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Prolyl endopeptidase is involved in the degradation of neural cell adhesion molecules in vitro

Reference: Jaako Külli, Waniek Alexander, Parik Keiti, Klimaviciusa Linda, Aonurm-Helm Anu, Noortoots Aveli, Anier Kaili, van Elzen Roos, Gerard Melanie, Lambeir Anne-Marie, ....- Prolyl endopeptidase is involved in the degradation of neural cell adhesion molecules in vitro Journal of cell science - ISSN 0021-9533 - 129:20(2016), p. 3792-3802 Full text (Publisher's DOI): http://dx.doi.org/doi:10.1242/JCS.181891 To cite this reference: http://hdl.handle.net/10067/1396760151162165141

Institutional repository IRUA

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 Morawski§2, Alexander Zharkovsky§1.

* Corresponding author § contributed equally

1. Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu 19, Ravila Str. 50411, Tartu, Estonia

* Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu 19, Ravila Str. 50411, Tartu, Estonia Phone: +372-7 374 354; GSM: +372-51 42 425; Fax: +372-7 374 352 e-mail: [email protected]

2. University of Leipzig, Paul Flechsig Institute for Brain Research Liebigstr. 19. 04103, Leipzig, Germany

3. Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences University of Antwerp, B-2610, Antwerp, Belgium

4. Interdisciplinary Research Centre KU Leuven-Kortrijk B-8500, Kortrijk, Belgium

Key words: Neural cell adhesion molecule, Prolyl endopeptidase, Neuronal plasticity, Metalloproteinase-9.

Summary statement Neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) are important regulators of the brain plasticity. We demonstrate that prolyl endopeptidase is involved in the regulation of NCAM and PSA-NCAM.

Abstract Neural cell adhesion molecule (NCAM), a membrane-associated glycoprotein and its polysialylated form (PSA-NCAM) play an important role in brain plasticity via regulating cell-cell interactions. Here, we demonstrate that the cytosolic serine protease prolyl endopeptidase (PREP) is able to regulate NCAM and PSA-NCAM. Using SH- SY5Y neuroblastoma cell line with stable overexpression of PREP we found a remarkable loss in PSA-NCAM and reduced levels of NCAM 180 kDa and NCAM 140 kDa protein species and a significant increase in the NCAM immunoreactive band migrating at an apparent molecular weight of 120 kDa in PREP-overexpressing cells. Moreover, increased levels of NCAM fragments in the concentrated medium derived from PREP-overexpressing cells were found. PREP overexpression selectively induced an activation of matrix metalloproteinase-9 (MMP-9), which could be involved in the observed degradation of NCAM, as MMP-9 neutralization reduced the levels of NCAM fragments in cell culture medium. We propose that increased PREP levels promote epidermal growth factor (EGF)-receptor signaling, which in turn activates MMP-9. In conclusion, our findings provide evidence for the novel roles for PREP in mechanisms regulating cellular plasticity via NCAM and PSA-NCAM.

Introduction The development of the nervous system, and its structural remodeling in the adult, rely on molecules mediating the structural plasticity of neurons, especially those involved in cell adhesion, cytoskeletal dynamics or synapse formation (Amoureux et al., 2000; Cremer et al., 1998; Kiss and Muller, 2001). Among these molecules, isoforms of the neural cell adhesion molecules (NCAMs) are of particular interest. NCAM is a cell surface glycoprotein that is represented by at least three isoforms (NCAM 120 kDa, NCAM 140 kDa and NCAM 180 kDa) which differ in their cytoplasmatic domains and attachment to the plasma membrane (Cunningham et al., 1987). The extracellular part of NCAM consists of five immunoglobulin (IgI-V)-like modules followed by two fibronectin type III domains (Finne et al., 1983; Rutishauser, 2008). NCAM establishes cell-cell adhesion through homo- and heterophilic interactions and thereby regulates processes like cell migration, neurite outgrowth and targeting, axonal branching, synaptogenesis, and synaptic plasticity (see (Walmod et al., 2004) for review). Neural plasticity, mediated through the NCAM protein is facilitated by posttranslational modifications, the most important and prevalent of which is addition of α-2,8-polysialic acid (PSA) chains to the IgV module of the extracellular NCAM domain. Addition of PSA homopolymers to NCAM abates interaction between NCAM molecules, decreases NCAM-mediated adhesion, thus allowing plasticity changes (Johnson et al., 2005; Kiselyov et al., 2003; Rutishauser, 2008; Seki and Arai, 1993a). The addition of PSA to NCAM occurs through two Golgi-associated polysialyltransferases ST8Sia2 and ST8Sia4 (Angata et al., 1997; Hildebrandt et al., 1998b; Nakayama et al., 1998). However, the physiological PSA- NCAM degradation and endogenous enzymes involved in this physiological turnover are not yet fully elucidated. It has been demonstrated that NCAM can be cleaved extracellularly by metalloproteinases and other proteolytical enzymes (Brennaman et al., 2014; Hinkle et al., 2006; Hübschmann et al., 2005; Kalus et al., 2006). Moreover, metalloproteinase- dependent shedding of NCAM induces release of soluble forms of NCAM consisting of several polypeptides with molecular weights ranging from 80 kDa to 200 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 the cleavage product have been associated with neuropsychiatric disorders such as schizophrenia (Vawter et al., 1998) and dementia (Strekalova et al., 2006). The cytosolic serine protease prolyl endopeptidase (PREP) hydrolyses small (<3 kDa) proline-containing peptides at the carboxy terminus of proline residue (Fülöp et al., 1998; Polgár, 2002; Rawlings et al., 1991). PREP is widely distributed in brain (Irazusta et al., 2002; Myöhänen et al., 2007; Myöhänen et al., 2008) and increased activity of PREP has been associated with cell death processes in various neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease (Brandt et al., 2008; Mantle et al., 1996). PREP is believed to act in the extracellular space being involved in the maturation and degradation of peptide hormones and neuropeptides (Bellemère et al., 2004; Cunningham and O’Connor, 1997; Shishido et al., 1999) which has been proposed as underlying mechanism of some beneficial effects of PREP inhibitors in animal memory models (Shishido et al., 1998; Toide et al., 1997; Yoshimoto et al., 1987) and in aged mice (Kato et al., 1997). Besides its extracellular action, PREP has been shown to act intracellularly and important roles of PREP has been demonstrated in signaling pathways or in transport and secretion of proteins and peptides associated with neurodegeneration (Brandt et al., 2008; Di Daniel et al., 2009; Rossner et al., 2005; Savolainen et al., 2015; Schulz et al., 2002; Schulz et al., 2005). Within recent years PREP has been suggested to be a contributor to neuroinflammation (Penttinen et al., 2011). PREP has been shown to be involved in mechanisms responsible for the transduction, and amplification of inflammatory processes, and this leads to the production of neurotoxic mediators, which in turn mediate pathology progression (for rev. see (Penttinen et al., 2011)). In this report, for the first time, we demonstrate that PREP is able to regulate the levels of NCAM via activation of MMP-9.

