Prolyl Endopeptidase Is Involved in the Degradation of Neural Cell Adhesion Molecules in Vitro

RESEARCH ARTICLE Prolyl endopeptidase is involved in the degradation of neural cell adhesion molecules in vitro Külli Jaako1,‡, Alexander Waniek2, Keiti Parik1, Linda Klimaviciusa1, Anu Aonurm-Helm1, Aveli Noortoots1, Kaili Anier1, Roos Van Elzen3, Melanie Gérard4, Anne-Marie Lambeir3, Steffen Roßner2, Markus Morawski2,* and Alexander Zharkovsky1,*

ABSTRACT membrane (Cunningham et al., 1987). The extracellular part of Membrane-associated glycoprotein neural cell adhesion molecule – (NCAM) and its polysialylated form (PSA-NCAM) play an important NCAM consists of five immunoglobulin-like modules (IgI IgV) – followed by two fibronectin type III domains (Finne et al., 1983; role in brain plasticity by regulating cell cell interactions. Here, we – demonstrate that the cytosolic serine protease prolyl endopeptidase Rutishauser, 2008). NCAM establishes cell cell adhesion through (PREP) is able to regulate NCAM and PSA-NCAM. Using a SH-SY5Y homo- and heterophilic interactions and thereby regulates processes neuroblastoma cell line with stable overexpression of PREP, we found like cell migration, neurite outgrowth and targeting, axonal a remarkable loss of PSA-NCAM, reduced levels of NCAM180 and branching, synaptogenesis, and synaptic plasticity (see Walmod NCAM140 protein species, and a significant increase in the NCAM et al., 2004 for review). Neural plasticity, mediated through the NCAM protein, is facilitated by post-translational modifications, the immunoreactive band migrating at an apparent molecular weight of α 120 kDa in PREP-overexpressing cells. Moreover, increased levels of most important and prevalent of which is addition of -2,8- NCAM fragments were found in the concentrated medium derived polysialic acid (PSA) chains to the IgV module of the extracellular from PREP-overexpressing cells. PREP overexpression selectively NCAM domain. Addition of PSA homopolymers to NCAM abates induced an activation of matrix metalloproteinase-9 (MMP-9), which interaction between NCAM molecules and decreases NCAM- could be involved in the observed degradation of NCAM, as MMP-9 mediated adhesion, thus allowing plasticity changes (Johnson et al., neutralization reduced the levels of NCAM fragments in cell culture 2005; Kiselyov et al., 2003; Rutishauser, 2008; Seki and Arai, medium. We propose that increased PREP levels promote epidermal 1993a). The addition of PSA to NCAM occurs through two Golgi- growth factor receptor (EGFR) signaling, which in turn activates associated polysialyltransferases, ST8Sia2 and ST8Sia4 (Angata MMP-9. In conclusion, our findings provide evidence for newly- et al., 1997; Hildebrandt et al., 1998b; Nakayama et al., 1998). discovered roles for PREP in mechanisms regulating cellular However, the physiological PSA-NCAM degradation and plasticity through NCAM and PSA-NCAM. endogenous enzymes involved in this physiological turnover are not yet fully elucidated. KEY WORDS: Neural cell adhesion molecule, Prolyl endopeptidase, It has been demonstrated that NCAM can be cleaved extracellularly Neuronal plasticity, Metalloproteinase-9 by metalloproteinases and other proteolytic enzymes (Brennaman et al., 2014; Hinkle et al., 2006; Hübschmann et al., 2005; Kalus INTRODUCTION et al., 2006). Moreover, metalloproteinase-dependent shedding of The development of the nervous system and its structural NCAM induces release of soluble forms of NCAM consisting of remodeling in the adult rely on molecules mediating the structural several polypeptides with molecular weights ranging from 80 kDa to plasticity of neurons, especially those involved in cell adhesion, 200 kDa (Krog et al., 1992; Nybroe et al., 1989; Sadoul et al., 1986). cytoskeletal dynamics or synapse formation (Amoureux et al., The functional significance of proteolytic shedding of membrane- 2000; Cremer et al., 1998; Kiss and Muller, 2001). Among these bound NCAM is unknown. However, increased amounts of the molecules, isoforms of the neural cell adhesion molecules cleavage product have been associated with neuropsychiatric (NCAMs) are of particular interest. NCAM (also known as disorders such as schizophrenia (Vawter et al., 1998) and dementia NCAM1) is a cell surface glycoprotein that is represented by at (Strekalova et al., 2006). least three isoforms (NCAM120, NCAM140 and NCAM180, with The cytosolic serine protease prolyl endopeptidase (PREP) molecular weights of 120, 140 and 180 kDa, respectively) that differ hydrolyses small (<3 kDa) proline-containing peptides at the in their cytoplasmic domains and attachment to the plasma carboxy terminus of proline residues (Fülöp et al., 1998; Polgár, 2002; Rawlings et al., 1991). PREP is widely distributed in the brain (Irazusta et al., 2002; Myöhänen et al., 2007, 2008) and increased 1Department of Pharmacology, Institute of Biomedicine and Translational Medicine, 2 activity of PREP has been associated with cell death processes in University of Tartu, Tartu 50411, Estonia. Paul Flechsig Institute for Brain Research, ’ University of Leipzig, Leipzig 04103, Germany. 3Laboratory of Medical various neurodegenerative diseases, including Alzheimer s and Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Parkinson’s diseases (Brandt et al., 2008; Mantle et al., 1996). 4 Antwerp B-2610, Belgium. Interdisciplinary Research Centre KU Leuven-Kortrijk, PREP is believed to act in the extracellular space with involvement Kortrijk B-8500, Belgium. *These authors contributed equally to this work in the maturation and degradation of peptide hormones and ̀ ‡ neuropeptides (Bellemere et al., 2004; Cunningham and Author for correspondence ([email protected]) O’Connor, 1997; Shishido et al., 1999), which has been proposed K.J., 0000-0003-4675-5188; L.K., 0000-0001-7085-3891 to be the mechanism underlying some beneficial effects of PREP inhibitors in animal memory models (Shishido et al., 1998; Toide

Received 11 October 2015; Accepted 18 August 2016 et al., 1997; Yoshimoto et al., 1987) and in aged mice (Kato et al., Journal of Cell Science

