Accepted Article Diseases, Xijing Hospital of Digestive Diseases, Xi’an, Shaanxi, China China Shaanxi, Xi’an, Diseases, Digestive of Hospital Xijing Diseases, Liberation Army, Kunming, Yunnan, China University, Yinchuan,Ningxia, China 1 Su This article This protectedis by Allcopyright. rights reserved. cite this article as an 'Accepted Article', doi: 10.1111/jnc.12765 which may lead to differences between thisversion and the Version of Record. Please beennot throughthe copyediting,typesetting, pagination and proofreading process This article hasbeen accepted for publicationand undergone fullpeer review but has 4 3 2 Article : type Original Article Accepted Date : 14-May-2014 : 28-Apr-2014 Date Received Bao Wang Medical University, Xi’an, Shaanxi, China China Shaanxi, Xi’an, University, Medical State Key Laboratory of Cancer Biology and Institute of Digestive of Digestive and Institute Biology of Cancer Laboratory Key State Department of Neurology, KunMing General Hospital Theof People’s Ningxia Key Laboratory of Cerebrocranial Diseases, Ningxia Medical Tang Neurosurgery, of Department 4 , GuodongGao Destabilization of Survival ofDestabilization in byNeurotoxin MEF2DmRNA Factor 1 * , ZhibiaoCai 1‡ , QianYang Models of Parkinson’s Disease Disease Parkinson’s of Models 1 * , Fangfang Lu 1‡ du Hospital, the MilitaryFourth

1 , Chen Li , Chen 2 , XiaofeiZhu 3 , Linna Accepted Article This article This protectedis by Allcopyright. rights reserved. ‡ *These authorscontributed equally tothis study. loss of DA neurons in SNc andthe pathogenic process of PD. isIt known (MEF2D), a key neuronal survival factor, has shownbeen to underlie the disease(PD).Loss transcription of factor myocyte enhancerfactor 2D pars compacta (SNc) is an important pathological feature in Parkinson’s Progressiveloss of dopaminergic (DA) neurons in the substantial nigra Abstract [email protected] 710038Tel: China; 2986 84777435; Fax:29 84777435; 86 Email: FourthMilitary Medical University,Xinsi Road, 569 Xi’an, Shannxi, Guodong Gao, Department of Neurosurgery, TangduHospital, the [email protected] China; Tel: 86 29 84717823; Fax: 86 29 84777435; Email: Military Medical University, 569Xinsi Road, Xi’an, Shannxi, 710038 Qian Yang, Department of Neurosurgery, Tangdu Hospital,the Fourth trigger neuronal death. Although neurotoxins clearly destabilize MEF2D MEF2D destabilize neurotoxins clearly Although death. neuronal trigger that PD-associated neurotoxins reduce the level of MEF2Dprotein to orsodnet: Correspondence to: Accepted Article This article This protectedis by Allcopyright. rights reserved. disease.The diseaseis characterized pathologically by the lossof Parkinson’s disease (PD)the is second most common neurodegenerative Introduction myocyte enhancerfactor 2D,mRNA. Keywords: neuronal viability. responsiblepart for neurotoxin-induceddecrease MEF2Din proteinand in is mRNA MEF2D of reduction and destabilization that suggest results toxicity. These MPP+-induced to the and cells SN4741 cells sensitized of the viability reduced alone mRNA MEF2D of regulation Down controls. to DA inSNc neurons MEF2D mRNA compared of level ofthe reduction a marked to led to MPTP exposure that showed microdissection analysis ofthe SNc DA neurons captured immune-laserby capture SN4741 cells. DA line, Quantitative cell neuronal ina mRNA MEF2D PCR decrease asignificant MPTP, caused of metabolite thetoxic MPP+, that we showed this work, In . MEF2D in decrease neurotoxin-induced to contributes mRNA MEF2D posttranslationalby mechanisms, it isnot known whetherregulation of Parkinson’s disease, MPTP/MPP+, in thehalf-life and total level of Accepted Article This article This protectedis by Allcopyright. rights reserved. paradigms by manystudies (Mao in experimental several survival a to in vital role neuronal play shown four isoforms ofMEF2s firstlyidentified in mammalian cells, hasbeen and function localization. inlevel, factors transcription of changes mechanisms that accounts for the alteration in expression is Albéri pathogenic process of PD (Zetterström the accompanies expression gene in alteration that shown have studies Przedborski 2003, Snyder &D'Amato 1986, Yan triggering orfacilitatingthe pathogenic process (Nagatsu 1997, Dauer& considered toone be the of important etiological factors either is to neurotoxins exposure clear, not entirely are DA SNc neurons of loss selective the for reasons precise the Although 1997). & Kish Tatton compacta (SNc) (de Lau & Breteler 2006, Tanner Goldman& 1996, pars nigra substantial the in neurons (DA) dopaminergic pigmented toxicity. Enhancing MEF2D activity protects DA neurons in SNc from from SNc in neurons DA protects activity MEF2D Enhancing toxicity. thatshown loss of MEF2D protein function underliesMPTP-induced 1,2,3,6-tetrahydropyridine(MPTP) mouse model of PD, it has been Mount 2004, Linseman & Transcription factor myocyte enhancer factor (MEF2D),2D one the of et et al. 2004, Salih &Brunet 2008, Maxwell et et al. 2013). In 1-methyl-4-phenyl et al. et al. 1999, Liu 1999, 1997, Smits 1997, et al. et al. et al. 2012). Previous Previous 2012). 2005). One of the the of One 2005). 2003, Heidenreich Heidenreich 2003, et al. 2003, Accepted Article This article This protectedis by Allcopyright. rights reserved. toxin-induced death(She 2003, Wang multipleby pathways indifferent survival and death signals(Gong proc pathogenic in PD rolecritical a 2013). These findings suggestthat dysregulation MEF2Dof protein plays neurotoxins significantly affected the level of MEF2D mRNA and MPTP/MPP+-induced oxidative stress. Ourstudy showed that MEF2DstabilitymRNA of and level the examined we Here unknown. remains mRNA MEF2D whether neurotoxins can preferentially regulatethelevel and stability of proteinand level function (Schwanhäusser modifications. toxic signals reduce MEF2D protein involves post-translational Together,these studies reveal that one of keythe mechanisms by which experimentaland idiopathic PD (Yang et al. 2009, Thomas al.et 2011). in activity MEF2D blocks and MEF2D of non-functional degradation in viability neuronal undermines which process, this block can (Yang (CMA) non-functional MEF2D is degraded by chaperone-mediated autophagy mRNA stability influencesgene expression, and has an effect on et al. et al. 2009, Li 2009, Thomas 2009, et al. in vitro in et al. 2011, Smith 2011, 2001). Our recent studies showed that that showed studies recent Our 2001). et et al. and ess. MEF2D protein level is controlled level MEF2D protein ess. in vivo 2011). Aggregation of et al. et et al. under neurotoxin neurotoxin under 2011).However, 2006, Mount 2006, α etal. -synuclein -synuclein etal.

