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Published in final edited form as: J Neurochem. 2011 Oct 20;119(5):945–956. doi: 10.1111/j.1471-4159.2011.07479.x

CHARACTERIZATION OF NECTIN PROCESSING MEDIATED BY PRESENILIN-DEPENDENT GAMMA-SECRETASE

Jinsook Kim 1, Allison Chang 1, Amanda Dudak 1, Howard J Federoff 1,2, Seung T Lim 1
PMCID: PMC3217175  NIHMSID: NIHMS324053  PMID: 21910732

Abstract

Nectins play an important role in forming various intercellular junctions including synapses. This role is regulated by several secretases present at intercellular junctions. We have investigated presenilin (PS)-dependent secretase mediated processing of nectins in PS1 KO cells and primary hippocampal neurons. The loss of PS1/gamma-secretase activity delayed the processing of nectin-1 and caused the accumulation of its full-length and C-terminal fragments. Overexpression of PS2 in PS1 KO cells compensated for the loss of PS1, suggesting that PS2 also has the ability to regulate nectin-1 processing. In mouse brain slices, a pronounced increase in levels of 30 and 24 kDa CTFs in response to chemical LTP was observed. The mouse brain synaptosomal fractionation study indicated that nectin-1 localized to postsynaptic and preferentially presynaptic membranes and that shedding occurs in both compartments. These data suggest that nectin-1 shedding and PS-dependent intramembrane cleavage occur at synapses, and is a regulated event during conditions of synaptic plasticity in the brain. Point mutation analysis identified several residues within the transmembrane domain that play a critical role in the positioning of cleavage sites by ectodomain sheddases. Nectin-3, which forms hetero-trans-dimers with nectin-1, also undergoes intramembrane cleavage mediated by PS1/γ-secretase, suggesting that PS1/γ-secreatse activity regulates synapse formation and remodeling by nectin processing.

Keywords: ectodomain shedding, nectin-1, nectin-3, PS1, PS2, secretase

INTRODUCTION

Alzheimer's disease (AD) is a neurodegenerative disorder associated with progressive functional decline, dementia and neuronal loss initiating and progressing in a specific region and manner. In AD, synapse loss is an early event and is a structural correlate of cognitive dysfunction. Synapse loss exhibits a more prominent correlation with the severity of cognitive impairment than amyloid plaques or neurofibrillary tangles (Terry et al. 1991). Therefore, it is important to decipher the regulatory mechanisms governing the subtle alteration in synaptic function and synaptic connectivity for AD pathogenesis.

Nectins are members of immunoglobulin-like adhesion molecules involved in cell-cell adherens junctions (AJs) (Takai & Nakanishi 2003, Takai et al. 2003). In the central nervous system (CNS), nectin-1 and 3 localize at the pre- and postsynaptic sides of synapses formed in the CA3 pyramidal region of adult mouse hippocampi (Mizoguchi et al. 2002). Nectins link to the actin cytoskeleton via afadin. The nectin-1/afadin adhesion system co-localizes with components of the cadherin/catenin system in the stratum lucidum (Mizoguchi et al. 2002, Nishioka et al. 2000) as well as at excitatory synapses in rat hippocampal cultures (Lim et al. 2008). Mutations in the nectin-1 gene cause cleft lip/palate-ectodermal dysplasia and, in some cases, mental retardation (Suzuki et al. 2000, Sozen et al. 2001). Both nectin-1 and nectin-3 knockout mice exhibit an abnormal mossy fiber trajectory and a reduction in the number of puncta adherentia junctions (PAJs) at the synapses between mossy fiber terminals and dendrites of CA3 pyramidal cells (Honda et al. 2006), as well as a developmental defect of the vitreous body (Inagaki et al. 2005). Furthermore, hippocampal neurons derived from nectin-1-null mice exhibit a reduction in spine head width and an increase in spine length (Togashi et al. 2006). The trans-interaction between nectin-1 and nectin-3 plays a critical role in sustaining the normal association between axons and dendrites (Togashi et al. 2006). Nectin-1 is initially expressed at excitatory and inhibitory synapses but is progressively lost at inhibitory synapses during their maturation (Lim et al. 2008). These data suggest that nectins play an important role in synapse formation and synaptic plasticity.