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1997). Besides its extracellular action, PREP has been shown to act isoforms. To test this, we measured NCAM degradation fragments intracellularly and important roles of PREP have been demonstrated in concentrated cell culture medium from PREP-overexpressing and in signaling pathways or in transport and secretion of proteins and wild-type cells. Two fragments recognized by the NCAM-specific peptides associated with neurodegeneration (Brandt et al., 2008; Di antibody were found in the concentrated medium; one of ∼46– Daniel et al., 2009; Rossner et al., 2005; Savolainen et al., 2015; 48 kDa, and another of ∼38–40 kDa. In the medium of PREP- Schulz et al., 2002, 2005). Within recent years PREP has been overexpressing cells, there was a significant increase in the level of the suggested to be a contributor to neuroinflammation (Penttinen et al., 38–40 kDa fragment compared with medium from wild-type cells 2011). PREP has been shown to be involved in mechanisms (P=0.0043, t-test, n=4; Fig. 2A,B), whereas no changes were responsible for the transduction and amplification of inflammatory observed in the levels of the 46–48 kDa fragment. These data processes leading to the production of neurotoxic mediators, which support our hypothesis that the overexpression of PREP induces in turn mediate pathology progression (for review, see Penttinen increased degradation of the NCAM180 and/or NCAM140 isoforms. et al., 2011). Subsequently, we aimed to evaluate whether the observed In this report, for the first time, we demonstrate that PREP is able changes in PSA-NCAM levels in PREP-overexpressing cells are to regulate levels of NCAM through activation of MMP-9. PREP-specific. PREP overexpression was knocked down in PREP- overexpressing cells through transfection with shRNA against RESULTS PREP (shPREP) or a control vector (shNC), together with a plasmid Levels of PSA-NCAM and NCAM protein and mRNA in expressing green fluorescent protein (GFP). After 72 h cells were neuroblastoma SH-SY5Y cells overexpressing PREP fixed and processed for PSA-NCAM immunocytochemistry. GFP- Increased levels of PREP protein and its activity in PREP- positive cells from the shPREP and shNC groups were selected for overexpressing SH-SY5Y cell lysates were confirmed by western PSA-NCAM fluorescent signal intensity analysis. Quantification blot and activity measurements (Fig. S1A,B). revealed an increase in PSA-NCAM immunopositive signals in PREP was detected on SDS-PAGE as a band at ∼80 kDa PREP-overexpressing cells transfected with shPREP compared with (Fig. S1A). A 30-fold increase in PREP protein level was found in shNC-transfected PREP-overexpressing cells (P=0.043, t-test, SH-SY5Y cells overexpressing PREP compared with wild-type n=10; Fig. 3A,B). This result confirms that alterations in cells (P<0.001, t-test, n=4). In these PREP-overexpressing cells, PSA-NCAM are PREP-specific. PREP activity assays demonstrated an 11-fold increase in enzymatic activity (P<0.0001, t-test, n=3; Fig. S1B). Addition of recombinant PREP to the culture medium To evaluate whether increased PREP levels are associated with decreases PSA-NCAM expression in wild-type SH-SY5Y altered levels of PSA-NCAM and/or NCAM, the proteins were neuroblastoma cells and primary cortical neurons quantified using western blot analysis. In wild-type cells, PSA- Human recombinant PREP (rPREP) was added to the cell culture NCAM was detected as a high-molecular-weight smear typical for medium of wild-type SH-SY5Y cells (1 nM or 10 nM) and primary highly polysialylated NCAM (Fig. 1A), whereas by comparison, cortical neurons (1 nM) for 72 h. A marked decrease in PSA- PSA-NCAM signal was almost absent in PREP-overexpressing NCAM level was found in wild-type SH-SY5Y neuroblastoma cells cells (P<0.0001, t-test, n=5–6; Fig. 1A). In wild-type cells, (F=10.26, P=0.001, one-way ANOVA followed by Bonferroni immunocytochemistry identified membrane-bound extracellular post-hoc test, n=3) after addition of rPREP (Fig. 3C). A similar PSA-NCAM molecules, which assembled in small bunches along decrease in PSA-NCAM level (P=0.03, t-test, n=5–6) was found in neurite-like processes and in larger patches associated with the primary cortical neurons (Fig. 3D). perikaryon. In PREP-overexpressing cells, however, PSA-NCAM signal was almost absent (Fig. 1C). Quantification of protein expression levels of We further investigated levels of the NCAM protein isoforms polysialyltransferase ST8Sia2 and ST8Sia4 in wild-type and (NCAM180, NCAM140 and NCAM120). A significant decrease in PREP-overexpressing neuroblastoma SH-SY5Y cells levels of the NCAM 180 kDa immunoreactive band (P=0.01, t-test, The formation of PSA-NCAM is dependent on the n=5–6) and the NCAM 140 kDa immunoreactive band (P=0.01, polysialyltransferases ST8Sia2 and ST8Sia4, which attach PSA t-test, n=5–6) was found in PREP-overexpressing neuroblastoma residues to the NCAM molecule. To exclude the possibility that cells compared with wild-type cells (Fig. 1B). In contrast, a PREP affects ST8Sia2 and ST8Sia4, wild-type and PREP- significant increase in the NCAM immunoreactive band migrating overexpressing cells were subjected to ST8Sia2 and ST8Sia4 at an apparent molecular weight of 120 kDa was found in PREP- immunocytochemistry and protein expression analysis (Fig. 4A,B). overexpressing cells (P=0.009, t-test, n=5-6; Fig. 1B). In wild-type No differences in expression of either ST8Sia2 or ST8Sia4 were cells, immunostaining for NCAM demonstrated an intensive positive observed by immunocytochemistry (Fig. 4A), and western blot signal for NCAM, whereas only a moderate NCAM-positive signal analysis demonstrated no statistically significant difference in could be detected in PREP-overexpressing cells (Fig. 1D). ST8Sia2 or ST8Sia4 protein levels between wild-type and PREP- Next, we aimed to elucidate whether the observed changes in the overexpressing cells (Fig. 4B). levels of NCAM protein isoforms were caused by the changes in transcription level. We measured mRNA levels of the 120, 140 and Impaired differentiation of PREP-overexpressing cells 180 isoforms as well as total NCAM using qPCR. No significant As NCAM and PSA-NCAM are involved in neurite outgrowth and differences in mRNA level between wild-type and PREP- neuronal differentiation (Seidenfaden et al., 2006; Williams et al., overexpressing cells were found for any form of NCAM 1994), it was of interest to evaluate whether or not an excess of (Table S1). Thus, the increase in the NCAM 120 kDa PREP, accompanied with altered NCAM expression, might impair immunoreactive band in PREP-overexpressing cells does not differentiation. To elucidate this, wild-type and PREP- result from increased expression of NCAM120 mRNA. overexpressing cells were grown with sequential exposure to Based on these results, we proposed that the increase in NCAM120 retinoic acid and brain-derived neurotrophic factor (BDNF) for 5 results from the degradation of NCAM140 and/or NCAM180 and 7 days, respectively, to achieve long-term survival of cells and a Journal of Cell Science

