Research Article Comparative mRNA Expression of eEF1A...

Research ArticleComparative mRNA Expression of eEF1A Isoformsand a PI3KAktmTOR Pathway in a Cellular Model ofParkinsonrsquos Disease

Kawinthra Khwanraj Suriyat MadlahKhwanthana Grataitong and Permphan Dharmasaroja

Department of Anatomy Faculty of Science Mahidol University Bangkok 10400 Thailand

Correspondence should be addressed to Permphan Dharmasaroja permphandhamahidolacth

Received 14 October 2015 Revised 23 December 2015 Accepted 19 January 2016

Academic Editor Helio Teive

Copyright copy 2016 Kawinthra Khwanraj et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The PI3KAktmTOR pathway is one of dysregulated pathways in Parkinsonrsquos disease (PD) Previous studies in nonneuronal cellsshowed that Akt regulation can be increased by eukaryotic protein elongation factor 1 alpha 2 (eEF1A2) eEF1A2 is proposed tocontribute protection against apoptotic death likely through activation of the PI3KAkt pathway Whether eEF1A2 plays a role inthe prevention of cell death in PD has not been investigated Recently gene profiling on dopaminergic neurons from postmortemPD patients showed both upregulation and downregulation of some PI3K andmTOR genes In this paper the expression of all genemembers of the PI3KAktmTOR pathway in relation to those of the eEF1A isoforms in a cellular model of PD was investigatedat the mRNA level The results showed a similar trend of upregulation of genes of the eEF1A isoforms (eEF1A1 and eEF1A2) andof the PI3K (classes IndashIII)Akt (Akt1 Akt2 and Akt3)mTOR (mTORC1 and mTORC2) pathway in both nondifferentiated anddifferentiated SH-SY5Y dopaminergic cells treated with 1-methyl-4-phenylpyridinium (MPP+) Upregulation of eEF1A2 Akt1 andmTORC1 was consistent with the relative increase of eEF1A2 Akt phospho-Akt and mTORC1 proteinsThe possible role of eEF1Aisoforms in the regulation of the PI3KAktmTOR pathway in PD is discussed

1 Introduction

The phosphatidylinositol 3-kinase (PI3K)Akt serinethreo-nine protein kinase (Akt)mammalian target of rapamycinkinase (mTOR) pathway is a crucial signaling mediatorwhich seems to serve as a connection between aspects ofneuronal proliferation differentiation and programmedapoptotic death [1 2] The PI3KAktmTOR system is alsoviewed as one amongst the key neuroprotective signalingpathways [3] Activation of the PI3KAkt pathway as aneuroprotective effect of caffeine has been shown in a cellularmodel of Parkinsonrsquos disease (PD) [4] Treatment with 1-methyl-4-phenylpyridinium (MPP+) leads to increaseddephosphorylation of mTOR and its substrate p70S6K inboth control and G2019S LRRK2-mutant skin fibroblasts[5] Several studies also support possible pharmacologicalprevention of dopaminergic neuron death through Akt andmTOR [6ndash9] Inhibition of mTOR using rapamycin has

been reported to suppress PINK1 and Parkin pathology [10]After activation mTOR further activates its downstreameffectors that regulate the transcription of many genes andthe synthesis of proteins [11]

eEF1A (eukaryotic protein elongation factor 1 alpha)proteins are GTP-binding proteins that bind to amino-acylated tRNA and deliver them to the ribosome duringthe elongation process of protein translation There aretwo isoforms of eEF1A eEF1A1 and eEF1A2 which sharegt90 sequence identity [12] eEF1A1 is widely expressedwhereas eEF1A2 is specifically expressed in the brain heartand skeletal muscles [12ndash14] In addition to their canonicalfunction in translation elongation binding of eEF1A1 toactin filaments and microtubules suggests its role in actinbindingbundling and microtubule bundlingsevering [15]eEF1A2 has been reported to interact with M4 muscarinicacetylcholine receptors (mAChR) in the brain [16] eEF1A2is considered an important human oncogene Its increased

Hindawi Publishing CorporationParkinsonrsquos DiseaseVolume 2016 Article ID 8716016 11 pageshttpdxdoiorg10115520168716016

2 Parkinsonrsquos Disease

expression has been found in tumors of the ovary breastand lung [17ndash20] The molecular mechanisms underlyingprogressive dopaminergic cell death in the nigrostriatalpathway of PD patients are not completely understoodRecent genetic and functional analyses show that there arepathogenic overlaps between cancer and PD [21] In theBT549 human breast cancer cell line eEF1A2 regulates cancerformation through Akt and PI3K-dependent remodelingof the cytoskeleton [22] In mouse plasmacytomas Eef1a2regulates tumor growth and proliferation through activationof the JAKSTAT and Akt signaling pathways [23]

Overexpression of eEF1A2 also protects cells againstapoptotic death induced by oxidative stress [24] eEF1A2 isfound to interact with peroxiredoxin-I (Prdx-I) a typical 2-Cys peroxiredoxin widely expressed in all mammalian cells[25] Prdx-I functionally protects cells from oxidative stress-induced cell damage by reducing a range of reactive oxygenspecies (ROS) production The interaction of eEF1A2 withPrdx-I provides an increase in protection from oxidativestress-induced death This protection is correlated with thedecrease in cleavage of caspases 3 and 8 and elevated expres-sion of prosurvival Akt

Whether eEF1A1 or eEF1A2 plays a role in the preventionof cell death in PD has not been investigated The aim ofour study was to analyze the pattern of gene expression foreEF1A isoforms in relation to that of the PI3KAktmTORpathway Thus we evaluated real-time mRNA expression ofPI3K isoforms PI3K classes I II and III Akt isoforms Akt1and Akt2 and mTOR isoforms mTORC1 and mTORC2together with the expression of eEF1A1 and eEF1A2 mRNAsusing an MPP+-induced SH-SY5Y cellular model of PD Weexpected that there would be a similar trend of increasedexpression of eEF1A isoforms and of the PI3KAktmTORpathway Indirectly we expected the result to suggest whetherany eEF1A isoforms could become a novel prosurvival factorin protecting dopaminergic cells against toxin-induced neu-ronal death

2 Materials and Methods

21 Materials Eaglersquos Minimum Essential Medium (MEM)Nutrient Mixture Hamrsquos F12 medium (F12) fetal bovineserum (FBS) and other supplements for cell culture werepurchased from Gibco (Gaithersburg MD USA) Retinoicacid (RA) 1-methyl-4-phenylpyridinium (MPP+) 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) and DMSO were purchased from Sigma-Aldrich(St Louis MO USA) Primers for real-time RT-PCR weresynthesized by and purchased from Biolegio (NijmegenNetherlands) Antibodies against Akt phosphorylated Aktphosphorylated mTORC1 and tyrosine hydroxylase (TH)were purchased from Cell Signaling Technology (DanversMA USA) Antibody against eEF1A2 and anti-120573-actinwere purchased from Sigma-Aldrich (St Louis MO USA)Horseradish peroxidase- (HRP-) conjugated anti-rabbitIgG and HRP-conjugated anti-mouse IgG were purchasedfrom Abcam (Cambridge UK) and Invitrogen (Eugene ORUSA) respectively

22 Culture and Treatment of SH-SY5Y Cells SH-SY5Yhuman neuroblastoma cells were grown in 1 1 mixture ofMEM and F12 supplemented with 10 heat-activated FBSCells were separated into two groups nondifferentiated anddifferentiated cells For neuronal differentiation of SH-SY5Ycells RA was added a day after plating at final concentration10 120583M in MEM-F12 with 10 FBS and maintained forthree days Neuronal differentiation was demonstrated asan increase in tyrosine hydroxylase (TH) expression usingimmunostaining Thereafter nondifferentiated and differen-tiated SH-SY5Y cells were exposed to 125 250 500 1000and 2000120583M of MPP+ for 24 hours The control groupsfor nondifferentiated and differentiated SH-SY5Y cells weretreated with the same medium without MPP+