Nectin-1 undergoes ectodomain shedding upon treatment with SF/HGF or TPA in MDCK cells (Tanaka et al. 2002) and in CHO cells (Kim et al. 2002), generating a large soluble fragment and a small C-terminal fragment (CTF). Shedding of nectin-1 also occurs at the synapses in mature hippocampal neurons (Lim et al. 2008), depending on postsynaptic NMDA receptor activation and activation of calmodulin (Kim et al. 2010). ADAM10 is responsible for the α-secretase cleavage event (Kim et al. 2010). Interestingly, nectin-1 undergoes intramembrane proteolytic processing analogous to that of APP, mediated by a presenilin (PS)-dependent γ-secretase (Kim et al. 2002). This observation is consistent with a previously reported role for PS1/γ-secretase in AJ function involving cadherin cleavage (Marambaud et al. 2002, Baki et al. 2001, Marambaud et al. 2003).

PS1 is a component of γ-secretase, which is an aspartyl protease that cleaves membrane-anchored truncated C-terminal products produced by ectodomain shedding (Medina & Dotti 2003). Mutations in the genes of PS1 and its homologue, PS2, are responsible for most cases of early-onset autosomal dominant familiar AD (FAD) (Sherrington et al. 1995). Both PS1 and PS2 can function independently within the γ-secretase complexes (Beel & Sanders 2008, St George-Hyslop 2000) and act as the catalytic component in the hetero-multimeric γ-secretase enzyme complexes (Wolfe 2006, De Strooper 2003). PS1 is localized at synaptic and epithelial cell-cell contact sites and forms complexes with the N-cadherin-catenin system (Baki et al. 2001, Georgakopoulos et al. 1999). It has been shown that the proteolytic release of the extracellular domain of transmembrane proteins, followed by intramembrane PS/γ-secretase-mediated cleavage is a novel mechanism for signal transduction (Parks & Curtis 2007, De Strooper & Annaert).

FAD mutations in PS genes may directly perturb synaptic activity by aberrantly modulating cell adhesion molecule processing including nectins. This altered processing could ultimately lead to synaptic dysfunction, which is the earliest detectable abnormality in AD. Therefore, understanding nectin processes mediated by PS/γ-secretase at synapses is potentially of clinical relevance. In this study, we further characterized the nectin-1 and nectin-3 processing mediated by PS/γ-secretase.

EXPERIMENTAL PROCEDURES

Cell lines, antibodies, and plasmids

Wild-type (PS1 +/+), PS +/-, and PS1 -/- mouse embryonic fibroblast (MEF) were generated as described previously (Berechid et al. 1999, Wong et al. 1997). COS-7 and HEK-293 cells were purchased from American Type Culture Collection. All cell lines were maintained in DMEM supplied with 10% of fetal bovine serum with antibiotics (Invitrogen, Carlsbad, CA). Rabbit anti-nectin-1 and l-afadin antibodies were prepared as described (Lim et al. 2008). Mouse anti-AF-6 and anti-N-cadherin antibodies were from BD Transduction Laboratory. Mouse anti-flag (m2), anti-PS1 CTF, and rabbit anti-actin antibodies were purchased from Sigma. Rabbit anti-nectin-3 (PVRL3) antibody was purchased from ProteinTech Group. Human nectin-1 (a gift from Dr. Patricia G Spear, Northwestern University) was flag tagged at the C-terminus and inserted into the pVAX vector. Point mutants were made using the Quick-change site-directed mutagenesis kit (Stratagene, La Jolla, CA). Sequence fidelity of all constructs and PCR inserts was verified by sequencing. Nectin-3α was cloned from mouse brain cDNA library. V5 epitope tag was inserted at the C-terminus of nectin-3α. PS1 and PS2 (a gift from Dr. John Hardy, NIH, MD) were tagged with V5 and subcloned into the pVAX vector.

