Research Article Glycogen Synthase Kinase-3...

Research ArticleGlycogen Synthase Kinase-3 Regulates Production of Amyloid-120573Peptides and Tau Phosphorylation in Diabetic Rat Brain

Zhong-Sen Qu1 Liang Li2 Xiao-Jiang Sun1 Yu-Wu Zhao1 Jin Zhang1 Zhi Geng1

Jian-Liang Fu1 and Qing-Guo Ren3

1 Department of Neurology The Sixth Peoplersquos Hospital Affiliated Shanghai Jiao Tong University Yi Shan Road 600Shanghai 200233 China

2Department of Neurology The Central Hospital Affiliated Qingdao University Qingdao 266042 China3Department of Pathophysiology Key Laboratory of Neurological Diseases of Hubei Province Tongji Medical CollegeHuazhong University of Science and Technology Wuhan 430030 China

Correspondence should be addressed to Zhong-Sen Qu qvzhsen126com

Received 23 December 2013 Accepted 25 January 2014 Published 3 April 2014

Academic Editors T Arendt T Pannicke and U Tan

Copyright copy 2014 Zhong-Sen Qu et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The pathogenesis of diabetic neurological complications is not fully understood Diabetes mellitus (DM) and Alzheimerrsquos disease(AD) are characterized by amyloid deposits Glycogen synthase kinase-3 (GSK-3) plays an important role in the pathogenesis of ADand DM Here we tried to investigate the production of amyloid-120573 peptides (A120573) and phosphorylation of microtubule-associatedprotein tau in DM rats and elucidate the role of GSK-3 and Akt (protein kinase B PKB) in these processes Streptozotocin injection-induced DM rats displayed an increased GSK-3 activity decreased activity and expression of Akt And A12057340 and A12057342 were foundoverproduced and the microtubule-associated protein tau was hyperphosphorylated in the hippocampus Furthermore selectiveinhibition ofGSK-3 by lithium could attenuate the conditions of A120573 overproduction and tau hyperphosphorylation Taken togetherour studies suggest that GSK-3 regulates both the production of A120573 and the phosphorylation of tau in rat brain and may thereforecontribute to DM caused AD-like neurological defects

1 Introduction

Alzheimerrsquos disease (AD) is characterized by the presence oftwo pathological protein deposits extracellular senile plaques(SP) and intracellular neurofibrillary tangles (NFTs) Theformer is composed of 120573-amyloid (A120573) [1] and the latter isformed by bundles of paired helical filaments (PHFs) whicharemainly constituted by abnormal hyperphosphorylated tauprotein [2] Several protein kinases may take part in thesepathological processes including cyclin-dependent kinase 5(cdk-5) GSK-3 and protein kinase A (PKA) Activated GSK-3 increases the production of A120573 peptides by promotingof 120574-secretase activity and induces the aggregation anddeposition of A120573 [3] On the other hand GSK-3 regulatestau hyperphosphorylation at ser198ser199ser202 sites andser396ser404 sites [4] Furthermore as a downregulator ofinsulin signaling GSK-3 is regulated by Akt [5]

The available epidemiological data are largely inconclu-sive with regard to the contribution of diabetes mellitus tocognitive impairment andAD-type neurodegeneration [6 7]Both diabetes and AD are characterized by localized amyloiddeposits that progress during the course of the diseases [8]In hippocampal neurons of diabetic mice the stains of A12057340and A12057342 are increased but the expression of GSK-3 isweakened by immunohistochemistry [9] In AD-like Tg2576mice diet-induced insulin resistance promotes A12057340 andA12057342 generation in the brain Further study suggest that PI3-kinasePS473-AktPKB is reduced in these brains and suggestthat GSK-3 as a downsteam kinase of these kinases may affectthe production of A120573 [10] In the skeletal muscle of type IIdiabetes mellitus patients GSK-3 activity and its expressionlevel are significantly higher [11] Increased GSK-3 activityis also found in the development of insulin resistance and

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 878123 8 pageshttpdxdoiorg1011552014878123

2 The Scientific World Journal

type II diabetes fat tissue of C57BL6J mice [12] Immuno-histochemical results show that ser198ser199ser202 sites arehyperphosphorylated in the hippocampus of diabetic micebut the expression of GSK-3 is reduced [13] In the brainof insulin knockout mice hyperphosphorylation of tau atthreonine 231 and of the neurofilament is exhibited but GSK-3120573 activity was inhibited [14]

All of the above evidences imply that GSK-3 may belinked to both A120573 production and tau hyperphosphorylationduring the course of diabetes To better understand themechanisms of AD-like changes in diabetic rat brain weinvestigated the activity of GSK-3 and Akt and the expressionof Akt then A120573 production and tau phosphorylation wasdetermined Furthermore the role of GSK-3 was explored byusing LiCl as a specific inhibitor [15]