23 Immunostaining for TyrosineHydroxylase SH-SY5Y cellswere fixed in 4 paraformaldehyde for 15 minutes at roomtemperature followed by sequential incubation with perme-abilizing solution (02 Triton X-100) in PBS for 30 minutesat room temperature Then cultures were washed again withPBS and incubated in blocking solution (3 BSA in 05Tween 20 in PBS) for 30 minutes Cells were incubatedwith rabbit polyclonal antibody against TH (1 200 dilutionin blocking solution Merck Millipore AB152) overnight at4∘C After washing cells were incubated with 1 500 dilu-tion of Alexa 488-conjugate secondary antibody for 1 hourat room temperature Coverslips were then mounted withVectashield antifadingmountingmediumwithDAPI (Vectorlaboratories CA) Cells were visualized under a confocallaser-scanning microscope (Olympus model FV 1000 TokyoJapan)

24 Cell Viability Assay SH-SY5Y cells were seeded ontoa 96-well plate at a density of 1 times 104 cellswell in 200120583Lof medium and incubated at 37∘C under 5 CO

2 After

exposure to MPP+ cell viability was measured by MTTcolorimetric assay This method was based on the reductionof tetra ring of MTT by mitochondrial dehydrogenases withNADH in the active mitochondria yielding a blue formazanproduct which can be measured spectrophotometricallyAfter incubation 20120583L ofMTT (5mgmL)was added to eachwell and the cells were cultured for another 4 h thenmediumwas removed and 100 120583L of DMSO was added to each wellto dissolve the formazan The color reaction was measuredat wavelength 570 nm with a reference at 690 nm using theVERSAmax Tunable microplate reader with SoftMax Prosoftware (Molecular Devices Sunnyvale CA USA)

25 Real-Time Quantitative PCR Analysis SH-SY5Y cellswere seeded onto 6-well plates Total mRNA was extractedfrom the pellet using PARIS kit according to the sup-plierrsquos instructions DNase I treatment was performed afterRNA extraction followed by heat inactivation The quantityand purity of RNA were determined by optical densitymeasurements at OD A260A280 ratio with 18 or aboveusing Nanodrop 2000 spectrophotometer (Thermo FisherScientific Inc Wilmington DE USA) Later the cDNA wassynthesized from 1 120583g of RNA using Masterscript RT-PCR

Parkinsonrsquos Disease 3

System (5 PRIME GaithersburgMD USA) according to themanufacturerrsquos instructions and stored at minus20∘C until assayKAPA SYBR FAST qPCR kit (Kapa Biosystems WoburnMA USA) was used for real-time PCR quantification The20120583L real-time PCR reactionmixture contained 20 ng cDNAtemplate 10 120583L of 1x KAPA SYBR FAST qPCR master mix200 nM of forward and reverse primers and PCR-gradewater 120573-Actin was used as a reference gene The sequencesof the primers are shown in Table 1 [26ndash28] The reactionwas performed in the Applied Biosystems 7500 real-timePCR system (Applied Biosystems Foster City CA USA)with the PCR cycling conditions as follows 3 minutes forenzyme activation at 95∘C 40 cycles of 3 seconds for initialdenaturation at 95∘C and annealingextension at 60∘C for1 minute Melting curve analysis was performed to verifyspecificity of each primer after PCR to ensure amplificationspecificity The threshold cycle (Ct) number was determinedand used in the comparative Ct methodThe relative quantityof the target gene was estimated by the 2minusΔΔCt method Alldata was analyzed by the ABI 7500 software version 20

26 Western Blot Analysis SH-SY5Y cells were seeded at aninitial concentration of 2times 105 cellswell in 6-well plates Aftertreatment withMPP+ for 24 hours cells were trypsinized andcentrifuged to collect the pellet Cold RIPA buffer (50mMTris PH 74 150mM NaCl 1 triton X-100 01 SDS1 sodium deoxycholate 5mM EDTA 30mM Na

2HPO4

and 50mM NaF) was added to the pellets mixed andincubated on ice for 20 minutes followed by centrifugationThe supernatants were collected and kept at minus80∘C untilbeing used Sixty120583g of protein extracts was separated in8 sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) and then transferred onto a nitrocellulosemembrane The membrane was blocked with 5 nonfat drymilk in 1x Tris-buffered saline containing 01 Tween 20for 2 hours It was then incubated with rabbit polyclonalanti-EEF1A2 (1 2000 dilution) rabbit monoclonal anti-Akt(1 1000 dilution) rabbit monoclonal anti-phosphorylatedAkt at serine 473 (1 1000 dilution) rabbit monoclonal anti-phosphorylated mTORC1 at Ser 2448 (1 1000 dilution) ormouse monoclonal anti-120573-actin (1 5000 dilution) overnightat 4∘C Subsequently themembranewas incubatedwithHRP-conjugated secondary antibodies (1 2000ndash5000 dilution)for 2 hours at 4∘C The bands were visualized using ECLPrime Western Blotting Detection (GE Healthcare Bucking-hamshire UK) The signal intensities were determined bydensitometry using Image-J software (National Institutes ofHealth Bethesda Maryland USA)

27 Statistical Analysis Experiments on cell viability assayand real-time quantitative PCR were repeated three times intriplicate measurement Statistical analyses were performedwith one-way ANOVA test followed by a post hoc analysis(Tukeyrsquos multiple comparison test) using GraphPad Prism 5Software for Windows (GraphPad Software Inc San DiegoCA USA) All values were expressed as mean plusmn standarderror of themean (meanplusmn SEM) for each group A119875 less than005 was considered statistically significant

Table 1 List of primer pairs used for real-time quantitative PCR

Gene symbol Primers sequences 51015840-31015840

eEF1A1 GGCATACCCGAGAGCATGAGGCATGTTAGCACTTGGC

eEF1A2 GAGCCCTCCCCCAACATGCCATGTTCACTGGCGCAAAGGTCAC

PIK3CA TGGATGCTCTACAGGGCTTTGTCTGGGTTCTCCCAATTCA

PIK3CB GCATTAAAAGGGAGCGAGTGCATGCCGTCGTAAAATCAGA

PIK3CD CTGGCTGAAGTCCAAGAACCCTCGGATCATGATGTTGTCG

PIK3CG ATACCATGATAGCGCCCTTGAATCACAGCGAACCTCTGCT

PIK3C2A GAAAAACGAGGAATCCGACACAGGGTTACTCCACCCAAGA

PIK3C2B TTCCCTAGTCGCTTCGTGATCAGTGGGTGGAAGAAGGTGT

PIK3C2G TTCATCTCCCAGATGGCTCTAGTGGGGTCCGTACATTTTG

PIK3C3 TCAGCCAAGCATTGTTGAAGTCCACTTTCGCGTTGTACTG

Akt1 TCTATGGCGCTGAGATTGTGCTTAATGTGCCCGTCCTTGT

Akt2 TATACCGCGACATCAAGCTGGGTCCCACAGAAGGTTTTCA

Akt3 TGGACAAAGATGGCCACATAACTGCTCGGCCATAGTCATT

mTORC1 AGGCCGCATTGTCTCTATCAAGCAGTAAATGCAGGTAGTCATCCA

mTORC2 TCTACCACGACAGCCCGGCATGGGGGCCCCGTTCCATCAT

120573-Actin CATGTACGTTGCTATCCAGGCCTCCTTAATGTCACGCACGAT

3 Results

31 Effect of MPP+ on SH-SY5Y Cell Viability To investigatethe influence of MPP+ on cell viability in nondifferentiatedand differentiated SH-SY5Y neuroblastoma cells cells wereexposed to 125 250 500 1000 and 2000 120583M MPP+ for 24hours For differentiated cells the cells were incubated with10 120583M RA for 3 days to induce neuronal differentiation priorto exposure to various dosages of MPP+ Differentiationof SH-SY5Y cells to a neuronal phenotype was confirmedby an increase in the expression of tyrosine hydroxylaseand neuritic outgrowth (Figure 1) Cell viability was exam-ined with an MTT assay Cell viability was significantlyreduced by all dosages of MPP+ in both nondifferentiatedand differentiated cells (Figure 2) In nondifferentiated cellsexposure to 500120583M MPP+ led to reduction of viability byapproximately 50 However 1000 120583M MPP+ was requiredto reduce the viability of differentiated cells to about 50Overall higher dosages of MPP+ were required to induce cell