Results The levels of PSA-NCAM and NCAM and their mRNA levels in neuroblastoma SH- SY5Y cells, overexpressing PREP The increased levels of PREP protein and its activity in PREP-overexpressing SH-SY5Y cell lysates were confirmed by the Western blot and activity measurements (Fig. S1A, B). PREP appeared on SDS-PAGE as band at around 80 kDa (Fig. S1A). A 30-fold increase in PREP protein level was found in SH-SY5Y cells, overexpressing PREP compared to

wild-type cells (p<0.001, t-test, n=4). In PREP-overexpressing cells, PREP activity assays demonstrated an 11-fold increase in the enzymatic activity (p<0.0001, t-test, n=3) (Fig. S1B). To evaluate whether increased PREP levels are associated with altered levels of PSA- NCAM and/or NCAM, the proteins were quantified using Western blot analysis. In wild- type cells, PSA-NCAM appeared as a high-molecular weight smear typical for highly polysialylated NCAM (Fig. 1A), whereas in PREP-overexpressing cells, PSA-NCAM signal was almost absent compared to wild-type cells (Fig. 1A) and analysis of optical density confirmed this finding (Fig. 1A) (p<0.0001, t-test, n=5-6). In wild-type cells, immunocytochemistry demonstrated membrane-bound extracellular PSA-NCAM molecules, which assembled in small bunches along neurite like processes and in larger patches associated with the perikaryon. In PREP-overexpressing cells, however, PSA- NCAM signal was almost absent (Fig. 1C). We further investigated the levels of the NCAM protein patterns (NCAM 180 kDa, NCAM 140 kDa and NCAM 120 kDa). A significant decrease in the levels of NCAM 180 kDa immunoreactive band (p=0.01, t-test, n=5-6) and NCAM 140 kDa immunoreactive band (p=0.01, t-test, n=5-6) was found in PREP-overexpressing neuroblastoma cells compared to wild-type cells (Fig. 1B). In contrast, a significant increase in the NCAM immunoreactive band migrating at an apparent molecular weight of 120 kDa was found in PREP-overexpressing cells (p=0.009, t-test, n=5-6). In wild-type cells, immunostaining for NCAM demonstrated an intensive NCAM immunopositive signal, whereas only moderate NCAM immunopositive signals could be detected in PREP-overexpressing cells (Fig. 1D). Next we aimed to elucidate, whether the observed changes in the levels of NCAM protein patterns are caused by the changes in their expression. We measured the 120, 140 and 180 isoforms as well as total NCAM mRNA levels using qPCR. No significant differences in the NCAM120, NCAM140, NCAM180 and total NCAM mRNA levels between wild-type and PREP-overexpressing cells were found (Table S1). Thus, the observed increase in NCAM 120 kDa immunoreactive band is not due to the increased expression NCAM mRNA. Next, we proposed that the increase in 120 kDa NCAM is due to the degradation of NCAM 140 and/or NCAM 180 kDa isoforms. To test this proposal, we measured NCAM

degradation fragments in concentrated cell culture medium from PREP-overexpressing and wild-type cells. Two fragments recognized by the NCAM–specific antibody were found in the concentrated medium: one is about 46-48 kDa, and another fragment is about 38-40 kDa. In the medium of PREP-overexpressing cells, there was a significant increase in the levels of 38-40 kDa fragment (p=0.0043, t-test, n=4, Fig. 2A, 2B), whereas no changes were observed in the levels of 46-48 kDa fragment. These data support our hypothesis that the overexpression of PREP induces an increased degradation of NCAM 180 and/or NCAM 140 kDa isoforms. Next, we aimed to evaluate whether the observed changes in the PSA-NCAM levels are PREP-specific in PREP-overexpressing cells. For that, PREP-overexpressing cells were transfected with shPREP or shNC together with green fluorescent protein GFP plasmid and after 72 hours cells were fixed and processed for PSA-NCAM immunocytochemistry. GFP-positive cells, from the shPREP and shNC group were selected for PSA-NCAM fluorescent signal intensity analysis. Quantification revealed an increase in PSA-NCAM immunopositive signals in PREP-overexpressing cells, transfected with shPREP compared to shNC transfected PREP-overexpressing cells (p=0.043, t-test, n=10), (Fig. 3A, 3B).

The effect of recombinant PREP added to the culture medium on PSA-NCAM expression in wild-type SH-SY5Y neuroblastoma cells and primary cortical neurons In a series of experiments on wild-type SH-SY5Y cells and on primary cortical neurons, the human recombinant PREP (rPREP, 1 nM or 10 nM) was added for 72 hours into the cell culture medium of wild-type SH-SY5Y neuroblastoma cells. A marked decrease in PSA-NCAM level was found (F=10.26, p=0.001, one-way ANOVA, followed by Bonferroni post-hoc test, n=3) after addition of PREP (Fig. 3C). Similar decrease in PSA- NCAM level (p=0.03, t-test, n=5-6) was found in experiments on the primary cortical neurons (Fig. 3D).