Accepted Article This article This protectedis by Allcopyright. rights reserved. (Sigma,base MPTP St Louis, once(i.p.) daily MO, intraperitoneallyUSA) the numberof animals mg/kg used. free Adult C57bl/6 micereceived 30 China). Allefforts wereto mademinimize animalsuffering andreduceto Fourththe Military Medical Universi Gu the with accordance in used were ExperimentalAnimal CentertheFourth of MilitaryMedical University, At leastweeks 8 old C57bl/6 male mice (25~35g), purchased fromthe andAnimal tissue preparations Signaling). (Cell antibody caspase-3 anti-cleaved Denmark); Copenhagen, Cytomation (Dako antibody secondary “Universal” biotinylated CA, USA); Jose, San Bioscience, (BD antibody anti-MEF2D anti-TH antibody, anti- The following antibodies used in thisstudy were obtained commercially: Antibodies Materials and Methods neurotoxicity. MPTP/MPP+-induced part in underlies mRNA of MEF2D that destabilization revealing factors, transcription other for markedly shortened its half-life compared to control mRNAs encoding β -actin antibody(Sigma, St Louis, MO, USA); ty (Xi’an, People’s Republic of of Republic People’s (Xi’an, ty idelines for Animal Care and Use forAnimal andUse Care of idelines Accepted Article This article This protectedis by Allcopyright. rights reserved. 1999), were cultured at 33ºC with 5% CO SN4741,a mouse embryonic substantial nigraderived linecell (Son Cell culture analysis. further for collected et al. and processed as described in full detail (L'Episcopo phosphate buffer(PB, pHfor 7.4)35 min. brains The werethen removed 2 min followed byfixative a containing 4% paraformaldehyde in M0.1 injection. Mice were perfused transcardially with 0.9%NaCl solution for study. Mice were sacrificeddays 3 or5 days followingthe first MPTP salinevolumestheat 0.9% of sameas usedin frequency MPTP this 2007, Thomas for consecutive 5 days in subacute manner (Jackson-Lewis & Przedborski reached 50-60% confluence. cells when done usually were Experiments plates. three into split were L-glutamine).When reachedcells 80% confluence(usually days),3 they and D-glucose,140mM 1%penicillin-streptomycin 1% (FBS), modified Eagle’s medium supplementedwith 10% fetal bovineserum 2004). Serial coronal sections(30 et al. 2012). Control cohorts of mice received equivalent μ m thick) containing the SNc were 2 in RF medium (Dulbecco’s (Dulbecco’s medium RF in etal. 2011, Morale 2011, et al.