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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 and corresponding statistical analysis, which revealed reduced NCAM 180 kDa and NCAM 140 kDa protein variants but increased NCAM 120 kDa protein variant level in PREP-overexpressing SH-SY5Y neuroblastoma cells (PREP) compared with wild-type cells. (C,D) 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 were counterstained with DAPI (blue). Data are given as ratio of mean values in percentage±s.e.m. (wild-type=100%). β-actin was used as loading control. *P<0.05, **P<0.01, ***P<0.001; unpaired t-test, n=6 for wild-type and n=5 for PREP. Scale bar: 10 µm. Three independent experiments were performed. high degree of differentiation (Encinas et al., 2000). As cells compared with wild-type cells (P<0.0001, t-test, n=9; Fig. 5B), demonstrated in Fig. 5A, wild-type cells yielded homogeneous indicating that alterations in NCAM molecule integrity lead to populations of Tuj-1-immunopositive cells with neuronal impaired differentiation toward a neuron-like cell type. morphology and abundantly branched neurites. In contrast, only moderate numbers of PREP-overexpressing cells were positive for Increased MMP-9 protein level and activity in neuroblastoma Tuj-1 (Fig. 5A). Quantification revealed that the percentage of Tuj- SH-SY5Y cells overexpressing PREP 1-positive cells was significantly lower in PREP-overexpressing It is well documented that PREP is only able to degrade relatively small peptides (<3 kDa) (Fülöp et al., 1998; Polgár, 2002; Rawlings et al., 1991). Therefore, it seemed unlikely that PREP itself is able to degrade NCAM. It has been demonstrated previously that NCAM molecule degradation might be mediated by the matrix metalloproteases (MMPs) (Brennaman et al., 2014; Hinkle et al., 2006; Hübschmann et al., 2005; Shichi et al., 2011). Therefore, to test whether increased expression and activity of PREP might be involved in the activation of MMPs, we measured the expression levels and activity of MMP-9. Increased levels of the active form of MMP-9 was found in PREP-overexpressing SH-SY5Y neuroblastoma cells compared with wild-type cells (P=0.001, t-test, n=3), whereas no changes were found in levels of the inactive form of MMP-9 (Fig. 6A). Moreover, immunocytochemical labeling of MMP-9 in PREP-overexpressing cells revealed intense granulation of the immunopositive signal mostly at the perinuclear region, Fig. 2. Detection of NCAM fragments in culture medium derived from wild- whereas in wild-type cells MMP-9 signal was associated with a type and PREP-overexpressing cells. (A) Representative western blot and sparse granulation pattern (Fig. 6B). In addition, zymography corresponding levels of degradation product of NCAM fragment 38-40 kDa in demonstrated increased proteolytic activity of MMP-9 in the serum- the culture medium derived from PREP-overexpressing cells, compared with free medium from cultured PREP-overexpressing cells compared culture medium derived from wild-type cells. (B) Quantification of data from A. Data are given as mean optical density (OD) ratio as percentage of control±s.e.m. with wild-type cells (P=0.002, t-test, n=3; Fig. 6C). (wild-type=100%). **P=0.0043, unpaired t-test, n=4. Two independent To test whether the observed increases in MMP-9 levels and experiments were performed. activity in PREP-overexpressing cells is responsible for NCAM Journal of Cell Science

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Fig. 3. Analysis of PSA-NCAM level in PREP-overexpressing cells in the context of PREP-knockdown and the effect of recombinant PREP on PSA-NCAM protein level in wild-type SH-SY5Y neuroblastoma cells and primary cortical neurons. (A) PREP-overexpressing SH-SY5Y neuroblastoma cells were transfected with either control vector (shNC) or shPREP and cells were immunostained for PSA-NCAM (red). ShPREP- transfected cells demonstrated restoration of PSA-NCAM expression (indicated with arrows). (B) Signal intensity analysis corresponding to the images in A demonstrated restoration of PSA-NCAM expression in shPREP-transfected cells compared with shNC-transfected PREP-overexpressing SH-SY5Y neuroblastoma cells. PSA-NCAM immunopositive signal intensity data are given as mean±s.e.m. relative fluorescence units (RFU). *P<0.05, unpaired t-test, n=10. Scale bar: 5 µm. (C,D) Western blot analysis and representative images demonstrating significantly decreased level of PSA-NCAM in (C) wild-type SH-SY5Y neuroblastoma cells and (D) cultured primary rat neurons when rPREP was added into cell culture medium at concentrations of 1 nM and 10 nM compared with vehicle-treated cells. Data are given as mean optical density (OD) ratio as a percentage of control±s.e.m. (untreated primary neurons or wild-type neuroblastoma cells=100%). β-actin was used as loading control. *P<0.05, unpaired t-test or one-way ANOVA, n=3 for wild-type SH-SY5Y neuroblastoma cells, n=5 for untreated cortical neurons and n=6 for rPREP-treated cortical neurons. Two independent experiments were performed.

degradation, PREP-overexpressing cells were incubated with MMP- To elucidate whether PREP overexpression could affect 9-neutralizing antibody for 96 h. A reduced level of NCAM other MMPs that might be involved in NCAM degradation, the fragments was found in the cell culture medium (P<0.001, t-test, protein levels of MMP-2, MMP-3 and ADAM-10 were n=5-6) compared with vehicle-treated PREP-overexpressing cells measured. No changes were found in the levels of these MMPs (Fig. 7A,B), indicating that MMP-9 is involved in the regulation of in PREP-overexpressing cells as compared with wild-type cells NCAM degradation. (Fig. S2).

Fig. 4. Analysis of protein expression of sialyltransferases in PREP-overexpressing SH-SY5Y neuroblastoma cells. (A) Representative fluorescent microphotographs, demonstrating ST8Sia2- and ST8Sia4-immunopositive staining in wild-type and PREP-overexpressing SH-SY5Y neuroblastoma cells. Nuclei were counterstained with Hoechst (blue). Scale bar: 10 µm. (B) Representative western blot and corresponding levels of ST8Sia2 and ST8Sia4 protein expression in wild-type and PREP-overexpressing cells. Data are given as mean optical density (OD) ratio as a percentage of control±s.e.m. (wild type=100%). β-actin was used as loading control. Unpaired t-test, n=6. Two independent experiments were performed. Journal of Cell Science

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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 and PREP-overexpressing SH-SY5Y neuroblastoma cells. Nuclei were counterstained with DAPI (blue). Scale bar: 25 µm. (B) Corresponding analysis of Tuj-1-immunopositive cells. Data are expressed as mean percentage±s.e.m. of Tuj-1-immunopositive cells. ***P<0.0001, unpaired t-test, n=9.