TH

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Figure 1 Expression of tyrosine hydroxylase in undifferentiated and differentiated SH-SY5Y cells Cells were differentiated in 10 120583Mretinoic acid (RA) After differentiation for 3 days expression of tyrosine hydroxylase (TH) was visualized through immunostaining inundifferentiated cells and RA-differentiated cells Nuclei were stained using DAPI White arrows indicate areas of neurite outgrowth

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Figure 2 Effect ofMPP+ on cell viability of nondifferentiated SH-SY5Y cells (a) and effect ofMPP+ on cell viability of differentiated SH-SY5Ycells (b) Cells were exposed to 125 250 500 1000 and 2000 120583M of MPP+ for 24 hours and cell viability was measured with an MTT assayFor neuronal differentiation SH-SY5Y cells were treated with 10 120583Mretinoic acid for three days before exposure toMPP+The control groupsfor nondifferentiated and differentiated SH-SY5Y cells were treated with the same medium without MPP+ MTT assays were repeated threetimes in triplicate measurement Data are shown as mean plusmn SEM lowast indicates 119875 lt 005 and lowast lowast lowast indicates 119875 lt 0001 versus 0 120583m of MPP+



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Figure 3 mRNA expression levels of eEF1A1 and eEF1A2 in nondifferentiated SH-SY5Y cells (a) and mRNA expression levels of eEF1A1 andeEF1A2 in differentiated SH-SY5Y cells (b) For neuronal differentiation SH-SY5Y cells were treated with 10 120583M retinoic acid for three daysbefore exposure to MPP+ The control groups for nondifferentiated and differentiated SH-SY5Y cells were treated with the same mediumwithout MPP+ Real-time quantitative PCRs were repeated three times in triplicate measurement Data are expressed as relative to a controland shown as mean plusmn SEM lowast indicates 119875 lt 005 and lowast lowast lowast indicates 119875 lt 0001 versus control of each group

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Figure 4 Relative protein expression of eEF1A2 in nondifferentiated and differentiated SH-SY5Y cells by Western blot (a) For neuronaldifferentiation SH-SY5Y cells were treated with 10 120583M retinoic acid for three days before exposure to MPP+ The control groups fornondifferentiated and differentiated SH-SY5Y cells were treated with the same medium without MPP+ (b) represents the quantificationof the band density normalized to actin relative to a nondifferentiated control

death in differentiated cells compared with nondifferentiatedcells Thus 500 and 1000120583M MPP+ were used to treatnondifferentiated and differentiated cells respectively in laterexperiments

32 Expression of eEF1A1 and eEF1A2 Quantitative real-time PCR showed that eEF1A1 and eEF1A2 mRNAs weresignificantly increased in nondifferentiated SH-SY5Y cellsafter 24-hour exposure to MPP+ (119875 lt 0001 Figure 3(a)) Indifferentiated cells a significant increase was observed onlyfor eEF1A2 mRNA (119875 lt 005 Figure 3(b)) As the eEF1A2isoform has been reported to be involved in protectionfrom oxidative stress-induced death Western blotting wasperformed subsequently to evaluate eEF1A2 expression at

the protein level The result showed relatively increasedlevels of the eEF1A2 protein in both nondifferentiated anddifferentiated cells treated with MPP+ (Figure 4) which isconsistent with the mRNA result

33 Expression of Akt1 Akt2 and Akt3 After 24-hour expo-sure to MPP+ a tendency of increased expression of Akt1Akt2 and Akt3 mRNA was observed in differentiated SH-SY5Y cells although this could not be confirmed statisti-cally (Figure 5(b)) In nondifferentiated cells significantlyincreased expression was observed for Akt1 mRNA (119875 lt005) and relatively increased expression was observed forAkt3 mRNA (Figure 5(a)) Western blotting using antibodiesagainst Akt did not show significantly increased levels of



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Figure 5 mRNA expression levels of Akt1 Akt2 and Akt3 in nondifferentiated SH-SY5Y cells (a) and mRNA expression levels of Akt1 Akt2and Akt3 in differentiated SH-SY5Y cells (b) For neuronal differentiation SH-SY5Y cells were treated with 10 120583M retinoic acid for three daysbefore exposure to MPP+ The control groups for nondifferentiated and differentiated SH-SY5Y cells were treated with the same mediumwithout MPP+ Real-time quantitative PCRs were repeated three times in triplicate measurement Data are expressed as relative to a controland shown as mean plusmn SEM lowast indicates 119875 lt 005 versus control of each group

total Akt protein in both nondifferentiated and differentiatedcells treated with MPP+ (Figure 6) However when usingan antibody against phospho-Akt1 (Ser-473) which can alsobind to equivalent residues of Akt2 and Akt3 the resultshowed relatively increased levels of phospho-Akttotal Aktprotein in both nondifferentiated and differentiated cellstreated with MPP+

34 Expression of mTORC1 and mTORC2 RelativelyincreasedmRNA expression levels ofmTORC1 andmTORC2were observed in both nondifferentiated and differentiatedSH-SY5Y cells after exposure to MPP+ for 24 hours com-pared to untreated cells (Figure 7) Significantly increasedexpression of mTORC1 mRNA was seen in nondifferentiatedcells (119875 lt 005 Figure 6(a)) Western blotting using anantibody against Ser-2448 residue of phospho-mTORC1 alsoshowed relatively increased levels of phospho-mTORC1 inboth nondifferentiated and differentiated cells (Figure 8)which is consistent with the mTORC1 mRNA result

35 mRNA Expression of PI3Ks All genes encoding themembers of the three human PI3K classes were studied ClassI included PIK3CA PIK3CB PIK3CD and PIK3CG genesClass II included PIK3C2A PIK3C2B and PIK3C2G ClassIII included PIK3C3 Quantitative real-time PCR showedthat all of the genes encoding class I and II PI3Ks wererelatively increased in differentiated SH-SY5Y cells treatedwith MPP+ for 24 hours when compared to untreated cells(Figure 9(b)) Expression of PIK3C was relatively decreasedin both differentiated and nondifferentiated cells (Figure 9)Expression of class I and II PI3K genes was somewhat variedin nondifferentiated cells (Figure 9(a)) It should be noted thatexpression of PIK3C2A was significantly increased in bothnondifferentiated and differentiated cells treated with MPP+(119875 lt 005)

4 Discussion

As expected we observed similar mRNA expression betweenboth eEF1A isoforms and PI3KAktmTOR in both nondif-ferentiated and differentiated SH-SY5Y cells Differentiatedand nondifferentiated SH-SY5Y cells have been widely usedas a cell model of dopaminergic neurons for PD research Aprevious study suggested that differentiated SH-SY5Y cellsinduced by low serum and RA treatment represent a moresuitable experimental model for studying the molecular andcellular mechanisms underlying the pathophysiology of PDas shown by increasing sensitivity to 6-hydroxydopamine(6-OHDA) toxicity during the differentiation process [29]In contrast another study suggested that nondifferentiatedSH-SY5Y is more appropriate [30] In the latter study non-differentiated SH-SY5Y cells were susceptible to 6-OHDAand MPP+ while RA-differentiated SH-SY5Y cells conferredhigher tolerance potentially by upregulating survival signal-ing including the Akt pathway Thus we used both nondif-ferentiated and differentiated SH-SY5Y cells for comparisonin the present study