Accepted Article This article This protectedis by Allcopyright. rights reserved. visualized by chemiluminescence detection. peroxidase-conjugated secondary antibody. Protein bands were washed three times withTBS-T and incubated witha awithprimary antibody with gentle shaking at 4ºC, and then they were (pH 7.6), 150mM NaCl,0.1%Tween-20), membraneswereincubated Tris (50mM (TBS-T) TBS-Tween in milk skimmed 5% with blocking After MA,USA). Bedford, corporation, (Millipore membranes (PVDF) fluoride totransferred polyvinylidene and (SDS-PAGE) gel electrophoresis The were separated by sodium dodecyl sulfate polyacrylamide Immunoblotting confocal microscope (Nikon, Si,C2 Japan). counterstained with DAPIfor 5 minutes and photographed using a room temperature for 2h, washed 3 times with PBS. Samples were were washed 3 times with PBS, incubated with a secondary antibody in were incubated withprimary a antibody at 4ºC overnight, and then they serum inPhosphate BufferSolution (PBS) for 30min. Afterwardssamples incubated with 0.1% Triton X-100 for 30min, blocked with 5% goat brain orCells sections with fixed were formaldehyde4% solution, Immunofluorescence

Accepted Article This article This protectedis by Allcopyright. rights reserved. forward, En2 5´-TCTTCTTTAGCTTCCTGGTGCG-3´; 5´-CGCCTGGGTCTACTGCACA-3´; En1reverse, En1 forward, 5´-GTGGTGAGCGAGTGGGTAGA-3´; reverse, MEF2D 5´-ATGGCAACAGCCTAAACAAAGT-3´; forward, MEF2D follow: as sequences Primer cycle. every during step the 55ºC of end the at measured then 40 cycles of 95ºC for 30s and 55ºC for30s. Fluorescence was mixtures. The thermal profile was 50ºC for 30min, 95ºC for 15min, and master mix (Stratagene) with 10ng of total RNA in 25 performed using BrilliantII SYBRgreen single-step quantitative RT-PCR quantitative PCR system (Stratagene). Quantization of mRNA was RT-PCR(qRT-PCR) was performed using anMx3005 or Mx4000 multiplex specific mRNA was normalized to for 30 min andvisualized under LEDlight. The abundance of each products wereseparated agarose in1.2% TBEingels buffer 1× at100V a semiquantitative determinationof the originalamount. RNA The PCR DNA, specific the amplify to template master(Stratagene). mix The products(1 transcribed using Brilliant II SYBR green single-step quantitative RT-PCR TRIzol Reagent (Invitrogen,Carlsbad, CA, USA). RNA (1 Total RNA was extracted according to the manufacturer’s protocol for RT-PCRandSemiquantitative real-timeqRT-PCR β -actin mRNA. Real-time quantitative using 25-cycleregular PCR to give μ l of cDNA) were used theas μ l reaction μ g) was reverse reverse was g) Accepted Article This article This protectedis by Allcopyright. rights reserved. 5´-TTGTGCCGGATGGAGTTCTT-3´; 5´-ACGGCTCACTTTGTCCCAGAT-3´; FoxO3reverse, 5´-TTCTCCGAGTCACTGTGCTC-3´; FoxO3forward, reverse, Pitx3 5´-GACGCAGGCACTCCACACC-3´; 5´-GTAAACGACCTCTCCGGCC-3´; Pitx3 forward, Nurr1 reverse, 5´-GTGCCTAGCTGTTGGGATGG-3´; 5´-GGCCGCTTGTCCTCTTTGT-3´; Nurr1forward, 5´-GACCGGCCTTCTTCAGGTC-3´; En2 reverse, Eight-micrometermembrane-mounted sections on (Microdissectslide capture microdissectionImmune-laser 5´-ATCTTCTCCATGTCGTCCCAGTTG-3´. 5´-AAGGACTCCTATAGTGGGTGACGA-3´; (Sigma, St Louis, MO, USA) for 1 to 2 min and counterstained with with counterstained and min to 2 for 1 USA) MO, Louis, St (Sigma, washed twice, all the sections were developed with diaminobenzidine secondary antibody for20 min at room temperature. After being Afterwards, allsections were incuba amin inhumid chamber, and washedtwice for 3 min each in PBS. then incubated with anti-TH primary antibody in the PBS solution for 30 the sections were incubated in 0.1%Triton X-100 withPBSfor 5min and 20 for 100%ethanol min; fixed in immediately prechilled and Germany) GmbH,Germany) were collected inthe cryostat(Leica CM 1900, β -actin forward, ted withbiotinylated “Universal” β -actin reverse, reverse, -actin Accepted Article were: spot size = 4 = spot size were: parameters cut Laser cells. interested the out cut and around” “draw to beam laser ultraviolet narrow a supply USA), CA, Devices, Molecular treatment for 24 h,apoptosis was measured ApopNexinby (50 MPP+ plates. in six-well were plated with lentivirus preinfected Cells apoptosis Measurement byof cytometry flow analysis. forfurther harvested fo lentivirus with infected were Cells medium. antibiotic-free with plate six-well in seeded were cells obtained commercially fromNeuronBiotech, Shanghai, SN4741China. Recombinantlentivirus vector for MEF2D gene (shRNA-MEF2D) was Lentivirus infection between pulses = 400 ms. This article This protectedis by Allcopyright. rights reserved. Varitas A LCM. StLo (Sigma, hematoxylin in a flow cytometer (CytomicsFC 500, Beckman Coulter, Brea, CA). Temecula, CA). Fluorescence due toFITC andstaining PI was measured (FITC) isothiocyanate Apoptosis μ M,Sigma, St Louis, MO, wereUSA) added tothe culture. After TM