Decreased EGFR and increased pEGFR expression in cortical cells isolated from mouse brain were enriched with human neuroblastoma SH-SY5Y cells overexpressing PREP recombinant PREP – this induced a decrease in PSA-NCAM It has previously been demonstrated that EGFR signaling is expression levels. Not only PSA-NCAM was altered in the presence involved in release and activation of MMP-9 (da Rosa et al., of PREP; we also found alterations in the levels of all NCAM 2014; Pei et al., 2014; Qiu et al., 2004). Therefore EGFR and protein variants tested. We found a reduction in NCAM 180 kDa phosphorylated EGFR (pEGFR) levels were measured and in and 140 kDa immunoreactive bands and an increase in the NCAM PREP-overexpressing cells overall decrease in total EGFR was 120 kDa immunoreactive band. As PREP might affect PSA-NCAM found (P=0.001, t-test, n=4; Fig. 8). In contrast, the levels of pEGFR and NCAM through different mechanisms including reduced were increased (P=0.001, t-test, n=4; Fig. 8). expression and/or increased breakdown of the NCAM protein, we explored these mechanisms in more detail. DISCUSSION In neuroblastoma cells overexpressing PREP, we did not find any This study shows for the first time that PREP is involved in changes in NCAM120, NCAM140, NCAM180 or in total NCAM the regulation of neural cell adhesion molecules. A PREP- mRNA levels. These data suggest that PREP does not affect the overexpressing neuroblastoma SH-SY5Y cell line was used as an transcription of NCAM. We also failed to find any changes in the in vitro model to mimic pathological conditions resulting from expression levels of two Golgi-associated polysialyltransferases, increased PREP expression. When PSA-NCAM protein levels were ST8Sia2 and ST8Sia4, which mediate the addition of PSA to quantified, we found a remarkable reduction of PSA-NCAM in NCAM (Eckhardt et al., 1995), suggesting that PREP does not PREP-overexpressing cells compared with wild-type cells. The affect the polysialylation of NCAM. Therefore, we propose that the finding that PSA-NCAM levels are altered in the context of higher reduction in the 140 kDa and 180 kDa protein variants of NCAM PREP levels was further confirmed in a series of experiments where and the reduction in PSA-NCAM result from their increased cell culture media for wild-type neuroblastoma cells or for primary degradation in the presence of PREP. To explore this proposal in

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) and PREP-overexpressing SH-SY5Y neuroblastoma cells. Quantitative western blot analysis demonstrating increased expression level of the active form of MMP-9, but not proMMP-9, in PREP-overexpressing SH-SY5Y cells compared with wild-type cells. β-actin was used as loading control. (B) Immunocytochemistry demonstrated intracellular distribution of MMP-9-immunopositive signal (red) in wild-type and PREP-overexpressing SH-SY5Y neuroblastoma cells. Perinuclear distribution and localization of MMP-9 in PREP-overexpressing cells is indicated by arrows. Nuclei were counterstained with DAPI. Scale bar: 5 µm. (C) Representative gelatin zymogram and corresponding MMP-9 zymogram analysis demonstrated increased activity of MMP-9 in serum-free cell culture medium of PREP-overexpressing SH-SY5Y neuroblastoma cells, compared with wild-type cells. Recombinant MMP-9 protein was used as an identification marker. Data are given as optical density (OD) ratios±s.e.m., **P<0.01, unpaired t-test, n=3. Two independent experiments were performed. Journal of Cell Science

