Prolyl endopeptidase is involved in the degradation of ... · RESEARCH ARTICLE Prolyl endopeptidase is involved in the degradation of neural cell adhesion molecules in vitro Külli

RESEARCH ARTICLE

Prolyl endopeptidase is involved in the degradation of neural celladhesion molecules in vitroKulli Jaako1Dagger Alexander Waniek2 Keiti Parik1 Linda Klimaviciusa1 Anu Aonurm-Helm1 Aveli Noortoots1Kaili Anier1 Roos Van Elzen3 Melanie Gerard4 Anne-Marie Lambeir3 Steffen Roszligner2 Markus Morawski2and Alexander Zharkovsky1

ABSTRACTMembrane-associated glycoprotein neural cell adhesion molecule(NCAM) and its polysialylated form (PSA-NCAM) play an importantrole in brain plasticity by regulating cellndashcell interactions Here wedemonstrate that the cytosolic serine protease prolyl endopeptidase(PREP) is able to regulate NCAMand PSA-NCAM Using a SH-SY5Yneuroblastoma cell linewith stable overexpression of PREP we founda remarkable loss of PSA-NCAM reduced levels of NCAM180 andNCAM140 protein species and a significant increase in the NCAMimmunoreactive band migrating at an apparent molecular weight of120 kDa in PREP-overexpressing cells Moreover increased levels ofNCAM fragments were found in the concentrated medium derivedfrom PREP-overexpressing cells PREP overexpression selectivelyinduced an activation of matrix metalloproteinase-9 (MMP-9) whichcould be involved in the observed degradation of NCAM as MMP-9neutralization reduced the levels of NCAM fragments in cell culturemedium We propose that increased PREP levels promote epidermalgrowth factor receptor (EGFR) signaling which in turn activatesMMP-9 In conclusion our findings provide evidence for newly-discovered roles for PREP in mechanisms regulating cellularplasticity through NCAM and PSA-NCAM

KEY WORDS Neural cell adhesion molecule Prolyl endopeptidaseNeuronal plasticity Metalloproteinase-9

INTRODUCTIONThe development of the nervous system and its structuralremodeling in the adult rely on molecules mediating the structuralplasticity of neurons especially those involved in cell adhesioncytoskeletal dynamics or synapse formation (Amoureux et al2000 Cremer et al 1998 Kiss and Muller 2001) Among thesemolecules isoforms of the neural cell adhesion molecules(NCAMs) are of particular interest NCAM (also known asNCAM1) is a cell surface glycoprotein that is represented by atleast three isoforms (NCAM120 NCAM140 and NCAM180 withmolecular weights of 120 140 and 180 kDa respectively) that differin their cytoplasmic domains and attachment to the plasma

membrane (Cunningham et al 1987) The extracellular part ofNCAM consists of five immunoglobulin-like modules (IgIndashIgV)followed by two fibronectin type III domains (Finne et al 1983Rutishauser 2008) NCAM establishes cellndashcell adhesion throughhomo- and heterophilic interactions and thereby regulates processeslike cell migration neurite outgrowth and targeting axonalbranching synaptogenesis and synaptic plasticity (see Walmodet al 2004 for review) Neural plasticity mediated through theNCAMprotein is facilitated by post-translational modifications themost important and prevalent of which is addition of α-28-polysialic acid (PSA) chains to the IgV module of the extracellularNCAM domain Addition of PSA homopolymers to NCAM abatesinteraction between NCAM molecules and decreases NCAM-mediated adhesion thus allowing plasticity changes (Johnson et al2005 Kiselyov et al 2003 Rutishauser 2008 Seki and Arai1993a) The addition of PSA to NCAM occurs through two Golgi-associated polysialyltransferases ST8Sia2 and ST8Sia4 (Angataet al 1997 Hildebrandt et al 1998b Nakayama et al 1998)However the physiological PSA-NCAM degradation andendogenous enzymes involved in this physiological turnover arenot yet fully elucidated

It has been demonstrated that NCAMcan be cleaved extracellularlyby metalloproteinases and other proteolytic enzymes (Brennamanet al 2014 Hinkle et al 2006 Huumlbschmann et al 2005 Kaluset al 2006) Moreover metalloproteinase-dependent shedding ofNCAM induces release of soluble forms of NCAM consisting ofseveral polypeptides with molecular weights ranging from 80 kDa to200 kDa (Krog et al 1992 Nybroe et al 1989 Sadoul et al 1986)The functional significance of proteolytic shedding of membrane-bound NCAM is unknown However increased amounts of thecleavage product have been associated with neuropsychiatricdisorders such as schizophrenia (Vawter et al 1998) and dementia(Strekalova et al 2006)

The cytosolic serine protease prolyl endopeptidase (PREP)hydrolyses small (lt3 kDa) proline-containing peptides at thecarboxy terminus of proline residues (Fuumlloumlp et al 1998 Polgaacuter2002 Rawlings et al 1991) PREP is widely distributed in the brain(Irazusta et al 2002 Myoumlhaumlnen et al 2007 2008) and increasedactivity of PREP has been associated with cell death processes invarious neurodegenerative diseases including Alzheimerrsquos andParkinsonrsquos diseases (Brandt et al 2008 Mantle et al 1996)PREP is believed to act in the extracellular space with involvementin the maturation and degradation of peptide hormones andneuropeptides (Bellemere et al 2004 Cunningham andOrsquoConnor 1997 Shishido et al 1999) which has been proposedto be the mechanism underlying some beneficial effects of PREPinhibitors in animal memory models (Shishido et al 1998 Toideet al 1997 Yoshimoto et al 1987) and in aged mice (Kato et alReceived 11 October 2015 Accepted 18 August 2016

1Department of Pharmacology Institute of Biomedicine and Translational MedicineUniversity of Tartu Tartu 50411 Estonia 2Paul Flechsig Institute for Brain ResearchUniversity of Leipzig Leipzig 04103 Germany 3Laboratory of MedicalBiochemistry Department of Pharmaceutical Sciences University of AntwerpAntwerp B-2610 Belgium 4Interdisciplinary Research Centre KU Leuven-KortrijkKortrijk B-8500 BelgiumThese authors contributed equally to this work

Dagger

Author for correspondence (KulliJaakoutee)

KJ 0000-0003-4675-5188 LK 0000-0001-7085-3891

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1997) Besides its extracellular action PREP has been shown to actintracellularly and important roles of PREP have been demonstratedin signaling pathways or in transport and secretion of proteins andpeptides associated with neurodegeneration (Brandt et al 2008 DiDaniel et al 2009 Rossner et al 2005 Savolainen et al 2015Schulz et al 2002 2005) Within recent years PREP has beensuggested to be a contributor to neuroinflammation (Penttinen et al2011) PREP has been shown to be involved in mechanismsresponsible for the transduction and amplification of inflammatoryprocesses leading to the production of neurotoxic mediators whichin turn mediate pathology progression (for review see Penttinenet al 2011)In this report for the first time we demonstrate that PREP is able

to regulate levels of NCAM through activation of MMP-9

RESULTSLevels of PSA-NCAM and NCAM protein and mRNA inneuroblastoma SH-SY5Y cells overexpressing PREPIncreased levels of PREP protein and its activity in PREP-overexpressing SH-SY5Y cell lysates were confirmed by westernblot and activity measurements (Fig S1AB)PREP was detected on SDS-PAGE as a band at sim80 kDa

(Fig S1A) A 30-fold increase in PREP protein level was found inSH-SY5Y cells overexpressing PREP compared with wild-typecells (Plt0001 t-test n=4) In these PREP-overexpressing cellsPREP activity assays demonstrated an 11-fold increase in enzymaticactivity (Plt00001 t-test n=3 Fig S1B)To evaluate whether increased PREP levels are associated with

altered levels of PSA-NCAM andor NCAM the proteins werequantified using western blot analysis In wild-type cells PSA-NCAM was detected as a high-molecular-weight smear typical forhighly polysialylated NCAM (Fig 1A) whereas by comparisonPSA-NCAM signal was almost absent in PREP-overexpressingcells (Plt00001 t-test n=5ndash6 Fig 1A) In wild-type cellsimmunocytochemistry identified membrane-bound extracellularPSA-NCAM molecules which assembled in small bunches alongneurite-like processes and in larger patches associated with theperikaryon In PREP-overexpressing cells however PSA-NCAMsignal was almost absent (Fig 1C)We further investigated levels of the NCAM protein isoforms

(NCAM180 NCAM140 and NCAM120) A significant decrease inlevels of the NCAM 180 kDa immunoreactive band (P=001 t-testn=5ndash6) and the NCAM 140 kDa immunoreactive band (P=001t-test n=5ndash6) was found in PREP-overexpressing neuroblastomacells compared with wild-type cells (Fig 1B) In contrast asignificant increase in the NCAM immunoreactive band migratingat an apparent molecular weight of 120 kDa was found in PREP-overexpressing cells (P=0009 t-test n=5-6 Fig 1B) In wild-typecells immunostaining for NCAM demonstrated an intensive positivesignal for NCAM whereas only a moderate NCAM-positive signalcould be detected in PREP-overexpressing cells (Fig 1D)Next we aimed to elucidate whether the observed changes in the

levels of NCAM protein isoforms were caused by the changes intranscription level We measured mRNA levels of the 120 140 and180 isoforms as well as total NCAM using qPCR No significantdifferences in mRNA level between wild-type and PREP-overexpressing cells were found for any form of NCAM(Table S1) Thus the increase in the NCAM 120 kDaimmunoreactive band in PREP-overexpressing cells does notresult from increased expression of NCAM120 mRNABased on these results we proposed that the increase in NCAM120

results from the degradation of NCAM140 andor NCAM180

isoforms To test this we measured NCAM degradation fragmentsin concentrated cell culture medium from PREP-overexpressing andwild-type cells Two fragments recognized by the NCAM-specificantibody were found in the concentrated medium one of sim46ndash48 kDa and another of sim38ndash40 kDa In the medium of PREP-overexpressing cells therewas a significant increase in the level of the38ndash40 kDa fragment compared with medium from wild-type cells(P=00043 t-test n=4 Fig 2AB) whereas no changes wereobserved in the levels of the 46ndash48 kDa fragment These datasupport our hypothesis that the overexpression of PREP inducesincreased degradation of the NCAM180 andor NCAM140 isoforms