Biotin labeling of cell surface proteins

PS1 -/- cells were cultured for 24 h in Dulbecco's modified Eagle's medium containing 10% FBS. Cells were washed twice with PBS, and surface proteins were labeled with Sulfo-NHS-SS-Biotin (Pierce) in PBS while gently shaking at 4 °C for 30 min. Fifty μl of quenching solution was added to cells at 4 °C and washed twice with TBS. Cells were collected in 1000 μl of lysis buffer, disrupted by sonication on ice, incubated for 5 min on ice, and clarified by centrifugation (10,000 × g, 2 min). To isolate biotin-labeled proteins, lysate was added to immobilized NeutrAvidin™ gel (50 μl) and incubated for 1 h at room temperature. Gels were washed three times with wash buffer and incubated for 10 min with SDS-PAGE sample buffer. Samples were analyzed by immunoblotting.

Transient transfection and immunoblot Analysis

Cells were transfected using LipoD293™ (SignaGen, Ijamsville, MD) according to manufacturer's protocol. For each experiment, 24 h after plating, cells were transfected with a freshly prepared DNA-LipoD293™ complex that contained the DNA of interest at a final concentration of 20 mg/ml. After 1 h incubation at 37°C, the media was exchanged with fresh DMEM. Transfected cells were lysed in reducing sample buffer 48 h after transfection. Samples were loaded and fractionated on 12% SDS-PAGE gels and transferred onto a nitrocellulose membrane. Anti-nectin-1 cytoplasmic antibodies were used for immunoblotting. All experiments were repeated three or four times.

Viral production and transduction

Nectin-1 with a C-terminus flag tag, nectin-3 with v5 epitope tagged at its C-terminus, and v5-epitope tagged PS1 and PS2 were subcloned into the pAd-Track vector (a kind gift from Dr. Vogelstein, Johns Hopkins University School of Medicine, Baltimore MD). Recombinant adenoviruses were generated as previously described (He et al. 1998).

Ionomycin (IM) treatments

To block the γ-secretase dependent cleavage of nectin-1 CTFs, PS1 +/+ and -/- MEF cells were treated with 1 μM γ-secretase inhibitor (Calbiochem, San Diego, CA) for 1 h before inducing the shedding process with 5 μM IM for 5 min. Cell homogenates of PS +/+ and -/- fibroblasts were prepared by directly lysing in RIPA buffer. After quantification with a BCA protein assay, 100 μg of proteins were loaded on 12% SDS-PAGE gels, blotted onto nitrocellulose membranes (Bio-Rad, Hercules, CA) and probed with antibodies as indicated in the figure legends.

Immunostaining and microscopy

MEF cells were fixed with methanol for 10 min at -20°C. COS-7 cells transfected with nectin-1 and its mutants were fixed in 3% PFA. After fixations, non-specific binding sites were blocked by incubation for 30 min at room temperature in Blotto-T (4% non-fat dry milk powder in 20 mM Tris, pH7.5, 150 mM NaCl, 1% TritonX-100). Cells were incubated for 1 h at room temperature with primary antibodies as described in legends. Cells were washed three times in Blotto-T to remove excess primary antibodies. Immunostaining was visualized by incubation with the following appropriate secondary antibodies at a 1:2,000 dilution: Alexa Fluor 488-goat anti-mouse IgG and Alexa Fluor 594-goat anti-rabbit IgG. Coverslips were mounted on microscope slides in mowiol (Calbiochem). Fluorescent images were captured using Scion imaging 1.60 software on an Axioskop camera (Zeiss) with a 40X, 1.4 N. A. lens. Images were prepared for presentation using Adobe Photoshop software.