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physiological events related to cancer transformation (Miyagi et al., 2003; Proshin et al., 2002; Tringali et al., 2012; Wada et al., 2007) and in developmental processes, such as lamination of newly generated hippocampal granule cells through the modulation of PSA (Sajo et al., 2016). The question of whether or not PREP overexpression affects sialidases needs further investigation. PREP is peptidase that cleaves small peptides (<3 kDa) (Fülöp et al., 1998; Polgár, 2002; Rawlings et al., 1991) and it seems unlikely that this enzyme can directly catalytically degrade the NCAM molecule. Previous studies have demonstrated that several metalloproteases, such as MMP-2 and MMP-9, as well as the ADAM family of metalloproteases, target NCAM (Brennaman et al., 2014; Hinkle et al., 2006; Hübschmann et al., 2005; Shichi et al., 2011). It has also been shown that inhibition of MMP-2 and Fig. 7. Analysis of the effect of MMP-9-neutralizing antibody on the levels MMP-9 prevents NCAM shedding, indicating the roles of these of NCAM fragments in PREP-overexpressing SH-SY5Y neuroblastoma MMPs in NCAM cleavage (Hübschmann et al., 2005; Shichi et al., cells. Representative western blot (A) and corresponding quantification of 2011). Moreover, ADAM-10-dependent shedding of NCAM has expression levels (B) demonstrating reduced appearance of NCAM been extensively studied, and the second fibronectin-type III – degradation fragment at 38 40 kDa in cell culture medium from SH-SY5Y domain of NCAM has been shown to be a target for ADAM-10 neuroblastoma cells treated with MMP-9-neutralizing antibody for 96 h as compared with vehicle-treated PREP-overexpressing cells. Data are given as cleavage (Brennaman et al., 2014). Based on these findings, we mean optical density (OD) ratio percentage of control±s.e.m. (PREP- explored in more detail the impact of metalloproteases on the PREP- overexpressing cell culture medium serves as a control). ***P<0.001, unpaired mediated decrease in PSA-NCAM and NCAM. We measured levels t-test, n=6 for PREP-overexpressing cell culture medium and n=5 for PREP- of MMP-2, MMP-3, MMP-9 and ADAM-10 in the lysates of wild- overexpressing cell culture medium treated with MMP-9-neutralizing antibody. type and PREP-overexpressing cells and found an increased level of Two independent experiments were performed. MMP-9 but not the other metalloproteases. Furthermore, we found that this increased level of MMP-9 was specific to its active form. more detail, we measured NCAM degradation products in Using zymography, we also observed increased MMP-9 activity. concentrated medium derived from PREP-overexpressing cells. Thus, it seems that PREP overexpression induces a selective Indeed, an increased level of peptide fragments at 38–40 kDa, increase in the expression and activity of MMP-9, which is probably recognized by anti-NCAM antibody, was found. Thus, it seems that involved in the observed degradation of NCAM. This proposal was the observed decrease in NCAM immunoreactive bands of 180 kDa further confirmed in our experiments where MMP-9-neutralizing and 140 kDa does indeed result from their breakdown in the antibody prevented the degradation of NCAM, as evidenced by the presence of PREP, and the observed increase in the NCAM decrease in NCAM fragments in the culture medium. immunoreactive band migrating at an apparent molecular weight of Previous studies have demonstrated that cleavage of NCAM180 120 kDa might be explained by the accumulation of cleaved and NCAM140 isoforms by ADAM-10 and ADAM-17 results in fragments of NCAM. However, NCAM fragments at 38–40 kDa the release of 110–115 kDa fragments into the cell culture medium, indicate that the cleaved fragment is too small to include the associated with the appearance of 30 kDa membrane-associated polysialylated IgV domain and therefore additional mechanisms, fragments in cell lysates (Brennaman et al., 2014; Kalus et al., such as increased PSA cleavage by neuraminidases, in addition to 2006). In addition to the aforementioned fragments cleaved by NCAM degradation by MMP-9, might be involved. In mammals, ADAMs, a 65 kDa degradation product of NCAM has been four sialidases are present and are involved in several key demonstrated to result from the action of MMP-9 and/or MMP-2 after cerebral ischemic neuronal damage and the appearance of this fragment was reduced when MMPs were suppressed (Shichi et al., 2011). The precise cleavage site through which these smaller fragments are produced is not known; however, it might be proposed that the origin of the fragment found in cell culture medium might represent an N-terminal domain of NCAM. The appearance of fragments of ∼40 kDa, derived from extracellular domains of NCAM, into cell culture medium might also explain the shift in western blot bands from strong bands at 180 kDa and 140 kDa to bands with lower apparent molecular weight and their accumulation at the range of 120 kDa. The mechanism by which PREP induces MMP-9 activation is not known, although several explanations might be proposed. PREP might activate MMP-9 by degrading physiological tissue inhibitor metalloproteases (TIMPs), as cells secrete pro-MMPs bound to TIMPs in a complex and TIMP processing is needed for zymogen Fig. 8. Analysis of EGFR and pEGFR levels in wild-type and PREP- activation. It has been demonstrated that in vitro conditions, overexpressing SH-SY5Y neuroblastoma cells. Representative western cleavage, degradation or chemical modification by proteolytic and blot and corresponding quantification of EGFR and pEGFR expression levels. Data are given as mean optical density (OD) ratio as percentage of control±s.e.m. non-proteolytic mechanisms are responsible for inactivation of (wild-type=100%). β-actin was used as loading control. **P<0.01, unpaired t-test, TIMPs through the involvement of serine or thiol proteases and n=4. Two independent experiments were performed. reactive oxygen species, respectively (Frears et al., 1996; Okada Journal of Cell Science

