Membrane-associated glycoprotein neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) play an important role in brain plasticity by regulating cell–cell interactions. Here, we demonstrate that the cytosolic serine protease prolyl endopeptidase (PREP) is able to regulate NCAM and PSA-NCAM. Using a SH-SY5Y neuroblastoma cell line with stable overexpression of PREP, we found a remarkable loss of PSA-NCAM, reduced levels of NCAM180 and NCAM140 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 were found in the concentrated medium derived from PREP-overexpressing cells. 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 receptor (EGFR) signaling, which in turn activates MMP-9. In conclusion, our findings provide evidence for newly-discovered roles for PREP in mechanisms regulating cellular plasticity through NCAM and PSA-NCAM.
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 (also known as NCAM1) is a cell surface glycoprotein that is represented by at least three isoforms (NCAM120, NCAM140 and NCAM180, with molecular weights of 120, 140 and 180 kDa, respectively) that differ in their cytoplasmic domains and attachment to the plasma membrane (Cunningham et al., 1987). The extracellular part of NCAM consists of five immunoglobulin-like modules (IgI–IgV) 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 post-translational 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 and 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 proteolytic 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 residues (Fülöp et al., 1998; Polgár, 2002; Rawlings et al., 1991). PREP is widely distributed in the brain (Irazusta et al., 2002; Myöhänen et al., 2007, 2008) and increased activity of PREP has been associated with cell death processes in various neurodegenerative diseases, including Alzheimer's and Parkinson's diseases (Brandt et al., 2008; Mantle et al., 1996). PREP is believed to act in the extracellular space with involvement 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 to be the mechanism underlying 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 have 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, 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 leading to the production of neurotoxic mediators, which in turn mediate pathology progression (for review, see Penttinen et al., 2011).
In this report, for the first time, we demonstrate that PREP is able to regulate levels of NCAM through activation of MMP-9.
Levels of PSA-NCAM and NCAM protein and mRNA in neuroblastoma SH-SY5Y cells overexpressing PREP
Increased levels of PREP protein and its activity in PREP-overexpressing SH-SY5Y cell lysates were confirmed by western blot and activity measurements (Fig. S1A,B).
PREP was detected on SDS-PAGE as a band at ∼80 kDa (Fig. S1A). A 30-fold increase in PREP protein level was found in SH-SY5Y cells overexpressing PREP compared with wild-type cells (P<0.001, t-test, n=4). In these PREP-overexpressing cells, PREP activity assays demonstrated an 11-fold increase in 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 was detected as a high-molecular-weight smear typical for highly polysialylated NCAM (Fig. 1A), whereas by comparison, PSA-NCAM signal was almost absent in PREP-overexpressing cells (P<0.0001, t-test, n=5–6; Fig. 1A). In wild-type cells, immunocytochemistry identified 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 levels of the NCAM protein isoforms (NCAM180, NCAM140 and NCAM120). A significant decrease in levels of the NCAM 180 kDa immunoreactive band (P=0.01, t-test, n=5–6) and the NCAM 140 kDa immunoreactive band (P=0.01, t-test, n=5–6) was found in PREP-overexpressing neuroblastoma cells compared with 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; Fig. 1B). In wild-type cells, immunostaining for NCAM demonstrated an intensive positive signal for NCAM, whereas only a moderate NCAM-positive signal could be detected in PREP-overexpressing cells (Fig. 1D).
Next, we aimed to elucidate whether the observed changes in the levels of NCAM protein isoforms were caused by the changes in transcription level. We measured mRNA levels of the 120, 140 and 180 isoforms as well as total NCAM using qPCR. No significant differences in mRNA level between wild-type and PREP-overexpressing cells were found for any form of NCAM (Table S1). Thus, the increase in the NCAM 120 kDa immunoreactive band in PREP-overexpressing cells does not result from increased expression of NCAM120 mRNA.