2 Materials and Methods

21 Antibodies and Chemicals The primary A120573 antibodiesG2-10 (capture antibody for A120573

40) G2-11 (capture antibody

for A12057342) and WO

2(detection antibody) used for ELISA

were purchased from A120573eta Company (Germany) and stan-dard synthetic A120573 peptides were obtained from Sigma (StLouis MO USA) Antibodies against phospho-Ser473 Aktand total Akt were purchased fromCell Signaling technology(Beverly MA USA) Monoclonal antibodies PHF-1 whichlabels tau phosphorylated at Ser-396 andor Ser-404 andTau-1 which labels tau where neither serines 198 199 or 202are phosphorylated were gifts from Dr Peter Davies (AlbertEinstein College of Medicine Bronx NY USA) and DrLester Binder (Northwestern University Chicago IL USA)respectively Polyclonal antibody 111e which reacts with thetotal tau was a kind gift fromDrs Iqbal K andGrundke-IqbalI (New York State Institute for Basic Research Staten IslandNY USA) Phospho-GS peptide a specific GSK-3 substratewas obtained from Upstate Biotechnology Inc (Lake PlacidNY USA) Bicinchoninic acid (BCA) protein detection kitand TMB were obtained from Pierce (Rockford IL USA)[120574-32P] ATP was obtained from Beijing Yahui Biologic andMedicinal Engineering Co (Beijing China) Goat anti-rabbit or goat anti-mouse peroxidase-conjugated secondaryantibodies and phosphocellulose paper were obtained fromPierce Chemical Co (Rockford IL USA) Polyclonal andmonoclonal Histostain-SP kits were obtained from ZymedLaboratories Inc (South San Francisco CA USA) Strepto-zotocin (STZ) diaminobenzidine LiCl (a specific inhibitorof GSK-3) and other chemicals were purchased from Sigma(St Louis MO USA)

22 Establishment of DM Rat Model Four-month-oldSprague-Dawley rats (Grade II male weight 200ndash250 gsupplied by Experimental Animal Center of Tongji MedicalCollege) were divided into 4 groups control group diabetesmellitus (DM) group DM plus NaCl group and DMplus LiCl group by random Except for control rats theother rats received intraperitoneal injection with 55mgkgstreptozotocin (STZ) The DM rats were determined byfasting blood glucose ge167mmol72 h after streptozotocin

injection DM plus NaCl group 24 h following STZinjection the rats were administered by intraperitonealinjection 04mL of 03M NaCl for 10 days DM plus LiClgroup 24 h following STZ injection the rats were injectedintraperitoneally with 400 120583L of 03M LiCl for 10 days

23 Preparation of Rat Hippocampal Extracts Followingcontinuous injection of NaCl or LiCl for 10 days the ratswere killed The hippocampus was immediately removedand homogenized at 4∘C using a Teflon glass homogenizerin 50mmol Tris-HCl pH 74 150mmol NaCl 10mmolNaF 1mmol Na

3VO4 10mmol 120573-mercaptoethanol 5mmol

EDTA 2mmol benzamidine 10mmol phenylmethylsulfonylfluoride 5 120583gmL leupeptin 5120583gmL aprotinin and 2 120583gmLpepstatinThe tissue homogenates were then divided into twoportions One portion of each homogenate was centrifugedat 12000timesg for 20min at 4∘C and the resulting supernatantwas stored at minus80∘C for assaying activities of protein kinasesThe other portion was mixed in 2 1 (vv) ratio with lysisbuffer containing 200mmol Tris-HCl pH 76 8 SDS40 glycerol boiled for 10min in a water bath and thencentrifuged at 12000timesg for 30min and the supernatant wasstored at minus80∘C for Western blot analysis The concentrationof protein in the hippocampal extracts was measured byBCA kit according to themanufacturerrsquos instructions (PierceCheshire UK)

24 Assay of GSK-3 and Akt Activity The GSK-3 activityin rat hippocampal exacts was measured using phospho-GS peptide 2 (Upstate Lake Placid NY USA) as describedpreviously [16] Briefly tissue extracts 75120583g proteins wereincubated for 30min at 30∘C with 20120583M peptide sub-strate and 200120583mol [120574-32P] ATP (1500 cpmpmol ATP) in30mmol Tris pH 74 10mmol MgCl

2 10mmol NaF 1mmol

Na3VO4 2mmol EGTA and 10mmol 120573-mercaptoethanol

in a total volume of 25 120583L The reaction was stopped byaddition of 25120583L of 300mM119874-phosphoric acidThe reactionmixture was applied in triplicates on phosphocellulose paper(pierce) The filters were washed three times with 75mmol119874-phosphoric acid dried and counted by liquid scintillationcounter GSK-3 activities was calculated with picomolesof phosphate incorporatedmg proteinmin at 30∘C andexpressed as relative activity against control The Akt activitywas measured using histone 2B as a substrate as describedpreviously [17 18] Briefly after the immunoprecipitates werewashedwith lysis buffer and kinase buffer 40120583L kinase buffercontaining 200 120583M [120574-32P] (5 120583Ci) 100120583MATP 1 120583g120583L his-tone 2B then the samples were incubated at 30∘C for 15minand spotted onto P81 filter papers the filter papers werewashed by 75mM 119874-phosphoric acid dried and counted byliquid scintillation counter Akt activity was also expressed asrelative activity against control

25 Measurement of A120573 in Hippocampus of the Rats TheA12057340 and A12057342 in the hippocampus were measured bya Sandwich enzyme-linked immunosorbent assay (ELISA)using antibodies as described previously [19] Experimentswere performed in a 96-well plate Briefly affinity-purified

The Scientific World Journal 3

30

24

18

12

6

0

Con DM DM + Na+ DM + Li+

Seru

m g

luco

se (m

ML

)

lowastlowast

lowastlowastlowastlowast

Figure 1 The level of serum glucose of the rats 72 h followingstreptozotocin injection the levels of serum glucose in DM groupDM plus NaCl group and DM plus LiCl group increased obviouslylowastlowast

119875 lt 001 versus control group

mAb G2-10 (05120583gwell) was applied as the capture antibodyfor A120573

40 mAb G2-11 (1 120583gwell) was used as a capture

antibody for A12057342 and mAb WO

2was used as a detec-

tion antibody Neutravidin-horseradish peroxidase and TMBwere used for reporter system and absorbance values at450 nm were determined with microplate reader (TECANAustria) Levels of A120573 were expressed as a relative levelagainst control Each sample was tested in triplicate in eachexperiment