3797 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3792-3802 doi:10.1242/jcs.181891 et al., 1992). The involvement of PREP in the processing of TIMPs, selective PREP inhibitors in a dose-dependent matter (Klegeris however, remains unclear as PREP is able to cleave only small et al., 2008). Moreover, studies in PREP-knockout mice have peptide fragments of 30 amino acids. There is a possibility that some demonstrated the association of PREP in the processes modulating small, as yet unknown, peptides, substrates of PREP, might directly neuroplasticity through inflammatory response. In PREP-knockout modulate MMP-9 activity. mouse hippocampus, alterations in the microglia activation in Moreover, it cannot be excluded that PREP might be involved in response to systemic administration of repeated doses of the regulation of MMP-9 release. It is known that EGF, through an lipopolysaccharide were found in addition to increased levels of interaction with its receptors, increases the release and activation of PSA-NCAM, and these data indirectly support the functional MMP-9 (da Rosa et al., 2014; Pei et al., 2014; Qiu et al., 2004). In significance of PREP in the regulation of PSA-NCAM (Höfling PREP-overexpressing cells, despite the overall decrease in total et al., 2016). Moreover, in clinical studies, increased PREP activity EGFR, the levels of phosphorylated (active form) EGFR increased, has been found in knee-joint synovial membranes, indicating its which indicates an activation of EGF signaling and consequent association with rheumatoid arthritis (Kamori et al., 1991). release of MMP-9; however, the precise mechanism through which Considering the demonstrated effects of PREP in inflammation PREP is involved in the regulation of EGF-mediated MMP-9 and possible role in neuroinflammation, resulting in changes in release remains unknown and certainly needs further investigation. neural plasticity as well as the role of NCAMs on brain plasticity, the The PREP-mediated mechanism of the regulation of PSA- interplay between these molecules should be considered plausible, NCAM and NCAM might be relevant for neuronal function and as NCAM-mediated neural plasticity is altered in pathological plasticity. It has been demonstrated that NCAM180 and NCAM140 conditions associated with inflammation. Decreased full-length are key regulators of neuronal development and maintenance as they NCAM180 has been described in mice 1 day after middle cerebral are widely expressed in post-synaptic densities of neurons, whereas artery occlusion (Shichi et al., 2011). Moreover, it has been shown NCAM140 is also expressed in migratory growth cones (Dityatev that in the processes of demyelinating neuroinflammation resulting et al., 2000; Persohn et al., 1989). The main function of these from the autoimmune encephalomyelitis, the level of MMP-2 was isoforms is activity-dependent sprouting (Schuster et al., 1998), significantly increased in hippocampus, which was accompanied by synaptic stability (Muller et al., 1996) and neurite outgrowth reduced levels of NCAM (Jovanova-Nesic and Shoenfeld, 2006). In (Sandig et al., 1996). Therefore, a prolonged deficiency in NCAM addition to previously mentioned increased cleavage of NCAM180 and/or its polysialylation might impair cellular functioning as it is during ischemic stress, similar effects in the decrease of NCAM180 well documented that NCAM in its polysialylated form is essential and increased content of the cleaved form of NCAM was described for neurons to migrate and establish new synaptic contacts (Muller after oxidative stress (Fujita-Hamabe and Tokuyama, 2012). et al., 1996; Rougon, 1993; Rutishauser and Landmesser, 1996; In conclusion, our study demonstrates that increased expression Seki and Arai, 1993a,b). In accordance with these studies, our level and activity of PREP is involved in mechanisms regulating experiments showed that the PREP-overexpressing neuroblastoma degradation of neural adhesion molecules, most likely by MMP-9 cells exhibit impaired differentiation, induced by retinoic acid and activation. These findings open up a new avenue for the exploration BDNF. The role of PSA in differentiation is important as PSA is of the roles of PREP in processes involved in NCAM degradation, highly re-expressed during progression of several malignant human which, in turn, is believed to be one major contributor for altered tumors, neuroblastoma among others and therefore could be neuroplasticity (Brusés and Rutishauser, 2001; Cremer et al., 1998; considered as an oncodevelopmental antigen. It has been Gnanapavan and Giovannoni, 2013). Moreover, these mechanisms demonstrated that in malignant human tumors polysialylation of are of importance regarding synaptic plasticity in the (re-) NCAM seems to increase the metastatic potential and has been organization of neuronal circuits. correlated with tumor progression and a poor prognosis (Glüer et al., 1998; Tanaka et al., 2001). In vitro studies in neuroblastoma cells MATERIALS AND METHODS have shown that application of endoneuraminidase (EndoN) Generation of PREP-overexpressing SH-SY5Y cell line treatment for PSA removal triggers the cell to cease proliferation The PREP-overexpressing SH-SY5Y cell line was generated as described in and to differentiate (Seidenfaden et al., 2003). Moreover, Gerard et al. (2010). Wild-type and PREP-overexpressing cells were grown neuroblastoma cells differentiating toward the neuron-like cell in DMEM/GlutaMAX-I medium (Thermo Scientific) containing 15% type express high amounts of NCAM and PSA, although reduction heat-inactivated fetal calf serum, 1% NEAA and 50 µg/ml gentamycin for wild-type cells; and additionally 200 µg/ml hygromycin B for PREP- in PSA expression was found after differentiation (Hildebrandt overexpressing cells. Cells were maintained at 37°C in a saturated humidity et al., 1998a). During regular differentiation, removal of PSA has atmosphere containing 95% air and 5% CO2. For differentiation studies, been demonstrated to increase the number of cell–cell contacts alltrans-retinoic acid (RA; Tocris Cookson, Bristol, UK) was added the day (Hildebrandt et al., 1998a), which relay on the basis for signal after plating at a final concentration of 10 µM in DMEM with 15% fetal calf transduction through second messenger pathways in addition to cell serum. After 5 days in the presence of RA, cells were washed three times adhesion per se; however, in PREP-overexpressing cells where with DMEM and incubated with 50 ng/ml BDNF (Sigma-Aldrich) in NCAM extracellular domains are partly degraded and NCAM DMEM (without serum) for 7 days. molecule integrity is disrupted, impaired differentiation toward neuron-like cell type, as well as formation of neurites, might be an PREP activity assay outcome of these alterations. PREP activity in SH-SY5Y cells was measured according to the method An increasing body of evidence from clinical and preclinical described by Klimaviciusa et al., (2012). Briefly, cells were washed twice with PBS and lysed by chilled hypotonic buffer (pH 7.5) containing 50 mM studies suggests an involvement of PREP in neuroinflammation. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 20 mM Recent in vitro studies demonstrated that PREP contributes to the NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM toxic effects of reactive microglial cells, as it was demonstrated dithiothreitol (DTT; Sigma-Aldrich). The obtained cell lysate was that activated microglia cells expressed high levels of PREP and centrifuged and supernatants were transferred to new tubes. Equal amounts the supernatant of these cells demonstrated toxic effects on SH- of protein samples (10 µg) were mixed with assay buffer containing

SY5Y neuroblastoma cells. This toxic effect was reduced by chromogenic PREP substrate 250 µM Z-Gly-Pro-p-nitroanilide (Bachem Journal of Cell Science

3798 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3792-3802 doi:10.1242/jcs.181891

