g for1h at4◦C.The super-natants containing intracellular A␤were neutralized with an addition of0.1volume0.5mol/L Tris–Cl(pH6.8),and stored at−70◦C until the ELISA detection of A␤.Tissue fromfive mice were mixed and subject to A␤ELISA using Mouse Amyloid1–40and A␤1–42Colorimetric Immunoassay Kit (Biosource Inc.).2.6.ImmunoblottingThe procedure for immunoblotting has been previously described(Chen et al.,2004).Briefly,aliquots of cyto-plasmic proteins(40␮g)were loaded into the lanes of a sodium dodecyl sulfate polyacrylamide gel.The proteins were separated by electrophoresis and transferred to nitrocel-lulose membranes(0.45or0.22␮m,Schleicher&Schuell, Boston,MA,USA).The membranes were blocked with5% nonfat dry milk in0.01M PBS(pH7.4)and0.05%Tween-20(TPBS)at room temperature for1h.Subsequently,the membrane was incubated with primary antibodies directed against target proteins overnight at4◦C.Thefinal dilutions for primary antibodies were:APP22c11,1:1,000;BACE1, 1:20,000;A␤6E10,1:600and␤-actin,1:10,000.After two quick washes in TPBS,the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (Amersham,Arlington Heights,IL,USA)diluted at1:5000 in TPBS for1h.The immuno-complexes were detected by the enhanced chemiluminescence method(Amersham).The density of immunoblotting was quantified with the soft-ware of Quantity One(Bio-Rad Laboratories,Hercules,CA, USA).2.7.β-andγ-secretase activityActivity of␤-secretase and␥-secretase was determined using commercial kits according to the manufacturer’s instructions.(Biovision Inc.,Mountain View,CA,USA). Briefly,the cells were washed twice with PBS,and65␮L extraction buffer was added to the dish.After10min incu-bation on ice,the extract was centrifuged at10,000×g for 5min.Fifty microliters of supernatant was mixed with an equal volume of2×reaction buffer and2microliters sub-strate.The plates were kept in the dark at37◦C for1h,and thefluorescence was recorded at FlexStation384(Molecular Devices,Sunnyvale,CA,USA).The protein concentrations were quantified by BCA method(Pierce)and an equal amount of cellular protein was used for measuring␤-or␥-secretase activity.2.8.ROS assayROS levels were monitored by thefluorescent dye5-(and-6-)-chloromethyl-2,7-dichlorodi-hydrofluorescein diacetate acetyl ester(H2DCFDA)(Molecular Probe)as previously described(Chen et al.,2008).Oxidation of H2DCFDA by peroxides yielded thefluorescent derivative dichlorofluo-rescein(DCF).Briefly,cells were incubated with10␮M H2DCFDA in PBS for30min at37◦C.Cells were collected by centrifugation and washed three times.Intracellular ROS levels(DCF signals)were measured with aflow cytometer (FACSAriaTM,BD Biosciences,San Jose,CA,USA)atλex of488nm andλem of520nm.Data acquisition and pro-cessing were performed using the Cellquest program(BD Biosciences).2.9.Statistical analysisAnalysis of variance(ANOV A)was performed,fol-lowed by the Newman–Keuls multiple comparison test using the software Prism(GraphPad software,Inc. La Jolla,CA).All the data were expressed as the mean±S.E.M.,where p<0.05was considered statistically significant.46Q.Zhang et al./Neurobiology of Aging 32(2011)42–53Fig.2.TD-induced A ␤accumulation in SH-SY5Y cells over-expressing APPsw.Secreted A ␤40(A)and A ␤42(B)in the culture medium and intracellular A ␤40(C)and A ␤42(D)in cell lysates were determined by ELISA as described under the Section 2.For thiamine reversal experiment (5d-R),cells were grown in TD medium for 5days and then switched to control medium containing 4mg/L thiamine for 24h.The experiments were replicated three times.