Subsequently we aimed to evaluate whether the observedchanges in PSA-NCAM levels in PREP-overexpressing cells arePREP-specific PREP overexpression was knocked down in PREP-overexpressing cells through transfection with shRNA againstPREP (shPREP) or a control vector (shNC) together with a plasmidexpressing green fluorescent protein (GFP) After 72 h cells werefixed and processed for PSA-NCAM immunocytochemistry GFP-positive cells from the shPREP and shNC groups were selected forPSA-NCAM fluorescent signal intensity analysis Quantificationrevealed an increase in PSA-NCAM immunopositive signals inPREP-overexpressing cells transfected with shPREP compared withshNC-transfected PREP-overexpressing cells (P=0043 t-testn=10 Fig 3AB) This result confirms that alterations inPSA-NCAM are PREP-specific

Addition of recombinant PREP to the culture mediumdecreases PSA-NCAM expression in wild-type SH-SY5Yneuroblastoma cells and primary cortical neuronsHuman recombinant PREP (rPREP) was added to the cell culturemedium of wild-type SH-SY5Y cells (1 nM or 10 nM) and primarycortical neurons (1 nM) for 72 h A marked decrease in PSA-NCAM level was found in wild-type SH-SY5Y neuroblastoma cells(F=1026 P=0001 one-way ANOVA followed by Bonferronipost-hoc test n=3) after addition of rPREP (Fig 3C) A similardecrease in PSA-NCAM level (P=003 t-test n=5ndash6) was found inprimary cortical neurons (Fig 3D)

Quantification of protein expression levels ofpolysialyltransferase ST8Sia2 and ST8Sia4 in wild-type andPREP-overexpressing neuroblastoma SH-SY5Y cellsThe formation of PSA-NCAM is dependent on thepolysialyltransferases ST8Sia2 and ST8Sia4 which attach PSAresidues to the NCAM molecule To exclude the possibility thatPREP affects ST8Sia2 and ST8Sia4 wild-type and PREP-overexpressing cells were subjected to ST8Sia2 and ST8Sia4immunocytochemistry and protein expression analysis (Fig 4AB)No differences in expression of either ST8Sia2 or ST8Sia4 wereobserved by immunocytochemistry (Fig 4A) and western blotanalysis demonstrated no statistically significant difference inST8Sia2 or ST8Sia4 protein levels between wild-type and PREP-overexpressing cells (Fig 4B)

Impaired differentiation of PREP-overexpressing cellsAs NCAM and PSA-NCAM are involved in neurite outgrowth andneuronal differentiation (Seidenfaden et al 2006 Williams et al1994) it was of interest to evaluate whether or not an excess ofPREP accompanied with altered NCAM expression might impairdifferentiation To elucidate this wild-type and PREP-overexpressing cells were grown with sequential exposure toretinoic acid and brain-derived neurotrophic factor (BDNF) for 5and 7 days respectively to achieve long-term survival of cells and a

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high degree of differentiation (Encinas et al 2000) Asdemonstrated in Fig 5A wild-type cells yielded homogeneouspopulations of Tuj-1-immunopositive cells with neuronalmorphology and abundantly branched neurites In contrast onlymoderate numbers of PREP-overexpressing cells were positive forTuj-1 (Fig 5A) Quantification revealed that the percentage of Tuj-1-positive cells was significantly lower in PREP-overexpressing

cells compared with wild-type cells (Plt00001 t-test n=9 Fig 5B)indicating that alterations in NCAM molecule integrity lead toimpaired differentiation toward a neuron-like cell type

IncreasedMMP-9 protein level and activity in neuroblastomaSH-SY5Y cells overexpressing PREPIt is well documented that PREP is only able to degrade relativelysmall peptides (lt3 kDa) (Fuumlloumlp et al 1998 Polgaacuter 2002 Rawlingset al 1991) Therefore it seemed unlikely that PREP itself is ableto degrade NCAM It has been demonstrated previously thatNCAM molecule degradation might be mediated by the matrixmetalloproteases (MMPs) (Brennaman et al 2014 Hinkle et al2006 Huumlbschmann et al 2005 Shichi et al 2011) Thereforeto test whether increased expression and activity of PREP mightbe involved in the activation of MMPs we measured the expressionlevels and activity of MMP-9 Increased levels of the activeform of MMP-9 was found in PREP-overexpressing SH-SY5Yneuroblastoma cells compared with wild-type cells (P=0001 t-testn=3) whereas no changes were found in levels of the inactive formof MMP-9 (Fig 6A) Moreover immunocytochemical labeling ofMMP-9 in PREP-overexpressing cells revealed intense granulationof the immunopositive signal mostly at the perinuclear regionwhereas in wild-type cells MMP-9 signal was associated with asparse granulation pattern (Fig 6B) In addition zymographydemonstrated increased proteolytic activity of MMP-9 in the serum-free medium from cultured PREP-overexpressing cells comparedwith wild-type cells (P=0002 t-test n=3 Fig 6C)

To test whether the observed increases in MMP-9 levels andactivity in PREP-overexpressing cells is responsible for NCAM

Fig 1 Analysis and quantification of PSA-NCAM and NCAM protein level by western blot and immunocytochemistry in wild-type and PREP-overexpressing neuroblastoma cells (A) Representative western blot and corresponding statistical analysis of PSA-NCAM in wild-type (WT) and PREP-overexpressing SH-SY5Y neuroblastoma cells demonstrating substantial reduction in PSA-NCAM protein level (B) Representative western blot andcorresponding statistical analysis which revealed reduced NCAM 180 kDa and NCAM 140 kDa protein variants but increased NCAM 120 kDa protein variantlevel in PREP-overexpressing SH-SY5Y neuroblastoma cells (PREP) compared with wild-type cells (CD) Illustrative photomicrographs highlighting the(C) reduced PSA-NCAM (red) and (D) NCAM immunopositive signal (green) in PREP-overexpressing cells compared with wild-type cells Nuclei werecounterstained with DAPI (blue) Data are given as ratio of mean values in percentageplusmnsem (wild-type=100) β-actin was used as loading control Plt005Plt001 Plt0001 unpaired t-test n=6 for wild-type and n=5 for PREP Scale bar 10 microm Three independent experiments were performed

Fig 2 Detection of NCAM fragments in culturemediumderived fromwild-type and PREP-overexpressing cells (A) Representative western blot andcorresponding levels of degradation product of NCAM fragment 38-40 kDa inthe culture medium derived from PREP-overexpressing cells compared withculture medium derived from wild-type cells (B) Quantification of data from ADataare given asmeanoptical density (OD) ratio as percentage of controlplusmnsem(wild-type=100) P=00043 unpaired t-test n=4 Two independentexperiments were performed

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degradation PREP-overexpressing cells were incubated withMMP-9-neutralizing antibody for 96 h A reduced level of NCAMfragments was found in the cell culture medium (Plt0001 t-testn=5-6) compared with vehicle-treated PREP-overexpressing cells(Fig 7AB) indicating that MMP-9 is involved in the regulation ofNCAM degradation

To elucidate whether PREP overexpression could affectother MMPs that might be involved in NCAM degradation theprotein levels of MMP-2 MMP-3 and ADAM-10 weremeasured No changes were found in the levels of these MMPsin PREP-overexpressing cells as compared with wild-type cells(Fig S2)

Fig 3 Analysis of PSA-NCAM level in PREP-overexpressingcells in the context of PREP-knockdown and the effect ofrecombinant PREP on PSA-NCAM protein level in wild-typeSH-SY5Y neuroblastoma cells and primary cortical neurons(A) PREP-overexpressing SH-SY5Y neuroblastoma cells weretransfected with either control vector (shNC) or shPREP andcells were immunostained for PSA-NCAM (red) ShPREP-transfected cells demonstrated restoration of PSA-NCAMexpression (indicated with arrows) (B) Signal intensity analysiscorresponding to the images in A demonstrated restoration ofPSA-NCAM expression in shPREP-transfected cells comparedwith shNC-transfected PREP-overexpressing SH-SY5Yneuroblastoma cells PSA-NCAM immunopositive signalintensity data are given as meanplusmnsem relative fluorescenceunits (RFU) Plt005 unpaired t-test n=10 Scale bar 5 microm(CD) Western blot analysis and representative imagesdemonstrating significantly decreased level of PSA-NCAM in(C) wild-type SH-SY5Y neuroblastoma cells and (D) culturedprimary rat neurons when rPREP was added into cell culturemedium at concentrations of 1 nM and 10 nM compared withvehicle-treated cells Data are given as mean optical density(OD) ratio as a percentage of controlplusmnsem (untreated primaryneurons or wild-type neuroblastoma cells=100) β-actin wasused as loading control Plt005 unpaired t-test or one-wayANOVA n=3 for wild-type SH-SY5Y neuroblastoma cells n=5 foruntreated cortical neurons and n=6 for rPREP-treated corticalneurons Two independent experiments were performed

Fig 4 Analysis of protein expression ofsialyltransferases in PREP-overexpressingSH-SY5Y neuroblastoma cells(A) Representative fluorescentmicrophotographs demonstrating ST8Sia2- andST8Sia4-immunopositive staining in wild-typeand PREP-overexpressing SH-SY5Yneuroblastoma cells Nuclei were counterstainedwith Hoechst (blue) Scale bar 10 microm(B) Representative western blot andcorresponding levels of ST8Sia2 and ST8Sia4protein expression in wild-type andPREP-overexpressing cells Data are given asmean optical density (OD) ratio as a percentageof controlplusmnsem (wild type=100) β-actin wasused as loading control Unpaired t-test n=6Two independent experiments were performed