26 Evaluation of Tau Phosphorylation and Expression of AktThe phosphorylation of tau at various sites was determinedbyWestern blot as described formerly [20] For immunoblot-ting about 20 120583g of proteins were loaded in each laneProteins were separated by SDS-PAGE and transferred topolyvinylidene difluoride membranes (Amersham Pharma-cia Biotech NJ USA) After being blocked for 1 h in a solutionof 5 nonfat dry milk in TBSTween 20 membranes wereimmunoblotted using primary antibodies PHF-1 (1 500)Tau-1 (1 30000) and 111e (1 3000) at 4∘C overnight anddeveloped with alkaline phosphatase-labeled IgG (05 gmLAmersham Pharmacia NJ USA) as secondary antibodies 5-bromo-4-chloro-3-indolyl-phosphatenitro blue tetrazolium(BCIPNBT) was used as substrate [2] Detection of thephosphorylation of Akt was performed with antibodies tophospho-Ser473 Akt and total Akt (Cell Signaling technology)(dilution 1 800) immunoblots were developed using horseradish peroxidase-conjugated goat anti-rabbit IgG (1 2000)followed by detection with enhanced chemiluminescence

27 Statistical Analysis The intensity of the protein bandsfrom Western blot was analyzed by Image-Pro Plus softwareand ID Image Analysis software (KODAK USA) respec-tively The data were presented as mean plusmn SD and wereanalyzed by analysis of variance

2

25

15

1

05

0


Relat

ive a

ctiv

ity o

f GSK

-3


Figure 2 The activity of GSK-3 The activities of GSK-3 in hippo-campal extracts from rats injected with STZ STZ plus NaCl orSTZ plus LiCl were determined by using specific peptide substrateslowastlowast

119875 lt 001 versus control group 119875 lt 001 versus DM group

02

04

06

08

1

12

0


Akt

activ

ity


lowastlowast

Figure 3 The activity of Akt The activities of Akt in hippocampalextracts from rats injected with STZ STZ plus NaCl or STZ plusLiCl were determined by using specific peptide substrates lowastlowast119875 lt001 versus control group

3 Results

31 Measurement the Level of Serum Glucose in Rats 72 hafter STZ injection the fasting blood glucose (FBG) of the ratsamong DM group DM plus NaCl group and DM plus LiClgroup was over 167mmolL and it was increased obviouslyas compared with controls The fasting blood glucose of therats in DM plus LiCl group was not induced a significantdecrease when compared with DM group but there was atendency toward decrease (Figure 1)


2

25

3

35

15

1

05

0


Relat

ive l

evel

of A

beta

40

lowastlowast

lowastlowast

(a)

1

18

16

14

12

08

06

04

02

0


Relat

ive l

evel

of A

beta

42

lowast

lowast

(b)

Figure 4 Production of A12057340 (a) and A12057342 (b) generation STZ treatment increased A12057340 and A12057342 production significantly and LiClcould reverse the change induced by STZ treatment lowast119875 lt 005 and lowastlowast119875 lt 001 versus control group

119875 lt 005 and 119875 lt 001 versus DM

group

2

16

12

08

04

0


Relat

ive l

evel

of P

HF-

1

lowastlowast

(a)

12

1

08

04

06

02

0


Relat

ive l

evel

of t

au-1


(b)

09

06

12

03

0


Relat

ive l

evel

of 1

11e

(c)

Figure 5 Tau phosphorylation lowast119875 lt 005 and lowastlowast119875 lt 001 versus control group 119875 lt 005 and

119875 lt 001 versus DM group

32 Assay of the Activity of GSK-3 and Akt To reveal therole of GSK-3 and Akt during the course of diabetes activityof GSK-3 and Akt was measured (Figure 3) GSK-3 is animportant kinase in the process of A120573 production andtau hyperphosphorylation [3 4] GSK-3 was inactivated byphosphorylation of serine 9 in GSK-3120573 and serine 21 in GSK-3120572 [18] Akt appears to be the predominant kinase mediatingthis phosphorylation of GSK-3 Both brain GSK-3 and Aktcan be regulated by blood glucose in mice [18] In our studywe found the strong activity of GSK-3 and lower activityin rat hippocampus when fasting blood glucose was highlyincreased by STZ intraperitoneal injection After treating

the DM rats with LiCl GSK-3 activity was decreased 46approximately No apparent inhibition of GSK-3 activity wasobserved after treating the DM rats with NaCl (Figure 2)Therewere not obvious changes ofAkt activity after treatmentof LiCl or NaCl with DM rats These results implied thathyperglycemia induced strong activity of GSK-3 and loweractivity of Akt in rat brain LiCl could directly inhibit theactivity of GSK-3 rather than Akt

33 Assay Production of A120573 and Tau Phosphorylation and theRole of LiCl A120573 deposition is an important mechanism in


both AD and diabetes and GSK-3 plays an important role inregulation of A120573 productionThe high level of GSK-3 activityand low level of Akt activity were found in our study Toelucidate the effects of GSK-3 and Akt on A120573 productionA120573 production was determined following measurement ofGSK-3 andAkt activity As shown in Figure 4 the productionof A12057340 (Figure 4(a)) and A12057342 (Figure 4(b)) was increasedsignificantly while activation of GSK-3 and inhibition of Aktwere induced in DM group After treating the DM rats withLiCl the production of A12057340 and A120573 42 was reduced by 60and 21 respectively There was not significant reductionof A12057340 and A12057342 following NaCl treatment These datashowed that production of A12057340 and A12057342 is increased inthe hippocampus of DM rats both activation of GSK-3 andinhibition of Aktmight play an important role in this process