Primary rat hippocampal culture and drug treatment

Primary rat hippocampal neurons were prepared from Sprague-Dawley E18 rat embryos (Taconic Labs, Germantown, NY). For biochemical experiments, single cell suspensions were plated on PEI-coated 12-well tissue culture plates (3 × 105 cells per well) and maintained in Neurobasal Medium with B27 supplement with 1:1 media changes every 3-4 days. Ara-C (5 μM) was added at DIV 4 to prevent proliferation of non-neuronal cells. For proteasomal inhibition, neurons at 28 days in vitro (DIV) were treated with 10 μm lactacystin in the presence or absence of 1 μm γ-secretase inhibitor X. Twenty-four h after treatment, cells were collected in 100 μl reducing sample buffer. For N-Methyl-D-aspartic acid (NMDA) treatment, neurons at 28 DIV were pretreated with 100 μM APV and 2 mM EGTA for 30 min, followed by 50 μM NMDA for 15 min. Cells were collected in 100 μl reducing sample buffer. For nectin-3 experiments, neurons at 14 DIV were infected with recombinant adenovirus expressing nectin-3α-v5 at an MOI of 200. Twenty-four h after infection, neurons were treated with 1 μM γ-secretase inhibitor X for 24 h. Cell homogenates for primary neurons were prepared by directly solubilizing them in 100 μl reducing SDS sample buffer. Equal volumes of cell lysates were separated on 12% Tris-Glycine gels, blotted onto nitrocellulose membranes (Bio-Rad, Hercules, CA) and probed with antibodies as indicated in the figure legends. Chemiluminescence signals were detected by BioMAX (Kodak, Rochester, NY) or a cooled-CCD device, BioChemi System (UVP Inc., Upland, CA).

Preparation of hippocampal slices and induction of LTP

4-6 week old mice were anesthetized by intraperitoneal injections of ketamine/xylazine at 80/10 mg/kg and submitted to hypoxia for 5-10 min prior to sacrifice. Hippocampal slices were dissected out from 400 μm thick whole brain coronal sections in cold a cerebrospinal fluid (aCSF), bubbled with 95% O2/5% CO2. Slices were maintained in aCSF containing (in mM) NaCl, 125; KCl, 2.5; MgSO4, 1.3; NaH2PO4, 1.0; NaHCO3, 26.2; CaCl2, 2.5; glucose, 11.0, with oxygenation for 1 h prior to the experiment. For chemical induction of LTP, Sp-cAMP (50 μM, Sigma) was bath applied for 15 min; slices were then transferred to aCSF and harvested after the time indicated in the figure legends.

Mouse brain subcellular fractionation

Cerebral cortices from ten adult mice were homogenized and fractionated (Carlin et al. 1980, Lee et al. 2001). Brains were homogenized in buffered sucrose (320 mM sucrose, 2 mM DTT, 1 mM EGTA, 1 mM EDTA, 4 mM HEPES·KOH, pH 7.4), supplemented with protease inhibitors, by twelve strokes in a Teflon-glass homogenizer. Cell debris and nuclei (P1) were removed by centrifugation for 10 min at 1,100g. The postnuclear supernatant (S1) was centrifuged for 10 min at 9,200g, and the resulting pellet was resuspended in the buffered sucrose. The resuspended pellet was further centrifuged for 15 min at 10,200g to obtain the washed synaptosomal fraction (P2). The washed P2 was lysed by addition of 10 volumes of water and homogenization in a Teflon-glass homogenizer. The synaptosomal membrane-enriched fraction (LP1) was collected by centrifugation at 25,000g for 20 min. The supernatant (LS1) was further centrifuged at 165,000g for 2 h to sediment LP2 (synaptic vesicle-enriched fraction), leaving the remaining soluble fraction, LS2.

Immunoblot analysis and statistical data analysis

Densitometry was preformed on VisionWorksLS (UVP BioImaging Systema, Cambridge, UK). Average densities of samples were calculated relative to the control from three independent Western blots, and ratios of relative densities were determined. Y-error bars were determined by calculating the standard deviations. P-values were calculated by utilizing T-tests, which determine if the ratios of densities were significantly different. P-values of less than 0.05 are typically considered significant.