An interesting finding of this study is the significantlyupregulated mRNA expression of eEF1A2 isoform of eEF1Ain SH-SY5Y cells after exposure to MPP+ The upregulationwas observed in both nondifferentiated and differentiatedcells In contrast significant upregulation of eEF1A1 wasobserved in nondifferentiated cells but not in differentiatedcells The explanation for eEF1A1 upregulation in nondiffer-entiated cells afterMPP+ exposure remains to be investigatedNondifferentiated SH-SY5Y cells contain characteristics ofcancer Upregulation of eEF1A1may reflect a cellular responseto increased synthesis of several intracellular proteins involv-ing pro- and antisurvival processes The same explanationmay be applied to the upregulation of eEF1A2 in nondiffer-entiated cells Another hypothesis for eEF1A2 upregulations



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Figure 6 Relative protein expression of total Akt and phospho-Akt in nondifferentiated and differentiated SH-SY5Y cells byWestern blot Forneuronal differentiation SH-SY5Y cells were treated with 10120583M retinoic acid for three days before exposure to MPP+ The control groups fornondifferentiated and differentiated SH-SY5Y cells were treatedwith the samemediumwithoutMPP+ (b) and (c) represent the quantificationof the band density of total Akt and phospho-Akt respectively The band density was normalized to actin and relative to a nondifferentiatedcontrol

observed in differentiated cells after MPP+ exposure is itsrole in the protection of cells against oxidative stress Thedifferentiated SH-SY5Y cells acquire neuronal phenotypesafter treatment with RA [29 30] RA treatment confers SH-SY5Y cells with higher tolerance to MPP+ potentially byupregulating the survival Akt signaling pathway Significantupregulation of eEF1A2 but not eEF1A1 in MPP+-treated dif-ferentiated SH-SY5Y cells whose phenotype is dopaminergicmay reflect differential functions of these eEF1A isoforms

eEF1A2 has been shown to activate prosurvival Akt ina PI3K-dependent manner in a breast cancer cell line [22]eEF1A2 has also been shown to involve a reduction ofapoptosis in mouse plasmacytoma cell lines via activationof Akt [23] The role of eEF1A2 in resistance to apoptosishas been supported in other cell types [25 31] Recentlyexpression profiling and pathway study using microarrayanalysis of dopaminergic neurons removed from the frozenbrains of patients with PD has shown that there are several

significantly dysregulated pathways Among these pathwaysthe PI3KAktmTOR signaling pathway is considered a cen-tral hub To associate the prosurvival role of eEF1A2 with thatof the PI3KAktmTOR pathway we investigated the relativeexpression of isoforms further for PI3K Akt and mTORfamilies at the mRNA level

In the present study we found that all of the three Aktisoforms were relatively increased in their expression in dif-ferentiated SH-SY5Y cells after exposure to MPP+ Althoughthe isoforms share a high degree of structural homologyseveral studies suggested distinct roles for each of the Aktfamily kinases [32ndash35] It is recognized thatAkt1 is involved inthe prosurvival processes of the cell including antiapoptosisThe role of Akt2 and Akt3 in the induction of caspase-dependent apoptosis has been reported [36 37] Studies ofthe role of eEF1A2 on activation of Akt are mainly focusedon the Akt1 isoform whose phosphorylation sites are at Thr308 and Ser 473 [22 23] Western blotting using the antibody



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Figure 7 mRNA expression levels ofmTORC1 andmTORC2 in nondifferentiated SH-SY5Y cells (a) andmRNA expression levels ofmTORC1andmTORC2 in differentiated SH-SY5Y cells (b) For neuronal differentiation SH-SY5Y cells were treated with 10120583M retinoic acid for threedays before exposure toMPP+The control groups for nondifferentiated and differentiated SH-SY5Y cells were treated with the samemediumwithout MPP+ Real-time quantitative PCRs were repeated three times in triplicate measurement Data are expressed as relative to a controland shown as mean plusmn SEM lowast indicates 119875 lt 005 versus control of each group

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Figure 8 Relative protein expression of phospho-mTORC1 in nondifferentiated and differentiated SH-SY5Y cells by Western blot Forneuronal differentiation SH-SY5Y cells were treated with 10120583M retinoic acid for three days before exposure to MPP+ The control groupsfor nondifferentiated and differentiated SH-SY5Y cells were treated with the same medium without MPP+ (b) represents the quantificationof the band density normalized to actin relative to a nondifferentiated control

against phospho-Akt (Ser 473) in our study also showedan increased expression of phospho-Akt protein in MPP+-treated SH-SY5Y cells those being both nondifferentiatedand differentiated similar to previous studies [4 38] Itshould be noted that antibody against the Ser-473 residueof Akt1 might bind to equivalent residues of Akt2 and Akt3There is evidence of differential regulation of the isoformsafter induction using a common Akt activator [39] WhethereEF1A2 is a common Akt activator and possibly contributesto isoform specific regulation of Akt in PD and other cellularprocesses remains unknown at this time

We investigated the downstream signaling of Akt fur-ther Activation of the PI3KAktpathway promotes survivaland neuronal protection by mTOR activation [40] mTOR

the mammalian target of rapamycin exists in two distinctcomplexes mTORC1 and mTORC2 [41] The PI3KAktpathway is a major upstream modulator of mTORC1 [42]mTORC1 is the key player in the modulation of theautophagic process with mTORC2 implicated in Akt activa-tion [40] Studies on neuroblastoma cellular models of PDshowed the reduced phosphorylation of the major autophagyrepressor mTORC1 after exposure to either 2ndash12 hours of1000 120583MMPP+ for differentiated cells or 8ndash16 hours of 50120583M6-OHDA for nondifferentiated cells [43 44] Less is knownabout the role of mTORC2 in PD Our Western blottingusing the antibody against phospho-mTORC1 also showedan increased expression of the phospho-mTORC1 proteinin SH-SY5Y cells after 24-hour exposure to 500120583M MPP+



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leve

l(b)

Figure 9 mRNA expression levels of genes of PI3K classes I II and III in nondifferentiated SH-SY5Y cells (a) and mRNA expression levelsof genes of PI3K classes I II and III in differentiated SH-SY5Y cells (b) For neuronal differentiation SH-SY5Y cells were treated with 10 120583Mretinoic acid for three days before exposure to MPP+ The control groups for nondifferentiated and differentiated SH-SY5Y cells were treatedwith the same medium without MPP+ Real-time quantitative PCRs were repeated three times in triplicate measurement Data are expressedas relative to a control and shown as mean plusmn SEM lowast indicates 119875 lt 005 versus control of each group

and 1000 120583M MPP+ for nondifferentiated and differentiatedcells respectively The protein results were consistent withthe mRNA results Discrepancies between our and previousstudies remain to be investigated There has not been anystudy of the relationship between eEF1A isoforms andmTORactivation in PD Evidence from acute brain slices hasshown that the induction of protein synthesis-dependenthippocampal long-term potentiation that increases the localexpression of eEF1A1 is mediated by the mTORC1 signaling[45] A study of pheochromocytoma cells also showed thatstimulation with a nerve growth factor causes preferentialsynthesis of eEF1A1 over eEF1A2This process is mediated bymTORC1 [46] Our results provide additional informationat the mRNA level regarding the correlation between theexpression of isoforms of eEF1A and mTOR in PD

Next we investigated the PI3Ks the upstream regulatorfor Akt activation There are three classes (IndashIII) of PI3Ks inmammals Involvement of PI3Ks in various cellularmodels ofPD is usually investigated using a PI3K inhibitor LY294002which inhibits most of members of the PI3K classes [46]Data is lacking with regard to the predominant class of PI3Ksinvolved in neuroprotection Our present data showed at themRNA level that almost all members of PI3Ks except classIII were likely to be upregulated when SH-SY5Y cells wereexposed to MPP+

