Neuroprotective changes in degeneration-related gene expression in the substantia nigra ...

14 Pages·2015·1.3 MB·English

Neuroprotective changes in degeneration-
related gene expression in the substantia nigra
following acupuncture in an MPTP mouse model of
Parkinsonism: Microarray analysis
Citation
Yeo, Sujung, Keon Sang An, Yeon-Mi Hong, Yeong-Gon Choi, Bruce Rosen, Sung-Hoon Kim, and
Sabina Lim. 2015. “Neuroprotective changes in degeneration-related gene expression in the
substantia nigra following acupuncture in an MPTP mouse model of Parkinsonism: Microarray
analysis.” Genetics and Molecular Biology 38 (1): 115-127. doi:10.1590/S1415-475738120140137.
http://dx.doi.org/10.1590/S1415-475738120140137.
Published Version
doi:10.1590/S1415-475738120140137
Permanent link
http://nrs.harvard.edu/urn-3:HUL.InstRepos:16121020
Terms of Use
This article was downloaded from Harvard University’s DASH repository, and is made available
under the terms and conditions applicable to Other Posted Material, as set forth at http://
nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Share Your Story
The Harvard community has made this article openly available.
Please share how this access benefits you. Submit a story .
Accessibility
GeneticsandMolecularBiology,38,1,115-127(2015)
Copyright©2015,SociedadeBrasileiradeGenética.PrintedinBrazil
DOI:http://dx.doi.org/10.1590/S1415-475738120140137
ResearchArticle
Neuroprotective changes in degeneration-related gene expression in the
substantia nigra following acupuncture in an MPTP mouse model of
Parkinsonism: Microarray analysis
SujungYeo1,2,KeonSangAn2,Yeon-MiHong1,2,Yeong-GonChoi1,2,BruceRosen2,3,
Sung-HoonKim2andSabinaLim1,2
1ResearchGroupofPainandNeuroscience,WHOCollaboratingCenterforTraditionalMedicine,
East-WestMedicalResearchInstitute,KyungHeeUniversity,Seoul,RepublicofKorea.
2DepartmentofBasicKoreanMedicalScience,CollegeofKoreanMedicine,KyungHeeUniversity,
Seoul,RepublicofKorea.
3DepartmentofRadiology,AthinoulaA.MartinosCenterforBiomedicalImaging,
MassachusettsGeneralHospital,HarvardMedicalSchool,Boston,USA.
Abstract
Parkinson’sdisease(PD)isaneurodegenerativedisordercharacterizedbythedeathofdopamine-generatingcells
inthesubstantianigra(SN).Acupuncturestimulationresultsinanenhancedsurvivalofdopaminergicneuronsinthe
SNinParkinsonismanimalmodels.Thepresentstudyinvestigatedchangesingeneexpressionprofilesmeasured
usingwholetranscriptarrayintheSNregionrelatedtotheinhibitoryeffectsofacupunctureinachronic1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)Parkinsonismmodel.Inthismodel,acupuncturestimulationatGB34
andLR3attenuatedthedecreaseintyrosinehydroxylaseintheSNregion;stimulationatnon-acupointsdidnotsup-
pressthisdecrease.Genearrayanalysisrevealedthat22(10annotatedgenes:Cdh1,Itih2,Mpzl2,Rdh9,Serping1,
Slc6a13,Slc6a20a,Slc6a4,Tph2,andUcma)probesthatwereup-regulatedinMPTPanimalsrelativetocontrols
wereexclusivelydown-regulatedbyacupuncturestimulation.Inaddition,17(twoannotatedgenes:4921530L21Rik
and Gm13931) probes that were down-regulated in MPTP animals compared to controls were exclusively up-
regulatedbyacupuncturestimulation.Thesefindingsindicatethatthe39probes(12annotatedgenes)affectedby
MPTPandacupuncturemayberesponsiblefortheinhibitoryeffectsofacupunctureondegeneration-relatedgene
expression in the SN following damage induced by MPTP intoxication.
Keywords:Parkinson’sdisease,genearray,differentiallyexpressedgenes,neurodegeneration,acupoints.
Received:May8,2014;Accepted:September17,2014.
Introduction Using a 6-hydroxydopamine (6-OHDA)-induced
Parkinsonismmodel,apreviousstudyfoundthatacupunc-
Parkinson’sdisease(PD)isaneurodegenerativedis-
turestimulationatacupointsGB34andLR3resultsinthe
ordercharacterizedbymajorbehavioralsymptoms,which
enhancedsurvivalofdopaminergicneuronsintheSNanda
include tremor, akinesia, bradykinesia, and stiffness (Jan-
~87.7% improvement in motor dysfunction (Park et al.,
kovic, 2008). PD results from the death of dopaminergic
2003).Similarfindingwerefoundfollowingtheadminis-
neurons in the substantia nigra (SN; (Goto et al., 1989;
tration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
Chauhanetal.,2001;Kimetal.,2003;Fernandez-Espejo
(MPTP), where the acupuncture-induced enhancement of
2004),whichplaysanimportantroleinseveralbrainfunc-
synaptic dopamine availability may play a critical role in
tions, particularly, motor planning, eye movement, learn-
the observed motor function improvement (Kim et al.,
ing,reward-seekingandaddiction.Notsurprisingly,gross
2011).Acupuncturestimulationatthesameacupointsalso
movementdysfunctioncausedbydamagetothisareaisa
attenuates the decrease in tyrosine hydroxylase (TH)
symptomofPD.