*P <0.05;**P <0.01;***P <0.001,compared with control group (Ctrl);#P <0.05,##P <0.01;###P <0.001compared with TD 5d group.3.Results3.1.TD causes death of SH-SY5Y cells overexpressing APPswTD causes regionally selective neuron death in mouse brain (Gibson et al.,1999),and apoptotic cell death in human neuroblastoma cells,SH-SY5Y (Wang et al.,2000).We demonstrated that treatment with thiamine deficient medium (TDM)plus thiamine antagonist,pyrithiamine (Py),induced cell death in SH-SY5Y cells overexpressing APPsw after 7days culture (Fig.1A).The apoptosis assay revealed that approximately 30%of the cells underwent apoptosis (Annexin-V positive)after 5days cultured in TDM plus Py,compared to 8.8%apoptotic cells in control groups (Fig.1B).MTT assay indicated that cell viability was significantly reduced in the TDM group and TDM +Py group after 5days in culture (Fig.1C).In parental SH-SY5Y cells,there were about 20%Annexin-V positive cells after 5days TD treatment (data not shown).Thus,the APPsw overexpres-sion increased the sensitivity of SH-SY5Y cells to thiamine deficiency.Five days of TD was selected to investigate the change of APP processing during TD.3.2.TD induces A βaccumulation in vitroThe concentration of A ␤is very low in cultured neu-rons and SH-SY5Y cells.In this study,SH-SY5Y cells overexpressing APPsw were employed to better quantify the level of A ␤.TD significantly increased the accu-mulation of A ␤in both the culture medium (secreted)and the cellular extract (intracellular)of SH-SY5Y cells overexpressing APPsw (Fig.2).After 5days of TD treatment (TD5),the concentrations of secreted and intra-cellular A ␤40were increased from 5.52±0.12pg/mg and 23.4±5.17pg/mg to 18.57±2.31pg/mg and 86.03±8.19pg/mg,respectively (Fig.2A and C).The levels of secreted and intracellular A ␤42were increased from 0.66±0.23pg/mg and 7.06±1.03pg/mg to 3.85±0.52pg/mg and 23.52±1.02pg/mg,respectivelyQ.Zhang et al./Neurobiology of Aging32(2011)42–5347Fig.3.TD-induced accumulation of A␤in the brains of C57BL/6J mice.Mice were fed either a control or TD diet for specified times.The thalamus(A and B)and hippocampus(C and D)were dissected and assayed for A␤40(A and C)and A␤42(B and D)production.For thiamine reversal experiment(8d-R),the mice were TD-induced for8days,and then switched to the control diet for24h.*P<0.05;**P<0.01;***P<0.001,compared with control group(Ctrl).(Fig.2B and D).To determine whether the increase of A␤caused by TD is reversible,we added thiamine back to the medium after5days of TD treatment.Both secreted and intracellular A␤were measured24h after thiamine re-administration.As shown in Fig.2,re-administration of thiamine reversed the TD-induced alterations in A␤production.3.3.TD induces Aβaccumulation in vivoThe effect of TD on the accumulation of A␤in the thalamus and hippocampus was examined.As shown in Fig.3,TD dramatically increased both A␤40and A␤42in the thalamus after8days of TD;the increase peaked on day10after TD.However,the increase in the hippocam-pus was quite modest.This is consistent with the fact that the thalamus is the most vulnerable region affected by TD. TD-induced accumulation of A␤was partially reversed by feeding animals a diet containing thiamine after8days of TD(Fig.3).3.4.TD decreases the mature APP and increases the activity ofβ-secretaseTo determine whether A␤accumulation was caused by changes in APP protein level,we examined APP expres-sion in SH-SY5Y cells overexpressing APPsw.TD treatment decreased the mature form of APP(Fig.4A and B).We fur-ther investigated the effect of TD on the activity of␤-secretase and␥-secretase,the enzymes involved in the amyloidogenic pathway.The activity of␤-secretase elevated after3days of TD and doubled on TD5(Fig.4C).TD-induced increase of ␤-secretase activity was inhibited with the supplementation of thiamine(Fig.4C).However,there was no statistically significant change in the activity of␥-secretase(Fig.4D).3.5.TD promotes the maturation of BACE1To further understand TD’s action,we examined the effect of TD on the protein levels of BACE1and␤-CTF(a product of␤-secretase cleavage).As shown in Fig.5,the protein lev-els of the mature form of BACE1increased after3days of48Q.Zhang et al./Neurobiology of Aging32(2011)42–53Fig.4.TD decreased mature APP and increased the activity of␤-secretase.(A)APP expression in SH-SY5Y cells over-expressing APPsw was determined by immunoblotting after the indicated time of TD.