ArcturusLCM, combined with TR/UV system(Arcturus μ m, power80m,pulse mw,Time= duration 15ms. = uis, MO, USA) and the sections were ready for ready were sections the and MO, USA) uis, r 72h. Afterwards, the cells were were cells the Afterwards, r 72h. Detection Kit (APT750, Millipore, Millipore, (APT750, Kit Detection TM fluorescein fluorescein Accepted Article This article This protectedis by Allcopyright. rights reserved. 10 Briefly, manufacturer. The assay was performed accordingto the specificationof the MTT assay experiments. Statistical significance was determined using Student usingStudent determined was significance Statistical experiments. independent three least at from SEM ± mean as expressed were Data Statistical analyses wavelength of 570 nm with 655 nmas a reference wavelength. theincubation, the absorbanceof the samples was measured at a the platewas incubated for 4h inahumidified incubator at 37ºC. After addedto SN4741 cells cultured in 96-well platein 100 modulate transcription factors on , we examined the the examined we expression, gene on factors transcription modulate survivaland death understress. To test whether neurotoxins may pl factors transcription of Regulation treatment in SN4741 ofAnalysis gene expression ofprofiles transcription factorsafter MPP+ Results wasstatistically significant. considered of p<0.05 value t-test. Statistical analyses were carried outusing SPSS version 19.0. A μ l of 5 mg/ml MTT labeling reagent were were reagent labeling mg/ml MTT 5 l of ays an important role in neuronal inneuronal role animportant ays μ l of medium, and Accepted Article This article This protectedis by Allcopyright. rights reserved. leveltheMEF2D ofproteins in models(SmithPDal. of et Tang 2006, Previous studies have shownthat exposure to neurotoxic stressreduces SN4741 in MPP+ The level of MEF2D mRNA and protein was significantly reduced by gene expressionneurotoxic bystress. regulated by neurotoxins, we chose to focuson MEF2D mRNA to study and DA neurons SNc of survival the for required is activity MEF2D that sensitiveto MPP+-induced stress. Since our previous studies haveshown Compared to other transcription factors, the MEF2D mRNA is more treatmentreduced only mRNA levels MEF2Dof andEn1 (Fig. 1a-f ). Nurr1,for MEF2D, levels mRNA of reduction clear a to led MPP+ with treatment short The PCR. real-time mRNA was extracted andanalyzed for mRNAlevels by quantitative 50 with treatment Following PD. in injury neuronal (Son etal. 1999, She et al. 2011). MPP+/MPTP have been used to study toused study the neuronalresponse induced by PD related stress signals dopaminergicneuronalline, SN4741, cell becausewidely been ithas Brunet 2008,Maxwellal. et 2005).We to chose a mouse use midbrain (Zetterströmal. et 1997, Smits etal. 2003, Albérial. et 2004, Salih& maintenance and survivalof DA neurons after neurotoxin treatment of mRNA levels key factors known several to requiredbe for the En1/2and Pitx3prolonger while μ M MPP+, the total total the M MPP+, et Accepted Article This article This protectedis by Allcopyright. rights reserved. Immunofluorescence analysis ofthe midbrainsections from the MPTP afterthe first injection (Day3,Day5) for subsequent analysis. sub-acute regimen and prepared the midbrainregion atand 3 days5 afollowing MPTP with mice intraperitoneally injected this, we For PD. To confirm the above finding neuronsMPTP-PD in mouse model levelmRNA The of MEF2D wassignificantly decreasedDA in theSNc level. protein reduction of MEF2D mRNA correlated closely withthe decrease inits the and mRNA MEF2D of level the decreases clearly MPP+ neurotoxin similar time-dependent manner as MEF2D protein(Fig. 2cand 2d). Thus, a in reduced markedly were mRNA MEF2D of levels the that showed semi-quantitativeand PCR quantitative real-time These PCR.data treatment paradigm and determined the MEF2D mRNAlevel by same the we employed mRNA, MEF2D affect may PD with associated decline in the level ofMEF2D protein. Totest whether neurotoxin gradual time-dependent a to led treatment MPP+ 2b). (Fig. immunoblot levels MEF2D of protein immunocytochemistryby (Fig.2a) and MPP+. At different times following this treatment,we measuredthe al. 2005).To establish this model, we treated SN4741 cells with 50 in vivo , we used the MPTP mouse modelof μ M Accepted Article ertxnMP etblzdMFDmN nD ernlS44 Neurotoxin MPP+ destabilized MEF2D mRNA in DA neuronal SN4741 life of MEF2D mRNA. We treated SN4741 cells with transcription of MEF2D mRNA byincreasing its degradation, we determined the half (Dreyfuss itand can provide an efficientand rapid wayto blockgene transcription Actinomycin D is one ofthe most frequently transcriptionused inhibitors abundance (Ross 1995, Wang half-lives of mRNA anplay import Although the level of mRNA isaffected itsby rate of synthesis,the inalmost organisms. all gene expression influences mRNA stability This article This protectedis by Allcopyright. rights reserved. protein MEF2D of decrease the with correlated which neurons, DA SNc the in mRNA MEF2D of level the reduced ThisPCR. an real-time quantitative theRNA fromdissected neuronsand measured MEF2DmRNA by total then extracted We 3b). (Fig. neurons TH positive isolate to (LCM) TH positive neuronsin the SNcand applied laser capture microdissection neurons atthe SNc region, we used immunohistochemistry to identify positive TH in mRNA MEF2D of levels the determine To 3a). (Fig. group control the to compared Day5) (Day3, neurons TH positive SNc the in protein MEF2D of level the in decline asignificant revealed mice treated et al. 1984). To determine whether MPP+ decreased the levels levels the decreased MPP+ whether To determine 1984). et al. ant role indetermination of mRNA ant alysisshowed that MPTP greatly 2002, Guhaniyogi & Brewer 2001). 2001). Brewer & Guhaniyogi 2002, in vivo (<.1 Fg c. 3c). (Fig. (p<0.01) Accepted Article This article This protectedis by Allcopyright. rights reserved. a increase marked caused level mRNA MEF2D in decrease that showed Analysis (Fig. 5a). lentivirus-shMEF2D of the effect shMEF2D confirmed group, which in level mRNA quantitative PCR showed that the obvious down-regulation of MEF2D shRNAto MEF2D for 72h. Semi-quantitativeRT-PCR and Real-time expressing lentivirus with cells SN4741 viability, incubated we neuronal affects mRNA MEF2D of down-regulation whether test To 2012). (Yamanaka mRNA target of degradation the guide could shRNA stability MEF2Dof mRNA level in PD models.Study has shown that Our aboveresults showed that neurotoxin MPTP/MPP+ reduced the neuronal lineSN4741 cells and enhances MPP+-induced toxicity Down-regulation of MEF2Dreduces levelthe mRNA DAsurvival of cells. SN4741 in mRNA mRNA wasshortened to 0.7 h, suggesting thatMPP+ destabilizesMEF2D MEF2D mRNA level (Fig. 4a and 4b). The estimated half-lifeof MEF2D simultaneously toSN4741 half-lifeawith of about Addition1.5 h. ofactinmoycinandDMPP+ PCR. ActinomycinDtreatment alonerevealed that MEF2D mRNA decays measuredMEF2Dlevelsthereal-time mRNA of over bytime quantitative then and treatment MPP+ or D without with actinomycin inhibitor cells significantly accelerated the decrease in the decrease accelerated significantly cells of cell viability by flow cytometry cytometry byflow viability of cell etal.