Quantification of protein expression levels of polysialyltransferase ST8Sia2 and ST8Sia4 in wild-type and PREP-overexpressing neuroblastoma SH-SY5Y cells The formation of PSA-NCAM is dependent on the polysialyltransferases, ST8Sia2 and ST8Sia4, which attach PSA-residues to the NCAM molecule. To exclude a possibility

that PREP affects ST8Sia2 and ST8Sia4, wild-type and PREP-overexpressing cells were subjected for ST8Sia2 and ST8Sia4 immunocytochemistry and protein expression analysis (Fig. 4A, 4B). Western blot analysis demonstrated no statistically significant differences between wild-type and PREP-overexpressing cells, in either ST8Sia2 or ST8Sia4 protein levels (Fig. 4B).

Impaired differentiation of PREP-overexpressing cells As NCAM and PSA-NCAM are involved in the neurite outgrowth and neuronal differentiation (Seidenfaden et al., 2006; Williams et al., 1994), it was of interest to evaluate whether or not an excess of PREP, accompanied with altered NCAM expression might impair differentiation. To elucidate this question, wild-type and PREP- overexpressing cells were grown with sequential exposure to retinoic acid and brain derived neurotrophic factor (BDNF) for 5 and 7 days, respectively to obtain long-term survival of cells and high degree of differentiation (Encinas et al., 2000). As demonstrated in Fig. 5A, wild-type cells yielded homogeneous populations of Tuj-1 immunopositive cells with neuronal morphology and abundantly branched neurites. In contrast, only moderate number of PREP-overexpressing cells demonstrated Tuj-1 immunopositive signal (Fig. 5A). Quantification of Tuj-1 positive cells revealed that the percentage of cells possessing Tuj-1 immunopositive staining was significantly lower (p<0.0001, t-test, n=9) in PREP-overexpressing cells as compared to wild-type cells (Fig. 5B), and these results indicate that alterations in NCAM molecule integrity lead to impaired differentiation toward neuron-like cell type.

Increased MMP-9 protein level and activity in neuroblastoma SH-SY5Y cells, overexpressing PREP It is well documented that PREP is 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 was demonstrated previously that NCAM molecule degradation might be mediated by the metalloproteases (Brennaman et al., 2014; Hinkle et al., 2006; Hübschmann et al., 2005; Shichi et al., 2011). Therefore, the question arose 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. An

increased level of active form of MMP-9 was found in PREP-overexpressing SH-SY5Y neuroblastoma cells compared to wild-type cells (p=0.001, t-test, n=3), whereas no changes were found in the levels of inactive form of MMP-9 (Fig. 6A). Moreover, in PREP-overexpressing cells immunocytochemical labeling of MMP-9 revealed intense granulation of the immunopositive signal mostly at perinuclear region, whereas in wild- type cells MMP-9 signal was associated with sparse granulation pattern on immunocytochemistry (Fig. 6B). In addition, a zymography demonstrated an increased proteolytic activity of MMP-9 in the serum free medium from cultured PREP- overexpressing cells (p=0.002, t-test, n=3) (Fig. 6C). To test whether the observed increase in MMP-9 levels and activity in PREP- overexpressing cells is responsible for NCAM degradation, PREP-overexpressing cells were incubated with MMP-9 neutralizing antibody for 96 hours and we found a reduced levels of NCAM fragments in the cell culture medium (p<0.001, t-test, n=5-6) compared to vehicle-treated PREP-overexpressing cells (Fig. 7A, 7B). To elucidate whether PREP overexpression could affect other MMPs which might be involved in NCAM degradation, the protein levels of MMP-2, MMP-3 and ADAM-10 were measured. No changes were found in the levels of those MMPs in PREP- overexpressing cells as compared to wild-type cells (Fig. S2).

Decreased EGF-receptor level and increased pEGF-receptor level in neuroblastoma SH-SY5Y cells, overexpressing PREP It has been demonstrated that EGF-receptor signaling is involved in release and activation of MMP-9 (da Rosa et al., 2014; Pei et al., 2014; Qiu et al., 2004). Therefore EGF- receptor and phosphorylated EGF-receptor levels were measured and in PREP- overexpressing cells overall decrease in total EGF-receptor was found (Fig. 8), (p=0.001, t-test, n=4). In contrast, the levels of phosphorylated form of EGF-receptor were increased (Fig.8) (p=0.001, t-test, n=4).

Discussion This study shows for the first time that PREP is involved in the regulation of neural cell adhesion molecules.

The PREP-overexpressing neuroblastoma SH-SY5Y cell line was used as an in vitro model to mimic pathological conditions showing increased PREP expression. When PSA-NCAM protein levels were quantified, we found a remarkable reduction of PSA- NCAM in PREP-overexpressing cells compared with wild-type cells. The finding that PSA-NCAM levels are altered in the context of higher PREP levels was further confirmed in a series of experiments where cell culture media for wild-type neuroblastoma cells or for primary cortical cells isolated from mouse brain were enriched with human recombinant PREP – this induced a decrease in PSA-NCAM expression levels. Not only PSA-NCAM was altered in the presence of PREP but we also found alterations in the levels of all NCAM protein variants. We found a reduction in NCAM 180 kDa and 140 kDa immunoreactive bands and an increase in NCAM 120 kDa immunoreactive band. Since PREP might affect PSA-NCAM and NCAM via different mechanisms including reduced expression and/or increased breakdown of the NCAM protein, we explored these mechanisms in more detail. In neuroblastoma cells overexpressing PREP, we did not find any changes in NCAM120, NCAM140, NCAM180 and in total NCAM mRNA levels. These data suggest that PREP does not affect the transcription of NCAM. We also failed to find any changes in the expression levels of two Golgi-associated polysialyltransferases, ST8Sia2 and ST8Sia4, which mediate the addition of PSA to NCAM (Eckhardt et al., 1995). These data suggest that PREP does not affect the polysialylation of NCAM. Therefore we propose that the reduction in 140 kDa and 180 kDa protein variants of NCAM and the reduction in PSA- NCAM are due to their increased degradation in the presence of PREP. To explore this proposal in more detail, we measured NCAM degradation products in concentrated medium derived from PREP-overexpressing cells. Indeed, an increased level of peptide fragments at 38-40 kDa, recognized by NCAM antibody, was found. Thus, it seems that the observed decrease in NCAM immunoreactive bands of 180 kDa and 140 kDa are due to their breakdown in the presence of PREP and the observed increase in the NCAM immunoreactive band migrating at an apparent molecular weight of 120 kDa might be explained by the accumulation of bigger fragments of NCAM. However, NCAM fragments at 38-40 kDa indicate that the cleaved fragment is too small to include the polysialylated Ig5 domain and therefore additional mechanisms, like increased PSA cleavage via neuraminidases, in addition to NCAM degradation by MMP-9 might be