Based on these results, we proposed that the increase in NCAM120 results from the degradation of NCAM140 and/or NCAM180 isoforms. To test this, 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 of ∼46–48 kDa, and another of ∼38–40 kDa. In the medium of PREP-overexpressing cells, there was a significant increase in the level of the 38–40 kDa fragment compared with medium from wild-type cells (P=0.0043, t-test, n=4; Fig. 2A,B), whereas no changes were observed in the levels of the 46–48 kDa fragment. These data support our hypothesis that the overexpression of PREP induces increased degradation of the NCAM180 and/or NCAM140 isoforms.
Subsequently, we aimed to evaluate whether the observed changes in PSA-NCAM levels in PREP-overexpressing cells are PREP-specific. PREP overexpression was knocked down in PREP-overexpressing cells through transfection with shRNA against PREP (shPREP) or a control vector (shNC), together with a plasmid expressing green fluorescent protein (GFP). After 72 h cells were fixed and processed for PSA-NCAM immunocytochemistry. GFP-positive cells from the shPREP and shNC groups 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 with shNC-transfected PREP-overexpressing cells (P=0.043, t-test, n=10; Fig. 3A,B). This result confirms that alterations in PSA-NCAM are PREP-specific.
Addition of recombinant PREP to the culture medium decreases PSA-NCAM expression in wild-type SH-SY5Y neuroblastoma cells and primary cortical neurons
Human recombinant PREP (rPREP) was added to the cell culture medium of wild-type SH-SY5Y cells (1 nM or 10 nM) and primary cortical neurons (1 nM) for 72 h. A marked decrease in PSA-NCAM level was found in wild-type SH-SY5Y neuroblastoma cells (F=10.26, P=0.001, one-way ANOVA followed by Bonferroni post-hoc test, n=3) after addition of rPREP (Fig. 3C). A similar decrease in PSA-NCAM level (P=0.03, t-test, n=5–6) was found in 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 the possibility that PREP affects ST8Sia2 and ST8Sia4, wild-type and PREP-overexpressing cells were subjected to ST8Sia2 and ST8Sia4 immunocytochemistry and protein expression analysis (Fig. 4A,B). No differences in expression of either ST8Sia2 or ST8Sia4 were observed by immunocytochemistry (Fig. 4A), and western blot analysis demonstrated no statistically significant difference in ST8Sia2 or ST8Sia4 protein levels between wild-type and PREP-overexpressing cells (Fig. 4B).
Impaired differentiation of PREP-overexpressing cells
As NCAM and PSA-NCAM are involved in 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, 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 achieve long-term survival of cells and a 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 numbers of PREP-overexpressing cells were positive for Tuj-1 (Fig. 5A). Quantification revealed that the percentage of Tuj-1-positive cells was significantly lower in PREP-overexpressing cells compared with wild-type cells (P<0.0001, t-test, n=9; Fig. 5B), indicating that alterations in NCAM molecule integrity lead to impaired differentiation toward a 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 only able to degrade relatively small peptides (<3 kDa) (Fülöp et al., 1998; Polgár, 2002; Rawlings et al., 1991). Therefore, it seemed unlikely that PREP itself is able to degrade NCAM. It has been demonstrated previously that NCAM molecule degradation might be mediated by the matrix metalloproteases (MMPs) (Brennaman et al., 2014; Hinkle et al., 2006; Hübschmann et al., 2005; Shichi et al., 2011). Therefore, to test whether increased expression and activity of PREP might be involved in the activation of MMPs, we measured the expression levels and activity of MMP-9. Increased levels of the active form of MMP-9 was found in PREP-overexpressing SH-SY5Y neuroblastoma cells compared with wild-type cells (P=0.001, t-test, n=3), whereas no changes were found in levels of the inactive form of MMP-9 (Fig. 6A). Moreover, immunocytochemical labeling of MMP-9 in PREP-overexpressing cells revealed intense granulation of the immunopositive signal mostly at the perinuclear region, whereas in wild-type cells MMP-9 signal was associated with a sparse granulation pattern (Fig. 6B). In addition, zymography demonstrated increased proteolytic activity of MMP-9 in the serum-free medium from cultured PREP-overexpressing cells compared with wild-type cells (P=0.002, t-test, n=3; Fig. 6C).