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Decreased EGFR and increased pEGFR expression inneuroblastoma SH-SY5Y cells overexpressing PREPIt has previously been demonstrated that EGFR signaling isinvolved in release and activation of MMP-9 (da Rosa et al2014 Pei et al 2014 Qiu et al 2004) Therefore EGFR andphosphorylated EGFR (pEGFR) levels were measured and inPREP-overexpressing cells overall decrease in total EGFR wasfound (P=0001 t-test n=4 Fig 8) In contrast the levels of pEGFRwere increased (P=0001 t-test n=4 Fig 8)

DISCUSSIONThis study shows for the first time that PREP is involved inthe regulation of neural cell adhesion molecules A PREP-overexpressing neuroblastoma SH-SY5Y cell line was used as anin vitro model to mimic pathological conditions resulting fromincreased PREP expression When PSA-NCAM protein levels werequantified we found a remarkable reduction of PSA-NCAM inPREP-overexpressing cells compared with wild-type cells Thefinding that PSA-NCAM levels are altered in the context of higherPREP levels was further confirmed in a series of experiments wherecell culture media for wild-type neuroblastoma cells or for primary

cortical cells isolated from mouse brain were enriched with humanrecombinant PREP ndash this induced a decrease in PSA-NCAMexpression levels Not only PSA-NCAMwas altered in the presenceof PREP we also found alterations in the levels of all NCAMprotein variants tested We found a reduction in NCAM 180 kDaand 140 kDa immunoreactive bands and an increase in the NCAM120 kDa immunoreactive band As PREP might affect PSA-NCAMand NCAM through different mechanisms including reducedexpression andor increased breakdown of the NCAM protein weexplored these mechanisms in more detail

In neuroblastoma cells overexpressing PREP we did not find anychanges in NCAM120 NCAM140 NCAM180 or in total NCAMmRNA levels These data suggest that PREP does not affect thetranscription of NCAM We also failed to find any changes in theexpression levels of two Golgi-associated polysialyltransferasesST8Sia2 and ST8Sia4 which mediate the addition of PSA toNCAM (Eckhardt et al 1995) suggesting that PREP does notaffect the polysialylation of NCAM Therefore we propose that thereduction in the 140 kDa and 180 kDa protein variants of NCAMand the reduction in PSA-NCAM result from their increaseddegradation in the presence of PREP To explore this proposal in

Fig 5 Analysis of differentiation in wild-type and PREP-overexpressing SH-SY5Y neuroblastoma cells(A) Representative microphotographs demonstrating Tuj-1-immunopositive staining (green) in differentiated wild-type andPREP-overexpressing SH-SY5Y neuroblastoma cells Nuclei werecounterstained with DAPI (blue) Scale bar 25 microm(B) Corresponding analysis of Tuj-1-immunopositive cells Data areexpressed as mean percentageplusmnsem of Tuj-1-immunopositivecells Plt00001 unpaired t-test n=9

Fig 6 Western blot analysis and representative immunocytochemical images and quantified enzymatical activity of MMP-9 in wild-type and PREP-overexpressing neuroblastoma cells (A) Representative western blots of both intact (inactive) and cleaved (active) forms of MMP-9 in wild-type (WT) andPREP-overexpressing SH-SY5Y neuroblastoma cells Quantitative western blot analysis demonstrating increased expression level of the active form of MMP-9but not proMMP-9 in PREP-overexpressing SH-SY5Y cells compared with wild-type cells β-actin was used as loading control (B) Immunocytochemistrydemonstrated intracellular distribution of MMP-9-immunopositive signal (red) in wild-type and PREP-overexpressing SH-SY5Y neuroblastoma cells Perinucleardistribution and localization of MMP-9 in PREP-overexpressing cells is indicated by arrows Nuclei were counterstained with DAPI Scale bar 5 microm(C) Representative gelatin zymogramand correspondingMMP-9 zymogramanalysis demonstrated increased activity of MMP-9 in serum-free cell culturemediumof PREP-overexpressing SH-SY5Y neuroblastoma cells compared with wild-type cells Recombinant MMP-9 protein was used as an identification marker Dataare given as optical density (OD) ratiosplusmnsem Plt001 unpaired t-test n=3 Two independent experiments were performed

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more detail we measured NCAM degradation products inconcentrated medium derived from PREP-overexpressing cellsIndeed an increased level of peptide fragments at 38ndash40 kDarecognized by anti-NCAM antibody was found Thus it seems thatthe observed decrease in NCAM immunoreactive bands of 180 kDaand 140 kDa does indeed result from their breakdown in thepresence of PREP and the observed increase in the NCAMimmunoreactive band migrating at an apparent molecular weight of120 kDa might be explained by the accumulation of cleavedfragments of NCAM However NCAM fragments at 38ndash40 kDaindicate that the cleaved fragment is too small to include thepolysialylated IgV domain and therefore additional mechanismssuch as increased PSA cleavage by neuraminidases in addition toNCAM degradation by MMP-9 might be involved In mammalsfour sialidases are present and are involved in several key

physiological events related to cancer transformation (Miyagiet al 2003 Proshin et al 2002 Tringali et al 2012 Wadaet al 2007) and in developmental processes such as lamination ofnewly generated hippocampal granule cells through the modulationof PSA (Sajo et al 2016) The question of whether or not PREPoverexpression affects sialidases needs further investigation

PREP is peptidase that cleaves small peptides (lt3 kDa) (Fuumlloumlpet al 1998 Polgaacuter 2002 Rawlings et al 1991) and it seemsunlikely that this enzyme can directly catalytically degrade theNCAM molecule Previous studies have demonstrated that severalmetalloproteases such as MMP-2 and MMP-9 as well as theADAM family of metalloproteases target NCAM (Brennamanet al 2014 Hinkle et al 2006 Huumlbschmann et al 2005 Shichiet al 2011) It has also been shown that inhibition of MMP-2 andMMP-9 prevents NCAM shedding indicating the roles of theseMMPs in NCAM cleavage (Huumlbschmann et al 2005 Shichi et al2011) Moreover ADAM-10-dependent shedding of NCAM hasbeen extensively studied and the second fibronectin-type IIIdomain of NCAM has been shown to be a target for ADAM-10cleavage (Brennaman et al 2014) Based on these findings weexplored in more detail the impact of metalloproteases on the PREP-mediated decrease in PSA-NCAM and NCAMWemeasured levelsof MMP-2 MMP-3 MMP-9 and ADAM-10 in the lysates of wild-type and PREP-overexpressing cells and found an increased level ofMMP-9 but not the other metalloproteases Furthermore we foundthat this increased level of MMP-9 was specific to its active formUsing zymography we also observed increased MMP-9 activityThus it seems that PREP overexpression induces a selectiveincrease in the expression and activity of MMP-9 which is probablyinvolved in the observed degradation of NCAM This proposal wasfurther confirmed in our experiments where MMP-9-neutralizingantibody prevented the degradation of NCAM as evidenced by thedecrease in NCAM fragments in the culture medium

Previous studies have demonstrated that cleavage of NCAM180and NCAM140 isoforms by ADAM-10 and ADAM-17 results inthe release of 110ndash115 kDa fragments into the cell culture mediumassociated with the appearance of 30 kDa membrane-associatedfragments in cell lysates (Brennaman et al 2014 Kalus et al2006) In addition to the aforementioned fragments cleaved byADAMs a 65 kDa degradation product of NCAM has beendemonstrated to result from the action of MMP-9 andor MMP-2after cerebral ischemic neuronal damage and the appearance of thisfragment was reduced when MMPs were suppressed (Shichi et al2011) The precise cleavage site through which these smallerfragments are produced is not known however it might beproposed that the origin of the fragment found in cell culturemedium might represent an N-terminal domain of NCAM Theappearance of fragments of sim40 kDa derived from extracellulardomains of NCAM into cell culture medium might also explain theshift in western blot bands from strong bands at 180 kDa and140 kDa to bands with lower apparent molecular weight and theiraccumulation at the range of 120 kDa

The mechanism by which PREP inducesMMP-9 activation is notknown although several explanations might be proposed PREPmight activate MMP-9 by degrading physiological tissue inhibitormetalloproteases (TIMPs) as cells secrete pro-MMPs bound toTIMPs in a complex and TIMP processing is needed for zymogenactivation It has been demonstrated that in vitro conditionscleavage degradation or chemical modification by proteolytic andnon-proteolytic mechanisms are responsible for inactivation ofTIMPs through the involvement of serine or thiol proteases andreactive oxygen species respectively (Frears et al 1996 Okada

Fig 7 Analysis of the effect of MMP-9-neutralizing antibody on the levelsof NCAM fragments in PREP-overexpressing SH-SY5Y neuroblastomacells Representative western blot (A) and corresponding quantification ofexpression levels (B) demonstrating reduced appearance of NCAMdegradation fragment at 38ndash40 kDa in cell culture medium from SH-SY5Yneuroblastoma cells treated with MMP-9-neutralizing antibody for 96 h ascompared with vehicle-treated PREP-overexpressing cells Data are given asmean optical density (OD) ratio percentage of controlplusmnsem (PREP-overexpressing cell culture medium serves as a control) Plt0001 unpairedt-test n=6 for PREP-overexpressing cell culture medium and n=5 for PREP-overexpressing cell culture medium treated with MMP-9-neutralizing antibodyTwo independent experiments were performed

Fig 8 Analysis of EGFR and pEGFR levels in wild-type and PREP-overexpressing SH-SY5Y neuroblastoma cells Representative westernblot and corresponding quantification of EGFR and pEGFR expression levelsData are given asmean optical density (OD) ratio as percentage of controlplusmnsem(wild-type=100) β-actin was used as loading control Plt001 unpaired t-testn=4 Two independent experiments were performed