involved. In mammals, four sialidases are present and these sialidases involved in several key physiological events related to cancer transformation (Miyagi et al., 2004; Proshin et al., 2002; Tringali et al., 2012; Wada et al., 2007) and in developmental processes, like lamination of newly generated hippocampal granule cells through the modulation of PSA (Sajo et al., 2016). The question 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 was also shown that inhibition of MMP- 2 and MMP-9 prevented NCAM shedding, indicating the roles of these MMPs in NCAM cleavage (Hübschmann et al., 2005; Shichi et al., 2011). Moreover, ADAM-10-dependent shedding of NCAM has been extensively studied, and the second fibronectin-type III domain of NCAM has been shown to be a target for ADAM-10 cleavage (Brennaman et al., 2014). Based on these findings, we explored in more detail the impact of metalloproteases in the PREP-mediated decrease in PSA-NCAM and NCAM. We measured the levels of MMP-2, MMP-3, MMP-9 and ADAM-10 in the lysates of wild- type and PREP-overexpressing cells and found an increased level of MMP-9 but not the other metalloproteases. Furthermore, we found that the increased levels of MMP-9 were due to its active form. Using zymography, we also found increased MMP-9 activity. Thus, it seems that PREP overexpression induces a selective increase in the levels and activity of MMP-9, which is most probably involved in the observed degradation of NCAM. This proposal was further confirmed in our experiments where MMP-9-neutralizing antibody prevented the degradation of NCAM, as evidenced by the decrease in NCAM fragments in the culture medium. Previous studies have demonstrated that cleavage of NCAM 180 kDa isoforms and NCAM 140 kDa isoforms by ADAM-10 and ADAM-17 results in the release of 110- 115-kDa fragments into the cell culture medium, associated with the appearance of 30 kDa membrane-associated fragments in cell lysates (Brennaman et al., 2014; Kalus et al., 2006). In addition to aforementioned fragments cleaved by ADAMs, a 65 kDa

degradation product of NCAM has been demonstrated to be the result of the action of MMP-9 and/or MMP-2 after cerebral ischemic neuronal damage and 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 domains of NCAM. Appearance of fragments ~40 kDa, derived from extracellular domains of NCAM into cell culture medium might explain also 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. 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 activation. It has been demonstrated that in vitro conditions cleavage, degradation, or chemical modification via proteolytic and nonproteolytic mechanisms are responsible for TIMPs inactivation via involving serine or thiol proteases and reactive oxygen species, respectively (Frears et al., 1996; Okada et al., 1992). The involvement of PREP in the processing of TIMPs, however, remains unclear as PREP is able to cleave only small peptide fragments of 30 amino acids. There is a possibility that some small, as yet unknown, peptides, substrates of PREP, might directly modulate MMP-9 activity. Moreover, it cannot be excluded that PREP might be involved in the regulation of MMP- 9 release. It is known that EGF, via an interaction with its receptors, increases the release and activation of MMP-9 (da Rosa et al., 2014; Pei et al., 2014; Qiu et al., 2004). In PREP-overexpressing cells, despite the overall decrease in total EGF receptor, the levels of phosphorylated (active form) EGF receptor increased, which indicates an activation of EGF signaling and consequent release of MMP-9, however the precise mechanism through which PREP is involved in the regulation of EGF-mediated MMP-9 release remains unknown and certainly needs further investigation. The PREP-mediated mechanism of the regulation of PSA-NCAM and NCAM might be relevant for neuronal function and plasticity. It has been demonstrated that NCAM180 and NCAM140 are key regulators of neuronal development and maintenance as they are widely expressed in postsynaptic densities of neurons, whereas NCAM140 is also

expressed in migratory growth cones (Dityatev et al., 2000; Persohn et al., 1989). The main function of these isoforms 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 NCAM and/or its polysialylation may impair cellular functioning as it is well documented that NCAM in its polysialylated form is essential for neurons to migrate and establish new synaptic contacts (Muller et al., 1996; Rougon, 1993; Rutishauser and Landmesser, 1996; Seki and Arai, 1993a; Seki and Arai, 1993b). In accordance with these studies, our experiments showed that the PREP- overexpressing neuroblastoma cells exhibit impaired differentiation, induced by retinoic acid and BDNF. The role of PSA in differentiation is important as PSA is highly reexpressed during progression of several malignant human tumors, neuroblastoma among others and therefore could be considered as an oncodevelopmental antigen. It has been demonstrated that in malignant human tumors polysialylation of NCAM appears to increase the metastatic potential and has been correlated with tumor progression and a poor prognosis (Glüer et al., 1998; Tanaka et al., 2001). In vitro studies in neuroblastoma cells, where endoneuraminidase (EndoN) treatment was applied for PSA removal trigger the cell to cease proliferation and to differentiate (Seidenfaden et al., 2003). Moreover, neuroblastoma cells differentiating toward the neuron-like cell type express high amounts of NCAM and PSA, although reduction in PSA expression was found after differentiation (Hildebrandt et al., 1998a). During the regular differentiation, removal of PSA has been demonstrated to increase the number of cell-cell contacts (Hildebrandt et al., 1998a), which relay on the basis for signal transduction via second messenger pathways in addition to cell adhesion per se, however in PREP-overexpressing cells where NCAM extracellular domains are partly degraded and NCAM molecule integrity is disrupted, impaired differentiation toward neuron-like cell type, as well as formation of neurites might be outcome of these alterations. An increasing body of evidence from clinical and preclinical studies suggests an involvement of PREP in neuroinflammation. Recent in vitro studies demonstrated that PREP contributes to the toxic effects of reactive microglial cells, as it was demonstrated that activated microglia cells expressed high levels of PREP and the 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 (Klegeris et al., 2008).