immunoreactivity (IR) and generates neuroprotective ef-
fects in the SN in an MPTP-induced mouse model of
Parkinsonism(Kangetal.,2007).Themechanismsunder-
SendcorrespondencetoSabinaLim.ResearchGroupofPainand
lyingtheneuroprotectiveeffectsofacupunctureontheSN
Neuroscience,WHOCollaboratingCenterforTraditionalMedicine,
arethoughttobeinpartrelatedtodecreasedmicroglialac-
East-West Medical Research Institute, Kyung Hee University,
130-701 Seoul, Republic of Korea. E-mail: [email protected]. tivationandreducedinflammatoryresponses(Kangetal.,
116 Yeoetal.
2007); these, in turn, inhibit ferric iron and ferritin heavy 2011b).Briefly,miceintheacupointsgroupwereimmobi-
chaindeposition(Choietal.,2009). lizedbyhand2hfollowingMPTPadministration,andthe
SeveralgenesthatareaffectedbyMPTPandstimula- acupuncture needles were inserted bilaterally to depths of
tionatacupointsmayberesponsiblefortheinhibitoryef- 1mmatacupointLR3and3mmatacupointGB34before
fects of acupuncture on MPTP-induced degeneration in beingturnedatarateoftwospinspersecondfor15s,asre-
striatalregions(Choietal.,2011b).Additionally,geneex- portedpreviously(Kangetal.,2007).Inthenon-acupoints
pression in thalamic regions damaged by MPTP intoxica- group,theneedleswereinsertedtoadepthof3mmonboth
tion may be affected, at least in part, by acupuncture at sidesofthehips,andaprocedureidenticaltothatdescribed
specificacupoints(Yeoetal.,2013).Changesingeneex- abovewasperformed.
pressionprofilesintheSNfollowingacupuncturestimula-
Immunohistochemistry
tion were identified in an acute Parkinsonism model over
threedays(Hongetal.,2010);however,nosuchchanges After four weeks, the brains were removed, post-
havebeendemonstratedinachronicParkinsonismanimal fixed in 0.05 M sodium-phosphate buffer containing 4%
model. Because PD is a chronic neurodegenerative disor- paraformaldehydefor12hat4°C,rinsedwith0.05Mso-
der, changes in gene expression in the SN in a chronic dium-phosphatebuffer,dehydratedwithsucrosefor12hat
Parkinsonismanimalmodelshouldbeinvestigatedtoiden- 4°C,andthencryosectioned.Coronalsectionsofthebrains
tify the neuroprotective mechanisms underlying acupunc- (30-(cid:2)mthickness)werecutusingacryomicrotome.Immu-
turetreatment. nohistochemical analyses were carried out using an ABC
Thus, we investigated changes in gene expression kitandaMouseonMouse(M.O.M)immunodetectionkit
profiles in the SN region in a relatively chronic Parkin- (Vector Laboratories, CA) using a modification of the
sonismmodelandidentifiedthegenesrelatedtotheinhibi- avidin-biotin-peroxidasemethod.Briefly,sectionsencom-
toryeffectofacupunctureonalterationsingeneexpression passingtheentirestriatalandSNregionswereincubatedin
inducedbyMPTPintoxication. phosphate-buffered saline (PBS; pH 7.4) with 3% H O ,
2 2
exposedto3%bovineserumalbumin(BSA)and0.3%Tri-
Materials and Methods tonX-100inPBSfor1h,andthentreatedwithanavidin-
biotinblockingkit(VectorLaboratories).Whenusingthe
MPTP model of Parkinsonism
mouse anti-TH antibody, sections were treated with an
Six-week-old male inbred C57BL/6 mice (20-22 g; M.O.M.mouseIg-blockingreagent(VectorLaboratories)
Samtaco, Korea) were divided into the following four atroomtemperaturefor1hpriortoincubationwiththepri-
groups: control (CTL); MPTP-treatment only (MPTP); maryantibody.Thereafter,eachsectionwasstainedover-
MPTP and acupuncture treatment at GB34 and LR3 night at 4 °C with a mouse anti-TH antibody (1:1,500;
(MPTP-A);andMPTPandacupuncturetreatmentatnon- SantaCruzBiotechnology,USA)toidentifydopaminergic
acupoints (MPTP-NA). Mice in the control group (n = 9) neurons in the striatal and SN regions. The sections were
were injected intraperitoneally with saline 0.9% (100 (cid:2)L) then sequentially treated with a biotinylated anti-mouse
oncedailyforfourweeks,whilemiceintheMPTPgroup IgGfollowedbyanavidin-biotin-peroxidasecomplexand
(n = 9) were injected intraperitoneally with MPTP-HCl developed using a diaminobenzidine-hydrogen peroxide
(20mg/kgoffreebase)insaline0.9%(100(cid:2)L)at24-hin- solution(0.003%3,3-diaminobenzidineand0.03%hydro-
tervals over four weeks to produce a sustained chronic genperoxidein0.05MTris,pH7.0).