RESULTS

PS1 dependent γ-secretase regulates nectin-1 processing

To investigate the effects of the loss of PS1 activity on the regulation of nectin-1 processing, we examined the endogenous expression level of full-length and CTFs of nectin-1 in homogenates prepared from PS1 +/+ (homozygous), +/- (heterozygous), and -/- (nullizygous) MEF cells. The absence of PS1 in the nullizygous cells was confirmed by immunoblotting with PS1 C-terminal specific antibody (Fig 1A). High expression of full-length nectin-1 was detected in PS1 -/- cells compared to both homozygous and heterozygous cell lines (Fig. 1A and 1B) under basal conditions. Densitometry analysis indicated that the expression level of full-length nectin-1 is 15 and 26 times higher in PS1 +/- and -/- cells, respectively, than that of PS1 +/+ cells (Fig. 1B). The accumulation of a 30 kDa band (we named as α-CTF), which is a product of ADAM10 activity (Kim et al. 2010), was also detected in PS1 +/- and -/- cells, whereas it was less prominent in homozygous cells (Fig. 1A), indicating that it is rapidly processed by a down-stream processor in homozygous cells. Interestingly, treating wild-type and PS1 +/- MEF cells with γ-secretase inhibitor for 24 h did not significantly increase the expression level of full-length nectin-1 (Fig. 1C), suggesting that the accumulation of nectin-1 in PS1 +/- and -/- MEF cells may be not solely due to the loss of γ-secretase activity, but perhaps to changes in gene expression of nectin-1. A 36 kDa-CTF was more prominent in homozygous cells than in PS1 +/- and -/- cells (Fig. 1A). We named the 36 kDa-CTF as CTF1. We also examined the level of afadin, which links nectin to the F-actin cytoskeleton. Two major isoforms of afadin have been identified: l-afadin and the shorter splice variant, s-afadin (Mandai et al. 1997). S-afadin lacks the actin filament binding domain and the third proline-rich domain (Mandai et al. 1997, Takahashi et al. 1999). In MEF cells, a single band of afadin was detected with AF-6 antibody (Fig. 1D), which recognizes both isoforms of afadin. This band was also recognized by l-afadin specific antibody (Fig. 1D), indicating that these MEF cells mainly express l-afadin. The level of l-afadin expression was similar in these MEF cells (Fig. 1E), indicating that the increased amount of nectin-1 did not affect the expression level or stability of l-afadin.

Figure 1. Full-length and α-CTF of nectin-1 are accumulated in PS1 -/- cells.

Figure 1

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A. The comparison of full-length and CTFs of nectin-1 among PS1 +/+, +/-, -/- MEF cells. Equal amounts of cell lysate (50 μg) were loaded for Western blot analysis. Compared with wild-type MEFs, a significant accumulation of full-length nectin-1 (100 kDa) and the α~-CTF (30 kDa) were observed in both PS1 +/- and -/- fibroblasts. Equal loading was shown by beta-actin immunoblot. An immunoblot was also probed with PS1 C-terminal fragment specific antibody to confirm the loss of PS in PS1 -/- cells. B. Relative density ratio of full-length nectin-1. The intensity of full-length nectin-1 was measured by densitometric analysis in three independent immunoblots. The level of full-length nectin-1 in PS -/- cells increased by 25 fold compared to that in wild-type MEF cells. C. Wildtype and PS1 +/- MEF cells were treated with 1 or 2 μM gamma-secretase inhibitor for 24 h, cells were lysed in reducing sample buffer and analyzed on 12% SDS-PAGE. Samples were transferred to nitrocellulose, and the blot was probed with anti-nectin-1 antibody. D. The expression level of AF-6 among PS1 +/+, +/-, and -/- MEF cells. Equal amounts of cell lysate (50 μg) were loaded for Western blot analysis. The blot was initially incubated with AF-6, which recognizes both l- and s-afadin, then, the same blot was stripped and reprobed with l-afadin specific antibody. Equal loading was shown by beta-actin immunoblot. E. Relative ratio of lafadin. The intensity of l-afadin was measured by densitometric analysis in three independent immunoblots. The level of l-afadin was similar in all three. F. Subcellular distribution of nectin-1 (left panel; red) and N-cadherin (middle panel; green) in PS1 +/- and -/- cells. Nectin-1 immunoreactivity was more intense in PS1 -/- cells as compared to the staining in the PS +/-cells. Nectin-1 immunostaining was colocalized with that of N-cadherin at cell-cell contact sites in the merged image (right panel; yellow). G. Subcellular distribution of l-afadin (left panel; red) and N-cadherin (middle panel; green) in PS1 +/- and -/- cells. L-afadin immunostaining was highly enriched at the cell-cell adhesion sites, visualized by N-cadherin staining. The experiment was repeated three times with similar results. Scale bar = 20 μm.