GSK-3 is also an important kinase in the regula-tion of tau phosphorylation When itwas activated tauwas prone to phosphorylation at ser198ser199ser202 sitesand ser396ser404 sites Hence we examined the state oftau phosphorylation in DM rat by Western blot usingphosphorylation-dependent and site-specific tau antibodiesWe found that inDMrat hippocampus the immunoreactivityof PHF-1 (detection of ser396ser404 phosphorylated sites)was increased obviously as compared with the control groupbut the staining of PHF-1 was reversed after treatment ofLiCl (Figure 5(a)) Moreover the immunoreactivity of Tau-1(detection of ser198ser199ser202 nonphosphorylated sites)was decreased in DM rat as compared with controls and LiClcould reverse this staining (Figure 5(b)) NaCl treatment didnot change the staining of PHF-1 and Tau-1 in the DM groupThe total level of tau measured by R111e was not changedsignificantly in all the four groups (Figure 5(c))

34 Assay of Akt Expression GSK-3 was inactivated by Aktby phosphorylation at serine 9 and serine 21 To test ifAkt was regulated by streptozotocin-induced hyperglycemiaand was regulated by activation of GSK-3 we examinedphosphorylation level of Akt Our results show that thephosphorylation of Akt at Ser473 site was deceased in DM rathippocampus and both LiCl and NaCl administration couldnot recover the decrease and the total level of Akt was notchanged These results suggest that GSK-3 was inhibited byLiCl instead of Akt inhibition (Figure 6)

35 Behavioral Testing To further explore the cognitiondysfunction caused by the changes of Akt andGSK-3 and Tauhyperphosphorylation in DM rats the step-down electronicinhibitory avoidance task was assessed in rats To examinewhich rats have lower ability of learning andmemory we firsttrained all rats to stay on the platform for 3min andnot to stepdown Ninety-four hours after the training latencies to step-down during training session were not significantly differentacross the groups (data are not shown because the latencyto step-down in this session was basically nonexistent) Theresults suggest significantly shorter latencies and more errortimes to step-down in DM rat when compared to controlgroups (119875 lt 005) Longer latencies and less error times wereobserved in LiCl administration to DM rat as compared with

1

12

Total-Akt

08

06

04

02

0

Con DM DM + Na+ DM + Li+Re

lativ

e lev

el o

f Akt lowastlowast


P-Ser-473-Akt

Figure 6 Akt expression lowastlowast119875 lt 001 versus control group

DM rat (119875 lt 005) There is no significant difference betweenNaCl administration to DM rats and DM rats (119875 gt 005)These results suggest that inhibition of LiCl on GSK-3 mightimprove the memory of DM rat (Figure 7)

4 Discussion

Study and memory dysfunction are the main phenomenaof central nervous system complications in type I diabetesmellitus [21] Cerebral atrophy which is characterized inAD patients is also found in young patients with type 1diabetes who are otherwise healthy [22] In experimentalanimal models an increase of stains with A12057340 and A12057342antibody is induced in DMmice hippocampus [9] Tau is alsohyperphosphorylated at ser199ser202while the expression ofGSK-3 is decreased in DMmice hippocampus [13] WhetherA120573 overproduction and tau hyperphosphorylation happensynchronously in the hippocampus ofDMrat is puzzling andthe role of GSK-3 and Akt in these processes is not clear

Both GSK-3 and Akt in mice brain could be regulated byalterations of blood glucose streptozotocin-induced hyper-glycemia brain increases Akt activity and decreases GSK-3activity which could be reversed by lowering blood glucosewith insulin administration [18] In an experimental modelrelated to sporadicAlzheimerrsquos disease after intracerebroven-tricular injection of streptozotocin for 1 month there is adecrease of GSK-3 alphabeta activity in the rat hippocam-pus [5] In our study the hyperglycemia was induced byintraperitoneal injection of rats with streptozotocin but anincrease of GSK-3 activity and a decrease of Akt activitywere induced in the rat hippocampus Hence we proposed


Erro

r tim

e (3 m

in)

4

3

2

1

0

1 2

(day)3 4

lowastlowast

(a)

200

150

100

50

0

1 2

(day)3 4

Late

ncy

(s3

min

)

lowastlowast

(b)

4

3

2

1

0

Con DM DM + NaCl DM + LiCl

Erro

r tim

e (3 m

in)

lowast

998779

(c)


200

150

250

100

50

0

Late

ncy

(s3

min

)lowast

998779

(d)

Figure 7 Step-down electronic inhibitory avoidance task lowast119875 lt 005 119875 lt 005 versus Con group 119875 lt 005 versus DM group (a) The

error time before modelingThe error time reached the similar time level after the rats were trained for 4 d lowastlowast119875 lt 001 119875 lt 001 versus 1 d

(b) The latency before modeling The latency was increased after the rats were trained for 3 d and 4 d lowastlowast119875 lt 001 119875 lt 001 versus 4 d (c)

The error time at 10 days after LiCl administration The error time increased in DM group but decreased in group with LiCl administrationfor 10 days lowast119875 lt 005

119875 lt 005 versus Con group 119875 lt 005 versus DM group (d) The latency at 10 days after LiCl administrationThe latency decreased in DM group but increased in group treated with LiCl administration for 10 days lowast119875 lt 005