Accepted Article This article This protectedis by Allcopyright. rights reserved. functions. and levels their regulate to modifications post-translational such as neurodegenerative disorders oftentarget key proteins by Oxidative stress and toxic signals known to cause age-related diseases Discussion Control and FUGW groups (Fig. bottom).5d MPP+ treatment in shMEF2D group wa after and (%) before cell ratio ofsurviving The top). 5d (Fig. assay MTT also sensitized SN4741 cellsto MPP+-induced toxicityas determined by Consistent withthese findings, pre-inhibitionofMEF2D function mRNA level activatedof caspase-3 following MPP+ treatment (Fig. 5c). Pre-inhibitionoffunction MEF2D mRNA also markedly increasedthe caspase-3. activate to sufficient was alone mRNA MEF2D interfering caspase-3.Analysis ofthe level of cleaved caspase-3 indicated that MPP+.with viability Cell was determ expressing shRNAto MEF2D as described above and then treated cells to sensitivity cell neurotoxin, we prei viability. to reduceneuronal sufficient is level mRNA MEF2D down-regulated suggest data These 5b). (Fig. (0.5%) group FUGW in cell death(14%) compared tothe controlgroups (0.4%) andthe To test the effects of down-regulation of MEF2D mRNA level on the the on level mRNA MEF2D of down-regulation of effects the test To ined by western blot for cleaved cleaved blotfor western by ined nfectedSN4741 with cells lentivirus s significantly lower than inthe lower significantly s Accepted Article This article This protectedis by Allcopyright. rights reserved. signals inhibitMEF2D activity byreducing MEF2D levelin neurons. (McKinsey survival neuronal including processes cell different in roles critical of MEF2D under stress. inrate its increase to This of decay. the contribute reductionmay overall MPP+reduced MEF2D mRNA in time-dependent manner viasignificant stress. oxidative to response in profiles different display factors key al.2009). Our current datarevealed nowthat the mRNAlevels of these (Zetterströmal. et 1997, Smits etal. 2003, Maxwell et al. 2005, Yang et factors playingkeya rolein maintenance and survival of DA neurons En1, En2, Nurr1, MEF2D, Pitx3 and FoxO3 have all been identified as gene expression(Heintz of thelevel to contributes significantly rate decay mRNA eukaryotes, Stability of mRNA influencesgene expression in organisms.For delineated. fully be to remains pathogenesis neurotoxins targetmay critical mRNAs or genetranscription in PD mRNA in many physiological and pathological processes.Whether of and levels expression gene by controlled also are function and level Protein death. to neuronal leading pathways and damage mitochondrial For PD, previousstudies have mainly focused on proteins involved in Many studies have shown that transcription factor MEF2D plays et al. 2002,Potthoff& Olson al.Mao 2007, et 1999).Stress et al. 1983). The transcription factors including including factors transcription The 1983). Accepted Article This article This protectedis by Allcopyright. rights reserved. and murine (Dodou focused on single Drosophila (Bour process of PD. Previous studies on mef2gene transcription mainly that decrease of MEF2D mRNA maycontribute tothe pathogenic stability to down-regulate MEF2D protein level.These findings suggest Our current studies show that neurotoxin reduces MEF2D mRNA undetermined. still is expression gene mef2d of regulation the However, 2005). al. et Tang 2003, al. et Gong 2009, al. et (Yang PD of models autophagy also participates in degradation of non-functional MEF2D in promotesits degradation caspases.by In addition, chaperon-mediated phosphorylationof MEF2DCdk5 byfollowing excitotoxicitystress that shown it been has For example, identified. been have Several mechanisms by whichstress signals reduceMEF2D protein level The stabilityof short-lived ( The abundance of mRNA isregulated by MEF2D. partof the mechanism contributing tostress-induced dysregulation of as mRNA mef2d of regulation establish help now findings Our family. abundance. Our data showedthat MPP+ reduced MEF2D mRNAa in such mRNAs isknown to cause significant changesin their protein pharmacological (de Nadal such stimuli, external and internal