The analysis of nectin-1 immunostaining further supported the accumulation of nectin-1 at cell-cell adhesion sites in PS1 -/- MEF cells. Nectin-1 immunoreactivity was much stronger in PS1 -/- cells compared to the immunostaining in PS1 +/- cells, and was distributed in a continuous belt along the regions of cell-cell contact (Fig. 1F). We were not able to detect nectin-1 by immunocytochemistry in wild-type cells due to low expression level of endogenous nectin-1 (data not shown). In PS1 -/- cells, nectin-1 and N-cadherin were detected at cell-cell contacts sites, and both molecules exhibited punctate immunostaining and co-localization (Fig. 1F). To examine whether the accumulation of nectin-1 at the cell-cell contact site was due to the full-length nectin-1 or CTFs, we measured cell surface proteins in PS1 -/- cells by biotin-labeling live cells and isolated biotin-labeled nectin-1 from lysates with avidin beads. The biotinylated nectin-1 was analyzed by immunoblotting. We only detected full-length nectin-1, but no CTFs (Supplemental Fig. 1), indicating that the nectin-1 immunostaining at the cell-cell surface was mainly due to the accumulation of the full-length nectin-1.

A similar subcellular distribution was observed for l-afadin (Fig. 1G; lower panel), suggesting that accumulated nectin-1 further recruited afadin to cell-cell contact sites. Afadin immunoreactivity was frequently located in the cytoplasm of the PS1 +/- cells (Fig. 1G; upper panel). These data indicate that the PS1/γ-secretase activity is involved in the processing of nectin-1 and consequently the recruitment of afadin at cell-cell junctions.

PS1/γ-secretase-mediated cleavage event regulates the metabolism of nectin-1 α-CTF in both constitutive and inducible manners in MEF cells

To determine whether PS1/γ-secrease activity regulates the metabolism of nectin-1 CTFs, PS1 +/+ and -/- cells were stimulated with ionomycin (IM), a calcium ionophore, in the presence or absence of γ-secretase inhibitor. We previously have shown that treating wild-type MEF cells with IM induced nectin-1 ectodomain shedding and caused the accumulation of a 30 kDa α-CTF (Kim et al. 2010). We analyzed the levels of CTFs generated in both PS1 +/+ and -/- cells.

Immunoblot prepared from unstimulated PS1 +/+ cells exhibited very faint 30 kDa α-CTF and 36 kDa CTF1 bands, and an additional band at 24 kDa (we named as γ-CTF) appeared in a longer exposure immunoblot (Fig. 2A; lane 1). Treating PS1 +/+ cells with γ-secretase inhibitor alone increased the level of α-CTF (Fig. 2A; lane 2) but not the CTF1. IM treatment for 5 min rapidly increased the level of α-CTF, which was further increased by γ-secretase inhibitor treatment, whereas the level of CTF1 was not affected by IM treatment. These data suggest that the α-CTF is a preferred substrate of PS1/γ-secretase in fibroblast cells. More prominent γ-CTF appeared in IM treated cell lysates (Fig. 2A; lane 3; longer exposure blot), whereas this band disappeared in the presence of γ-secretase inhibitor (lane 4; longer exposure blot), indicating that the 24 kDa band is a product of γ-secretase activity. Since the CTF1 was not affected by IM treatment, the CTF1 might be a constitutive cleavage product of nectin-1. Unlike homozygous cells, treating PS1 -/- cells with γ-secretase inhibitor had no effect on the levels of α-CTF (Fig. 2B). IM treatment also substantially increased the level of α-CTF in PS1 -/- cells. However, unlike in PS1 +/+ cells, the 24 kDa band was not detected upon IM stimulation. The addition of γ-secretase inhibitor had no effect on the level of α-CTF (Fig. 2B) even after IM stimulation, indicating that α-CTF is mainly processed by PS1/ γ-secretase.