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et al 1992) The involvement of PREP in the processing of TIMPshowever remains unclear as PREP is able to cleave only smallpeptide fragments of 30 amino acids There is a possibility that somesmall as yet unknown peptides substrates of PREP might directlymodulate MMP-9 activityMoreover it cannot be excluded that PREP might be involved in

the regulation of MMP-9 release It is known that EGF through aninteraction with its receptors increases the release and activation ofMMP-9 (da Rosa et al 2014 Pei et al 2014 Qiu et al 2004) InPREP-overexpressing cells despite the overall decrease in totalEGFR the levels of phosphorylated (active form) EGFR increasedwhich indicates an activation of EGF signaling and consequentrelease of MMP-9 however the precise mechanism through whichPREP is involved in the regulation of EGF-mediated MMP-9release remains unknown and certainly needs further investigationThe PREP-mediated mechanism of the regulation of PSA-

NCAM and NCAM might be relevant for neuronal function andplasticity It has been demonstrated that NCAM180 and NCAM140are key regulators of neuronal development and maintenance as theyare widely expressed in post-synaptic densities of neurons whereasNCAM140 is also expressed in migratory growth cones (Dityatevet al 2000 Persohn et al 1989) The main function of theseisoforms is activity-dependent sprouting (Schuster et al 1998)synaptic stability (Muller et al 1996) and neurite outgrowth(Sandig et al 1996) Therefore a prolonged deficiency in NCAMandor its polysialylation might impair cellular functioning as it iswell documented that NCAM in its polysialylated form is essentialfor neurons to migrate and establish new synaptic contacts (Mulleret al 1996 Rougon 1993 Rutishauser and Landmesser 1996Seki and Arai 1993ab) In accordance with these studies ourexperiments showed that the PREP-overexpressing neuroblastomacells exhibit impaired differentiation induced by retinoic acid andBDNF The role of PSA in differentiation is important as PSA ishighly re-expressed during progression of several malignant humantumors neuroblastoma among others and therefore could beconsidered as an oncodevelopmental antigen It has beendemonstrated that in malignant human tumors polysialylation ofNCAM seems to increase the metastatic potential and has beencorrelated with tumor progression and a poor prognosis (Gluumler et al1998 Tanaka et al 2001) In vitro studies in neuroblastoma cellshave shown that application of endoneuraminidase (EndoN)treatment for PSA removal triggers the cell to cease proliferationand to differentiate (Seidenfaden et al 2003) Moreoverneuroblastoma cells differentiating toward the neuron-like celltype express high amounts of NCAM and PSA although reductionin PSA expression was found after differentiation (Hildebrandtet al 1998a) During regular differentiation removal of PSA hasbeen demonstrated to increase the number of cellndashcell contacts(Hildebrandt et al 1998a) which relay on the basis for signaltransduction through second messenger pathways in addition to celladhesion per se however in PREP-overexpressing cells whereNCAM extracellular domains are partly degraded and NCAMmolecule integrity is disrupted impaired differentiation towardneuron-like cell type as well as formation of neurites might be anoutcome of these alterationsAn increasing body of evidence from clinical and preclinical

studies suggests an involvement of PREP in neuroinflammationRecent in vitro studies demonstrated that PREP contributes to thetoxic effects of reactive microglial cells as it was demonstratedthat activated microglia cells expressed high levels of PREP andthe supernatant of these cells demonstrated toxic effects on SH-SY5Y neuroblastoma cells This toxic effect was reduced by

selective PREP inhibitors in a dose-dependent matter (Klegeriset al 2008) Moreover studies in PREP-knockout mice havedemonstrated the association of PREP in the processes modulatingneuroplasticity through inflammatory response In PREP-knockoutmouse hippocampus alterations in the microglia activation inresponse to systemic administration of repeated doses oflipopolysaccharide were found in addition to increased levels ofPSA-NCAM and these data indirectly support the functionalsignificance of PREP in the regulation of PSA-NCAM (Houmlflinget al 2016) Moreover in clinical studies increased PREP activityhas been found in knee-joint synovial membranes indicating itsassociation with rheumatoid arthritis (Kamori et al 1991)

Considering the demonstrated effects of PREP in inflammationand possible role in neuroinflammation resulting in changes inneural plasticity as well as the role of NCAMs on brain plasticity theinterplay between these molecules should be considered plausibleas NCAM-mediated neural plasticity is altered in pathologicalconditions associated with inflammation Decreased full-lengthNCAM180 has been described in mice 1 day after middle cerebralartery occlusion (Shichi et al 2011) Moreover it has been shownthat in the processes of demyelinating neuroinflammation resultingfrom the autoimmune encephalomyelitis the level of MMP-2 wassignificantly increased in hippocampus which was accompanied byreduced levels of NCAM (Jovanova-Nesic and Shoenfeld 2006) Inaddition to previously mentioned increased cleavage of NCAM180during ischemic stress similar effects in the decrease of NCAM180and increased content of the cleaved form of NCAM was describedafter oxidative stress (Fujita-Hamabe and Tokuyama 2012)

In conclusion our study demonstrates that increased expressionlevel and activity of PREP is involved in mechanisms regulatingdegradation of neural adhesion molecules most likely by MMP-9activation These findings open up a new avenue for the explorationof the roles of PREP in processes involved in NCAM degradationwhich in turn is believed to be one major contributor for alteredneuroplasticity (Bruseacutes and Rutishauser 2001 Cremer et al 1998Gnanapavan and Giovannoni 2013) Moreover these mechanismsare of importance regarding synaptic plasticity in the (re-)organization of neuronal circuits

MATERIALS AND METHODSGeneration of PREP-overexpressing SH-SY5Y cell lineThe PREP-overexpressing SH-SY5Y cell line was generated as described inGerard et al (2010) Wild-type and PREP-overexpressing cells were grownin DMEMGlutaMAX-I medium (Thermo Scientific) containing 15heat-inactivated fetal calf serum 1 NEAA and 50 microgml gentamycin forwild-type cells and additionally 200 microgml hygromycin B for PREP-overexpressing cells Cells were maintained at 37degC in a saturated humidityatmosphere containing 95 air and 5 CO2 For differentiation studiesalltrans-retinoic acid (RA Tocris Cookson Bristol UK) was added the dayafter plating at a final concentration of 10 microM in DMEMwith 15 fetal calfserum After 5 days in the presence of RA cells were washed three timeswith DMEM and incubated with 50 ngml BDNF (Sigma-Aldrich) inDMEM (without serum) for 7 days

PREP activity assayPREP activity in SH-SY5Y cells was measured according to the methoddescribed by Klimaviciusa et al (2012) Briefly cells were washed twicewith PBS and lysed by chilled hypotonic buffer (pH 75) containing 50 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 20 mMNaCl 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mMdithiothreitol (DTT Sigma-Aldrich) The obtained cell lysate wascentrifuged and supernatants were transferred to new tubes Equal amountsof protein samples (10 microg) were mixed with assay buffer containingchromogenic PREP substrate 250 microM Z-Gly-Pro-p-nitroanilide (Bachem

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AG Bubendorf ) The product absorbance was continuously measured for30 min at 405 nm using an ELISA plate reader (Tecan CrailsheimGermany) and PREP activity was calculated using a p-nitroanilide (Sigma-Aldrich) standard curve

Primary culture of rat cortical neuronsPrimary neuronal cultures were prepared from 1-day-old Wistar rat pupsaccording to the method of Alho and colleagues (1988) with minormodifications Briefly cortices were dissected in ice-cold Krebs-Ringersolution (135 mM NaCl 5 mM KCl 1 mM MgSO4 04 mM K2HPO215 mM glucose 20 mM HEPES pH 74 containing 03 bovine serumalbumin) and trypsinized in 08 trypsin-EDTA (Invitrogen UK) for10 min at 37degC followed by trituration in 0008 DNAse I solutioncontaining 005 soybean trypsin inhibitor (both from Surgitech ASEstonia) Cells were resuspended in Eaglersquos basal medium with Earlersquos salts(BME Invitrogen UK) containing 10 heat-inactivated fetal calf serum(Invitrogen) 25 mM KCl 2 mM GlutaMAX-I (Invitrogen UK) and100 microgml gentamycin (KRKA) Cells were plated onto poly-L-lysine-coated (Sigma-Aldrich) cell culture dishes at a density of 18times105 cellscm2After 25 h the medium was changed to Neurobasal-A medium (Gibco)Cultures were incubated for 4 days in an atmosphere of 95 air and 5 CO2

at 37degC

Treatment with rPREPWild-type SH-SY5Y neuroblastoma cells or primary cortical neurons atDIV4 were grown in DMEM cell culture medium plus 10 fetal calf serumor Neurobasal-A medium containing 2 mM GlutaMAX-I B-27 supplement(Gibco) and 100 μgml gentamycin respectively Human recombinant PREPprotein (rPREP) (a generous gift from Dr Zoltan Szeltner Institute ofEnzymology Research Centre for Natural Sciences Hungarian Academy ofScience) stock solution was dissolved in buffered saline 1 mM DTT pH 75and added into cell culture medium at concentrations of 1 nM and 10 nM forwild-type SH-SY5Y neuroblastoma cells and 1 nM for primary corticalneurons Cells were incubated for 72 h and then processed for western blotanalysis

Treatment with MMP-9-neutralizing antibody and NCAMcleavage assayMMP-9-neutralizing antibody (clone 6-6B) was purchased from Millipore(IM09L) and dissolved according to manufacturerrsquos instructions PREP-overexpressing SH-SY5Y neuroblastoma cells were treated with MMP-9-neutralizing antibody or appropriate vehicle for 96 h at a concentration of50 microgml in serum free DMEMGlutaMAX-I medium Detection of NCAMfragments was done according to the method of Brennaman and colleagues(2014) After 96 h cell culture medium was collected treated with proteaseinhibitors (Roche) centrifuged for 5 min (173 g) followed by concentrationwith Millipore centrifugal concentrators according to the manufacturerrsquosinstructions (30000 MW cutoff 10times concentration) Protein concentrationsfrom each concentrated cell culture media sample were measured byBradford method and equivalent amount of proteins from media sampleswere resolved by electrophoresis on 10 SDS-polyacrylamide gel asdescribed below In addition a blank control sample was prepared andresolved by electrophoresis The blank control consisted of pure DMEM cellculture medium and MMP-9-neutralizing antibody concentrated undersimilar conditions to the experimental samples