Moreover, studies in PREP knockout (k/o) mice have demonstrated association of PREP in the processes modulating neuroplasticity via inflammatory response. In PREP k/o mice hippocampus, alterations in the microglia activation in response to systemic administration of repeated doses of lipopolysaccharide were found in addition to increased levels of PSA-NCAM, and these data indirectly support the functional significance of PREP in the regulation of PSA-NCAM (Höfling et al., 2016). Moreover, in clinical studies, increased PREP activity has been found in knee-joint synovial membranes indicating its association with rheumatoid arthritis (Kamori et al., 1991). Considering the demonstrated effects of PREP in inflammation and possible role in neuroinflammation, resulting in changes in neural plasticity as well as the role of NCAMs on brain plasticity, the interplay between these molecules should be considered plausible, as NCAM-mediated neural plasticity is altered in pathological conditions associated with inflammation. Decreased full-length NCAM180 has been described in mice 1 day after middle cerebral artery occlusion (Shichi et al., 2011). Moreover, it has been shown that in the processes of demyelinating neuroinflammation due to the autoimmune encephalomyelitis, the level of MMP-2 was significantly increased in hippocampus, which was accompanied by reduced levels of NCAM (Jovanova-Nesic and Shoenfeld, 2006). In addition to previously mentioned increased cleavage of NCAM180 during ischemic stress, similar effects in the decrease of NCAM180 and increased content of cleaved form of NCAM was described after oxidative stress (Fujita-Hamabe and Tokuyama, 2012). In conclusion, our study demonstrates that increased expression level and activity of PREP is involved in mechanisms regulating degradation of neural adhesion molecules most likely via MMP-9 activation. These findings open up a new avenue for the exploration of the roles of PREP in processes involved in NCAM degradation which in turn, is believed to be one major contributor for altered neuroplasticity (Brusés and Rutishauser, 2001; Cremer et al., 1998; Gnanapavan and Giovannoni, 2013). Moreover, these mechanisms are of importance regarding synaptic plasticity in the (re-)organization of neuronal circuits.

Materials and methods Generation of PREP-overexpressing SH-SY5Y cell line

Generation of PREP-overexpressing SH-SY5Y cell line was generated as described in (Gerard et al., 2010). Wild-type and PREP-overexpressing cells were grown in DMEM/GlutaMAX-I medium (Thermo Scientific) containing 15% 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-overexpressing cells. Cells were maintained at 37 °C in a saturated humidity atmosphere containing 95% air and 5% CO2. For differentiation studies, alltrans-retinoic acid (RA, Tocris Cookson, Bristol, U.K.) was added the day after plating at a final concentration of 10 μM in DMEM with 15% fetal calf serum. After 5 days in the presence of RA, cells were washed three times with DMEM and incubated with 50 ng/ml BDNF (Sigma, Aldrich) in DMEM (without serum) for 7 days.

PREP activity assay PREP activity in SH-SY5Y cells was measured according to the method 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 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), 20 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM dithiothreitol (DTT, Sigma-Aldrich). The obtained cell lysate was centrifuged and supernatants were transferred to new tubes. Equal amount of protein samples (10 µg) were mixed with assay buffer containing chromogenic PREP substrate 250 µM Z-Gly-Pro-p-nitroanilide (Bachem AG, Bubendorf). The product absorbance was continuously measured 30 minutes at 405 nm using ELISA plate reader (Tecan, Crailsheim, Germany) and PREP activity was calculated using p-nitroanilide (Sigma- Aldrich) standard curve.

Primary culture of rat cortical neurons Primary neuronal cultures were prepared from 1-day-old Wistar rat pups according to the method of Alho and colleagues (Alho et al., 1988) with minor modifications. Briefly, cortices were dissected in ice-cold Krebs-Ringer solution (135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 0.4 mM K2HPO2, 15 mM glucose, 20 mM HEPES, pH 7.4, containing 0.3% bovine serum albumin) and trypsinized in 0.8% trypsin-EDTA (Invitrogen, U.K.) for 10 minutes at 37 °C followed by trituration in 0.008% DNAse I solution containing 0.05%

soybean trypsin inhibitor (both from Surgitech AS, Estonia). Cells were resuspended in Eagle’s basal medium with Earle’s salts (BME, Invitrogen, U.K.) containing 10% heat- inactivated fetal bovine serum (FBS, Invitrogen, U.K.), 25 mM KCl, 2 mM GlutaMAX™-I (Invitrogen, U.K.) and 100 μg/ml gentamycin (KRKA). Cells were plated onto poly-L-lysine (Sigma Chemical Co, MO, USA) coated cell culture dishes at the density 1.8 × 105 cells/cm2. 2.5 hours later, the medium was changed for Neurobasal™-A medium (Gibco, 10888-022). Cultures were incubated for 4 days in an atmosphere of

95% air and 5% CO2 at 37 °C.