et al. et et al. t =2 h or less) mRNA changes in response to to response in changes mRNA less) or h =2 2004), another member of MEF2s MEF2s of member another 2004), 2011). A small fluctuation in half-lives of as as developmenta a change in the mRNA half-life. thehalf-life. mRNA in change a et et al. 1995, Taylor l, nutritional, l, nutritional, et al. 1995) Accepted Article This article This protectedis by Allcopyright. rights reserved. Simunovic 2002, (Bustin demonstrated theadvantages of analyzing samplesdissected LCMby great heterogeneity of neural phenotypes.Many studies have a is there where system nervous central the in researches for important for information and specific relevant cellular population fromawell-defined tissue region provides more toxicity. to MPP+-induced and sensitized cells death cell induced sh-MEF2D by alone mRNA MEF2D of Down-regulation mechanism by which neurotoxin reduces MEF2D protein. findings support destabilization MEF2Dof mRNA as animportant markedly shortened its half-lives from to1.5 h 0.7 h. Together, these Neurotoxin MPTP/MPP+ destabilized short-livedMEF2D mRNA and Based data, on our half-lifethe ofMEF2D is about mRNA h. 1.5 level. inprotein decrease a with correlating manner, time-dependent of the changes of MEF2D mRNA levels in TH positive neurons in the SNc. SNc. the in neurons THpositive in levels mRNA MEF2D of the changes of neurons andto obtain a muchmore precise andreliable quantification the contaminating signalsfrom non-neuronal cells or THnegative eliminate to significantly us allowed approach This SNc. from neurons combined immunohistochemistryand LCMto isolate TH positiveDA Compared toCompared samplestissuesisolationof whole or sections, of desired et al. 2009, Finak 2009, the targets. This is particularly the Thisis targets. particularly etal. 2006).this In study, we Accepted Article This article This protectedis by Allcopyright. rights reserved. The authors declarethat no competinginterests exist. interests Competing (Q.Y). National Natural Science Foundation of China, GrantNo. 31371400 Grant 2011CB510000(Q.Y), FMMU Research Foundation (Q.Y) and manuscript. This work was supported by Chinese National 973 Projects and Neurology,Emory University, USA, forthecritical reading theof Acknowledgements regulation of MEF2D mRNA stability under PD-related stress. in areinvolved pathways decay mRNA which to test important be will It MEF2D proteindegradation for and destabilizing MEF2D (Fig.6). mRNA targeting pathways: two by activity MEF2D repress PD to related signals relevanceregulatingof MEF2DPD modelin mRNA the it establishes significant isTH since of neurons SNc inpositive mRNA Our finding that MPTP/MPP+ specifically reduces thelevels of MEF2D The authors thankZixu Maofrom the Department Pharmacologyof neurotoxic that propose we study, this in findings allthe on Base in vivoin . Accepted Article Dauer, W. and Przedborski,Dauer, and W. (2003) Parkinson's S. disease: mechanisms models.and PCR transcription reverse real-time using mRNA of Quantification (2002) S. Bustin, This article This protectedis by Allcopyright. rights reserved. Re (2001) G. Brewer, and J. Guhaniyogi, a is Mef2c (2004) L. B. Black, and S.-M. Xu, P., J. Anderson, P., M. Verzi, E., Dodou, de Nadal, E.,Ammerer, and G. Posas, F. (2011) Controlling gene expression in de Lau, M. andL. Breteler,M. (2006) Epidemiology ofParkinson's disease. Bour,B. A., O'Brien,M. A., Lockwood, W.L. cell-autonomously are (2004) H. H. Simon, and P. Sgadò, L., Albéri, References Gong, X., Tang, X., Wiedmann, M., Wang, X., Peng, J., Zheng, D., Blair, L. A., Marshall, A., Marshall, L. Blair, D., Zheng, J., Peng, X., Wang, M., Wiedmann, X., Tang, X., Gong, Finak, G., Sadekova,Pepin, (2006)GeneS.,et al. expression F. signaturesof Y. Choi, and A. S. Adam, G., Dreyfuss, cells. Neuron, 23-39. problems. and trends (RT-PCR): factor that isessential myogenesis. for H., Abmayr, S. M. and Nguyen, T.(1995)H. Drosophila MEF2, transcriptiona 33-46. transcription. mRNA of inhibitors with treated cells in ribonucleoproteins messenger development. embryonic mouse during field heart anterior the in factors and GATA ISL1 of target transcriptional direct response to stress. Neurology, Development, neurons. dopaminergic mesencephalic in apoptosis prevent to required transcriptionneurotoxicity-induced in factor MEF2 apoptosis. Cdk5-mediated(2003)Mao, Z. andJ. inhibitionprotective theof of effects Cancer Research, tumors. basal-like identify tissue breast normal morphologically Gene,