PREP expression knockdown in SH-SY5Y neuroblastoma cellsand image analysisFor transfection of wild-type and PREP-overexpressing SH-SY5Yneuroblastoma cells grown on glass-bottomed cell culture dishes (35 cmdiameter) the conditioned medium was replaced with 120 microl Opti-MEMmedium (Gibco) containing 2 Lipofectamine 2000 transfection reagent(Thermo Scientific) with either empty vector (control) or validated PREP(shPREP) shRNA plasmid (KH20237N SA Biosciences MD USA) andpGFP (632370 Clontech) The dishes were incubated for 3 h after whichfresh DMEM medium was added The transfected cells were allowed to

express the shRNA for 72 h before immunocytochemical detection for PSA-NCAM was performed Immunopositive signals were detected with aconfocal microscope LSM 510 (Zeiss Denmark) equipped with an argonndashkrypton laser 3D images were constructed from a series (10ndash13) of scans ofthe GFP-positive cells at 1 microm intervals using a 40times (water) objective andfurther analyzed for signal intensity Fluorescence quantification analysiswas performed using ImageJ software (NIH) Each cell to be analyzed wasmanually defined and three regions were selected just beside the cell in anarea without fluorescent objects to be used for background adjustmentSubsequently the corrected total cell fluorescence (CTCF) was obtainedaccording to Gavet and Pines (2010) CTCF was calculated by multiplyingthe area (in square pixels) of the selected cell of interest bymean backgroundfluorescence intensity and subtracting the result from whole-cellfluorescence intensity Overlapping of cells was excluded by checking allchannels in each image PSA-NCAM-immunopositive signal intensity isgiven in relative fluorescence units (RFU)

Immunocytochemistry and image analysisSH-SY5Y neuroblastoma cells were fixed by using 4 paraformaldehyde inphosphate-buffered saline (PBS) for 10 min at room temperaturepermeabilized in 005 Triton X-100 (Sigma-Aldrich) for 30 min andrinsed in PBS Non-specific binding was blocked by 2 normal goat serum(Vector Laboratories) in PBS containing 03 Triton X-100 for 1 hfollowed by 48 h of incubation at 4degC with different primary antibodiesdiluted in blocking buffer based on the requirement of the experimentPrimary antibodies used were mouse anti-PSA-NCAM (1500 MercMillipore MAB5324) rabbit anti-NCAM (11000 Merc MilliporeAB5032) rabbit anti-MMP-9 (1700 Merc Millipore AB19016) rabbitanti-ST8Sia4 (1500 Thermo Scientific PA5-26774) rabbit anti-ST8Sia2(1200 Proteintech Europe 19736-1-AP) mouse anti-Tuj-1 (1700 MercMillipore MAB1637) After being washed in PBS cells were incubated inAlexa Fluor 594 goat anti-mouse IgM (11000 A-21044) Alexa Fluor 488goat anti-rabbit IgG (11000 A-11008) Alexa Fluor 594 goat anti-rabbitIgG (11000 A-11012) or Alexa Fluor 488 goat anti-mouse IgG (11000A-11001) (all from Life Technologies) in blocking buffer for 1 h Nucleiwere counterstained with DAPI solution (Sigma-Aldrich 300 nM in PBS)

A laser scanning confocal microscope (LSM 510 Duo Zeiss Germany)equipped with a C-Apocromat 40times120 water immersion M27 objective(Zeiss Germany) was used for image acquiring and acquisition parameters(magnification laser intensity gain pinhole aperture) were kept constantbetween different experimental groups For differentiation analysis ninerandomly taken fields from three cell culture dishes (35 cm diameter) weretaken at magnification times40 3D images were constructed from a series (10ndash12) of scans at 1 microm intervals for image analysis The number of Tuj-1-immunopositive cells and the total number of DAPI-positive nuclei werecounted and the datawere expressed as a percentage of cells positive for Tuj-1 signal in wild-type and PREP-overexpressing cells

Western blot analysisPrimary neurons or SH-SY5Y cells were lysed in RIP-A cell lysis buffer(20 mM Tris-HCl pH 80 137 mM NaCl 10 glycerol 1 NP-40 and2 mM EDTA containing protease and phosphatase inhibitor cocktail)incubated on ice for 20 min and centrifuged (15900 g for 20 min at 4degC)Equivalent amounts of protein were resolved by electrophoresis on 8 or10 SDS-polyacrylamide gels Resolved proteins were transferred toHybond-P PVDF membranes or onto Immobilon-FL or Immobilon-FLPVDF membranes (Merck Millipore) in 01 M Tris-base pH 83 0192 Mglycine and 20 (vv) methanol using an electrophoretic transfer system(Bio-Rad) The membranes were blocked by using 05 (wv) nonfat driedmilk (BD Biosciences) in TBS containing 0025 (vv) Tween-20 (Sigma-Aldrich) or Odyssey blocking buffer (Li-Cor Bioscience) After blockingthe membranes were incubated overnight with different primary antibodiesbased on the requirement of the experiment Antibodies used were mouseanti-PSA-NCAM (11000 Millipore MAB5324) rabbit anti-NCAM(11000 Millipore AB5032) rabbit anti-NCAM (1800 extracellulardomain H-300 sc-10735 Santa Cruz Biotechnology) rabbit anti-MMP-9(11000 Merc Millipore AB19016) rabbit anti-MMP-3 (11000 Abcam

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ab52915) rabbit anti-ADAM-10 (1500 Abcam ab1997) rabbit anti-MMP-2 (1500 Merc Millipore AB19015) rabbit anti-ST8Sia4 (1750Thermo Scientific PA5-26774) rabbit anti-ST8Sia2 (1500 ProteintechEurope 19736-1-AP) rabbit anti-EGFR (11000 Abcam EP38Y) rabbitanti-pEGFR (1800 Abcam EP774Y) chicken anti-PREP (11000 agenerous gift from Dr Arturo Garcia-Horsman Division of Pharmacologyand Toxicology University of Helsinki Finland) Incubations werefollowed by washing and incubation with the goat anti-rabbit or goat anti-mouse horseradish-peroxidase (HRP)-conjugated secondary antibodies(14000 Thermo Scientific 31460 and 32430 respectively) goat anti-rabbit IRDye 800 CW (926-32211) or 680 LT (926-68021) goat anti-mouseIRDye 800 CW (926-32210) or 680 LT (926-68020) donkey anti-chickenIRDye 680 CW (926-68028) (all at 110000 Li-Cor Biosciences) for 1 h atroom temperature Immunoreactive bands were detected by medical X-rayfilm (AGFA EAS4Y) or using the Odyssey Infrared Imaging System(Odyssey CLxreg Li-Cor Biosciences) To normalize immunoreactivity ofthe proteins β-actin was measured on the same blot by using a mousemonoclonal anti-β-actin antibody (11000 Li-Cor Biosciences 926-42212)followed by incubating with the same HRP-conjugated secondary antibody(14000) goat anti-mouse IRDye 680 LT or 800 CW (110000) as aboveThe ratios of proteins of interest to β-actin were calculated and expressed asthe mean OD ratio in arbitrary unitsplusmnsem or expressed as a percentage ofcontrolplusmnsem

RNA isolation and real-time quantitative PCRTotal RNA was extracted from wild-type and PREP-overexpressing SH-SY5Y neuroblastoma cells using the RNeasy Mini Kit (QIAGEN)according to the manufacturersrsquo protocol cDNA was synthesized from1 microg of total RNA using the First Strand cDNA Synthesis Kit (FermentasInc Burlington Canada) Real-time quantitative PCR (qPCR) wasperformed using QuantStudio 12K Flex Real-Time PCR System equippedwith QuantStudio 12K Flex Software (ThermoFisher Scientific) Primerswere synthesized by TAG Copenhagen AS (Copenhagen Denmark) andprimer sequences were follows NCAM 120 kDa forward 5prime-CATGTCACCACTCACAGATACTTTTG-3prime reverse 5prime-CTCTGTAAATCTAGCATGATGGTTTTT-3prime NCAM 140 kDa forward 5prime-AACGAGACCACGCCACTGA-3prime reverse 5prime-CGTTTCTGTCTCCTGGCACTCT-3prime NCAM 180 kDaforward 5prime-GACTTTAAAATGGACGAAGGGAAC-3prime reverse 5prime-CCCAGGGCTGCAAAAACA-3prime (adapted from Winter et al 2008) NCAM totalforward 5prime-GAGATCAGCGTTGGAGAGTCC-3prime reverse 5prime-GGAGAACCAGGAGATGTCTTTATCTT-3prime (adapted fromValentineret al 2011)HPRT1(hypoxanthine phosphoribosyltransferase 1) forward 5prime-CTTTGCTGACCTGCTGGATTAC-3prime reverse 5prime-GTCCTTTTCACCAGCAAGCTTG-3primeHMBS (hydroxymethylbilane synthase) forward 5prime-GGCAATGCGGCTGCAA-3prime reverse 5prime-GGGTACCCACGCGAATCAC-3prime SDHA(succinate dehydrogenase complex subunit A) forward 5prime-GGCAATGCGGCTGCAA-3prime reverse 5prime-GGGTACCCACGCGAATCAC-3prime PCRamplification was performed in a total reaction volume of 10 microl in threeparallels The reaction mixture consisted of 1 microl First Strand cDNA dilutedtemplate 5 microl 2times Master SYBR Green qPCR Master Mix (AppliedBiosystems) 3 microl H2O and 1 microl gene-specific 10 microM PCR primer pairstock The PCR amplification was performed as follows denaturation stepat 95degC for 10 min followed by denaturation at 95degC for 15 s andannealing and extension at 60degC for 1 min repeated for 40 cycles SYBRGreen fluorescence was measured after each extension step andamplification specificity was confirmed by melting curve analyses andgel electrophoresis of the PCR products Relative mRNA levels werecalculated by normalization of target gene mRNA level to the geometricmean of the expression level of three endogenous reference genes (HPRT1HMBS and SDHA)