119875 lt 005 versus Con

119875 lt 005 versus DM

that hyperglycemia might affect the activities of GSK-3 andAkt in DM rat brain In AD-like Tg2576 mice diet-inducedinsulin resistance promotes Abeta40 and Abeta42 peptidegeneration in the brain that corresponds with increasedgamma-secretase activities Further exploration of the appar-ent interrelationship of insulin resistance to brain amyloi-dosis reveals a functional decrease in insulin receptor- (IR-)mediated signal transduction in the brain AktPKB inhibitsglycogen synthase kinase (GSK-3 alpha) activity [3] Wealso found that there was an increase of A120573 productionwhen GSK-3 activity was increased and PKB activity wasdecreased in the hippocampus of DM rats Lithium a specificinhibitor of GSK-3120572 and GSK-3120573 reduces A120573 productionby interfering with APP cleavage at the 120574-secretase step andis found to reduce A120573 production in the mice expressingpathogenic familial Alzheimerrsquos disease [3] In our study aftertreating DM rats with LiCl GSK-3 activity was decreasedsignificantly in the hippocampus and A12057340 and A12057342 werereduced by 60 and 21 but administration of DM ratswith NaCl reduced neither the activation of GSK-3 nor the

overproduction of A12057340 and A12057342 The low level activity ofAkt maintained in LiCl administration showed that lithiumcould not inhibit Akt in DM rat brain These data suggestedthat GSK-3 played an important role in A120573 overproductionof DM rat hippocampus

On the other hand LiCl as a specific GSK-3 inhibitorhas been confirmed to block tau hyperphosphorylation eitherin culture neuron or in rat brain [23 24] The major kinasefor tau phosphorylation is GSK3120573 Smaller contributionsof GSK3120572 cdk-5 and MAPK are suggested [25] In DMmice hippocampus tau is hyperphosphorylated at ser199ser202 sites with lower GSK-3 expression [13]

In our study

we found that tau hyperphosphorylation was induced atser198ser199ser202 Ser396Ser404 sites just when the activ-ity of GSK-3 increased highly When the DM rats weretreated with LiCl GSK-3 activity decreased about 46 andhyperphosphorylation tau was reversed at ser396ser404ser198ser199ser202 sites in the hippocampusThese sites arejust the targets of GSK-3 [26] while NaCl treatment showedno apparent changes in these sites as compared with DM rats


These data also suggested that LiCl reduced tau hyperphos-phorylation at ser396ser404 ser198ser199ser202 in DMrat hippocampus by inhibition of GSK-3 rather than of Akt(PKB)

Tau hyperphosphorylation may be associated with cog-nitive impairment [27 28] Whether the changes of Aktand GSK-3 may reduce memory retention in DM rat is notsure Our results suggest that the increase of GSK-3 maybe response for the impairment of step-down inhibitoryavoidance task rather than Akt because the activities and theexpression of Akt were not significantly different among DMgroup DM + NaCl group and DM + LiCl group but otherstudies show that learning impairment and hippocampalERK and Akt inactivation are induced by scopolamine inmale Sprague-Dawley rats [29]

In conclusion our results demonstrate that GSK-3 has animportant role in the pathogenesis of diabetic neurologicalcomplications byregulation of A120573 production and tau hyper-phosphorylation and the present data suggest that GSK-3might be a key target in the therapy of central nervous systemneuropathy of diabetes mellitus

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Zhong-Sen Qu and Liang Li contributed to this work equally

Acknowledgment

This work was supported by the Grants from China Postdoc-toral Science Foundation (20060390635)

References

[1] D J Selkoe ldquoThe cell biology 120573-amyloid precursor protein andpresenilin in Alzheimerrsquos diseaserdquo Trends in Cell Biology vol 8no 11 pp 447ndash453 1998

[2] I Grundke-Iqbal K Iqbal and Y-C Tung ldquoAbnormal phos-phorylation of the microtubule-associated protein 120591 (tau) inAlzheimer cytoskeletal pathologyrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 83 no13 pp 44913ndash4917 1986

[3] C J Phiel C A Wilson V M-Y Lee and P S KleinldquoGSK-3120572 regulates production of Alzheimerrsquos disease amyloid-120573 peptidesrdquo Nature vol 423 no 6938 pp 435ndash438 2003

[4] J L Shi Y Z Jia L L Hong et al ldquoTau becomes a morefavorable substrate for GSK-3 when it is prephosphorylated byPKA in rat brainrdquoThe Journal of Biological Chemistry vol 279no 48 pp 50078ndash50088 2004

[5] M Salkovic-Petrisic F Tribl M Schmidt S Hoyer and PRiederer ldquoAlzheimer-like changes in protein kinase B andglycogen synthase kinase-3 in rat frontal cortex and hippocam-pus after damage to the insulin signalling pathwayrdquo Journal ofNeurochemistry vol 96 no 4 pp 1005ndash1015 2006

[6] W L Xu C X Qiu A Wahlin B Winblad and L FratiglionildquoDiabetes mellitus and risk of dementia in the Kungsholmenproject a 6-year follow-up studyrdquo Neurology vol 63 no 7 pp1181ndash1186 2004

[7] A Ott R P Stolk F van Harskamp H A Pols A Hofman andM M Breteler ldquoDiabetes mellitus and the risk of dementia theRotterdam studyrdquo Neurology vol 53 no 9 pp 1937ndash1942 1999

[8] M R Nicolls ldquoThe clinical and biological relationship betweenType II diabetes mellitus and Alzheimerrsquos diseaserdquo CurrentAlzheimer Research vol 1 no 1 pp 47ndash54 2004

[9] S Shuli Z Yongmei Z Zhijuan and J Zhiwei ldquo120573-Amyloid andits binding protein in the hippocampus of diabetic mice effectof APP17 peptiderdquo NeuroReport vol 12 no 15 pp 3317ndash33192001

[10] L Ho W Qin P N Pompl et al ldquoDiet-induced insulinresistance promotes amyloidosis in a transgenic mouse modelof Alzheimerrsquos diseaserdquo The FASEB Journal vol 18 no 7 pp902ndash904 2004

[11] O Kaidanovich and H Eldar-Finkelman ldquoThe role of glycogensynthase kinase-3 in insulin resistance and Type 2 diabetesrdquoExpert Opinion onTherapeutic Targets vol 6 no 5 pp 555ndash5612002