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Accepted Article Yan, M. H., Wang, X.andZhu, X.(2012) Mitochondrial Defects and Oxidative Stress in This article This protectedis by Allcopyright. rights reserved. * n=3; β Nurr1, FoxO3and Pitx3 were quantifiedby qRT-PCRand normalized to 50 SN4741 cells aftertreatment. MPP+ (a-f) Fig.Analysis 1 ofgene expression profiles of transcription factorsin Figure legends T. andPerlmann, L. Olson, J., B. Hoffer, L., Jansson, L., Solomin, H., R. Zetterström, Yang, Q., She, H., Gearing, M., Colla, E.,Lee, M., Shacka, J. J. and Mao, Z. (2009) E. F. Reyes-Turcu, S., Mehta, Yamanaka, S., O. P. and Brown, D. J., Herschlag, R. Tibshirani, D., J. Storey, L., C. Liu, Y., Wang, Wang, X.,She, H.andMao, Z.(2009) Phosphorylation neuronalof survival factor -actin mRNA level as described in as described level mRNA -actin μ MMPP+ for 12 or 24h, and the mRNA levelsof MEF2D, En1, En2, Medicine Disease. Parkinson's and Disease Alzheimer's retrotransposons. and genes developmental silences machinery specialized by triggered RNAi (2012) I. S. Grewal, and B. G. 248-250. mice. Nurr1-deficient in agenesis neuron Dopamine (1997) autophagy. fa survival neuronal of Regulation Sciences, of Academy National decay. mRNA in specificity functional and Precision (2002) Chemistry, Biological 3 kinase synthase glycogen by MEF2D p < 0.05, ** . Science, p < 0.01). 0.01). <

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99, Materials and Methods. (mean±SEM; ctor MEF2D by chaperone-mediated MEF2D by ctor 5860-5865. , Zhuang, F., Fuchs, R. T., Rong, Y., Robb, Robb, Y., Rong, T., R. Fuchs, F., , Zhuang, β in neuronal apoptosis. SN4741 were cells to exposed Nature Free Radical Biology and and Biology Radical Free . Proceedings of the Science, Journal of of Journal