Gelatin zymographyEqual amounts of cell culture medium (normalized to cell count in cellculture dish) were separated by electrophoresis in SDS-PAGE gelscontaining 1 mgml porcine skin gelatin (Sigma-Aldrich) at 90 V Gelswere washed for 40 min in 25 Triton X-100 incubated for 48 h at 37degCwith gentle agitation in 1 Triton X-100 5 mM CaCl2 005 M Trisfollowed by staining with 05 Coomassie Blue R-250 (Sigma-Aldrich) for

1 h and destaining with 30methanol and 10 acetic acid in distilled waterZones of proteolysis appeared as clear bands against a blue backgroundBands were scanned and intensity of bands was quantified using ImageJsoftware

Data analysisData presented are the meanplusmnsem and experiments were repeated 2ndash3times Data were analyzed using GraphPad Prism 5 software Statisticalanalysis was performed by using two-tailed Studentrsquos t-test or one-wayANOVA followed by the multiple comparison Bonferroni test whereappropriate In all instances Plt005 was considered statistically significant

AcknowledgementsThe technical assistance of Mrs Olili Suvi was greatly appreciated The authorswould like to thank Dr Rajeev Jain (Department of Pharmacology Institute ofBiomedicine and Translational Medicine University of Tartu Estonia) for providingthe plasmids

Competing interestsThe authors declare no competing or financial interests

Author contributionsKJ AW conception and design of the study conduction of experimental workstatistical analysis and interpretation of the data drafting and revision of themanuscript KP LK AA-H KA AN participation in the experimental workconduction of immunocytochemistry western blot and qPCR MG generationPREP-overexpressing SH-SY5Y cell line RVE A-ML SR MM AZ approvalof study design interpretation of the data critical revision of the article approval ofthe final version of the manuscript

FundingThis study was supported by the 7th Framework Programme of Health of theEuropean Commission [project NEUROPRO HEALTH-F2-2008-223077 to SRA-ML AZ] Eesti Teadusfondi (Estonian Science Foundation) [grant number8740 to KJ] and Eesti Teadusagentuur (Estonian Research Council) [Institutionalresearch funding grant IUT2-3 to AZ] the Deutsche Forschungsgemeinschaft(German Research Foundation) [grants MO 22492-1 and MO 22492-2 to MMwithin the PP 1608] the Alzheimer Forschung Initiative [grant number 1186 toMM]Universiteit Antwerpen [special research fund grant FFB3551 to A-ML and RVE]and Sachsische Aufbaubank (SAB)European Social Fund (ESF) [grant numberSAB100154907 to SR MM AZ and AW]

Supplementary informationSupplementary information available online athttpjcsbiologistsorglookupdoi101242jcs181891supplemental

ReferencesAlho H Ferrarese C Vicini S and Vaccarino F (1988) Subsets of GABAergic

neurons in dissociated cell cultures of neonatal rat cerebral cortex show co-localization with specific modulator peptides Dev Brain Res 39 193-204

Amoureux M C Cunningham B A Edelman G M and Crossin K L (2000)N-CAM binding inhibits the proliferation of hippocampal progenitor cells andpromotes their differentiation to a neuronal phenotype J Neurosci 203631-3640

Angata K Nakayama J Fredette B Chong K Ranscht B and Fukuda M(1997) Human STX polysialyltransferase forms the embryonic form of the neuralcell adhesion molecule Tissue-specific expression neurite outgrowth andchromosomal localization in comparison with another polysialyltransferasePST J Biol Chem 272 7182-7190

Bellemere G Vaudry H Mounien L Boutelet I and Jegou S (2004)Localization of the mRNA encoding prolyl endopeptidase in the rat brain andpituitary J Comp Neurol 471 128-143

Brandt I Gerard M Sergeant K Devreese B Baekelandt V AugustynsK Scharpe S Engelborghs Y and Lambeir A-M (2008) Prolyloligopeptidase stimulates the aggregation of alpha-synuclein Peptides 291472-1478

Brennaman L H Moss M L and Maness P F (2014) EphrinAEphA-inducedectodomain shedding of neural cell adhesion molecule regulates growth conerepulsion through ADAM10 metalloprotease J Neurochem 128 267-279

Bruses J L and Rutishauser U (2001) Roles regulation and mechanism ofpolysialic acid function during neural development Biochimie 83 635-643

Cremer H Chazal G Carleton A Goridis C Vincent J-D and Lledo P-M(1998) Long-term but not short-term plasticity at mossy fiber synapses is impairedin neural cell adhesion molecule-deficient mice Proc Natl Acad Sci USA 9513242-13247

3800


Journal

ofCe

llScience

Cunningham D F and OrsquoConnor B (1997) Identification and initialcharacterization of a N-benzyloxycarbonyl-prolyl-prolinal (Z-Pro-prolinal)-insensitive 7-(N-benzyloxycarbonyl-glycyl-prolyl-amido)-4-methylcoumarin (Z-Gly-Pro-NH-Mec)-hydrolysing peptidase in bovine serum Eur J Biochem 244900-903

CunninghamB A Hemperly J J Murray B A Prediger E A BrackenburyR and Edelman G M (1987) Neural cell adhesion molecule structureimmunoglobulin-like domains cell surface modulation and alternative RNAsplicing Science 236 799-806

da Rosa M R P Falcao A S C Fuzii H T da Silva Kataoka M S RibeiroA L R Boccardo E de Siqueira A S Jaeger R G de Jesus VianaPinheiro J and de Melo Alves Junior S (2014) EGFR signaling downstreamof EGF regulates migration invasion and MMP secretion of immortalized cellsderived from human ameloblastoma Tumour Biol 35 11107-11120

Di Daniel E Glover C P Grot E Chan M K Sanderson T H White J HEllis C L Gallagher K T Uney J Thomas J et al (2009) Prolyloligopeptidase binds to GAP-43 and functions without its peptidase activity MolCell Neurosci 41 373-382

Dityatev A Dityateva G and Schachner M (2000) Synaptic strength as afunction of post- versus presynaptic expression of the neural cell adhesionmolecule NCAM Neuron 26 207-217

Eckhardt M Muhlenhoff M Bethe A Koopman J Frosch M and Gerardy-Schahn R (1995) Molecular characterization of eukaryotic polysialyltransferase-1Nature 373 715-718

Encinas M Iglesias M Liu Y Wang H Muhaisen A Cen a V Gallego Cand Comella J X (2000) Sequential treatment of SH-SY5Y cells with retinoicacid and brain-derived neurotrophic factor gives rise to fully differentiatedneurotrophic factor-dependent human neuron-like cells J Neurochem 75991-1003

Finne J Finne U Deagostini-Bazin H and Goridis C (1983) Occurrence ofalpha 2-8 linked polysialosyl units in a neural cell adhesion molecule BiochemBiophys Res Commun 112 482-487

Frears E R Zhang Z Blake D R OrsquoConnell J P andWinyard P G (1996)Inactivation of tissue inhibitor of metalloproteinase-1 by peroxynitrite FEBS Lett381 21-24

Fujita-Hamabe W and Tokuyama S (2012) The involvement of cleavage ofneural cell adhesion molecule in neuronal death under oxidative stress conditionsin cultured cortical neurons Biol Pharm Bull 35 624-628

Fulop V Bocskei Z and Polgar L (1998) Prolyl oligopeptidase an unusualbeta-propeller domain regulates proteolysis Cell 94 161-170

Gavet O and Pines J (2010) Progressive activation of CyclinB1-Cdk1coordinates entry to mitosis Dev Cell 18 533-543

Gerard M Deleersnijder A Daniels V Schreurs S Munck S Reumers VPottel H Engelborghs Y Van den Haute C Taymans J-M et al (2010)Inhibition of FK506 binding proteins reduces alpha-synuclein aggregation andParkinsonrsquos disease-like pathology J Neurosci 30 2454-2463

Gluer S Schelp C Madry N von Schweinitz D Eckhardt M and Gerardy-Schahn R (1998) Serum polysialylated neural cell adhesion molecule inchildhood neuroblastoma Br J Cancer 78 106-110

Gnanapavan S and Giovannoni G (2013) Neural cell adhesion molecules inbrain plasticity and disease Mult Scler Relat Disord 2 13-20

Hildebrandt H Becker C Gluer S Rosner H Gerardy-Schahn R andRahmann H (1998a) Polysialic acid on the neural cell adhesion moleculecorrelates with expression of polysialyltransferases and promotes neuroblastomacell growth Cancer Res 58 779-784

Hildebrandt H Becker C Murau M Gerardy-Schahn R and Rahmann H(1998b) Heterogeneous expression of the polysialyltransferases ST8Sia II andST8Sia IV during postnatal rat brain development J Neurochem 71 2339-2348

Hinkle C L Diestel S Lieberman J and Maness P F (2006)Metalloprotease-induced ectodomain shedding of neural cell adhesionmolecule (NCAM) J Neurobiol 66 1378-1395

Hofling C Kulesskaya N Jaako K Peltonen I Mannisto P T Nurmi AVartiainen N Morawski M Zharkovsky A Votildeikar V et al (2016)Deficiency of prolyl oligopeptidase in mice disturbs synaptic plasticity andreduces anxiety-like behaviour body weight and brain volume EurNeuropsychopharmacol 26 1048-1061

Hubschmann M V Skladchikova G Bock E and Berezin V (2005) Neuralcell adhesion molecule function is regulated by metalloproteinase-mediatedectodomain release J Neurosci Res 80 826-837