Parkinsonrsquos disease is a complex disorder Data frommicroarray-based gene expression profiling on human sub-stantia nigra dopaminergic neurons dissected using lasermicrodissection showed both upregulation and downregula-tion of genes of the PI3KAktmTOR signaling pathway inthe neurons of PD patients [6 47] For example PIK3C2G

and PIK3R2 members of class II and I PI3Ks respectivelyare upregulated in human dopaminergic neurons [47] Weobserved considerable variability of mRNA expression levelsacross the sample populations The results derived fromwithin each sample represented as mean plusmn SD were statis-tically significant (data not shown) while those derived fromanalysis across samples represented as mean plusmn SEM weremostly insignificant However there was overall consistencyamong each gene studiedThis observation was similar to theresults of a previous study performedusingTaqMan real-timePCR to validate the microarray data [47] It should be alsoemphasized that mRNA expression levels reveal informationat the level of transcriptional activation of genes but donot provide much about actual protein levels or functionsAdditional work on the protein expression for each isoformof eEF1A and members of the PI3KAktmTOR pathway isnecessary to confirm the involvement of these genes andproteins in the mechanistic network of PD

5 Conclusions

OurmRNA expression analysis is the first study grounded onthe expression of gene members of the PI3KAktmTOR sig-naling pathway in relation to those of the eEF1A isoforms in atoxin-induced cellularmodel of PD Both eEF1A1 and eEF1A2may be involved in the response to MPP+ of dopaminergiccells although eEF1A2 seems to be predominant Associationbetween eEF1A2 and PI3KAktmTOR has been supportedin other cell types [22 48] Whether or not eEF1A2 couldbe a potential candidate for pharmacological interventionremains to be seen Further studies to address the relationship


or interaction between eEF1A isoforms and PI3KAktmTORmembers including other cell survival pathways should beconducted in available cellular or animal models of PD Inaddition overexpression study for eEF1A may accentuate theeffects of the PI3KAktmTOR pathway The current resultscould be a useful adjunct to support previous studies usingthe microarray-based gene profiling analysis although val-idation of the gene expression in human dopaminergic neu-rons of PDpatients remains to be performed Lastly our studyon the PI3KAktmTOR genes in SH-SY5Y dopaminergiccells also provides a reference gene framework for researchersworking in PD The expression pattern will offer useful cluesabout the function of the PI3KAktmTOR pathway duringPD pathogenesis and regulation of neuroprotection

Conflict of Interests

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

Acknowledgments

This research is under the research framework of MahidolUniversity This research was supported by grants fromThai-land Research Fund and Mahidol University (IRG5780011 toP Dharmasaroja) andThailand Research Fund Royal GoldenJubilee PhD Scholarship (PHD00582556 to K Khwanraj)

References

[1] J A Engelman J Luo and L C Cantley ldquoThe evolutionof phosphatidylinositol 3-kinases as regulators of growth andmetabolismrdquoNature Reviews Genetics vol 7 no 8 pp 606ndash6192006

[2] P T Hawkins K E Anderson K Davidson and L R StephensldquoSignalling through Class I PI3Ks in mammalian cellsrdquo Bio-chemical Society Transactions vol 34 no 5 pp 647ndash662 2006

[3] A Chen L-J Xiong Y Tong and M Mao ldquoNeuroprotec-tive effect of brain-derived neurotrophic factor mediated byautophagy through the PI3KAktmTOR pathwayrdquo MolecularMedicine Reports vol 8 no 4 pp 1011ndash1016 2013

[4] K Nakaso S Ito and K Nakashima ldquoCaffeine activates thePI3KAkt pathway and prevents apoptotic cell death in aParkinsonrsquos disease model of SH-SY5Y cellsrdquo NeuroscienceLetters vol 432 no 2 pp 146ndash150 2008

[5] S M S Yakhine-Diop J M Bravo-San Pedro R Gomez-Sanchez et al ldquoG2019S LRRK2mutant fibroblasts from Parkin-sonrsquos disease patients show increased sensitivity to neurotoxin 1-methyl-4-phenylpyridinium dependent of autophagyrdquo Toxicol-ogy vol 324 pp 1ndash9 2014

[6] M Elstner C M Morris K Heim et al ldquoExpression analysisof dopaminergic neurons in Parkinsonrsquos disease and aginglinks transcriptional dysregulation of energy metabolism to celldeathrdquo Acta Neuropathologica vol 122 no 1 pp 75ndash86 2011

[7] H Aleyasin M W C Rousseaux P C Marcogliese et al ldquoDJ-1 protects the nigrostriatal axis from the neurotoxin MPTP bymodulation of the AKT pathwayrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no7 pp 3186ndash3191 2010

[8] C Malagelada Z H Jin V Jackson-Lewis S Przedborski andL A Greene ldquoRapamycin protects against neuron death inin vitro and in vivo models of Parkinsonrsquos diseaserdquo Journal ofNeuroscience vol 30 no 3 pp 1166ndash1175 2010

[9] S Timmons M F Coakley A M Moloney and C Orsquo NeillldquoAkt signal transduction dysfunction in Parkinsonrsquos diseaserdquoNeuroscience Letters vol 467 no 1 pp 30ndash35 2009

[10] L S Tain H Mortiboys R N Tao E Ziviani O Bandmannand A J Whitworth ldquoRapamycin activation of 4E-BP preventsparkinsonian dopaminergic neuron lossrdquo Nature Neurosciencevol 12 no 9 pp 1129ndash1135 2009

[11] C K TsangHQi L F Liu andX F S Zheng ldquoTargetingmam-malian target of rapamycin (mTOR) for health and diseasesrdquoDrug Discovery Today vol 12 no 3-4 pp 112ndash124 2007

[12] S M Knudsen J Frydenberg B F C Clark and H LeffersldquoTissue-dependent variation in the expression of elongationfactor-1 alpha isoforms isolation and characterisation of acDNA encoding a novel variant of human elongation-factor 1alphardquoEuropean Journal of Biochemistry vol 215 no 3 pp 549ndash554 1993

[13] S Kahns A Lund P Kristensen et al ldquoThe elongation factor 1A-2 isoform from rabbit cloning of the cDNA and characteri-zation of the proteinrdquo Nucleic Acids Research vol 26 no 8 pp1884ndash1890 1998

[14] D M Chambers J Peters and C M Abbott ldquoThe lethalmutation of the mouse wasted (wst) is a deletion that abolishesexpression of a tissue-specific isoform of translation elongationfactor 1120572 encoded by the Eef1a2 generdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 95 no 8 pp 4463ndash4468 1998

[15] J Condeelis ldquoElongation factor 1120572 translation and thecytoskeletonrdquo Trends in Biochemical Sciences vol 20 no 5 pp169ndash170 1995

[16] D B McClatchy C R Knudsen B F Clark R A Kahn RA Hall and A I Levey ldquoNovel interaction between the M

4

muscarinic acetylcholine receptor and elongation factor 1A2rdquoThe Journal of Biological Chemistry vol 277 no 32 pp 29268ndash29274 2002

[17] N Anand S Murthy G Amann et al ldquoGene encoding proteinelongation factor EEF1A2 is a putative oncogene in ovariancancerrdquo Nature Genetics vol 31 no 3 pp 301ndash305 2002

[18] VA L TomlinsonH JNewberyN RWray et al ldquoTranslationelongation factor eEF1A2 is a potential oncoprotein that isoverexpressed in two-thirds of breast tumoursrdquo BMC Cancervol 5 article 113 2005

[19] M-H Lee and Y-J Surh ldquoeEF1A2 as a putative oncogenerdquoAnnals of the New York Academy of Sciences vol 1171 pp 87ndash93 2009

[20] R Li H Wang B N Bekele et al ldquoIdentification of putativeoncogenes in lung adenocarcinoma by a comprehensive func-tional genomic approachrdquo Oncogene vol 25 no 18 pp 2628ndash2635 2006