Treatment with rPREP Wild-type SH-SY5Y neuroblastoma cells or primary cortical neurons at DIV4 were grown in DMEM cell culture medium plus 10% fetal calf serum or Neurobasal™-A medium, containing 2 mM GlutaMAX™-I, B-27 supplement (Gibco, 17504-044) and 100 μg/ml gentamycin, respectively. Human recombinant PREP protein ([rPREP], a generous gift from Dr. Zoltan Szeltner, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Science (Stock solution was dissolved in buffered saline, 1 mM DTT, pH 7.5) was added into cell culture medium at concentrations 1 and 10 nM for wild-type SH-SY5Y neuroblastoma cells and 1 nM for primary cortical neurons and cells were incubated for 72 hours and then processed for Western blot analysis.

Treatment with MMP-9 neutralizing antibody and NCAM cleavage assay MMP-9 neutralizing antibody (clone 6-6B) was purchased from Millipore (IM09L) and dissolved according to manufacturer’s instructions. PREP-overexpressing SH-SY5Y neuroblastoma cells were treated with MMP-9 neutralizing antibody or appropriate vehicle for 96 hours at concentrations 50 μg/ml in serum free DMEM/GlutaMAX-I medium. Detection of NCAM fragments was done according to the method of Brennaman and colleagues (Brennaman et al., 2014). After 96 hours, cell culture medium was collected, treated with protease inhibitors (Roche, 11697498001), centrifuged for 5 min (1100 rpm), followed by concentration by Millipore centrifugal concentrators according to the manufacturer’s instructions (30 000 MW cutoff - 10 x concentration;

Millipore). Protein concentrations from each concentrated cell culture media samples were measured by Bradford method and equivalent amount of proteins from media samples were resolved by electrophoresis on 10% SDS-polyacrylamide gel as described below. In addition, blank control sample was prepared and resolved by electrophoresis. Blank control consisted pure DMEM cell culture medium and MMP-9 neutralizing antibody concentrated at similar conditions as experimental samples.

Knocking down of PREP expression in SH-SY5Y neuroblastoma cells and image analysis For transfection of wild-type and PREP-overexpressing SH-SY5Y neuroblastoma cells grown on glass-bottomed cell culture dishes (d=3.5 cm), the conditioned medium was replaced with 120 µl Opti-MEM medium (Gibco, 51985-026) containing 2% Lipofectamine® 2000 transfection reagent (Thermo Scientific, 11668027) with either empty vector (control) or validated PREP (shPREP) shRNA plasmid (KH20237N, SA Biosciences, MD, USA) and pGFP (632370, Clontech). The dishes were incubated for 3 hours, after which fresh DMEM medium was added. The transfected DNAs were allowed to express 72 hours before the immunocytochemical detection for PSA-NCAM was performed. Immunopositive signals were detected with a confocal microscope LSM 510 (Zeiss, Denmark) equipped with an argon-krypton laser. 3D images were constructed from a series (10-13) of scans of the GFP-positive cells 1 μm intervals using a 40× (water) objective and further analyzed for signal intensity. Fluorescence quantification analysis was performed using ImageJ software. Each cell to be analyzed was manually defined and three regions were selected just beside the cell in an area without fluorescent objects to be used for background adjustment. Subsequently, the corrected total cell fluorescence (CTCF) was obtained according to (Burgess et al., 2010; Gavet and Pines, 2010). CTCF was calculated by multiplying the area (in square pixels) of selected cell of interest by mean fluorescence of PSA-NCAM (per pixel) of background readings and subtracting the result from whole cell fluorescence (in pixels). Overlapping of aforementioned cells was excluded by checking all channels in each image. PSA-NCAM immunopositive signal intensity is given in relative fluorescence units (RFU).

Immunocytochemistry and image analysis

SH-SY5Y neuroblastoma cells were fixed by using 4% paraformaldehyde in phosphate- buffered saline (PBS) for 10 minutes at room temperature, permeabilized in 0.05% Triton X-100 (Sigma-Aldrich) for 30 minutes, and rinsed in PBS. Non-specific binding was blocked by 2% normal goat serum (S-1000, Vector Laboratories) in PBS containing 0.3% Triton X-100 for 1 hour, followed by 48 hours of incubation at 4 °C with different primary antibodies diluted in blocking buffer based on the requirement of the experiment. Primary antibody dilutions used were mouse anti-PSA-NCAM 1:500 (Merc Millipore, MAB5324), rabbit anti-NCAM 1:1000 (Merc Millipore, AB5032), rabbit anti-MMP-9 1:700 (Merc Millipore, AB19016), rabbit anti ST8Sia4 1:500 (Thermo Scientific, PA5- 26774), rabbit anti ST8Sia2 1:200 (Proteintech Europe, 19736-1-AP), mouse anti Tuj-1 1:700 (Merc Millipore, MAB1637). After being washed in PBS, cells were incubated in Alexa-Fluor®594 goat anti-mouse IgM (Life Technologies, A-21044), Alexa-Fluor®488 goat anti-rabbit IgG (A-11008), Alexa-Fluor®594 goat anti-rabbit IgG (A-11012) or Alexa-Fluor®488 goat anti-mouse IgG (A-11001) in blocking buffer for 1 hour. Nuclei were counterstained with DAPI (Sigma-Aldrich, D-9542) solution (300 nM in PBS). A laser scanning confocal microscope (LSM 510 Duo, Zeiss, Germany) equipped with a C-Apocromat 40×/1.20 water immersion M27 objective (Zeiss, Germany) was used for image acquiring and acquisition parameters (magnification, laser intensity, gain, pinhole aperture) were kept constant between different experimental groups. For differentiation analysis, 9 randomly taken fields from 3 cell culture dishes (d=3.5 cm) were taken at magnification x40. 3D images were constructed from a series (10-12) of scans at 1 μm intervals for image analysis. The number of Tuj-1 immunopositive cells and the total number of DAPI-positive nuclei were counted and the data were expressed as a percentage of cells, positive for Tuj-1 signal in wild-type and PREP-overexpressing cells.