Irazusta J Larrinaga G Gonzalez-Maeso J Gil J Meana J J andCasis L(2002) Distribution of prolyl endopeptidase activities in rat and human brainNeurochem Int 40 337-345

Johnson C P Fujimoto I Rutishauser U and Leckband D E (2005) Directevidence that neural cell adhesion molecule (NCAM) polysialylation increasesintermembrane repulsion and abrogates adhesion J Biol Chem 280 137-145

Jovanova-Nesic K and Shoenfeld Y (2006) MMP-2 VCAM-1 and NCAM-1expression in the brain of rats with experimental autoimmune encephalomyelitisas a trigger mechanism for synaptic plasticity and pathology J Neuroimmunol181 112-121

Kalus I Bormann U Mzoughi M Schachner M and Kleene R (2006)Proteolytic cleavage of the neural cell adhesion molecule by ADAM17TACE isinvolved in neurite outgrowth J Neurochem 98 78-88

Kamori M Hagihara M Nagatsu T Iwata H and Miura T (1991) Activities ofdipeptidyl peptidase II dipeptidyl peptidase IV prolyl endopeptidase andcollagenase-like peptidase in synovial membrane from patients with rheumatoidarthritis and osteoarthritis Biochem Med Metab Biol 45 154-160

Kato A Fukunari A Sakai Y and Nakajima T (1997) Prevention of amyloid-like deposition by a selective prolyl endopeptidase inhibitor Y-29794 insenescence-accelerated mouse J Pharmacol Exp Ther 283 328-335

Kiselyov V V Skladchikova G Hinsby A M Jensen P H Kulahin NSoroka V Pedersen N Tsetlin V Poulsen F M Berezin V et al (2003)Structural basis for a direct interaction between FGFR1 and NCAM and evidencefor a regulatory role of ATP Structure 11 691-701

Kiss J Z and Muller D (2001) Contribution of the neural cell adhesion moleculeto neuronal and synaptic plasticity Rev Neurosci 12 297-310

Klegeris A Li J Bammler T K Jin J Zhu D Kashima D T Pan SHashioka S Maguire J McGeer P L et al (2008) Prolyl endopeptidase isrevealed following SILAC analysis to be a novel mediator of human microglial andTHP-1 cell neurotoxicity Glia 56 675-685

Klimaviciusa L Jain R K Jaako K Van Elzen R Gerard M van DerVeken P Lambeir A-M and Zharkovsky A (2012) In situ prolyloligopeptidase activity assay in neural cell cultures J Neurosci Methods 204104-110

Krog L Olsen M Dalseg A-M Roth J and Bock E (1992) Characterizationof soluble neural cell adhesion molecule in rat brain CSF and plasmaJ Neurochem 59 838-847

Mantle D Falkous G Ishiura S Blanchard P J and Perry E K (1996)Comparison of proline endopeptidase activity in brain tissue from normal casesand cases with Alzheimerrsquos disease Lewy body dementia Parkinsonrsquos diseaseand Huntingtonrsquos disease Clin Chim Acta 249 129-139

Miyagi T Wada T Yamaguchi K and Hata K (2003) Sialidase andmalignancy a minireview Glycoconj J 20 189-198

Muller D Wang C Skibo G Toni N Cremer H Calaora V Rougon G andKiss J Z (1996) PSAndashNCAM is required for activity-induced synaptic plasticityNeuron 17 413-422

Myohanen T T Venalainen J I Tupala E Garcia-Horsman J A MiettinenR and Mannisto P T (2007) Distribution of immunoreactive prolyloligopeptidase in human and rat brain Neurochem Res 32 1365-1374

Myohanen T T Venalainen J I Garcıa-Horsman J A Piltonen M andMannisto P T (2008) Distribution of prolyl oligopeptidase in the mouse whole-body sections and peripheral tissues Histochem Cell Biol 130 993-1003

Nakayama J Angata K Ong E Katsuyama T and Fukuda M (1998)Polysialic acid a unique glycan that is developmentally regulated by twopolysialyltransferases PST and STX in the central nervous system frombiosynthesis to function Pathol Int 48 665-677

Nybroe O Linnemann D and Bock E (1989) Heterogeneity of soluble neuralcell adhesion molecule J Neurochem 53 1372-1378

Okada Y Gonoji Y Naka K Tomita K Nakanishi I Iwata K Yamashita Kand Hayakawa T (1992) Matrix metalloproteinase 9 (92-kDa gelatinasetype IVcollagenase) from HT 1080 human fibrosarcoma cells Purification and activationof the precursor and enzymic properties J Biol Chem 267 21712-21719

Pei J Lou Y Zhong R and Han B (2014) MMP9 activation triggered byepidermal growth factor induced FoxO1 nuclear exclusion in non-small cell lungcancer Tumour Biol 35 6673-6678

Penttinen A Tenorio-Laranga J Siikanen A Morawski M Rossner S andGarcıa-Horsman J A (2011) Prolyl oligopeptidase a rising star on the stage ofneuroinflammation research CNS Neurol Disord Drug Targets 10 340-348

Persohn E Pollerberg G E and Schachner M (1989) Immunoelectron-microscopic localization of the 180 kD component of the neural cell adhesionmolecule N-CAM in postsynaptic membranes J Comp Neurol 288 92-100

Polgar L (2002) The prolyl oligopeptidase family Cell Mol Life Sci 59 349-362Proshin S Yamaguchi K Wada T and Miyagi T (2002) Modulation of

neuritogenesis by ganglioside-specific sialidase (Neu 3) in human neuroblastomaNB-1 cells Neurochem Res 27 841-846

Qiu Q Yang M Tsang B K and Gruslin A (2004) EGF-induced trophoblastsecretion of MMP-9 and TIMP-1 involves activation of both PI3K and MAPKsignalling pathways Reproduction 128 355-363

Rawlings N D Polgar L and Barrett A J (1991) A new family of serine-typepeptidases related to prolyl oligopeptidase Biochem J 279 907-908

Rossner S Schulz I Zeitschel U Schliebs R Bigl V and Demuth H-U(2005) Brain prolyl endopeptidase expression in aging APP transgenic mice andAlzheimerrsquos disease Neurochem Res 30 695-702

Rougon G (1993) Structure metabolism and cell biology of polysialic acidsEur J Cell Biol 61 197-207

Rutishauser U (2008) Polysialic acid in the plasticity of the developing and adultvertebrate nervous system Nat Rev Neurosci 9 26-35

Rutishauser U and Landmesser L (1996) Polysialic acid in the vertebratenervous system a promoter of plasticity in cell-cell interactions Trends Neurosci19 422-427

3801


Journal

ofCe

llScience

Sadoul K Meyer A Low M G and Schachner M (1986) Release of the 120kDa component of the mouse neural cell adhesion molecule N-CAM from cellsurfaces by phosphatidylinositol-specific phospholipase C Neurosci Lett 72341-346

Sajo M Sugiyama H Yamamoto H Tanii T Matsuki N Ikegaya Y andKoyama R (2016) Neuraminidase-dependent degradation of polysialic acid isrequired for the lamination of newly generated neurons PLoSONE 11 e0146398

Sandig M Rao Y Siu C-H and Kalnins V I (1996) Integrity of the homophilicbinding site is required for the preferential localization of NCAM in intercellularcontacts Biochem Cell Biol 74 373-381

Savolainen M H Yan X Myohanen T T and Huttunen H J (2015) Prolyloligopeptidase enhances α-synuclein dimerization via direct protein-proteininteraction J Biol Chem 290 5117-5126

Schulz I Gerhartz B Neubauer A Holloschi A Heiser U Hafner M andDemuth H-U (2002) Modulation of inositol 145-triphosphate concentration byprolyl endopeptidase inhibition Eur J Biochem 269 5813-5820

Schulz I Zeitschel U Rudolph T Ruiz-Carrillo D Rahfeld J-U GerhartzB Bigl V Demuth H-U and Rossner S (2005) Subcellular localizationsuggests novel functions for prolyl endopeptidase in protein secretionJ Neurochem 94 970-979

Schuster T Krug M Hassan H and Schachner M (1998) Increase inproportion of hippocampal spine synapses expressing neural cell adhesionmolecule NCAM180 following long-term potentiation J Neurobiol 37 359-372

Seidenfaden R Krauter A Schertzinger F Gerardy-Schahn R andHildebrandt H (2003) Polysialic acid directs tumor cell growth by controllingheterophilic neural cell adhesion molecule interactions Mol Cell Biol 235908-5918

Seidenfaden R Krauter A and Hildebrandt H (2006) The neural cell adhesionmolecule NCAM regulates neuritogenesis by multiple mechanisms of interactionNeurochem Int 49 1-11

Seki T and Arai Y (1993a) Distribution and possible roles of the highlypolysialylated neural cell adhesion molecule (NCAM-H) in the developing andadult central nervous system Neurosci Res 17 265-290

Seki T and Arai Y (1993b) Highly polysialylated neural cell adhesion molecule(NCAM-H) is expressed by newly generated granule cells in the dentate gyrus ofthe adult rat J Neurosci 13 2351-2358

Shichi K Fujita-Hamabe W Harada S Mizoguchi H Yamada KNabeshima T and Tokuyama S (2011) Involvement of matrixmetalloproteinase-mediated proteolysis of neural cell adhesion molecule in thedevelopment of cerebral ischemic neuronal damage J Pharmacol Exp Ther338 701-710

Shishido Y Furushiro M Tanabe S Taniguchi A Hashimoto SYokokura T Shibata S Yamamoto T and Watanabe S (1998) Effect of

ZTTA a prolyl endopeptidase inhibitor on memory impairment in a passiveavoidance test of rats with basal forebrain lesions Pharm Res 15 1907-1910