[21] M J Devine H Plun-Favreau and N W Wood ldquoParkinsonrsquosdisease and cancer twowars one frontrdquoNature ReviewsCancervol 11 no 11 pp 812ndash823 2011

[22] A Amiri F Noei S Jeganathan G Kulkarni D E Pinke andJ M Lee ldquoeEF1A2 activates Akt and stimulates Akt-dependentactin remodeling invasion and migrationrdquo Oncogene vol 26no 21 pp 3027ndash3040 2007

[23] Z Li C-F Qi D-M Shin et al ldquoEef1a2 promotes cell growthinhibits apoptosis and activates JAKSTAT and AKT signaling


in mouse plasmacytomasrdquo PLoS ONE vol 5 no 5 Article IDe10755 2010

[24] E Chen G Proestou D Bourbeau and E Wang ldquoRapid up-regulation of peptide elongation factor EF-1120572 protein levelsis an immediate early event during oxidative stress-inducedapoptosisrdquo Experimental Cell Research vol 259 no 1 pp 140ndash148 2000

[25] R Chang and E Wang ldquoMouse translation elongation factoreEF1A-2 interacts with Prdx-I to protect cells against apoptoticdeath induced by oxidative stressrdquo Journal of Cellular Biochem-istry vol 100 no 2 pp 267ndash278 2007

[26] H Yamaguchi S Yoshida E Muroi et al ldquoPhosphoinosi-tide 3-kinase signaling pathway mediated by p110120572 regulatesinvadopodia formationrdquo Journal of Cell Biology vol 193 no 7pp 1275ndash1288 2011

[27] D PachowN Andrae N Kliese et al ldquomTORC1 inhibitors sup-press meningioma growth in mouse modelsrdquo Clinical CancerResearch vol 19 no 5 pp 1180ndash1189 2013

[28] S Tripathy and D B Jump ldquoElovl5 regulates the mTORC2-Akt-FOXO1 pathway by controlling hepatic cis-vaccenic acidsynthesis in diet-induced obesemicerdquo Journal of Lipid Researchvol 54 no 1 pp 71ndash84 2013

[29] FM Lopes R SchroderM L C da Frota Jr et al ldquoComparisonbetween proliferative and neuron-like SH-SY5Y cells as an invitro model for Parkinson disease studiesrdquo Brain Research vol1337 pp 85ndash94 2010

[30] Y-T CheungW K-W Lau M-S Yu et al ldquoEffects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitromodel in neurotoxicity researchrdquo NeuroToxicology vol 30 no1 pp 127ndash135 2009

[31] L-B Ruest R Marcotte and E Wang ldquoPeptide elongation fac-tor eEF1A-2S1 expression in cultured differentiated myotubesand its protective effect against caspase-3-mediated apoptosisrdquoThe Journal of Biological Chemistry vol 277 no 7 pp 5418ndash5425 2002

[32] W S Chen P-Z Xu K Gottlob et al ldquoGrowth retardation andincreased apoptosis in mice with homozygous disruption of theakt1 generdquo Genes amp Development vol 15 no 17 pp 2203ndash22082001

[33] H Cho J Mu J K Kim et al ldquoInsulin resistance and a diabetesmellitus-like syndrome in mice lacking the protein kinase Akt2(PKB 120573)rdquo Science vol 292 no 5522 pp 1728ndash1731 2001

[34] T F Franke ldquoPI3KAkt getting it right mattersrdquo Oncogene vol27 no 50 pp 6473ndash6488 2008

[35] J Laine G Kunstle T Obata and M Noguchi ldquoDifferentialregulation of Akt kinase isoforms by the members of the TCL1oncogene familyrdquo Journal of Biological Chemistry vol 277 no5 pp 3743ndash3751 2002

[36] Q Huang F Lan Z Zheng et al ldquoAkt2 kinase sup-presses glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-mediated apoptosis in ovarian cancer cells via phosphorylatinggapdh at threonine 237 and decreasing its nuclear transloca-tionrdquo The Journal of Biological Chemistry vol 286 no 49 pp42211ndash42220 2011

[37] Y Shao and A E Aplin ldquoAkt3-mediated resistance to apoptosisin B-RAF-targeted melanoma cellsrdquo Cancer Research vol 70no 16 pp 6670ndash6681 2010

[38] W CuiW Li R Han et al ldquoPI3-KAkt and ERK pathways acti-vated by VEGF play opposite roles in MPP+-induced neuronalapoptosisrdquoNeurochemistry International vol 59 no 6 pp 945ndash953 2011

[39] J-I Okano I Gaslightwala M J Birnbaum A K Rustgi andH Nakagawa ldquoAktprotein kinase B isoforms are differentiallyregulated by epidermal growth factor stimulationrdquoThe Journalof Biological Chemistry vol 275 no 40 pp 30934ndash30942 2000

[40] D Heras-Sandoval J M Perez-Rojas J Hernandez-Damianand J Pedraza-Chaverri ldquoThe role of PI3KAKTmTOR path-way in themodulation of autophagy and the clearance of proteinaggregates in neurodegenerationrdquo Cellular Signalling vol 26no 12 pp 2694ndash2701 2014

[41] P T Bhaskar and N Hay ldquoThe two TORCs and Aktrdquo Develop-mental Cell vol 12 no 4 pp 487ndash502 2007

[42] B DManning and L C Cantley ldquoAKTPKB signaling navigat-ing downstreamrdquo Cell vol 129 no 7 pp 1261ndash1274 2007

[43] K Arsikin T Kravic-Stevovic M Jovanovic et al ldquoAutophagy-dependent and -independent involvement of AMP-activatedprotein kinase in 6-hydroxydopamine toxicity to SH-SY5Yneuroblastoma cellsrdquo Biochimica et Biophysica Acta (BBA)mdashMolecular Basis of Disease vol 1822 no 11 pp 1826ndash1836 2012

[44] J Deguil D Jailloux G Page et al ldquoNeuroprotective effects ofpituitary adenylate cyclasendashactivating polypeptide (PACAP) inMPP+-induced alteration of translational control in neuro-2aneuroblastoma cellsrdquo Journal of Neuroscience Research vol 85no 9 pp 2017ndash2025 2007

[45] P Tsokas E A Grace P Chan et al ldquoLocal protein synthesismediates a rapid increase in dendritic elongation factor 1Aafter induction of late long-term potentiationrdquo The Journal ofNeuroscience vol 25 no 24 pp 5833ndash5843 2005

[46] G Zhu XWang SWu andQ Li ldquoInvolvement of activation ofPI3KAkt pathway in the protective effects of puerarin againstMPP+-induced human neuroblastoma SH-SY5Y cell deathrdquoNeurochemistry International vol 60 no 4 pp 400ndash408 2012

[47] F Simunovic M Yi Y Wang et al ldquoGene expression profilingof substantia nigra dopamine neurons further insights intoParkinsonrsquos disease pathologyrdquo Brain vol 132 no 7 pp 1795ndash1809 2009

[48] E Petroulakis and E Wang ldquoNerve growth factor specificallystimulates translation of eukaryotic elongation factor 1A-1(eEF1A-1) mRNA by recruitment to polyribosomes in PC12cellsrdquo The Journal of Biological Chemistry vol 277 no 21 pp18718ndash18727 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


expression has been found in tumors of the ovary breastand lung [17ndash20] The molecular mechanisms underlyingprogressive dopaminergic cell death in the nigrostriatalpathway of PD patients are not completely understoodRecent genetic and functional analyses show that there arepathogenic overlaps between cancer and PD [21] In theBT549 human breast cancer cell line eEF1A2 regulates cancerformation through Akt and PI3K-dependent remodelingof the cytoskeleton [22] In mouse plasmacytomas Eef1a2regulates tumor growth and proliferation through activationof the JAKSTAT and Akt signaling pathways [23]