[12] H Eldar-Finkelman S A Schreyer M M Shinohara R CLeBoeuf and E G Krebs ldquoIncreased glycogen synthase kinase-3 activity in diabetes- and obesity- prone C57BL6J micerdquoDiabetes vol 48 no 8 pp 1662ndash1666 1999

[13] Y-M Zhao J-J Pei Z-J Ji Z-W Zhao Y-Y Qian and S-LSheng ldquoEffect of amyloid precursor protein 17mer peptide onmicrotubule structure and tau protein hyperphosphorylation inhippocampal neurons of experimental diabetic micerdquoNeuroRe-port vol 14 no 1 pp 61ndash66 2003

[14] R Schechter D Beju and K E Miller ldquoThe effect of insulindeficiency on tau and neurofilament in the insulin knockoutmouserdquo Biochemical and Biophysical Research Communicationsvol 334 no 4 pp 979ndash986 2005

[15] V Stambolic L Ruel and J R Woodgett ldquoLithium inhibitsglycogen synthase kinase-3 activity and mimics wingless sig-nalling in intact cellsrdquo Current Biology vol 6 no 12 pp 1664ndash1668 1996

[16] T Tanaka J Zhong K Iqbal E Trenkner and I Grundke-IqballdquoThe regulation of phosphorylation of 120591 in SY5Y neuroblastomacells the role of protein phosphatasesrdquo The FEBS Letters vol426 no 2 pp 248ndash254 1998

[17] A Jacob D Cooney S Tridandapani T Kelley and K MCoggeshall ldquoFc120574RIIb modulation of surface immunoglobulin-induced Akt activation in murine B cellsrdquo The Journal ofBiological Chemistry vol 274 no 19 pp 13704ndash13710 1999

[18] B Clodfelder-Miller P de Sarno A A Zmijewska L Song andR S Jope ldquoPhysiological and pathological changes in glucoseregulate brain Akt and glycogen synthase kinase-3rdquoThe Journalof Biological Chemistry vol 280 no 48 pp 39723ndash39731 2005

[19] Y-C Zhang Z-F Wang Q Wang Y-P Wang and J-Z WangldquoMelatonin attenuates 120573-amyloid-induced inhibition of neu-rofilament expressionrdquo Acta Pharmacologica Sinica vol 25 no4 pp 447ndash451 2004

[20] M Bennecib C-X Gong I Grundke-Iqbal and K Iqbal ldquoRoleof protein phosphatase-2A and -1 in the regulation of GSK-3cdk5 and cdc2 and the phosphorylation of tau in rat forebrainrdquoThe FEBS Letters vol 485 no 1 pp 87ndash93 2000

[21] A D Mooradian ldquoPathophysiology of central nervous systemcomplications in diabetes mellitusrdquo Clinical Neuroscience vol4 no 6 pp 322ndash326 1997


[22] G J Biessels L P van der Heide A Kamal R L A W Bleysand W H Gispen ldquoAgeing and diabetes implications for brainfunctionrdquo European Journal of Pharmacology vol 441 no 1-2pp 1ndash14 2002

[23] J R Munoz-Montano F J Moreno J Avila and J Dıaz-Nido ldquoLithium inhibits Alzheimerrsquos disease-like tau proteinphosphorylation in neuronsrdquo FEBS Letters vol 411 no 2-3 pp183ndash188 1997

[24] M Hong D C R Chen P S Klein and V M-Y Lee ldquoLithiumreduces tau phosphorylation by inhibition of glycogen synthasekinase-3rdquo The Journal of Biological Chemistry vol 272 no 40pp 25326ndash25332 1997

[25] Y Tomidokoro K Ishiguro Y Harigaya et al ldquoA120573 amyloidosisinduces the initial stage of tau accumulation in APPSw micerdquoNeuroscience Letters vol 299 no 3 pp 169ndash172 2001

[26] T Li and H K Paudel ldquoGlycogen synthase kinase 3120573 phos-phorylates Alzheimerrsquos disease-specific Ser396 of microtubule-associated protein tau by a sequential mechanismrdquo Biochem-istry vol 45 no 10 pp 3125ndash3133 2006

[27] Y-G Chen ldquoSpecific tau phosphorylation sites in hippocampuscorrelate with impairment of step-down inhibitory avoidancetask in ratsrdquo Behavioural Brain Research vol 158 no 2 pp 277ndash284 2005

[28] H Le Freche J Brouillette F-J Fernandez-Gomez et al ldquoTauphosphorylation and sevoflurane anesthesia an association topostoperative cognitive impairmentrdquo Anesthesiology vol 116no 4 pp 779ndash787 2012

[29] M Moosavi G Yadollahi Khales L Abbasi A Zarifkar andK Rastegar ldquoAgmatine protects against scopolamine-inducedwater maze performance impairment and hippocampal ERKand Akt inactivationrdquo Neuropharmacology vol 62 no 5-6 pp2018ndash2023 2012

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type II diabetes fat tissue of C57BL6J mice [12] Immuno-histochemical results show that ser198ser199ser202 sites arehyperphosphorylated in the hippocampus of diabetic micebut the expression of GSK-3 is reduced [13] In the brainof insulin knockout mice hyperphosphorylation of tau atthreonine 231 and of the neurofilament is exhibited but GSK-3120573 activity was inhibited [14]

All of the above evidences imply that GSK-3 may belinked to both A120573 production and tau hyperphosphorylationduring the course of diabetes To better understand themechanisms of AD-like changes in diabetic rat brain weinvestigated the activity of GSK-3 and Akt and the expressionof Akt then A120573 production and tau phosphorylation wasdetermined Furthermore the role of GSK-3 was explored byusing LiCl as a specific inhibitor [15]