(B)The expression of mature APP was quantified and normalized to␤-actin expression as described under the Section2.The activities of␤-secretase(C)and␥-secretase(D)were determined by the method in Section2.For thiamine reversal experiment(5d-R),cells were grown in TD medium for5days and then switched to control medium containing4mg/L thiamine for24h.The experiments were replicated three times. *P<0.05;**P<0.01,compared with Ctrl group;#P<0.05,compared with TD5days group.Fig.5.TD-induced alterations of BACE1expression.(A)Expression of BACE1and␤-CTF(the product of␤-secretase cleavage)were determined by immunoblotting.Both mature(larger molecular weight)and immature forms of BACE1were detected.The product of␤-secretase cleavage(␤-CTF)and ␣-secretase cleavage(␣-CTF)were also detected.Mature BACE1(B),immature BACE1(C),and␤-CTF were quantified and normalized to␤-actin expression as described under the Section2.For thiamine reversal experiment(5d-R),cells were grown in TD medium for5days and then switched to control medium containing4mg/L thiamine for24h.The experiments were replicated three times.*P<0.05;**P<0.01;***P<0.001,compared with Ctrl group;#P<0.05, ##P<0.01compared with TD5days group.Q.Zhang et al./Neurobiology of Aging32(2011)42–5349Fig.6.Effect of␤-secretase inhibitor on TD-induced increase of A␤.Cells were grown in TD medium for5days and then treated with␤-secretase inhibitor IV(2␮mol/L)for24h.A␤40(A)and A␤42(B)in the culture medium(secreted)and the cellular extract(intracellular)were determined by ELISA as described under the Section2.The CTFs were detected by immunoblotting(C).The experiments were replicated three times.*P<0.05;***P<0.001,compared with TD5days group.TD,while the immature form of BACE1decreased.Quan-titative analysis showed that the mature form of BACE1 increased by approximately100%while the immature form of BACE1decreased by50%on TD5(Fig.5B and C).Simi-larly,the levels of␤-CTF were significantly increased on TD5 (Fig.5A and D).TD-induced increase of BACE1maturation and␤-CTF production were inhibited by the supplementa-tion of thiamine(Fig.5).A real-time PCR study indicated that there was no significant alteration of BACE1mRNA during TD(data not shown).Therefore,TD increased the activity of␤-secretase through promoting the maturation of BACE1.To verify that TD-induced A␤production resulted from BACE1activation,we examined the levels of A␤in the presence of␤-secretase inhibitor IV.As shown in Fig.6,␤-secretase inhibitor IV significantly decreased TD-mediated up-regulation of both the secreted and intracellular A␤as well as␤-CTF.3.6.Antioxidant reduces TD-induced AβaccumulationSince TD-induced neuronal loss accompanies a mild and chronic impairment of oxidative metabolism(Ke and Gibson, 2004),we sought to determine if oxidative stress plays a role in TD-induced A␤accumulation.As shown in Fig.7A,TD significantly increased reactive oxygen species(ROS)pro-duction in SH-SY5Y cells expressing APPsw;TD causeda Fig.7.Effect of antioxidants on TD-induced ROS production.(A)SH-SY5Y cells over-expressing APPsw were TD-induced for specified times.Intracellular ROS accumulation was determined as described under the Section2.(B)SH-SY5Y cells over-expressing APPsw were TD-induced with/without Trolox (200␮mol/L)for5days.Intracellular ROS accumulation was measured.The experiments were replicated three times.*P<0.05;***P<0.001,compared with Ctrl group;###P<0.001compared with TD5days group.50Q.Zhang et al./Neurobiology of Aging 32(2011)42–53Fig.8.The effect of Trolox on TD-induced accumulation of both secreted and intracellular A ␤.A ␤40(A and C)and A ␤42(B and D)in the culture medium (secreted)(A and B)and the cellular extract (intracellular)(C and D)were determined by ELISA as described under the Section 2.SH-SY5Y cells over-expressing APPsw were TD-treated with/without Trolox (200␮mol/L)for 5days.The experiments were replicated three times.