AG, Bubendorf). The product absorbance was continuously measured for express the shRNA for 72 h before immunocytochemical detection for PSA- 30 min at 405 nm using an ELISA plate reader (Tecan, Crailsheim, NCAM was performed. Immunopositive signals were detected with a Germany) and PREP activity was calculated using a p-nitroanilide (Sigma- confocal microscope LSM 510 (Zeiss, Denmark) equipped with an argon– Aldrich) standard curve. krypton laser. 3D images were constructed from a series (10–13) of scans of the GFP-positive cells at 1 µm intervals using a 40× (water) objective and Primary culture of rat cortical neurons further analyzed for signal intensity. Fluorescence quantification analysis Primary neuronal cultures were prepared from 1-day-old Wistar rat pups was performed using ImageJ software (NIH). Each cell to be analyzed was according to the method of Alho and colleagues (1988) with minor manually defined and three regions were selected just beside the cell in an modifications. Briefly, cortices were dissected in ice-cold Krebs-Ringer area without fluorescent objects to be used for background adjustment. solution (135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 0.4 mM K2HPO2, Subsequently, the corrected total cell fluorescence (CTCF) was obtained 15 mM glucose, 20 mM HEPES, pH 7.4, containing 0.3% bovine serum according to Gavet and Pines (2010). CTCF was calculated by multiplying albumin) and trypsinized in 0.8% trypsin-EDTA (Invitrogen, UK) for the area (in square pixels) of the selected cell of interest by mean background 10 min at 37°C followed by trituration in 0.008% DNAse I solution fluorescence intensity and subtracting the result from whole-cell containing 0.05% soybean trypsin inhibitor (both from Surgitech AS, fluorescence intensity. Overlapping of cells was excluded by checking all Estonia). Cells were resuspended in Eagle’s basal medium with Earle’s salts channels in each image. PSA-NCAM-immunopositive signal intensity is (BME; Invitrogen, UK) containing 10% heat-inactivated fetal calf serum given in relative fluorescence units (RFU). (Invitrogen), 25 mM KCl, 2 mM GlutaMAX-I (Invitrogen, UK) and 100 µg/ml gentamycin (KRKA). Cells were plated onto poly-L-lysine- Immunocytochemistry and image analysis 5 2 coated (Sigma-Aldrich) cell culture dishes at a density of 1.8×10 cells/cm . SH-SY5Y neuroblastoma cells were fixed by using 4% paraformaldehyde in After 2.5 h, the medium was changed to Neurobasal-A medium (Gibco). phosphate-buffered saline (PBS) for 10 min at room temperature, Cultures were incubated for 4 days in an atmosphere of 95% air and 5% CO2 permeabilized in 0.05% Triton X-100 (Sigma-Aldrich) for 30 min, and at 37°C. rinsed in PBS. Non-specific binding was blocked by 2% normal goat serum (Vector Laboratories) in PBS containing 0.3% Triton X-100 for 1 h, Treatment with rPREP followed by 48 h of incubation at 4°C with different primary antibodies Wild-type SH-SY5Y neuroblastoma cells or primary cortical neurons at diluted in blocking buffer based on the requirement of the experiment. DIV4 were grown in DMEM cell culture medium plus 10% fetal calf serum Primary antibodies used were: mouse anti-PSA-NCAM (1:500; Merc or Neurobasal-A medium, containing 2 mM GlutaMAX-I, B-27 supplement Millipore, MAB5324), rabbit anti-NCAM (1:1000; Merc Millipore, (Gibco) and 100 μg/ml gentamycin, respectively. Human recombinant PREP AB5032), rabbit anti-MMP-9 (1:700; Merc Millipore, AB19016), rabbit protein (rPREP) (a generous gift from Dr. Zoltan Szeltner, Institute of anti-ST8Sia4 (1:500; Thermo Scientific, PA5-26774), rabbit anti-ST8Sia2 Enzymology, Research Centre for Natural Sciences, Hungarian Academy of (1:200; Proteintech Europe, 19736-1-AP), mouse anti-Tuj-1 (1:700; Merc Science) stock solution was dissolved in buffered saline, 1 mM DTT, pH 7.5 Millipore, MAB1637). After being washed in PBS, cells were incubated in and added into cell culture medium at concentrations of 1 nM and 10 nM for Alexa Fluor 594 goat anti-mouse IgM (1:1000; A-21044), Alexa Fluor 488 wild-type SH-SY5Y neuroblastoma cells and 1 nM for primary cortical goat anti-rabbit IgG (1:1000; A-11008), Alexa Fluor 594 goat anti-rabbit neurons. Cells were incubated for 72 h and then processed for western blot IgG (1:1000; A-11012) or Alexa Fluor 488 goat anti-mouse IgG (1:1000; analysis. A-11001) (all from Life Technologies) in blocking buffer for 1 h. Nuclei were counterstained with DAPI solution (Sigma-Aldrich; 300 nM in PBS). A laser scanning confocal microscope (LSM 510 Duo, Zeiss, Germany) Treatment with MMP-9-neutralizing antibody and NCAM equipped with a C-Apocromat 40×/1.20 water immersion M27 objective cleavage assay (Zeiss, Germany) was used for image acquiring and acquisition parameters MMP-9-neutralizing antibody (clone 6-6B) was purchased from Millipore ’ (magnification, laser intensity, gain, pinhole aperture) were kept constant (IM09L) and dissolved according to manufacturer s instructions. PREP- between different experimental groups. For differentiation analysis, nine overexpressing SH-SY5Y neuroblastoma cells were treated with MMP-9- randomly taken fields from three cell culture dishes (3.5 cm diameter) were neutralizing antibody or appropriate vehicle for 96 h at a concentration of taken at magnification ×40. 3D images were constructed from a series (10– 50 µg/ml in serum free DMEM/GlutaMAX-I medium. Detection of NCAM 12) of scans at 1 µm intervals for image analysis. The number of Tuj-1- fragments was done according to the method of Brennaman and colleagues immunopositive cells and the total number of DAPI-positive nuclei were (2014). After 96 h, cell culture medium was collected, treated with protease counted and the data were expressed as a percentage of cells positive for Tuj- inhibitors (Roche), centrifuged for 5 min (173 g), followed by concentration 1 signal in wild-type and PREP-overexpressing cells. with Millipore centrifugal concentrators according to the manufacturer’s instructions (30,000 MW cutoff, 10× concentration). Protein concentrations from each concentrated cell culture media sample were measured by Western blot analysis Bradford method and equivalent amount of proteins from media samples Primary neurons or SH-SY5Y cells were lysed in RIP-A cell lysis buffer were resolved by electrophoresis on 10% SDS-polyacrylamide gel as (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP-40 and described below. In addition, a blank control sample was prepared and 2 mM EDTA containing protease and phosphatase inhibitor cocktail), resolved by electrophoresis. The blank control consisted of pure DMEM cell incubated on ice for 20 min and centrifuged (15,900 g for 20 min at 4°C). culture medium and MMP-9-neutralizing antibody concentrated under Equivalent amounts of protein were resolved by electrophoresis on 8% or similar conditions to the experimental samples. 10% SDS-polyacrylamide gels. Resolved proteins were transferred to Hybond-P PVDF membranes or onto Immobilon-FL or Immobilon-FL PVDF membranes (Merck Millipore) in 0.1 M Tris-base, pH 8.3, 0.192 M PREP expression knockdown in SH-SY5Y neuroblastoma cells glycine and 20% (v/v) methanol using an electrophoretic transfer system and image analysis (Bio-Rad). The membranes were blocked by using 0.5% (w/v) nonfat dried For transfection of wild-type and PREP-overexpressing SH-SY5Y milk (BD Biosciences) in TBS containing 0.025% (v/v) Tween-20 (Sigma- neuroblastoma cells grown on glass-bottomed cell culture dishes (3.5 cm Aldrich) or Odyssey blocking buffer (Li-Cor Bioscience). After blocking, diameter), the conditioned medium was replaced with 120 µl Opti-MEM the membranes were incubated overnight with different primary antibodies medium (Gibco) containing 2% Lipofectamine 2000 transfection reagent based on the requirement of the experiment. Antibodies used were mouse (Thermo Scientific) with either empty vector (control) or validated PREP anti-PSA-NCAM (1:1000; Millipore, MAB5324), rabbit anti-NCAM (shPREP) shRNA plasmid (KH20237N; SA Biosciences, MD, USA) and (1:1000; Millipore, AB5032), rabbit anti-NCAM (1:800; extracellular pGFP (632370; Clontech). The dishes were incubated for 3 h, after which domain H-300, sc-10735, Santa Cruz Biotechnology), rabbit anti-MMP-9

fresh DMEM medium was added. The transfected cells were allowed to (1:1000; Merc Millipore, AB19016), rabbit anti-MMP-3 (1:1000; Abcam, Journal of Cell Science