2 Materials and Methods

21 Antibodies and Chemicals The primary A120573 antibodiesG2-10 (capture antibody for A120573

40) G2-11 (capture antibody

for A12057342) and WO

2(detection antibody) used for ELISA

were purchased from A120573eta Company (Germany) and stan-dard synthetic A120573 peptides were obtained from Sigma (StLouis MO USA) Antibodies against phospho-Ser473 Aktand total Akt were purchased fromCell Signaling technology(Beverly MA USA) Monoclonal antibodies PHF-1 whichlabels tau phosphorylated at Ser-396 andor Ser-404 andTau-1 which labels tau where neither serines 198 199 or 202are phosphorylated were gifts from Dr Peter Davies (AlbertEinstein College of Medicine Bronx NY USA) and DrLester Binder (Northwestern University Chicago IL USA)respectively Polyclonal antibody 111e which reacts with thetotal tau was a kind gift fromDrs Iqbal K andGrundke-IqbalI (New York State Institute for Basic Research Staten IslandNY USA) Phospho-GS peptide a specific GSK-3 substratewas obtained from Upstate Biotechnology Inc (Lake PlacidNY USA) Bicinchoninic acid (BCA) protein detection kitand TMB were obtained from Pierce (Rockford IL USA)[120574-32P] ATP was obtained from Beijing Yahui Biologic andMedicinal Engineering Co (Beijing China) Goat anti-rabbit or goat anti-mouse peroxidase-conjugated secondaryantibodies and phosphocellulose paper were obtained fromPierce Chemical Co (Rockford IL USA) Polyclonal andmonoclonal Histostain-SP kits were obtained from ZymedLaboratories Inc (South San Francisco CA USA) Strepto-zotocin (STZ) diaminobenzidine LiCl (a specific inhibitorof GSK-3) and other chemicals were purchased from Sigma(St Louis MO USA)

22 Establishment of DM Rat Model Four-month-oldSprague-Dawley rats (Grade II male weight 200ndash250 gsupplied by Experimental Animal Center of Tongji MedicalCollege) were divided into 4 groups control group diabetesmellitus (DM) group DM plus NaCl group and DMplus LiCl group by random Except for control rats theother rats received intraperitoneal injection with 55mgkgstreptozotocin (STZ) The DM rats were determined byfasting blood glucose ge167mmol72 h after streptozotocin

injection DM plus NaCl group 24 h following STZinjection the rats were administered by intraperitonealinjection 04mL of 03M NaCl for 10 days DM plus LiClgroup 24 h following STZ injection the rats were injectedintraperitoneally with 400 120583L of 03M LiCl for 10 days

23 Preparation of Rat Hippocampal Extracts Followingcontinuous injection of NaCl or LiCl for 10 days the ratswere killed The hippocampus was immediately removedand homogenized at 4∘C using a Teflon glass homogenizerin 50mmol Tris-HCl pH 74 150mmol NaCl 10mmolNaF 1mmol Na

3VO4 10mmol 120573-mercaptoethanol 5mmol

EDTA 2mmol benzamidine 10mmol phenylmethylsulfonylfluoride 5 120583gmL leupeptin 5120583gmL aprotinin and 2 120583gmLpepstatinThe tissue homogenates were then divided into twoportions One portion of each homogenate was centrifugedat 12000timesg for 20min at 4∘C and the resulting supernatantwas stored at minus80∘C for assaying activities of protein kinasesThe other portion was mixed in 2 1 (vv) ratio with lysisbuffer containing 200mmol Tris-HCl pH 76 8 SDS40 glycerol boiled for 10min in a water bath and thencentrifuged at 12000timesg for 30min and the supernatant wasstored at minus80∘C for Western blot analysis The concentrationof protein in the hippocampal extracts was measured byBCA kit according to themanufacturerrsquos instructions (PierceCheshire UK)

24 Assay of GSK-3 and Akt Activity The GSK-3 activityin rat hippocampal exacts was measured using phospho-GS peptide 2 (Upstate Lake Placid NY USA) as describedpreviously [16] Briefly tissue extracts 75120583g proteins wereincubated for 30min at 30∘C with 20120583M peptide sub-strate and 200120583mol [120574-32P] ATP (1500 cpmpmol ATP) in30mmol Tris pH 74 10mmol MgCl

2 10mmol NaF 1mmol

Na3VO4 2mmol EGTA and 10mmol 120573-mercaptoethanol

in a total volume of 25 120583L The reaction was stopped byaddition of 25120583L of 300mM119874-phosphoric acidThe reactionmixture was applied in triplicates on phosphocellulose paper(pierce) The filters were washed three times with 75mmol119874-phosphoric acid dried and counted by liquid scintillationcounter GSK-3 activities was calculated with picomolesof phosphate incorporatedmg proteinmin at 30∘C andexpressed as relative activity against control The Akt activitywas measured using histone 2B as a substrate as describedpreviously [17 18] Briefly after the immunoprecipitates werewashedwith lysis buffer and kinase buffer 40120583L kinase buffercontaining 200 120583M [120574-32P] (5 120583Ci) 100120583MATP 1 120583g120583L his-tone 2B then the samples were incubated at 30∘C for 15minand spotted onto P81 filter papers the filter papers werewashed by 75mM 119874-phosphoric acid dried and counted byliquid scintillation counter Akt activity was also expressed asrelative activity against control

25 Measurement of A120573 in Hippocampus of the Rats TheA12057340 and A12057342 in the hippocampus were measured bya Sandwich enzyme-linked immunosorbent assay (ELISA)using antibodies as described previously [19] Experimentswere performed in a 96-well plate Briefly affinity-purified