model of Parkinsonism (Bezard et al., 1997; Kuhn et al.,
Western blotting
2003;Choietal.,2011a;Yeoetal.,2013).Onthedayfol-
lowingthefinalMPTPtreatment,micewereanesthetized The bilateral striatal and SN regions were homoge-
using16.5%urethaneandperfusedtranscardiallywithcold nized in 20 mM HEPES-KOH buffer (pH 7.5) with
0.05 M sodium-phosphate buffer to enable immunohisto- 150mMNaCl,0.5%sodiumdeoxycholate,0.1%SDS,and
chemical evaluation. The Kyung Hee University Animal a protease inhibitor cocktail. After centrifugation at
ExperimentationCommitteeapprovedallanimalprotocols 15,000xgfor10min,solublesupernatantsamplesofequal
usedinthisstudy.Reagentsusedbutnotmentionedwere proteinconcentration(30(cid:2)gtotalprotein)wereseparated
purchasedfromSigma(USA). by 12% sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis(SDS-PAGE)andthentransferredtopolyviny-
Acupuncture administration
lidene difluoride (PVDF) membranes (Bio-Rad; USA).
Acupuncture was performed manually 2 h after the Themembraneswereblockedwith5%skimmilkin0.1%
first MPTP injection and at 48-h intervals thereafter for a Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.5] and
totalof14sessions.Theacupunctureprocedure(acupoints: 150mMNaClcontaining0.1%Tween-20;TBST)atroom
GB34 [Yanglingquan] and LR3 [Taichong]; non-acu- temperaturefor1handthenincubatedwithmouseanti-TH
points: both sides of the hips) was performed as reported (1:2,000) and mouse anti-actin (1:5,000, Santa Cruz Bio-
previously(Parketal.,2003;Kangetal.,2007;Choietal., technology) antibodies. After being washed with 0.1%
Acupunctureandgeneexpression 117
TBST,themembraneswereincubatedwithananti-mouse usingtheDEG-findingmodule;i.e.,log1.3/log2=0.379<
IgG-peroxidaseantibody(1:2,000,Bio-Rad),andtheanti- averagelog2fold-changevalue(X)fortheidentificationof
gen-antibody complexes were visualized using the Pierce up-regulated genes and - log1.3/log2 = -0.379 > average
ECLwesternblottingsubstrate(ThermoScientific,USA). log2fold-changevalue(X)fortheidentificationofdown-
regulated genes. A pathway analysis of all metabolic and
RNA extraction and microarray analysis KyotoEncyclopediaofGenesandGenomes(KEGG)path-
ways was performed using the pathway module, as de-
TotalRNAwasextractedfromthebilateralSNtissue
scribedpreviously(Hwangetal.,2009;Choietal.,2011a;
of each group (n = 2; both groups) using an RNeasy Plus
Yeoetal.,2013).ANationalCenterforBiotechnologyIn-
Minikit(QIAGEN,USA)accordingtothemanufacturer’s
formationGeneExpressionOmnibus(NCBIGEO)acces-
instructions.IsolatedRNAqualitywasestimatedandquan-
sion number was assigned to the raw microarray data
tified using a NanoDrop ND-1000 spectrophotometer
generatedinthisstudy.
(NanoDropTechnologies,USA).Analiquot(300ng)ofto-
talRNAwassubjectedtoanAffymetrixGeneChip®Mouse
Real time RT-PCR
Gene 1.0 ST Array (genome-wide expression profiling
Forreal-timereversetranscriptionpolymerasechain
chip;28,853genesof35,557probes;Affymetrix,USA)ac-
reaction(RT-PCR)analyses,thetotalRNA(500ng)ofthe
cording to the GeneChip Whole Transcript (WT) Sense
samples(CTL,MPTP,andMPTP-A)wassubjectedtothe
Target Labeling Assay manual, as reported previously
reaction using a SuperScript First-strand Synthesis Kit
(Choietal.,2011a;Hwangetal.,2009;Linetal.,2010).
(InvitrogenLifeTechnologies,USA)accordingtotheman-
Briefly,theprocedureswerecarriedoutasfollows:mixing
ufacturer’s instructions. The advanced relative expression
ofT7-(N)6primerandpoly-ARNAcontrolswith300ngof
levels of representative DEGs were monitored with a
totalRNAisolatedfromtheSNofeachgroup;synthesisof
LightCycler 480 II Real Time PCR Instrument and the
first-cycle, first-strand complementary DNA (cDNA) and
LightCycler 480 software ver. 1.5.0.39 (Roche Diagnos-
then second-strand cDNA; synthesis of first-cycle cRNA
tics; Germany) using a LightCycler 480 SYBR Green I
and cleanup of cRNA; synthesis of second-cycle, single-
Master(RocheDiagnostics).Meancrossingpoint(CP)val-
strand cDNA (ss cDNA) and cleanup of ss cDNA; frag-
ues were obtained and the expression levels of the target
mentation of ss cDNA and labeling of the fragmented ss
genesinthevariousgroupswerethencomparedtothoseof
cDNA;hybridizationoflabeledsscDNAtotheGeneChip;
the reference gene, glyceraldehyde-3-phosphate dehydro-
and finally, the staining, washing, and scanning of the
genase (GAPDH), using an advanced relative quantifica-
GeneChip using a Fluidics 450 station and the GeneChip
tion method. The ratios of the concentrations of target
OperatingSoftware(GCOS,Affymetrix).
genes to that of the reference gene were obtained; the
Microarray data analysis primersequencesforeachgeneareshowninTable1.The
specificityofeachprimersetwasconfirmedbydetermin-
Theusefulnessofthesignalintensitieswasfirsteval-
ingthemeltingtemperatureandsizeofeachproductbygel
uatedbyvisualexaminationofthescannedimages.Quality
electrophoresis.
controlofthescanneddatawasthenconductedbyconfirm-
ingtheorderofthesignalintensitiesofpoly-Acontrolsand Statistical analysis
hybridization controls using the Expression Console soft-
Statisticalanalyseswereperformedusingthestatisti-
ware (Affymetrix). Microarray data were analyzed using
cal package for the social sciences (SPSS) software, ver.