To test whether the observed increases 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 h. A reduced level of NCAM fragments was found in the cell culture medium (P<0.001, t-test, n=5-6) compared with vehicle-treated PREP-overexpressing cells (Fig. 7A,B), indicating that MMP-9 is involved in the regulation of NCAM degradation.
To elucidate whether PREP overexpression could affect other MMPs that 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 these MMPs in PREP-overexpressing cells as compared with wild-type cells (Fig. S2).
Decreased EGFR and increased pEGFR expression in neuroblastoma SH-SY5Y cells overexpressing PREP
It has previously been demonstrated that EGFR signaling is involved in release and activation of MMP-9 (da Rosa et al., 2014; Pei et al., 2014; Qiu et al., 2004). Therefore EGFR and phosphorylated EGFR (pEGFR) levels were measured and in PREP-overexpressing cells overall decrease in total EGFR was found (P=0.001, t-test, n=4; Fig. 8). In contrast, the levels of pEGFR were increased (P=0.001, t-test, n=4; Fig. 8).
This study shows for the first time that PREP is involved in the regulation of neural cell adhesion molecules. A PREP-overexpressing neuroblastoma SH-SY5Y cell line was used as an in vitro model to mimic pathological conditions resulting from 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; we also found alterations in the levels of all NCAM protein variants tested. We found a reduction in NCAM 180 kDa and 140 kDa immunoreactive bands and an increase in the NCAM 120 kDa immunoreactive band. As PREP might affect PSA-NCAM and NCAM through 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 or 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), suggesting that PREP does not affect the polysialylation of NCAM. Therefore, we propose that the reduction in the 140 kDa and 180 kDa protein variants of NCAM and the reduction in PSA-NCAM result from 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 anti-NCAM antibody, was found. Thus, it seems that the observed decrease in NCAM immunoreactive bands of 180 kDa and 140 kDa does indeed result from 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 cleaved fragments of NCAM. However, NCAM fragments at 38–40 kDa indicate that the cleaved fragment is too small to include the polysialylated IgV domain and therefore additional mechanisms, such as increased PSA cleavage by neuraminidases, in addition to NCAM degradation by MMP-9, might be involved. In mammals, four sialidases are present and are involved in several key physiological events related to cancer transformation (Miyagi et al., 2003; Proshin et al., 2002; Tringali et al., 2012; Wada et al., 2007) and in developmental processes, such as lamination of newly generated hippocampal granule cells through the modulation of PSA (Sajo et al., 2016). The question of whether or not PREP overexpression affects sialidases needs further investigation.
PREP is peptidase that cleaves small peptides (<3 kDa) (Fülöp et al., 1998; Polgár, 2002; Rawlings et al., 1991) and it seems unlikely that this enzyme can directly catalytically degrade the NCAM molecule. Previous studies have demonstrated that several metalloproteases, such as MMP-2 and MMP-9, as well as the ADAM family of metalloproteases, target NCAM (Brennaman et al., 2014; Hinkle et al., 2006; Hübschmann et al., 2005; Shichi et al., 2011). It has also been shown that inhibition of MMP-2 and MMP-9 prevents 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 on the PREP-mediated decrease in PSA-NCAM and NCAM. We measured 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 this increased level of MMP-9 was specific to its active form. Using zymography, we also observed increased MMP-9 activity. Thus, it seems that PREP overexpression induces a selective increase in the expression and activity of MMP-9, which is 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 NCAM180 and NCAM140 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 the aforementioned fragments cleaved by ADAMs, a 65 kDa degradation product of NCAM has been demonstrated to result from the action of MMP-9 and/or MMP-2 after cerebral ischemic neuronal damage and the appearance of this fragment was reduced when MMPs were suppressed (Shichi et al., 2011). The precise cleavage site through which these smaller fragments are produced is not known; however, it might be proposed that the origin of the fragment found in cell culture medium might represent an N-terminal domain of NCAM. The appearance of fragments of ∼40 kDa, derived from extracellular domains of NCAM, into cell culture medium might also explain the shift in western blot bands from strong bands at 180 kDa and 140 kDa to bands with lower apparent molecular weight and their accumulation at the range of 120 kDa.