30

24

18

12

6

0


Seru

m g

luco

se (m

ML

)

lowastlowast











2

25

15

1

05

0


Relat

ive a

ctiv

ity o

f GSK

-3




02

04

06

08

1

12

0


Akt

activ

ity


lowastlowast


3 Results



2

25

3

35

15

1

05

0


Relat

ive l

evel

of A

beta

40

lowastlowast

lowastlowast

(a)

1

18

16

14

12

08

06

04

02

0


Relat

ive l

evel

of A

beta

42

lowast

lowast

(b)



group

2

16

12

08

04

0


Relat

ive l

evel

of P

HF-

1

lowastlowast

(a)

12

1

08

04

06

02

0


Relat

ive l

evel

of t

au-1


(b)

09

06

12

03

0


Relat

ive l

evel

of 1

11e

(c)











1

12

Total-Akt

08

06

04

02

0


lativ

e lev

el o

f Akt lowastlowast


P-Ser-473-Akt



4 Discussion




Erro

r tim

e (3 m

in)

4

3

2

1

0

1 2

(day)3 4

lowastlowast

(a)

200

150

100

50

0

1 2

(day)3 4

Late

ncy

(s3

min

)

lowastlowast

(b)

4

3

2

1

0


Erro

r tim

e (3 m

in)

lowast

998779

(c)


200

150

250

100

50

0

Late

ncy

(s3

min

)lowast

998779

(d)











In our study










Acknowledgment


References











































Neural Plasticity










Psychiatry Journal









Journal of



30

24

18

12

6

0


Seru

m g

luco

se (m

ML

)

lowastlowast











2

25

15

1

05

0


Relat

ive a

ctiv

ity o

f GSK

-3




02

04

06

08

1

12

0


Akt

activ

ity


lowastlowast


3 Results



2

25

3

35

15

1

05

0


Relat

ive l

evel

of A

beta

40

lowastlowast

lowastlowast

(a)

1

18

16

14

12

08

06

04

02

0


Relat

ive l

evel

of A

beta

42

lowast

lowast

(b)



group

2

16

12

08

04

0


Relat

ive l

evel

of P

HF-

1

lowastlowast

(a)

12

1

08

04

06

02

0


Relat

ive l

evel

of t

au-1


(b)

09

06

12

03

0


Relat

ive l

evel

of 1

11e

(c)











1

12

Total-Akt

08

06

04

02

0


lativ

e lev

el o

f Akt lowastlowast


P-Ser-473-Akt



4 Discussion




Erro

r tim

e (3 m

in)

4

3

2

1

0

1 2

(day)3 4

lowastlowast

(a)

200

150

100

50

0

1 2

(day)3 4

Late

ncy

(s3

min

)

lowastlowast

(b)

4

3

2

1

0


Erro

r tim

e (3 m

in)

lowast

998779

(c)


200

150

250

100

50

0

Late

ncy

(s3

min

)lowast

998779

(d)











In our study










Acknowledgment


References











































Neural Plasticity










Psychiatry Journal









Journal of



2

25

3

35

15

1

05

0


Relat

ive l

evel

of A

beta

40

lowastlowast

lowastlowast

(a)

1

18

16

14

12

08

06

04

02

0


Relat

ive l

evel

of A

beta

42

lowast

lowast

(b)



group

2

16

12

08

04

0


Relat

ive l

evel

of P

HF-

1

lowastlowast

(a)

12

1

08

04

06

02

0


Relat

ive l

evel

of t

au-1


(b)

09

06

12

03

0


Relat

ive l

evel

of 1

11e

(c)











1

12

Total-Akt

08

06

04

02

0


lativ

e lev

el o

f Akt lowastlowast


P-Ser-473-Akt



4 Discussion




Erro

r tim

e (3 m

in)

4

3

2

1

0

1 2

(day)3 4

lowastlowast

(a)

200

150

100

50

0

1 2

(day)3 4

Late

ncy

(s3

min

)

lowastlowast

(b)

4

3

2

1

0


Erro

r tim

e (3 m

in)

lowast

998779

(c)


200

150

250

100

50

0

Late

ncy

(s3

min

)lowast

998779

(d)











In our study










Acknowledgment


References











































Neural Plasticity










Psychiatry Journal









Journal of







1

12

Total-Akt

08

06

04

02

0


lativ

e lev

el o

f Akt lowastlowast


P-Ser-473-Akt



4 Discussion




Erro

r tim

e (3 m

in)

4

3

2

1

0

1 2

(day)3 4

lowastlowast

(a)

200

150

100

50

0

1 2

(day)3 4

Late

ncy

(s3

min

)

lowastlowast

(b)

4

3

2

1

0


Erro

r tim

e (3 m

in)

lowast

998779

(c)


200

150

250

100

50

0

Late

ncy

(s3

min

)lowast

998779

(d)











In our study










Acknowledgment


References











































Neural Plasticity










Psychiatry Journal









Journal of



Erro

r tim

e (3 m

in)

4

3

2

1

0

1 2

(day)3 4

lowastlowast

(a)

200

150

100

50

0

1 2

(day)3 4

Late

ncy

(s3

min

)

lowastlowast

(b)

4

3

2

1

0


Erro

r tim

e (3 m

in)

lowast

998779

(c)


200

150

250

100

50

0

Late

ncy

(s3

min

)lowast

998779

(d)











In our study










Acknowledgment


References











































Neural Plasticity










Psychiatry Journal









Journal of










Acknowledgment


References











































Neural Plasticity










Psychiatry Journal









Journal of























Neural Plasticity










Psychiatry Journal









Journal of


Research Article Glycogen Synthase Kinase-3...

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Transcript of Research Article Glycogen Synthase Kinase-3...