© 2016. Published by The Company of Biologists Ltd.

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: Kulli.Jaako@ut.ee

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.

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JCS Advance Online Article. Posted on 26 August 2016

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.

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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;

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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

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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

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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

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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).

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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

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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.

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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.

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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

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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.

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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 g/ml gentamycin for wild-type cells; and additionally 200 g/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 M 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

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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 g/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 g/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

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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 m 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

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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 m 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-

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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

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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 l in three parallels. The

reaction mixture consisted of 1 l First Strand cDNA diluted template, 5 l 2x Master SYBR

Green qPCR Master Mix (Applied Biosystems Inc, USA), 3 l H20 and 1 l gene-specific 10

M 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

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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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Figures

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).

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Data are given as ratio of mean values in % ± s.e.m. (WT=100%). -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.

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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.

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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 (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

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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%). -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.

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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. -Actin was used as loading control. Unpaired t-test, n=6. Scale bar =

10 µm. Two independent experiments were performed.

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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 m. (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.

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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. -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 m. (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.

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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.

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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%). -Actin was

used as loading control. **p<0.01, unpaired t-test, n=4. Two independent experiments were

performed.

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Fig S.1

Quantification of PREP protein level and enzyme activity in wild-type and PREP-

overexpressing SH-SY5Y neuroblastoma cells

(A) Representative Western blot of total PREP protein in wild-type (WT) and PREP-

overexpressing (PREP) SH-SY5Y neuroblastoma cells. β-Actin was used as a loading

control for statistical analysis of the PREP expression level in WT and PREP-

overexpressing SH-SY5Y cells. Data are expressed as OD ratio ± s.e.m. (p<0.001, n=4, t-

test) (B) PREP activity measured by using substrate Z-Gly-Pro-pNA in WT and PREP-

overexpressing SH-SY5Y cell lysates. ***p<0.0001, unpaired t-test, n=3.

J. Cell Sci. 129: doi:10.1242/jcs.181891: Supplementary information

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Fig S.2

Quantification of MMP-2, MMP-3 and ADAM-10 protein levels in wild-type and

PREP-overexpressing SH-SY5Y neuroblastoma cells

Representative Western blot and corresponding analysis of total MMP-2 (left panel),

MMP-3 (central panel) and ADAM-10 (right panel) protein levels in wild-type (WT) and

PREP-overexpressing (PREP) SH-SY5Y neuroblastoma cells. β-Actin was used as a

loading control. Data are expressed as OD ratio % of control (WT=100%) ± s.e.m.

(p>0.05, unpaired t-test, n=6 for MMP-2, n=6 for MMP-3 and n=4 for ADAM-10). Two

independent experiments were performed.

J. Cell Sci. 129: doi:10.1242/jcs.181891: Supplementary information

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Table 1. The Ct/deltaCt values for NCAM120, NCAM140, NCAM180 and

totalNCAM, assessed by qPCR

WT

NCAM

120

PREP

NCAM

120

WT

NCAM

140

PREP

NCAM

140

WT

NCAM

180

PREP

NCAM

180

WT

NCAM

TOTAL

PREP

NCAM

TOTAL

Average

Ct(NCAM)

25.77 25.87 21.85 22.02 23.97 23.94 21.04 21.12

Ct(reference

genes)

20.37 20.36 20.37 20.36 20.37 20.36 20.37 20.36

delta Ct 5.40 5.51 1.48 1.66 3.60 3.58 0.67 0.76

N 6 6 6 6 6 6 6 6

J. Cell Sci. 129: doi:10.1242/jcs.181891: Supplementary information

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