The mechanism by which PREP induces MMP-9 activation is not known, although several explanations might be proposed. PREP might activate MMP-9 by degrading physiological tissue inhibitor metalloproteases (TIMPs), as cells secrete pro-MMPs bound to TIMPs in a complex and TIMP processing is needed for zymogen activation. It has been demonstrated that in vitro conditions, cleavage, degradation or chemical modification by proteolytic and non-proteolytic mechanisms are responsible for inactivation of TIMPs through the involvement of 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, through 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 EGFR, the levels of phosphorylated (active form) EGFR 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 post-synaptic 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 might 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,b). 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 re-expressed 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 seems 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 have shown that application of endoneuraminidase (EndoN) treatment for PSA removal triggers 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 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 through 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 an 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 mice have demonstrated the association of PREP in the processes modulating neuroplasticity through inflammatory response. In PREP-knockout mouse 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 resulting from 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 the 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 by 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
The 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, UK) 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 amounts 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 for 30 min at 405 nm using an ELISA plate reader (Tecan, Crailsheim, Germany) and PREP activity was calculated using a 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 (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, UK) for 10 min 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, UK) containing 10% heat-inactivated fetal calf serum (Invitrogen), 25 mM KCl, 2 mM GlutaMAX-I (Invitrogen, UK) and 100 µg/ml gentamycin (KRKA). Cells were plated onto poly-L-lysine-coated (Sigma-Aldrich) cell culture dishes at a density of 1.8×105 cells/cm2. After 2.5 h, the medium was changed to Neurobasal-A medium (Gibco). Cultures were incubated for 4 days in an atmosphere of 95% air and 5% CO2 at 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) 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 and added into cell culture medium at concentrations of 1 nM and 10 nM for wild-type SH-SY5Y neuroblastoma cells and 1 nM for primary cortical neurons. Cells were incubated for 72 h 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 h at a concentration of 50 µg/ml in serum free DMEM/GlutaMAX-I medium. Detection of NCAM fragments was done according to the method of Brennaman and colleagues (2014). After 96 h, cell culture medium was collected, treated with protease inhibitors (Roche), centrifuged for 5 min (173 g), followed by concentration with Millipore centrifugal concentrators according to the manufacturer's instructions (30,000 MW cutoff, 10× concentration). Protein concentrations from each concentrated cell culture media sample were measured by Bradford method and equivalent amount of proteins from media samples were resolved by electrophoresis on 10% SDS-polyacrylamide gel as described below. In addition, a blank control sample was prepared and resolved by electrophoresis. The blank control consisted of pure DMEM cell culture medium and MMP-9-neutralizing antibody concentrated under similar conditions to the experimental samples.
PREP expression knockdown 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 (3.5 cm diameter), the conditioned medium was replaced with 120 µl Opti-MEM medium (Gibco) containing 2% Lipofectamine 2000 transfection reagent (Thermo Scientific) with either empty vector (control) or validated PREP (shPREP) shRNA plasmid (KH20237N; SA Biosciences, MD, USA) and pGFP (632370; Clontech). The dishes were incubated for 3 h, after which fresh DMEM medium was added. The transfected cells were allowed to express the shRNA for 72 h before immunocytochemical detection for PSA-NCAM was performed. Immunopositive signals were detected with 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 at 1 µm intervals using a 40× (water) objective and further analyzed for signal intensity. Fluorescence quantification analysis was performed using ImageJ software (NIH). 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 Gavet and Pines (2010). CTCF was calculated by multiplying the area (in square pixels) of the selected cell of interest by mean background fluorescence intensity and subtracting the result from whole-cell fluorescence intensity. Overlapping of 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 min at room temperature, permeabilized in 0.05% Triton X-100 (Sigma-Aldrich) for 30 min, and rinsed in PBS. Non-specific binding was blocked by 2% normal goat serum (Vector Laboratories) in PBS containing 0.3% Triton X-100 for 1 h, followed by 48 h of incubation at 4°C with different primary antibodies diluted in blocking buffer based on the requirement of the experiment. Primary antibodies 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 (1:1000; A-21044), Alexa Fluor 488 goat anti-rabbit IgG (1:1000; A-11008), Alexa Fluor 594 goat anti-rabbit IgG (1:1000; A-11012) or Alexa Fluor 488 goat anti-mouse IgG (1:1000; A-11001) (all from Life Technologies) in blocking buffer for 1 h. Nuclei were counterstained with DAPI solution (Sigma-Aldrich; 300 nM in PBS).