Shishido Y Furushiro M Tanabe S Shibata S Hashimoto S andYokokura T (1999) Effects of prolyl endopeptidase inhibitors and neuropeptideson delayed neuronal death in rats Eur J Pharmacol 372 135-142

Strekalova H Buhmann C Kleene R Eggers C Saffell J Hemperly JWeiller C Muller-Thomsen T and Schachner M (2006) Elevated levels ofneural recognitionmolecule L1 in the cerebrospinal fluid of patients with Alzheimerdisease and other dementia syndromes Neurobiol Aging 27 1-9

Tanaka F Otake Y Nakagawa T Kawano Y Miyahara R Li MYanagihara K Inui K Oyanagi H Yamada T et al (2001) Prognosticsignificance of polysialic acid expression in resected non-small cell lung cancerCancer Res 61 1666-1670

Toide K Shinoda M Fujiwara T and Iwamoto Y (1997) Effect of a novel prolylendopeptidase inhibitor JTP-4819 on spatial memory and central cholinergicneurons in aged rats Pharmacol Biochem Behav 56 427-434

Tringali C Lupo B Silvestri I Papini N Anastasia L Tettamanti G andVenerando B (2012) The plasma membrane sialidase NEU3 regulates themalignancy of renal carcinoma cells by controlling 1 integrin internalization andrecycling J Biol Chem 287 42835-42845

Valentiner U Muhlenhoff M Lehmann U Hildebrandt H and SchumacherU (2011) Expression of the neural cell adhesion molecule and polysialic acid inhuman neuroblastoma cell lines Int J Oncol 39 417-424

Vawter M P Cannon-Spoor H E Hemperly J J Hyde T M VanderPuttenD M Kleinman J E and Freed W J (1998) Abnormal expression of cellrecognition molecules in schizophrenia Exp Neurol 149 424-432

Wada T Hata K Yamaguchi K Shiozaki K Koseki K Moriya S andMiyagi T (2007) A crucial role of plasma membrane-associated sialidase in thesurvival of human cancer cells Oncogene 26 2483-2490

Walmod P S Kolkova K Berezin V and Bock E (2004) Zippers makesignals NCAM-mediated molecular interactions and signal transductionNeurochem Res 29 2015-2035

Williams E J Furness J Walsh F S and Doherty P (1994) Activation of theFGF receptor underlies neurite outgrowth stimulated by L1 N-CAM and N-cadherin Neuron 13 583-594

Winter C Pawel B Seiser E Zhao H Raabe E Wang Q Judkins A RAttiyeh E and Maris J M (2008) Neural cell adhesion molecule (NCAM)isoform expression is associated with neuroblastoma differentiation statusPediatr Blood Cancer 51 10-16

Yoshimoto T Kado K Matsubara F Koriyama N Kaneto H and Tsuru D(1987) Specific inhibitors for prolyl endopeptidase and their anti-amnesic effectJ Pharmacobiodyn 10 730-735

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1997) Besides its extracellular action PREP has been shown to actintracellularly and important roles of PREP have been demonstratedin signaling pathways or in transport and secretion of proteins andpeptides associated with neurodegeneration (Brandt et al 2008 DiDaniel et al 2009 Rossner et al 2005 Savolainen et al 2015Schulz et al 2002 2005) Within recent years PREP has beensuggested to be a contributor to neuroinflammation (Penttinen et al2011) PREP has been shown to be involved in mechanismsresponsible for the transduction and amplification of inflammatoryprocesses leading to the production of neurotoxic mediators whichin turn mediate pathology progression (for review see Penttinenet al 2011)In this report for the first time we demonstrate that PREP is able

to regulate levels of NCAM through activation of MMP-9

RESULTSLevels of PSA-NCAM and NCAM protein and mRNA inneuroblastoma SH-SY5Y cells overexpressing PREPIncreased levels of PREP protein and its activity in PREP-overexpressing SH-SY5Y cell lysates were confirmed by westernblot and activity measurements (Fig S1AB)PREP was detected on SDS-PAGE as a band at sim80 kDa

(Fig S1A) A 30-fold increase in PREP protein level was found inSH-SY5Y cells overexpressing PREP compared with wild-typecells (Plt0001 t-test n=4) In these PREP-overexpressing cellsPREP activity assays demonstrated an 11-fold increase in enzymaticactivity (Plt00001 t-test n=3 Fig S1B)To evaluate whether increased PREP levels are associated with

altered levels of PSA-NCAM andor NCAM the proteins werequantified using western blot analysis In wild-type cells PSA-NCAM was detected as a high-molecular-weight smear typical forhighly polysialylated NCAM (Fig 1A) whereas by comparisonPSA-NCAM signal was almost absent in PREP-overexpressingcells (Plt00001 t-test n=5ndash6 Fig 1A) In wild-type cellsimmunocytochemistry identified membrane-bound extracellularPSA-NCAM molecules which assembled in small bunches alongneurite-like processes and in larger patches associated with theperikaryon In PREP-overexpressing cells however PSA-NCAMsignal was almost absent (Fig 1C)We further investigated levels of the NCAM protein isoforms

(NCAM180 NCAM140 and NCAM120) A significant decrease inlevels of the NCAM 180 kDa immunoreactive band (P=001 t-testn=5ndash6) and the NCAM 140 kDa immunoreactive band (P=001t-test n=5ndash6) was found in PREP-overexpressing neuroblastomacells compared with wild-type cells (Fig 1B) In contrast asignificant increase in the NCAM immunoreactive band migratingat an apparent molecular weight of 120 kDa was found in PREP-overexpressing cells (P=0009 t-test n=5-6 Fig 1B) In wild-typecells immunostaining for NCAM demonstrated an intensive positivesignal for NCAM whereas only a moderate NCAM-positive signalcould be detected in PREP-overexpressing cells (Fig 1D)Next we aimed to elucidate whether the observed changes in the

levels of NCAM protein isoforms were caused by the changes intranscription level We measured mRNA levels of the 120 140 and180 isoforms as well as total NCAM using qPCR No significantdifferences in mRNA level between wild-type and PREP-overexpressing cells were found for any form of NCAM(Table S1) Thus the increase in the NCAM 120 kDaimmunoreactive band in PREP-overexpressing cells does notresult from increased expression of NCAM120 mRNABased on these results we proposed that the increase in NCAM120

results from the degradation of NCAM140 andor NCAM180

isoforms To test this we measured NCAM degradation fragmentsin concentrated cell culture medium from PREP-overexpressing andwild-type cells Two fragments recognized by the NCAM-specificantibody were found in the concentrated medium one of sim46ndash48 kDa and another of sim38ndash40 kDa In the medium of PREP-overexpressing cells therewas a significant increase in the level of the38ndash40 kDa fragment compared with medium from wild-type cells(P=00043 t-test n=4 Fig 2AB) whereas no changes wereobserved in the levels of the 46ndash48 kDa fragment These datasupport our hypothesis that the overexpression of PREP inducesincreased degradation of the NCAM180 andor NCAM140 isoforms

Subsequently we aimed to evaluate whether the observedchanges in PSA-NCAM levels in PREP-overexpressing cells arePREP-specific PREP overexpression was knocked down in PREP-overexpressing cells through transfection with shRNA againstPREP (shPREP) or a control vector (shNC) together with a plasmidexpressing green fluorescent protein (GFP) After 72 h cells werefixed and processed for PSA-NCAM immunocytochemistry GFP-positive cells from the shPREP and shNC groups were selected forPSA-NCAM fluorescent signal intensity analysis Quantificationrevealed an increase in PSA-NCAM immunopositive signals inPREP-overexpressing cells transfected with shPREP compared withshNC-transfected PREP-overexpressing cells (P=0043 t-testn=10 Fig 3AB) This result confirms that alterations inPSA-NCAM are PREP-specific

Addition of recombinant PREP to the culture mediumdecreases PSA-NCAM expression in wild-type SH-SY5Yneuroblastoma cells and primary cortical neuronsHuman recombinant PREP (rPREP) was added to the cell culturemedium of wild-type SH-SY5Y cells (1 nM or 10 nM) and primarycortical neurons (1 nM) for 72 h A marked decrease in PSA-NCAM level was found in wild-type SH-SY5Y neuroblastoma cells(F=1026 P=0001 one-way ANOVA followed by Bonferronipost-hoc test n=3) after addition of rPREP (Fig 3C) A similardecrease in PSA-NCAM level (P=003 t-test n=5ndash6) was found inprimary cortical neurons (Fig 3D)

Quantification of protein expression levels ofpolysialyltransferase ST8Sia2 and ST8Sia4 in wild-type andPREP-overexpressing neuroblastoma SH-SY5Y cellsThe formation of PSA-NCAM is dependent on thepolysialyltransferases ST8Sia2 and ST8Sia4 which attach PSAresidues to the NCAM molecule To exclude the possibility thatPREP affects ST8Sia2 and ST8Sia4 wild-type and PREP-overexpressing cells were subjected to ST8Sia2 and ST8Sia4immunocytochemistry and protein expression analysis (Fig 4AB)No differences in expression of either ST8Sia2 or ST8Sia4 wereobserved by immunocytochemistry (Fig 4A) and western blotanalysis demonstrated no statistically significant difference inST8Sia2 or ST8Sia4 protein levels between wild-type and PREP-overexpressing cells (Fig 4B)

Impaired differentiation of PREP-overexpressing cellsAs NCAM and PSA-NCAM are involved in neurite outgrowth andneuronal differentiation (Seidenfaden et al 2006 Williams et al1994) it was of interest to evaluate whether or not an excess ofPREP accompanied with altered NCAM expression might impairdifferentiation To elucidate this wild-type and PREP-overexpressing cells were grown with sequential exposure toretinoic acid and brain-derived neurotrophic factor (BDNF) for 5and 7 days respectively to achieve long-term survival of cells and a

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Documents

Prolyl endopeptidase is involved in the degradation of ... · RESEARCH ARTICLE Prolyl endopeptidase is involved in the degradation of neural cell adhesion molecules in vitro Külli