GenPlexver.3.0(ISTECH,Korea,(Anetal.,2009;Choiet
18.Valuesaremeans(cid:3)SEofthreeindependentdetermina-
al.,2011a;Hwangetal.,2009;Yeoetal.,2013).Atotalof
tions.
eightCELfiles(twoCELfilesgeneratedfromeachgroupx
four experimental groups) were uploaded and normalized
underthefollowingconditions:perfectmatch(PM)-onlyas Table1-Thesequencesofprimers.
aPMintensityadjustment;theRobustMultichipAnalysis
(RMA)quantificationmethodasaprobesetsummarization Genesymbol GeneaccessionNo. Primersequences(5’(cid:4)3’)
algorithmforlogtransformationwithbase2(log2);andthe
Rdh9 NM_153133 F:gagcgtgttgggaacagag
quantile normalization method for evaluation of prelimi- R:gtttcttcatccactcgttgg
narydataqualityinthepreprocessingmodule,whichfunc- Slc6a4 NM_010484 F:acctggacactccattccac
tions as a data quality control, using the Expression R:cctggagtccctttgactga
Consolesoftware(Affymetrix).Themeansignalintensities Tph2 NM_173391 F:gagcttgatgccgaccat
of the 28,853 genes were obtained from two chips from R:tggccacatccacaaaatac
eachgroup.Followingnormalization,thedifferentiallyex- Ucma NM_001113558 F:cgtggaggaacagagagatga
pressed genes (DEGs) in the GeneChip that satisfied the R:aggccatcataatgccactg
conditionsofthefoldchangecutoff(1.3)aswellastheStu- GAPDH NM_008084 F:gtcttcaccaccatggagaagg
dent’st-testsignificancecriterion(p<0.05)wereidentified R:tcatggatgaccttggccag
118 Yeoetal.
Results and Discussion ble S2) DEGs were identified in the SN region following
chronicMPTPintoxication.
Chronic MPTP-induced Parkinsonism mouse model
and the preventive effects of acupuncture Changes in gene expression in the SN region
following acupuncture
BecauseTHexpressionissignificantlyattenuatedin
the brains of PD patients (Pardridge, 2005) and in Par- Of the 28,853 genes represented in the oligonucleo-
kinsonism animal models (Park et al., 2003; Kang et al., tidearray,thosewithlog2-transformedmeansignalinten-
2007;Choietal.,2009),weevaluatedTHlevelstoconfirm sitiesgreaterthan0.379(1.3-foldchange)andap<0.05by
the establishment of a chronic MPTP-induced Parkinso- Student’st-testbetweentheMPTP-AandMPTPgroupsor
nism mouse model and the mediating effects of acupunc- theMPTP-NAandMPTPgroupswereselectedandevalu-
tureatGB34andLR4.IntheSNparscompacta(SNpc)and ated.ComparedtotheMPTPgroup,genesintheSNregion
striatal regions, TH levels were noticeably reduced in the wereregardedasDEGsfollowingacupuncturestimulation
MPTP group (MPTP) relative to the control group (CTL; atacupointsandnon-acupoints,respectively.
Figures 1a, b, e, f, i, and j). Acupuncture stimulation at
Up-down-regulated genes following MPTP intoxication and
GB34andLR3(MPTP-A)attenuatedthedecreaseinTHin
acupuncture
both areas (Figures 1c, g, and k), but acupuncture at non-
acupoints(MPTP-NA)didnotsimilarlyinfluenceTH(Fig- Comparedtothecontrol,22(10annotatedgenes)of
ures 1d, h, and l). Immunohistochemical analyses using the244probeswereup-regulatedintheSNregionfollow-
WesternblotsconfirmedthatthedecreaseinTHlevelsin ing MPTP exposure and exclusively down-regulated by
the striatal and SNpc regions induced by MPTP intoxica- acupuncture at acupoints but not non-acupoints (“Up-
tionwassignificantlyinhibitedbyacupunctureatacupoints down”;Table2).
(MPTP-A)butnotatnon-acupoints(MPTP-NA;Figure2). Cadherin 1 (Cdh1) is a classic member of the
These results confirm that acupuncture stimulation sup- cadherin superfamily that is thought to contribute to the
presses the pathological change in TH levels induced by controlofsynapseformationandsynaptictransmissionand
MPTPintoxicationinthestriatalandSNpcregions,ofthe plasticity(Angstetal.,2001).Cdh1signalingisanimpor-
miceinourstudy. tant component of the regulation of GABAergic synapses
inbrainneurons(Fiederlingetal.,2011);indeed,elevated
Changes in gene expression in the SN region
GABAlevelshavebeendetectedinPD(Emiretal.,2012).
following chronic MPTP intoxication
Inthecurrentstudy,Cdh1wasup-regulatedintheMPTP
Asshownintheboxplotgraph(FigureS1)normal- groupcomparedtocontrols.However,acupuncturestimu-
ization through the preprocessing module was successful. lationatGB34andLR3maintainedCdh1expressionsimi-
Ofthe28,835genesrepresentedintheoligonucleotidear- lartolevelsincontrolanimals,suggestingthatthedown-
ray, genes were selected and evaluated if they displayed regulationofCdh1expressionbyacupuncturemayhavea
log2-transformedmeansignalintensitiesgreaterthan0.379 neuroprotectiveeffect.