A laser scanning confocal microscope (LSM 510 Duo, Zeiss, Germany) 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, nine randomly taken fields from three cell culture dishes (3.5 cm diameter) were taken at magnification ×40. 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 RIP-A cell 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 inhibitor cocktail), incubated on ice for 20 min and centrifuged (15,900 g for 20 min 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 Biosciences) in TBS containing 0.025% (v/v) Tween-20 (Sigma-Aldrich) or Odyssey blocking buffer (Li-Cor Bioscience). After blocking, the membranes were incubated overnight with different primary antibodies based on the requirement of the experiment. Antibodies 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 antibodies (1:4000; Thermo Scientific, 31460 and 32430, respectively), goat anti-rabbit IRDye 800 CW (926-32211) or 680 LT (926-68021), goat anti-mouse IRDye 800 CW (926-32210) or 680 LT (926-68020), donkey anti-chicken IRDye 680 CW (926-68028) (all at 1:10,000, Li-Cor Biosciences) for 1 h at 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 the same HRP-conjugated secondary antibody (1:4000), goat anti-mouse IRDye 680 LT or 800 CW (1:10,000) as above. The ratios 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 a percentage 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) according to the manufacturers' protocol. cDNA was synthesized 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). Primers were synthesized 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 2× Master SYBR Green qPCR Master Mix (Applied Biosystems), 3 µl H2O 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).
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 (Sigma-Aldrich) at 90 V. Gels were washed for 40 min in 2.5% Triton X-100, incubated for 48 h 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) for 1 h 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 presented are the mean±s.e.m., and experiments were repeated 2–3 times. Data were analyzed using GraphPad Prism 5 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.
The technical assistance of Mrs Olili Suvi was greatly appreciated. The authors would like to thank Dr. Rajeev Jain (Department of Pharmacology, Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia) for providing the plasmids.
The authors declare no competing or financial interests.
K.J., A.W.: conception and design of the study, conduction of experimental work, statistical analysis and interpretation of the data, drafting and revision of the manuscript. K.P., L.K., A.A.-H., K.A., A.N.: participation in the experimental work, conduction of immunocytochemistry, western blot and qPCR. M.G.: generation PREP-overexpressing SH-SY5Y cell line. R.V.E., A.-M.L., S.R., M.M., A.Z.: approval of study design, interpretation of the data, critical revision of the article, approval of the final version of the manuscript.
This study was supported by the 7th Framework Programme of Health of the European Commission [project NEUROPRO, HEALTH-F2-2008-223077 to S.R., A.-M.L., A.Z.], Eesti Teadusfondi (Estonian Science Foundation) [grant number 8740 to K.J.] and Eesti Teadusagentuur (Estonian Research Council) [Institutional research funding grant IUT2-3 to A.Z.], the Deutsche Forschungsgemeinschaft (German Research Foundation) [grants MO 2249/2-1 and MO 2249/2-2 to M.M within the PP 1608], the Alzheimer Forschung Initiative [grant number 1186 to M.M.], Universiteit Antwerpen [special research fund grant FFB3551 to A-M.L. and R.V.E] and Sächsische Aufbaubank (SAB)/European Social Fund (ESF) [grant number SAB100154907 to S.R., M.M., A.Z. and A.W.].
Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.181891.supplemental
- Received October 11, 2015.
- Accepted August 18, 2016.
- © 2016. Published by The Company of Biologists Ltd