Western blot analysis Primary neurons or SH-SY5Y cells were lysed in cell lysis buffer (RIP-A lysis buffer: 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% NP-40 and 2 mM EDTA containing protease and phosphatase inhibitors cocktail), and incubated on ice for 20 minutes and centrifuged (13 000 rpm for 20 minutes at 4 °C). Equivalent amounts of protein were resolved by electrophoresis on 8% or 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 glycine and 20% (v/v) methanol using an electrophoretic transfer system (Bio- Rad). The membranes were blocked by using 0.5% (w/v) nonfat dried milk (BD, 232100) in TBS containing 0.025% (v/v) Tween-20 (Sigma, P1379) or Odyssey® blocking buffer (Li-Cor Bioscience, 927-50000). After blocking, the membranes were incubated overnight with different primary antibodies based on the requirement of the experiment. Antibody dilutions used were mouse anti-PSA-NCAM 1:1000 (Millipore, MAB5324), rabbit anti-NCAM 1:1000 (Millipore, AB5032), rabbit anti-NCAM 1:800 (extracellular domain H-300, sc-10735, Santa Cruz Biotechnology), rabbit anti-MMP-9 1:1000 (Merc Millipore, AB19016), rabbit anti MMP-3 1:1000 (Abcam, ab52915), rabbit anti ADAM- 10 1:500 (Abcam, ab1997), rabbit anti MMP-2 1:500 (Merc Millipore, AB19015), rabbit anti ST8Sia4 1:750 (Thermo Scientific, PA5-26774), rabbit anti ST8Sia2 1:500 (Proteintech Europe, 19736-1-AP), rabbit anti EGFR 1:1000 (Abcam, EP38Y), rabbit anti pEGFR 1:800 (Abcam, EP774Y), chicken anti-PREP 1:1000 (a generous gift from Dr. Arturo Garcia-Horsman, Division of Pharmacology and Toxicology, University of Helsinki, Finland). Incubations were followed by washing and incubation with the goat anti-rabbit or goat anti-mouse horseradish-peroxidase (HRP)-conjugated secondary antibody (1:4000), goat anti-rabbit IR-Dye® 800 CW or 680 LT, goat anti-mouse IR- Dye® 800 CW or 680 LT (1:10 000, Li-Cor Biosciences, 926-32211 or 926-68021), donkey anti chicken IR-Dye® 680 CW (1:10 000, Li-Cor Biosciences, USA, 926-68028) for 1 hour at room temperature. Immunoreactive bands were detected by medical X-ray film (AGFA, EAS4Y) or using the Odyssey Infrared Imaging System (Odyssey CLx®, Li-Cor Biosciences). To normalize immunoreactivity of 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) followed by incubating with a horseradish-peroxidase (HRP)-conjugated secondary antibody (1:4000), goat anti-mouse IR-Dye® 680 LT or 800 CW (1:10 000, Li-Cor Biosciences, 926-68020 or 926-32210). The ratio of proteins of interest to β-actin were calculated and expressed as the mean OD ratio in arbitrary units ± s.e.m. or expressed as % of control ± s.e.m.

RNA isolation and real-time quantitative PCR

Total RNA was extracted from wild-type and PREP-overexpressing SH-SY5Y neuroblastoma cells using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturers’ protocol. cDNA was synthesised from 1 µg of total RNA using the First Strand cDNA Synthesis Kit (Fermentas Inc, Burlington, Canada). Real-time quantitative PCR (qPCR) was performed using QuantStudio 12K Flex Real-Time PCR System equipped with QuantStudio 12K Flex Software (ThermoFisher Scientific Inc, Waltham, MA, USA). Primers were synthesised by TAG Copenhagen AS (Copenhagen, Denmark) and primer sequences were follows: NCAM 120 kDa forward 5’- CATGTCACCACTCACAGATACTTTTG-3’, reverse 5’- CTCTGTAAATCTAGCATGATGGTTTTT-3’; NCAM 140 kDa forward 5’- AACGAGACCACGCCACTGA-3’, reverse 5’-CGTTTCTGTCTCCTGGCACTCT-3’; NCAM 180 kDa forward 5’-GACTTTAAAATGGACGAAGGGAAC-3’, reverse 5’- CCCAGGGCTGCAAAAACA-3’ (adapted from (Winter et al., 2008)); NCAM total forward 5’-GAGATCAGCGTTGGAGAGTCC-3’, reverse 5’- GGAGAACCAGGAGATGTCTTTATCTT-3’ (adapted from (Valentiner et al., 2011)); HPRT1 (Hypoxanthine phosphoribosyltransferase 1) forward 5’-

CTTTGCTGACCTGCTGGATTAC-3’, reverse 5’-GTCCTTTTCACCAGCAAGCTTG- 3’; HMBS (Hydroxymethylbilane synthase) forward 5’-GGCAATGCGGCTGCAA-3’, reverse 5’-GGGTACCCACGCGAATCAC-3’; SDHA (Succinate dehydrogenase complex, subunit A) forward 5’-GGCAATGCGGCTGCAA-3’, reverse 5’- GGGTACCCACGCGAATCAC-3’. PCR amplification was performed in a total reaction volume of 10 μl in three parallels. The reaction mixture consisted of 1 μl First Strand cDNA diluted template, 5 μl 2x Master SYBR Green qPCR Master Mix (Applied

Biosystems Inc, USA), 3 μl H20 and 1 μl gene-specific 10 μM PCR primer pair stock. The PCR amplification was performed as follows: denaturation step at 95 °C for 10 min, followed by denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 1 min, repeated for 40 cycles. SYBR Green fluorescence was measured after each extension step and amplification specificity was confirmed by melting curve analyses and gel electrophoresis of the PCR products. Relative mRNA levels were calculated by normalization of target gene mRNA level to the geometric mean of the expression level of three endogenous reference genes (HPRT1, HMBS and SDHA).