3799 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3792-3802 doi:10.1242/jcs.181891 ab52915), rabbit anti-ADAM-10 (1:500; Abcam, ab1997), rabbit anti- 1 h and destaining with 30% methanol and 10% acetic acid in distilled water. MMP-2 (1:500; Merc Millipore, AB19015), rabbit anti-ST8Sia4 (1:750; Zones of proteolysis appeared as clear bands against a blue background. Thermo Scientific, PA5-26774), rabbit anti-ST8Sia2 (1:500; Proteintech Bands were scanned and intensity of bands was quantified using ImageJ Europe, 19736-1-AP), rabbit anti-EGFR (1:1000; Abcam, EP38Y), rabbit software. anti-pEGFR (1:800; Abcam, EP774Y), chicken anti-PREP (1:1000; a generous gift from Dr Arturo Garcia-Horsman, Division of Pharmacology Data analysis and Toxicology, University of Helsinki, Finland). Incubations were Data presented are the mean±s.e.m., and experiments were repeated 2–3 followed by washing and incubation with the goat anti-rabbit or goat anti- times. Data were analyzed using GraphPad Prism 5 software. Statistical mouse horseradish-peroxidase (HRP)-conjugated secondary antibodies analysis was performed by using two-tailed Student’s t-test or one-way (1:4000; Thermo Scientific, 31460 and 32430, respectively), goat anti- ANOVA, followed by the multiple comparison Bonferroni test, where rabbit IRDye 800 CW (926-32211) or 680 LT (926-68021), goat anti-mouse appropriate. In all instances, P<0.05 was considered statistically significant. IRDye 800 CW (926-32210) or 680 LT (926-68020), donkey anti-chicken IRDye 680 CW (926-68028) (all at 1:10,000, Li-Cor Biosciences) for 1 h at Acknowledgements room temperature. Immunoreactive bands were detected by medical X-ray The technical assistance of Mrs Olili Suvi was greatly appreciated. The authors film (AGFA, EAS4Y) or using the Odyssey Infrared Imaging System would like to thank Dr. Rajeev Jain (Department of Pharmacology, Institute of (Odyssey CLx®, Li-Cor Biosciences). To normalize immunoreactivity of Biomedicine and Translational Medicine, University of Tartu, Estonia) for providing the plasmids. the proteins, β-actin was measured on the same blot by using a mouse monoclonal anti-β-actin antibody (1:1000, Li-Cor Biosciences, 926-42212) Competing interests followed by incubating with the same HRP-conjugated secondary antibody The authors declare no competing or financial interests. (1:4000), goat anti-mouse IRDye 680 LT or 800 CW (1:10,000) as above. β The ratios of proteins of interest to -actin were calculated and expressed as Author contributions the mean OD ratio in arbitrary units±s.e.m. or expressed as a percentage of K.J., A.W.: conception and design of the study, conduction of experimental work, control±s.e.m. statistical analysis and interpretation of the data, drafting and revision of the manuscript. K.P., L.K., A.A.-H., K.A., A.N.: participation in the experimental work, RNA isolation and real-time quantitative PCR conduction of immunocytochemistry, western blot and qPCR. M.G.: generation Total RNA was extracted from wild-type and PREP-overexpressing SH- PREP-overexpressing SH-SY5Y cell line. R.V.E., A.-M.L., S.R., M.M., A.Z.: approval SY5Y neuroblastoma cells using the RNeasy Mini Kit (QIAGEN) of study design, interpretation of the data, critical revision of the article, approval of the final version of the manuscript. according to the manufacturers’ protocol. cDNA was synthesized from 1 µg of total RNA using the First Strand cDNA Synthesis Kit (Fermentas Funding Inc, Burlington, Canada). Real-time quantitative PCR (qPCR) was This study was supported by the 7th Framework Programme of Health of the performed using QuantStudio 12K Flex Real-Time PCR System equipped European Commission [project NEUROPRO, HEALTH-F2-2008-223077 to S.R., with QuantStudio 12K Flex Software (ThermoFisher Scientific). Primers A.-M.L., A.Z.], Eesti Teadusfondi (Estonian Science Foundation) [grant number were synthesized by TAG Copenhagen AS (Copenhagen, Denmark) and 8740 to K.J.] and Eesti Teadusagentuur (Estonian Research Council) [Institutional primer sequences were follows: NCAM 120 kDa forward 5′-CATGTCACC research funding grant IUT2-3 to A.Z.], the Deutsche Forschungsgemeinschaft ACTCACAGATACTTTTG-3′, reverse 5′-CTCTGTAAATCTAGCATGA (German Research Foundation) [grants MO 2249/2-1 and MO 2249/2-2 to M.M TGGTTTTT-3′; NCAM 140 kDa forward 5′-AACGAGACCACGCCAC within the PP 1608], the Alzheimer Forschung Initiative [grant number 1186 to M.M.], TGA-3′, reverse 5′-CGTTTCTGTCTCCTGGCACTCT-3′; NCAM 180 kDa Universiteit Antwerpen [special research fund grant FFB3551 to A-M.L. and R.V.E] and Sächsische Aufbaubank (SAB)/European Social Fund (ESF) [grant number forward 5′-GACTTTAAAATGGACGAAGGGAAC-3′, reverse 5′-CCCA SAB100154907 to S.R., M.M., A.Z. and A.W.]. GGGCTGCAAAAACA-3′ (adapted from Winter et al., 2008); NCAM total ′ ′ ′ forward 5 -GAGATCAGCGTTGGAGAGTCC-3 , reverse 5 -GGAGAACC Supplementary information ′ AGGAGATGTCTTTATCTT-3 (adapted from Valentiner et al., 2011); HPRT1 Supplementary information available online at (hypoxanthine phosphoribosyltransferase 1)forward5′-CTTTGCTGA http://jcs.biologists.org/lookup/doi/10.1242/jcs.181891.supplemental CCTGCTGGATTAC-3′,reverse5′-GTCCTTTTCACCAGCAAGCTTG-3′; HMBS (hydroxymethylbilane synthase) forward 5′-GGCAATG References CGGCTGCAA-3′, reverse 5′-GGGTACCCACGCGAATCAC-3′; SDHA Alho, H., Ferrarese, C., Vicini, S. and Vaccarino, F. (1988). Subsets of GABAergic (succinate dehydrogenase complex, subunit A) forward 5′-GGCAAT neurons in dissociated cell cultures of neonatal rat cerebral cortex show co- GCGGCTGCAA-3′, reverse 5′-GGGTACCCACGCGAATCAC-3′. PCR localization with specific modulator peptides. Dev. Brain Res. 39, 193-204. Amoureux, M. C., Cunningham, B. A., Edelman, G. M. and Crossin, K. L. 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