(1.3foldchange)andp<0.05byStudent’st-testbetween Solutecarrierfamily6(neurotransmittertransporter,
the control and MPTP groups. Compared to the control, GABA), member 13 (Slc6a13) is a sodium- and chlo-
244up-regulated(TableS1)and255down-regulated(Ta- ride-dependent member of solute carrier family 6 (SLC6)
Figure1-THexpressionismaintainedbyacupunctureinachronicMPTP-inducedParkinsonismmodel.Thestriatum(ST,a-d)andsubstantianigra(SN,
e-l)wereimmunostainedusingananti-tyrosinehydroxylase(TH;a-d,40x;e-h,100x;i-l,400x)antibody.CTL,salinecontrol;MPTP,MPTPtreated
only;MPTP-A,acupuncturetreatedatacupointsGB34andLR3,withMPTP;MPTP-NA,acupuncturetreatedatnon-acupoints,withMPTP.TheTH
densityineachSTregionwasevaluatedbymeasuringthedensitometryinthesamearea(500(cid:2)m2)andthenumberofTH-positivecellsintheSNpc
(200x400(cid:2)m)countedinthesamearea.A*p<0.05comparedtothecontrolanda#p<0.05comparedtotheMPTPwereconsideredsignificant.
Acupunctureandgeneexpression 119
behighlylabiletooxidation(Kuhnetal.,1980).Therefore,
the oxidative processes that prevail in PD may cause a
misfoldingofTph2andresultinthemodificationofseroto-
ninfunctionsuchasisseenindopamineneurons.Tph2oxi-
dation inhibits its activity and leads to the formation of
high-molecular-weightaggregatesinadithiothreitol-rever-
sible manner (Kuhn et al., 2011). Alteration of serotonin
levels by Tph2 hyperinnervation affects the wiring of the
brainandcanproducelong-lastingchangesleadingtothe
developmentofneurodevelopmentaldisorderssuchasPD
(Migliarinietal.,2012).
Myelinproteinzero-like2(Mpzl2)isalsoknownas
EvaandEva1.Inhumanchoroidplexusepithelialcellsand
asubsetofCD4Tlymphocytes,Mpzl2isexpressedathigh
levels.Mpzl2expressedinchoroidplexuscellsmayregu-
latethepermeabilityoftheblood-cerebrospinalfluid(CSF)
barrier(Chatterjeeetal.,2008),suggestinganovelmecha-
nism of CNS immune surveillance regulation (Wojcik et
al.,2011).Inthecurrentstudy,Mpzl2wasup-regulatedin
theMPTPgroupcomparedtocontrols;however,acupunc-
turestimulationatGB34andLR3maintainedMpzl2gene
expression at levels similar to those in control animals.
Theseresultsalsosuggestthatacupuncturesuppressesthe
Figure2- InhibitionofthedecreaseinMPTP-inducedTHexpressionby Mpzl2 overexpression that can be caused by MPTP neu-
acupunctureatacupoints.Westernblotdeterminationoftyrosinehydrox-
ronalloss.
ylase (TH) expression levels in the striatum (ST) and substantia nigra
(SN).ActinintheSTwasusedasatotalproteinloadingcontrol(30(cid:2)g). Theserine(orcysteine)peptidaseinhibitor,cladeG,
Lane1,CTL;Lane2,MPTP;Lane3,MPTP-A;andLane4,MPTP-NA.A member1(Serping1)geneencodestheserineproteasein-
*p<0.05comparedtothecontrolanda#p<0.05comparedtotheMPTP hibitor(serpin)knownasC1inhibitor.TheC1inhibitoris
wereconsideredsignificant.
importantforregulationofseveralprocesses,includingin-
flammation,thatareinvolvedinmaintenanceofbloodves-
that mediates the rapid removal of GABA and maintains sels(Shagdarsurenetal.,2008).Serping1isup-regulatedin
low extracellular levels of this neurotransmitter (Chen et the microglial pathway upon stimulation by interferon-
al.,2004;Kanner,2006).Evidencesuggeststhatalteration gamma (IFN-gamma; (Moran et al., 2007) or lethal lipo-
ofGABAtransportersislinkedtoPD(Wangetal.,2007). polysaccharide-inducedendotoxicshock(Liuetal.,2007).