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Accepted Article described in (a) and MEF2D protein was testified (n =3). =3). (n testified was protein and MEF2D (a) in described treated as were SN4741 cells treatment. after MPP+ level protein This article This protectedis by Allcopyright. rights reserved. DissectionDA of neurons from mousebrain SNc region LCM.by µm. 25 = Bar injection). MPTP first the after days five or three byimmunofluorscence (green: MEF2D; red: TH; blue: DAPI; Day3 or 5: MPTP. Sections of SNcregion of mouse wereanalyzedbrain for MEF2D MPTPregionmouse of modelMice PD. oftreated were withsaline or SNc DA neurons. (a) MPTP-inducedFig. 3 the indecrease level ofMEF2D inmRNA mouse p values presented werecorrected for The PCR (d). quantitative real-time by and (c) PCR reverse-transcriptase fromSN4741 cells andanalyzed by semi-quantitative extracted was RNA Total (a). in as described MPP+ with treated were of MPP+ ontheof levels MEF2D mR µm. 200 = Bar MEF2D). green: DAPI; (blue: by immunofluorescence analyzed 24 and for then 12 or h 50 with treated were cells SN4741 treatment. MPP+ following cells. (a) MPP+-induceddecreaseSN4741inNeurotoxin inMEF2DFig. 2 mRNA antibody for immunohistochemicalanalysis (top panel). TH anti-TH with stained were mice treated saline/MPTP from sections brain <0.05, ** Immunofluorescence analysis of MEF2D in SN4741 cells p < 0.01). < Immunofluorescence analysisof MEF2D inthe SNc (b) Western blot analysis of MEF2D NA in SN4741 cells. SN4741 cells cells SN4741 cells. in SN4741 NA β -actin controls (mean±SEM; n=3; * (c) + and neurons in neurons (b) (b) μ (d)

M MPP+ Mouse Effect Accepted Article mRNA. (a) mRNA. InhibitionSN4741Fig. 5 cellof survival bydown-regulationof MEF2D (mean±SEM; n=3; MPP+ treatment following inMEF2D half-life mRNA changes times. three repeated were experiments The after mRNA normalization to thewas determined level of for MEF2D mRNA by real-time quantitative PCR.Half-life of MEF2D MPP+. Total RNA was isolated at theindicated time points andanalyzed 50 without or with µg/ml) (10 D actinomycin with treated were cells PCR. The values presented are corrected for shown(b) in and analyzed for MEF2D mRNA by real-time quantitative SN4741 cells wereincubated with lentivirus expressing shRNA MEF2Dto This article This protectedis by Allcopyright. rights reserved. photo for TH for brain sections after microdissection; bottom panel: a representative the SNcwere dissected LCM.by (middle panel: a representative photo analysis of MEF2D mRNA in LCM-dissected TH Bar µm. = 200 experiments. independent three from RNA was extractedfromthe microdissected TH Determination of the effect of ofthe effect Determination DestabilizationFig. 4 MEF2D of mRNA byMPP+SN4741 in cells. (a) ** n=3; p <0.01). Effects of of lentivirus-shMEF Effects + neurons harvested on the caps).The data were represents p =0.002). MPP+ on MEF2D mRNA stability. mRNA MPP+MEF2D on 2D on levelof MEF2D mRNA. β (b) (b) -actin control(mean±SEM; + neurons in SNc. Total Total SNc. in neurons + neurons in SNc as as SNc in neurons Quantification of (c) Quantitative β -actin mRNA. mRNA. -actin

SN4741 SN4741 μ M Accepted Article cells were treated as described in(a described as treated were cells down-regulation of MEF2D mRNA on MPP+-induced cell death. SN4741 stress via a posttranslational and mRNApathways. regulatory model in which MEF2D activity is reduced by neurotoxic MEF2D. regulation dual of SchematicMPTP/MPP+-mediated diagramFig. 6 of MPP+ treatment (bottom) (mean the MTT assay (top) and the ratio of surviving cell (%) before and after This article This protectedis by Allcopyright. rights reserved. immunoblot. by determined were caspase-3 cleaved shMEF2D were exposed to 50µM MPP+ for additional 24 h. The levels of cleavedcaspase-3. SN4741cells preinfected with lentivirus-control or < 0.01). p ** n=3; semi-quantitativeand PCR quantitative real-time (mean PCR. by determined were mRNA of MEF2D levels the and 72h for repeated three times. times. three repeated staining and quantified flowby cytometry. The experimentswere double V-FITC/PI by Annexin determined was viability Cell infected). was not sample (control for 72h shMEF2D or (FUGW) vector empty survival. SN4741 wereon the cells cell Data from currentand previous studiessupport a dual (b) Effectsof down-regulation MEF2D oflevelmRNA (c) Effectsof down-regulation of MEF2D mRNA on ± SEM; n=3; ** p < 0.01). p < ** n=3; SEM; ). Cell viability was measured using using measured was viability Cell ). incubated withlentivirus carrying (d) Effects of of Effects

± SEM; SEM; Accepted Article

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Accepted Article This article This protectedis by Allcopyright. rights reserved.

Accepted Article This article This protectedis by Allcopyright. rights reserved.