Overexpression of eEF1A2 also protects cells againstapoptotic death induced by oxidative stress [24] eEF1A2 isfound to interact with peroxiredoxin-I (Prdx-I) a typical 2-Cys peroxiredoxin widely expressed in all mammalian cells[25] Prdx-I functionally protects cells from oxidative stress-induced cell damage by reducing a range of reactive oxygenspecies (ROS) production The interaction of eEF1A2 withPrdx-I provides an increase in protection from oxidativestress-induced death This protection is correlated with thedecrease in cleavage of caspases 3 and 8 and elevated expres-sion of prosurvival Akt

Whether eEF1A1 or eEF1A2 plays a role in the preventionof cell death in PD has not been investigated The aim ofour study was to analyze the pattern of gene expression foreEF1A isoforms in relation to that of the PI3KAktmTORpathway Thus we evaluated real-time mRNA expression ofPI3K isoforms PI3K classes I II and III Akt isoforms Akt1and Akt2 and mTOR isoforms mTORC1 and mTORC2together with the expression of eEF1A1 and eEF1A2 mRNAsusing an MPP+-induced SH-SY5Y cellular model of PD Weexpected that there would be a similar trend of increasedexpression of eEF1A isoforms and of the PI3KAktmTORpathway Indirectly we expected the result to suggest whetherany eEF1A isoforms could become a novel prosurvival factorin protecting dopaminergic cells against toxin-induced neu-ronal death

2 Materials and Methods

21 Materials Eaglersquos Minimum Essential Medium (MEM)Nutrient Mixture Hamrsquos F12 medium (F12) fetal bovineserum (FBS) and other supplements for cell culture werepurchased from Gibco (Gaithersburg MD USA) Retinoicacid (RA) 1-methyl-4-phenylpyridinium (MPP+) 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) and DMSO were purchased from Sigma-Aldrich(St Louis MO USA) Primers for real-time RT-PCR weresynthesized by and purchased from Biolegio (NijmegenNetherlands) Antibodies against Akt phosphorylated Aktphosphorylated mTORC1 and tyrosine hydroxylase (TH)were purchased from Cell Signaling Technology (DanversMA USA) Antibody against eEF1A2 and anti-120573-actinwere purchased from Sigma-Aldrich (St Louis MO USA)Horseradish peroxidase- (HRP-) conjugated anti-rabbitIgG and HRP-conjugated anti-mouse IgG were purchasedfrom Abcam (Cambridge UK) and Invitrogen (Eugene ORUSA) respectively

22 Culture and Treatment of SH-SY5Y Cells SH-SY5Yhuman neuroblastoma cells were grown in 1 1 mixture ofMEM and F12 supplemented with 10 heat-activated FBSCells were separated into two groups nondifferentiated anddifferentiated cells For neuronal differentiation of SH-SY5Ycells RA was added a day after plating at final concentration10 120583M in MEM-F12 with 10 FBS and maintained forthree days Neuronal differentiation was demonstrated asan increase in tyrosine hydroxylase (TH) expression usingimmunostaining Thereafter nondifferentiated and differen-tiated SH-SY5Y cells were exposed to 125 250 500 1000and 2000120583M of MPP+ for 24 hours The control groupsfor nondifferentiated and differentiated SH-SY5Y cells weretreated with the same medium without MPP+

23 Immunostaining for TyrosineHydroxylase SH-SY5Y cellswere fixed in 4 paraformaldehyde for 15 minutes at roomtemperature followed by sequential incubation with perme-abilizing solution (02 Triton X-100) in PBS for 30 minutesat room temperature Then cultures were washed again withPBS and incubated in blocking solution (3 BSA in 05Tween 20 in PBS) for 30 minutes Cells were incubatedwith rabbit polyclonal antibody against TH (1 200 dilutionin blocking solution Merck Millipore AB152) overnight at4∘C After washing cells were incubated with 1 500 dilu-tion of Alexa 488-conjugate secondary antibody for 1 hourat room temperature Coverslips were then mounted withVectashield antifadingmountingmediumwithDAPI (Vectorlaboratories CA) Cells were visualized under a confocallaser-scanning microscope (Olympus model FV 1000 TokyoJapan)

24 Cell Viability Assay SH-SY5Y cells were seeded ontoa 96-well plate at a density of 1 times 104 cellswell in 200120583Lof medium and incubated at 37∘C under 5 CO

2 After

exposure to MPP+ cell viability was measured by MTTcolorimetric assay This method was based on the reductionof tetra ring of MTT by mitochondrial dehydrogenases withNADH in the active mitochondria yielding a blue formazanproduct which can be measured spectrophotometricallyAfter incubation 20120583L ofMTT (5mgmL)was added to eachwell and the cells were cultured for another 4 h thenmediumwas removed and 100 120583L of DMSO was added to each wellto dissolve the formazan The color reaction was measuredat wavelength 570 nm with a reference at 690 nm using theVERSAmax Tunable microplate reader with SoftMax Prosoftware (Molecular Devices Sunnyvale CA USA)

25 Real-Time Quantitative PCR Analysis SH-SY5Y cellswere seeded onto 6-well plates Total mRNA was extractedfrom the pellet using PARIS kit according to the sup-plierrsquos instructions DNase I treatment was performed afterRNA extraction followed by heat inactivation The quantityand purity of RNA were determined by optical densitymeasurements at OD A260A280 ratio with 18 or aboveusing Nanodrop 2000 spectrophotometer (Thermo FisherScientific Inc Wilmington DE USA) Later the cDNA wassynthesized from 1 120583g of RNA using Masterscript RT-PCR




2HPO4





















3 Results



TH

Non

diffe

rent

iate

d ce

llsD

iffer

entia

ted

cells

DAPI+TH



lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

25

50

75

100

Cel

l via

bilit

y (

of u

ntre

ated

cells

)

125 250 500 1000 20000MPP+ (120583M)

(a)



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

25

50

75

100

Cel

l via

bilit

y (

of u

ntre

ated

cells

)

125 250 500 1000 20000MPP+ (120583M)

(b)




lowastlowastlowast

lowastlowastlowast


00

05

10

15

20m

RNA

expr

essio

n le

vel

(a)


eEF1A1 eEF1A2

lowast


00

05

10

15

20

mRN

A ex

pres

sion

leve

l

(b)



eEF1A2

MPP+MPP+

120573-Actin

(a)

Control Control


MPP+ MPP+00

05

10

15

20

Relat

ive e

EF1A

2 pr

otei

n ex

pres

sion

(b)








Akt1 Akt2 Akt3

lowast


05

10

15

20m

RNA

expr

essio

n le

vel

(a)



00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l

(b)





4 Discussion





Phospho-Akt

Total Akt

MPP+ MPP+

120573-Actin

(a)


MPP+MPP+

00

05

10

15

Relat

ive t

otal

Akt

pro

tein

expr

essio

n



2

4

6

8

Relat

ive p

hosp

ho-A

ktto

tal A

kt ex

pres

sion

(c)








mTORC1 mTORC2

lowast


00

05

10

15

20

25

30m

RNA

expr

essio

n le

vel

(a)



00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l

(b)


Phospho-mTORC1


MPP+ MPP+

120573-Actin

(a)

Control Control


00

05

10

15

20

Relat

ive m

TORC

1 pr

otei

n ex

pres

sion

(b)







Control

lowast

MPP+PI

K3C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25m

RNA

expr

essio

n le

vel


(a)



lowast

ControlMPP+

PIK3

C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l(b)






5 Conclusions






Acknowledgments


References

















4









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2HPO4





















3 Results



TH

Non

diffe

rent

iate

d ce

llsD

iffer

entia

ted

cells

DAPI+TH



lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

25

50

75

100

Cel

l via

bilit

y (

of u

ntre

ated

cells

)

125 250 500 1000 20000MPP+ (120583M)

(a)



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

25

50

75

100

Cel

l via

bilit

y (

of u

ntre

ated

cells

)

125 250 500 1000 20000MPP+ (120583M)