Accordingly,theoverstimulationofGABAAreceptorscan Inter-alphatrypsininhibitor,heavychain2(Itih2)has
potentiateneuronalcelldamage;GABAtransportblockers alsobeenidentifiedduringinflammation(Scaveniusetal.,
are neuroprotective in these situations (Zeevalk and 2011),andmaycontributetothecascadeofeventsleading
Nicklas,1996,1997),suggestingthatoverexpressionofthe toneuronaldegeneration(HirschandHunot2009;Tufekci
GABA transporter can damage neurons. In the current et al., 2012). Therapeutic strategies aim to down-regulate
study,Slc6a13wasup-regulatedintheMPTPgroupcom- these inflammatory processes and may slow the progres-
paredtothecontrolgroup;thereforethismaybetheunder- sionofPD(HirschandHunot2009).Therefore,themainte-
lying cause of neuronal intoxication. However, acupunc- nance by acupuncture stimulation of expression levels of
turestimulationatGB34andLR3maintainedSlc6a13gene Itih2 and Serping1, which are involved in neuroinflam-
expressionatlevelssimilartothoseincontrolanimals,sug- mation, similar to those in control animals suggests that
gestingthatacupuncturehasaneuroprotectiveeffect. acupuncturemayhaveaneuroprotectiveeffect.
Tryptophan hydroxylase 2 (Tph2), with a molecular SLC6 (neurotransmitter transporter), member 20A
weightof56kDa,isthepredominantformofthisenzyme (Slc6a20a) is also known as Xt3s1, Xtrp3s1, AU022428,
present in brain extracts from the mesencephalic tegmen- andA730081N20Rik.Neurotransmittertransportersofthe
tum, striatum, and hippocampus (Sakowski et al., 2006). SLC6 family play an important role in the removal of
Tph2 initiates serotonin synthesis in mammals with Tph1 neurotransmitters in brain tissue and in amino acid trans-
(Aleninaetal.,2009),andplaysacriticalroleinthemain- portinepithelialcells(Kowalczuketal.,2005).Eitherthe
tenance of brain serotonin homeostasis (Beaulieu et al., IMINOsystemortheiminoacidcarriersupportsthemain
2008).Tph2-derivedserotoninisinvolvedintheregulation load of proline transport (Munck and Munck, 1994); this
ofbehaviorandautonomicpathwaysbutisnotessentialfor geneisexpressedathighlevelsinthebrain.Morespecifi-
adultlife(Aleninaetal.,2009).However,Tph2isknownto cally,theIMINOsystemispresentinthemicroglialcellsof
120 Yeoetal.
M
ap-valueMAvs. 0.004 0.036 0.027 0.048 0.026 0.011 0.004 0.015 0.006 0.033 0.046 0.044 0.033 0.004 0.024 0.015 0.027 0.007 0.020 0.004 0.028 0.014
n
i
M
Averagelog2(foldhange)inMAvs.(Down-regulated) -0.502 -0.392 -0.884 -0.539 -0.569 -0.561 -0.594 -1.233 -1.582 -0.607 -0.505 -0.617 -1.107 -0.457 -0.512 -0.594 -0.383 -1.296 -0.425 -0.402 -0.434 -1.013
c
C
ap-valuenMvs. 0.002 0.024 0.010 0.043 0.042 0.011 0.015 0.017 0.007 0.012 0.011 0.010 0.005 0.037 0.035 0.030 0.018 0.032 0.030 0.028 0.038 0.028
i
ge)d)
vs.MPTP. og2(foldchanC(Up-regulate 0.705 0.439 0.630 0.470 0.467 0.611 0.649 0.544 0.796 0.455 0.794 0.652 1.200 0.657 0.743 1.136 0.439 1.258 0.478 0.412 0.526 0.914
P-A gelvs.
PT eraM
M vn
Ai
n
i
nddown-regulated ProbeIDeneaccessionNo.) 10575052(NM_009864) 10480003(NM_010582) 10584827(NM_007962) 10367041(NM_153133) 10484463(NM_009776) 10541318(NM_144512) 10597960(NM_139142) 10378816(NM_010484) 10372443(NM_173391) 10469058NM_001113558) b10338456 b10338657 b10339444 b10342771 b10343150 b10343377 b10343396 b10343743 b10344355 b10423383 b10594513 b10598073
a G (
ol (
ntr
o
PTPvs.c mber13 A member4
Table2-Listof(up-down)substantianigralgeneswhichwereup-regulatedinM GenesymbolGenetitle Cdh1cadherin1 Itih2inter-alphatrypsininhibitor,heavychain2 Mpzl2myelinproteinzero-like2 Rdh9retinoldehydrogenase9 Serping1serine(orcysteine)peptidaseinhibitor,cladeG,member1 Slc6a13solutecarrierfamily6(neurotransmittertransporter,GABA),me Slc6a20asolutecarrierfamily6(neurotransmittertransporter),member20 Slc6a4solutecarrierfamily6(neurotransmittertransporter,serotonin),(Slc6a4),mRNA Tph2tryptophanhydroxylase2 Ucmaupperzoneofgrowthplateandcartilagematrixassociated aDeterminedusingStudent’st-test.bGeneaccessionNo.wasnotdetermined.