Gelatin zymography Equal amounts of cell culture medium (normalized to cell count in cell culture dish) were separated by electrophoresis in SDS-PAGE gels containing 1 mg/ml porcine skin gelatin (G2500, Sigma, USA) at 90 V. Gels were washed for 40 minutes in 2.5% Triton X-100, incubated for 48 hours at 37 °C with gentle agitation in 1% Triton X-100, 5 mM CaCl2, 0.05 M Tris, followed by staining with 0.5% Coomassie Blue R-250 (Sigma-Aldrich, USA) for 1 hour and destaining with 30% methanol and 10% acetic acid in distilled water. Zones of proteolysis appeared as clear bands against a blue background. Bands were scanned and intensity of bands was quantified using ImageJ software.

Data analysis Data presented are the mean ± s.e.m., and the experiments were repeated 2–3 times. Data were analysed using GraphPad 5 Prism software. Statistical analysis was performed by using two-tailed Student’s t-test or one-way ANOVA, followed by the multiple comparison Bonferroni test, where appropriate. In all instances, p<0.05 was considered statistically significant.

Acknowledgements and funding This study was supported by the EU grant NEUROPRO project No. 223077 (S.R., A- M.L., A.Z), the Estonian Science Foundation Grant 8740 (K.J.) and Estonian Science Council Grant (Institutional research founding) IUT2-3 (A.Z.), the German Research Foundation MO 2249/2-1 (M.M) within the PP 1608, GRK 1097 ‘‘INTERNEURO’’ (M.M.), the Alzheimer Forschungsinitiativee.V. (AFI #1186, M.M.), European Union and the Federal State of Saxony Funding (S.R., A.Z., M.M., A.W.), RVE, UAntwerp special research fund grant FFB3551 (A-M. L.).

Conflict of interest All authors declare that they have no conflicts of interest.

Contribution of authors Külli Jaako, PhD; Alexander Waniek, PhD. Conception and design of the study, conduction of experimental work, statistical analysis and interpretation of the data, drafting and revision of the manuscript. Keiti Parik, PhD Student; Linda Klimaviciusa, PhD; Anu Aonurm-Helm, PhD; Kaili Anier, PhD; Aveli Noortoots, PhD Student. Participitation in the experimental work, conduction of immunocytochemistry, Western blot and qPCR. Melanie Gérard, PhD. Generation PREP o/e SH-SY5Y cell line. Roos Van Elzen PhD; Anne-Marie Lambeir, PhD; Steffen Roßner PhD; Markus Morawski PhD, MD; Alexander Zharkovsky PhD, MD. Approval of study design, interpretation of the data, critical revision of the article, approval of the final version of the manuscript.

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Figure legends 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 WT and PREP-overexpressing SH-SY5Y neuroblastoma cells demonstrating tremendous 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 to wild- type cells (WT). Illustrative photomicrographs highlighting the (C) reduced PSA-NCAM (red) and (D) NCAM immunopositive signal (green) in PREP-overexpressing cells compared to wild- type cells. Nuclei were counterstained with DAPI (blue). Data are given as ratio of mean values in % ± s.e.m. (WT=100%). β-Actin was used as loading control. *p<0.05, **p<0.01, unpaired t-test, n=6 for WT and n=5 for PREP. Scale bar = 10 µm. Three independent experiments were performed.

Fig. 2 Detection of NCAM fragments in the culture medium, derived from wild-type and PREP-overexpressing cells Representative Western blot (A) and corresponding levels of degradation product of NCAM fragment 38-40 kDa in the culture medium derived from PREP-overexpressing cells, compared to culture medium derived from wild-type cells (B). Data are given as mean OD ratio as % of control ± s.e.m (WT=100%). **p=0.0043, unpaired t-test, n=4. Two independent experiments were performed.

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 (indicated with arrows). (B) Corresponding signal intensity analysis demonstrated restoration of PSA- NCAM in shPREP-transfected cells compared to 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. 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 1nM and 10 nM compared to vehicle-treated cells. Data are given as mean OD ratio as % of control ± s.e.m (untreated primary neurons or WT 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.

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). (B) Representative Western blot and corresponding levels of ST8Sia2 and ST8Sia4 protein expression in wild-type and PREP-overexpressing cells. β-Actin was used as loading control. Unpaired t-test, n=6. Scale bar = 10 µm. Two independent experiments were performed.

Fig. 5 Analysis of differentiation of 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 of Tuj-1 immunopositive cells ± s.e.m, ***p<0.0001, 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 WT and PREP-overexpressing SH-SY5Y neuroblastoma cells. Quantitative Western blot analysis demonstrating increased expression level of active form of MMP-9, but not proMMP-9 in PREP-overexpressing SH-SY5Y cells compared to 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 media of PREP-overexpressing SH-SY5Y neuroblastoma cells, compared to wild-type cells. Recombinant MMP-9 protein was used as an identification marker. Data are given as OD ratios ± s.e.m, **p<0.01, unpaired t-test, n=3. Two independent experiments were performed.

Fig. 7 Analysis of the effect of MMP-9 neutralizing antibody on the levels of NCAM fragments in PREP-overexpressing SH-SY5Y neuroblastoma cells Representative Western blot (A) and corresponding levels demonstrating reduced appearance of NCAM degradation fragment at 38-40 kDa in cell culture medium in SH- SY5Y neuroblastoma cells treated with neutralizing MMP-9 antibody for 96 hours as

compared to vehicle treated PREP-overexpressing cells (B). Data are given as mean OD ratio % of control ± s.e.m, (PREP-overexpressing cell culture medium serves as a control). ***p<0.001, unpaired t-test, n=6 for PREP-overexpressing cell culture medium and n=5 for PREP-overexpressing cell culture medium treated with MMP-9 neutralizing antibody. Two independent experiments were performed.

Fig. 8 Analysis of EGF and pEGF receptor levels in wild-type and PREP-overexpressing SH-SY5Y neuroblastoma cells Representative Western blot and corresponding levels of EGF-receptor and pEGF- receptor levels. Data are given as mean OD ratio as % of control ± s.e.m (WT=100%). β- Actin was used as loading control. **p<0.01, unpaired t-test, n=4. Two independent experiments were performed.