(b)




lowastlowastlowast

lowastlowastlowast


00

05

10

15

20m

RNA

expr

essio

n le

vel

(a)


eEF1A1 eEF1A2

lowast


00

05

10

15

20

mRN

A ex

pres

sion

leve

l

(b)



eEF1A2

MPP+MPP+

120573-Actin

(a)

Control Control


MPP+ MPP+00

05

10

15

20

Relat

ive e

EF1A

2 pr

otei

n ex

pres

sion

(b)








Akt1 Akt2 Akt3

lowast


05

10

15

20m

RNA

expr

essio

n le

vel

(a)



00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l

(b)





4 Discussion





Phospho-Akt

Total Akt

MPP+ MPP+

120573-Actin

(a)


MPP+MPP+

00

05

10

15

Relat

ive t

otal

Akt

pro

tein

expr

essio

n



2

4

6

8

Relat

ive p

hosp

ho-A

ktto

tal A

kt ex

pres

sion

(c)








mTORC1 mTORC2

lowast


00

05

10

15

20

25

30m

RNA

expr

essio

n le

vel

(a)



00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l

(b)


Phospho-mTORC1


MPP+ MPP+

120573-Actin

(a)

Control Control


00

05

10

15

20

Relat

ive m

TORC

1 pr

otei

n ex

pres

sion

(b)







Control

lowast

MPP+PI

K3C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25m

RNA

expr

essio

n le

vel


(a)



lowast

ControlMPP+

PIK3

C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l(b)






5 Conclusions






Acknowledgments


References

















4









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TH

Non

diffe

rent

iate

d ce

llsD

iffer

entia

ted

cells

DAPI+TH



lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

25

50

75

100

Cel

l via

bilit

y (

of u

ntre

ated

cells

)

125 250 500 1000 20000MPP+ (120583M)

(a)



lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

25

50

75

100

Cel

l via

bilit

y (

of u

ntre

ated

cells

)

125 250 500 1000 20000MPP+ (120583M)

(b)




lowastlowastlowast

lowastlowastlowast


00

05

10

15

20m

RNA

expr

essio

n le

vel

(a)


eEF1A1 eEF1A2

lowast


00

05

10

15

20

mRN

A ex

pres

sion

leve

l

(b)



eEF1A2

MPP+MPP+

120573-Actin

(a)

Control Control


MPP+ MPP+00

05

10

15

20

Relat

ive e

EF1A

2 pr

otei

n ex

pres

sion

(b)








Akt1 Akt2 Akt3

lowast


05

10

15

20m

RNA

expr

essio

n le

vel

(a)



00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l

(b)





4 Discussion





Phospho-Akt

Total Akt

MPP+ MPP+

120573-Actin

(a)


MPP+MPP+

00

05

10

15

Relat

ive t

otal

Akt

pro

tein

expr

essio

n



2

4

6

8

Relat

ive p

hosp

ho-A

ktto

tal A

kt ex

pres

sion

(c)








mTORC1 mTORC2

lowast


00

05

10

15

20

25

30m

RNA

expr

essio

n le

vel

(a)



00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l

(b)


Phospho-mTORC1


MPP+ MPP+

120573-Actin

(a)

Control Control


00

05

10

15

20

Relat

ive m

TORC

1 pr

otei

n ex

pres

sion

(b)







Control

lowast

MPP+PI

K3C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25m

RNA

expr

essio

n le

vel


(a)



lowast

ControlMPP+

PIK3

C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l(b)






5 Conclusions






Acknowledgments


References

















4









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















lowastlowastlowast

lowastlowastlowast


00

05

10

15

20m

RNA

expr

essio

n le

vel

(a)


eEF1A1 eEF1A2

lowast


00

05

10

15

20

mRN

A ex

pres

sion

leve

l

(b)



eEF1A2

MPP+MPP+

120573-Actin

(a)

Control Control


MPP+ MPP+00

05

10

15

20

Relat

ive e

EF1A

2 pr

otei

n ex

pres

sion

(b)








Akt1 Akt2 Akt3

lowast


05

10

15

20m

RNA

expr

essio

n le

vel

(a)



00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l

(b)





4 Discussion





Phospho-Akt

Total Akt

MPP+ MPP+

120573-Actin

(a)


MPP+MPP+

00

05

10

15

Relat

ive t

otal

Akt

pro

tein

expr

essio

n



2

4

6

8

Relat

ive p

hosp

ho-A

ktto

tal A

kt ex

pres

sion

(c)








mTORC1 mTORC2

lowast


00

05

10

15

20

25

30m

RNA

expr

essio

n le

vel

(a)



00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l

(b)


Phospho-mTORC1


MPP+ MPP+

120573-Actin

(a)

Control Control


00

05

10

15

20

Relat

ive m

TORC

1 pr

otei

n ex

pres

sion

(b)







Control

lowast

MPP+PI

K3C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25m

RNA

expr

essio

n le

vel


(a)



lowast

ControlMPP+

PIK3

C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l(b)






5 Conclusions






Acknowledgments


References

















4









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Akt1 Akt2 Akt3

lowast


05

10

15

20m

RNA

expr

essio

n le

vel

(a)



00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l

(b)





4 Discussion





Phospho-Akt

Total Akt

MPP+ MPP+

120573-Actin

(a)


MPP+MPP+

00

05

10

15

Relat

ive t

otal

Akt

pro

tein

expr

essio

n



2

4

6

8

Relat

ive p

hosp

ho-A

ktto

tal A

kt ex

pres

sion

(c)








mTORC1 mTORC2

lowast


00

05

10

15

20

25

30m

RNA

expr

essio

n le

vel

(a)



00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l

(b)


Phospho-mTORC1


MPP+ MPP+

120573-Actin

(a)

Control Control


00

05

10

15

20

Relat

ive m

TORC

1 pr

otei

n ex

pres

sion

(b)







Control

lowast

MPP+PI

K3C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25m

RNA

expr

essio

n le

vel


(a)



lowast

ControlMPP+

PIK3

C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l(b)






5 Conclusions






Acknowledgments


References

















4









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Phospho-Akt

Total Akt

MPP+ MPP+

120573-Actin

(a)


MPP+MPP+

00

05

10

15

Relat

ive t

otal

Akt

pro

tein

expr

essio

n



2

4

6

8

Relat

ive p

hosp

ho-A

ktto

tal A

kt ex

pres

sion

(c)








mTORC1 mTORC2

lowast


00

05

10

15

20

25

30m

RNA

expr

essio

n le

vel

(a)



00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l

(b)


Phospho-mTORC1


MPP+ MPP+

120573-Actin

(a)

Control Control


00

05

10

15

20

Relat

ive m

TORC

1 pr

otei

n ex

pres

sion

(b)







Control

lowast

MPP+PI

K3C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25m

RNA

expr

essio

n le

vel


(a)



lowast

ControlMPP+

PIK3

C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l(b)






5 Conclusions






Acknowledgments


References

















4









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















mTORC1 mTORC2

lowast


00

05

10

15

20

25

30m

RNA

expr

essio

n le

vel

(a)



00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l

(b)


Phospho-mTORC1


MPP+ MPP+

120573-Actin

(a)

Control Control


00

05

10

15

20

Relat

ive m

TORC

1 pr

otei

n ex

pres

sion

(b)







Control

lowast

MPP+PI

K3C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25m

RNA

expr

essio

n le

vel


(a)



lowast

ControlMPP+

PIK3

C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l(b)






5 Conclusions






Acknowledgments


References

















4









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Control

lowast

MPP+PI

K3C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25m

RNA

expr

essio

n le

vel


(a)



lowast

ControlMPP+

PIK3

C3

PIK3

C2G

PIK3

C2B

PIK3

C2A

PIK3

CG

PIK3

CD

PIK3

CB

PIK3

CA

00

05

10

15

20

25

mRN

A ex

pres

sion

leve

l(b)






5 Conclusions






Acknowledgments


References

















4









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References

















4









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Comparative mRNA Expression of eEF1A...

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