Acupunctureandgeneexpression 121
the brain (Kowalczuk et al., 2005), by which proline is ningham et al., 2009). The up-regulation of Rdh9 in the
taken up (Hubscher and Berkley, 1992; Mikulska and MPTP-treatedgrouprelativetothecontrolgroupisthought
Lisowski,2003).This,inturn,influencestheprolinelevel tobeoneaspectofthisconsequence.However,furtherevi-
inextracellularfluid(ECF).Prolineitselfhasbeenpostu- dence is needed to confirm this, and the maintenance of
latedtobeneurotoxicwhenitsextracellularconcentration Rdh9 expression following acupuncture may have neuro-
isincreasedinthebrain(CohenandNadler1997;Nadleret protectiveeffects.
al.,1988).Inthecurrentstudy,Slc6a20aexpressioninthe
Down-up regulated genes by MPTP and acupuncture
SNwasup-regulatedintheMPTP-inducedmousemodelof
PD.Thus,adisturbanceintheuptakeofprolineinfluences Of the 255 probes down-regulated in the SN region
the extracellular concentration of proline. Increasing pro- comparedtocontrols,17(twoannotatedgenes)wereexclu-
linelevelsinECFmayinduceneuronalcelldeath,suggest- sivelyup-regulatedfollowingacupunctureatacupointsbut
ing that down-regulation of Slc6a20a gene expression notatnon-acupoints(“Down-up”;Table3).
followingacupuncturemayhaveaneuroprotectiveeffect. ThefunctionoftheRIKENcDNA4921530L21gene
SLC6 (neurotransmitter transporter, serotonin), (4921530L21Rik)hasyettobecharacterized.Thefunction
member 4 (Slc6a4) is also known as Htt, Sert, 5-HTT, or ofthepredictedgene13931(Gm13931)isknownasolfac-
AI323329. Recently, it was reported that MPTP-induced tory receptor-related genes. The olfactory system may be
dopaminergic denervation is followed by serotoninergic particularlysuitablerouteforthepenetrationofxenobiotic
hyperinnervation(Hebertetal.,2005).Ina6-OHDAani- agentsintotheCNS,aswasshowninanintranasalMPTP
malmodelofPD,theeffectsofdestroyingascendingdopa- model(Predigeretal.,2009,2010).Thus,olfactoryrecep-
minepathwaysonextracellularlevelsofserotoninandse- tor-relatedgenesmayberelatedtothepathologicalmecha-
rotonin innervation in the rat striatum were examined nismsofMPTPneurotoxicity.
(Balciogluetal.,2003).Specifically,theSlc6a4promoter
Signal pathway analysis
maygovernthegeneticriskofPD(Albanietal.,2009).In
the current study, Slc6a4 was up-regulated in the MPTP Pathwayanalysesforthe22probesthatwereup-re-
groupcomparedtocontrols,andacupuncturestimulationat gulated by MPTP intoxication (vs. CTL) and down-regu-
GB34andLR3maintainedSlc6a4geneexpressionatlev- latedbyacupunctureatacupoints(vs.MPTP),aswellasfor
els similar to those in control animals. These results indi- the 17 probes down-regulated by MPTP intoxication (vs.
catethatacupuncturemaysuppresstheSlc6a4overexpres- CTL) and up-regulated by acupuncture at acupoints (vs.
sioncausedbyMPTPintoxication. MPTP; Table 4) were performed. Cdh1, which is “down-
Upperzoneofgrowthplateandcartilagematrixasso- up” regulated by MPTP and acupuncture stimulation at
ciated(Ucma)isahighlyconservedtyrosine-sulphatedse- acupoints, was involved in “thyroid, endometrial, bladder
creted protein with a molecular weight of 17 kDa that is cancer”,“bacterialinvasionofepithelialcells”,and“mela-
expressed by juvenile chondrocytes (Tagariello et al., noma” at significant levels according to the over-repre-
2008).Ucmamaybeinvolvedinthenegativecontrolofthe sentationanalysis(ORA).However,“pathwaysincancer”,
osteogenic differentiation of osteochondrogenic precursor “adherens junction” and “cell adhesion molecules
cellsinperipheralzonesoffetalcartilageandatthecarti- (CAMs)” were not significant. Tph2 was involved in
lage-boneinterface(Surmann-Schmittetal.,2008).Anim- “tryptophan metabolism” and “serotonergic synapse” at
balanceinisoformexpressionmay,therefore,beinvolved significant levels. Slc6a4 was involved in “serotonergic
in skeletal pathology (Le Jeune et al., 2010). PD patients synapse” at a significant level. Serping1 was involved in
havebeenreportedtohaveasignificantlyincreasedriskof “complementandcoagulationcascades”and“pertussis”at
fracture(Satoetal.,2001;Pouwelsetal.,2013),whichmay significant levels. Slc6a13 was involved in “GABAergic
becausedbyanimbalanceinUcmaexpression.Inthecur- synapse”, albeit not significantly so. Gm13931 was in-
rentstudy,Ucmaexpressionwasup-regulatedintheMPTP volvedin“olfactorytransduction”andwas“down-up”reg-
group compared to the control group, and acupuncture ulatedbyMPTPandacupuncturestimulationatacupoints,
stimulationatGB34andLR3maintainedUcmaexpression evenifnotsignificantaccordingtotheORA.
atlevelssimilartothoseincontrolanimals. ThesefindingsdemonstratethatbothMPTPandacu-
Retinol dehydrogenase 9 (Rdh9) is a short-chain puncture at acupoints influence Cdh1, Tph2, Slc6a4,
dehydrogenase/reductaseandconverts9-cis-retinolinto9- Serping1, Slc6a13, and Gm13931 expression in the indi-
cis-retinal and 3alpha-androstanediol into dihydrotestos- catedpathwaysintheSNregion.