**P <0.01;***P <0.001,compared with control group (Ctrl);#P <0.05,##P <0.01compared between TD-treated with/without Trolox after 5days.threefold increase in intracellular ROS accumulation after 5days of TD.The antioxidant Trolox,a water-soluble deriva-tive of vitamin E,significantly inhibited TD-induced ROS production (Fig.7B).We further examined the effect of Trolox on A ␤accumulation and APP processing.As shown in Fig.8,Trolox significantly inhibited TD-induced accumula-tion of both secreted and intracellular A ␤.Meanwhile,Trolox blocked TD-mediated BACE1maturation and suppressed the ␤-secretase activity (Fig.9).These results suggested that oxidative stress played an important role in TD-induced mat-uration of BACE1and A ␤production.3.7.Exogenous A βenhances TD-induced ROS productionThe neurotoxicity of synthetic A ␤has been well estab-lished in vitro ,and oxidative stress is proposed as animportant mechanism underlying A ␤toxicity (Hensley et al.,1994;Keller et al.,1997;Mattson,1995;Pereira et al.,1999;Reddy,2006).To determine whether A ␤could modulate TD-induced ROS accumulation,we examined the effect of A ␤at a physiologically relevant concentration (0.25nmol/L)on TD-induced ROS production.As shown in Fig.10,A ␤1–40but not A ␤40–1significantly promoted TD-induced ROS production.These results suggested that A ␤accumulation could exacerbate the oxidative stress induced by TD.4.DiscussionWe demonstrate that TD elevates A ␤production in both cultured neuronal cells and animal models.This finding is consistent with a recent study showing that TD enhancesQ.Zhang et al./Neurobiology of Aging32(2011)42–5351Fig.9.The effect of Trolox on TD-mediated BACE1maturation and suppression of␤-secretase activity.SH-SY5Y cells over-expressing APPsw were TD-treated with/without Trolox(200␮mol/L)for5days.(A)The activity of␤-secretase was determined by the method in the Section2.(B)Expression of BACE1,␤-CTF and␣-CTF were determined by immunoblotting.Mature BACE1(C)and immature BACE1(D)were quantified and normalized to␤-actin expression as described under the Section2.The experiments were replicated three times.*P<0.05;**P<0.01;***P<0.001,compared with control group(Ctrl);##P<0.01, ###P<0.001,compared between TD-treated with/without Trolox after5days.A␤plaque formation in a transgenic mouse AD model(Karuppagounder et al.,2008).Accumulation of A␤in the brain is a hallmark of AD.Several processes may be involved in A␤accumulation inthe brain.These include increased production of amyloid pre-cursor protein(APP),accelerated APP proteolyticprocessingFig.10.Effect of soluble synthetic A␤on TD-induced ROS production. A␤1–40or A␤40–1(0.25nmol/L)was added to the culture medium at the onset of TD.After incubation for5days,ROS production was measured as described under the Section2.TD-induced ROS production without A␤treatment was arbitrarily designated as one.The experiment was repli-cated three times.**P<0.01compared with TD5days group without A␤treatment.or inefficient clearance of A␤.A␤is generated by sequential proteolytic processing of APP;the activity of␤-secretase and its functional component,BACE1,is critical for the proteol-ysis of APP and acts as a rate-limiting step for the generation of A␤.APP is cleaved by␤-secretase(BACE1)to produce a secreted form of APP and a99-residue membrane-associated fragment(␤-CTF).␤-CTF is further cleaved by␥-secretase to generate A␤and APP intracellular domain fragments.Our results reveal that TD promotes the maturation of BACE1and increases␤-secretase activity,which results in more A␤pro-duction.The conclusion is further supported by thefindings that TD does not affect␥-secretase activity and that a␤-secretase inhibitor effectively blocks TD-induced alterations in A␤levels.Importantly,TD-induced A␤production and the maturation of BACE1is reversible upon supplementation with thiamine.It is noted that TD-induced changes in the thalamus are much more drastic compared to the hippocampal for-mation.This is consistent with the regional specificity of TD-induced brain damage(Calingasan et al.,1999;Ke et al.,2003;Karuppagounder et al.,2008;Navarro et al.,2008; Savage et al.,2007;Todd and Butterworth,1999;Zuccoli et al.,2008.).The thalamus,especially the submedial thalamic nuclei(SmTN),is most sensitive to TD-induced damage.For example,selective neuronal death occurred in SmTN after8 days of TD in mice,while the neurons in hippocampus and cortex are spared after11days of TD(Calingasan et al.,1999; Ke et al.,2003).It appears that the severity of TD-induced。