terone(Zhuangetal.,2002).WidespreadRdh9functionin
Validation of gene expression changes in the SN
steroid or retinoid metabolism begins mid-embryogenesis
region following MPTP and acupuncture at
(Hu et al., 2007), and may be an important link between
acupoints
neuroactive steroids and neurodegenerative disorders
(Melcangi et al., 2012). Moreover, testosterone has been Tovalidatethemicroarraydata,representativegenes
reported to be neurotoxic to dopaminergic neurons (Cun- wereselectedandtheirexpressionlevelsintheSNregion
122 Yeoetal.
nM
i
ap-valueMAvs. 0.002 0.048 0.007 0.028 0.042 0.002 0.017 0.046 0.018 0.034 0.003 0.016 0.024 0.002 0.043 0.042 0.036
n
i
hange)ulated)
cg
2(fold(Up-re 0.492 0.611 0.605 0.806 1.176 0.894 1.286 1.254 0.536 0.701 1.229 1.311 1.863 1.442 0.757 0.454 0.473
gM
o
gelvs.
aA
erM
v
A
P. C
PT vs.
M M
2 5 1 4 9 1 3 6 2 1 1 2 3 2 2 5 6
Avs. aine 0.02 0.01 0.02 0.02 0.00 0.00 0.04 0.03 0.03 0.01 0.01 0.04 0.02 0.02 0.04 0.01 0.03
MPTP- p-valu
n
i
d
PTPvs.controlandup-regulate Averagelog2(foldchange)inMvs.C(Down-regulated) -0.398 -0.711 -0.566 -0.829 -1.417 -0.982 -0.693 -0.936 -0.634 -1.097 -0.899 -1.936 -2.031 -1.053 -1.027 -1.306 -0.523
M
in 3)
weredown-regulated ProbeID(GeneaccessionNo.) 10416727(BC04957 10485792(XM_003086873) b10338220 b10338896 b10339243 b10340276 b10341589 b10342476 b10342786 b10342853 b10343119 b10343349 b10343551 b10343553 b10343684 b10344102 b10344601
h
c
hi
w 1
bstantianigralgenes Genetitle RIKENcDNA4921530L21gene predictedgene1393 st.etermined.
u ed
wn-up)s udentt-twasnot
Table3-Listof(do Genesymbol 4921530L21Rik Gm13931 aDeterminedusingStbGeneaccessionNo.
Acupunctureandgeneexpression 123
Figure3-Validationoftheexpressionofrepresentativegenesbyreal-timeRT-PCR.Rdh9(A),Slc6a4(B),Tph2(C)andUcma(D),whichwere
“up-down”regulatedinthemicroarraydata,wereamplifiedbyreal-timeRT-PCRandthenquantifiedrelativetothereferencegene(GAPDH).Valuesare
means(cid:3)SE.*p<0.05,**p<0.005,or***p<0.0001wereconsideredsignificant.
were evaluated by real time RT-PCR (Figure 3). Expres- areprimarycausesofneurodegenerativedisorders(Drou-
sionlevelsofRdh9,Slc6a4,Tph2andUcma,whichwere in-Ouellet and Cicchetti, 2012; Pradhan and Andreasson
“up-down” regulated in the microarray analysis, also 2013),andtheirexpressioncanbemaintainedatsimilarto
showed “up-down” regulation in real time RT-PCR (Fig-
normal levels by acupuncture. Therefore, the effect of
ure3).Thesefindingsdemonstratethattheregulationpat-
MPTP intoxication on gene expression in the SN region
ternsintheSNregionregulatedbyMPTPandacupuncture
maybeamelioratedbyacupunctureatacupoints.Thissug-
at acupoints determined by real-time RT-PCR correlate
gestsanattenuatingeffectofacupunctureonthedegenera-
withthosedeterminedusingmicroarrays.
tionofdopaminergicneuron-likecellsbyMPTPintheSN.
Acupuncture stimulation attenuates the reduction of
Previous research employing a mouse model of acute
TH induced by 6-OHDA intoxication (Park et al., 2003)
MPTP Parkinsonism has demonstrated that acupuncture
and MPTP (Kang et al., 2007; Choi et al., 2011b) in
treatmentsuppressedgenesrelatedtocytokine-cytokinere-
nigrostriataldopaminergicneurons.Behavioraltestsdem-
ceptorinteractionandoxidativephosphorylationpathways
onstrated that acupuncture stimulation improves motor
(Hongetal.,2010).Cellularresponsestodeleteriousevents
dysfunctionina6-OHDAParkinsonismmodelby~87.7%
like oxidative stress and cytokine receptor-mediated
(Parketal.,2003).TheSNisacrucialaspectofthemotor
circuit(Brazhniketal.,2012;Gaugleretal.,2012)andis apoptosismighteventuallyleadtodopaminergiccelldeath
oneoftheareasdamagedmostmarkedlyinPD(Castroet andhencediseaseprogression.Oxidativestressisanearly
al.,2013;Fahimetal.,2013),particularlyrelativetodys- eventthatmaydirectlykilldopaminergicneurons(Honget
tonia(Truongetal.,2009). al.,2010).Moreover,thecurrentstudyshowedthatatthe
chronicstage,theexpressionofgenesinvolvedinPDwas
Theneuroprotectiveeffectsofacupuncturehavebeen
confirmed at a genetic level. The microarray results indi- modulated by acupuncture stimulation. These results sug-
catethatSlc6a4andTph2,whichareinvolvedinPD,and gestthatavarietyofgenesmayberelatedtotheeffectof
Mpzl2andSerping1,whichareinvolvedininflammation, acupunctureonthetreatmentofPD.