Figure 2. Nectin-1 α-CTF is further processed through PS1/γ-secretase activity.

Figure 2

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PS1 +/+ and -/- MEF cells were stimulated with 5 μM IM for 5 min and then collected in cell lysate buffer. Equal amounts of cell lysate (100 μg) were loaded for Western blot analysis. A. IM stimulation increased nectin-1 γ-CTF and γ-CTF in WT MEFs cells. Pretreatment with γ-secretase inhibitor (1 μM) for 1 h resulted in further accumulation of α-CTF but a disappearance of γ-CTF after IM treatment. B. IM stimulation did not increase the α-CTF level and the γ-CTF was not detected in PS1 -/- cells, indicating that the 24 kDa is the product of γ-secretase activity. Beta-actin was used as a loading control for each experiment.

PS2 rescues the loss of PS1 in PS1 -/- cells in nectin-1 processing

PS2 is a close homologue of PS1, and there is some functional redundancy between them (Lai et al. 2003). To address whether PS2 is also involved in nectin-1 processing, we examined the effects of increasing PS2 expression in PS1 -/- cells on nectin-1 CTFs. Due to low level of endogenous nectin-1, we infected PS1 +/+ and -/- MEF cells with recombinant adenovirus expressing nectin-1. Two days after infection, cells were collected and the levels of nectin-1 CTFs were analyzed by immunoblotting. The viral mediated overexpression of nectin-1 exhibited all three CTFs in PS1 +/+ cells (Fig. 3A), whereas the γ-CTF was missing and the α-CTF and CTF1 were highly accumulated in PS1 -/- cells as expected (Fig. 3A). This data indicates that viral mediated expression of nectin-1 did not alter the nectin-1 processing. We then introduced v5 epitope-tagged PS1 or PS2 into infected PS1 -/- cells and analyzed the generation of CTFs by Western blotting. PS1-/- cells expressing exogenous nectin-1 and empty vector did not exhibit the γ-CTF, whereas PS1 -/- cells co-expressing nectin-1 and PS1 exhibited prominent 24 kDa γ-CTF bands (Fig. 3B), demonstrating that PS1 restored normal processing of nectin-1. PS1 -/- cells co-expressing both nectin-1 and PS2 also exhibited the γ-CTF (Fig. 3B), although the intensity of the band was substantially reduced compared to PS1 expressing cells (Fig. 3C). This low generating efficiency of the γ-CTF by PS2 was rescued by co-expressing with PS1 (Fig. 3B; lane 4). This data indicates that PS2 is also able to participate in nectin-1 processing, but less efficiently than PS1.

Figure 3. Overexpressed PS2 rescues the loss of PS1 in the PS1 -/- cells on nectin-1 processing.

Figure 3

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A. PS1 +/+ and -/- MEF cells were infected with recombinant adenovirus expressing flag-tagged nectin-1 at an MOI of 1000. Cell lysates were collected 48 h after infection and analyzed on 12% SDS-PAGE. The γ-CTF was not detected in PS1 -/-cells. B. PS1 -/- cells were co-infected with recombinant adenoviruses expressing flag-tagged nectin-1, PS1, and PS2 at an MOI of 500 each. Cell lysates were collected 72 h after infection and analyzed by Western blot. Immunoblots were probed with nectin-1 cyto-specific antibody to detect nectin-1 CTFs, and PS1 and PS2 antibodies to detect full-length PS1 and PS2 respectively. C. Relative ratio of the nectin-1 γ-CTF. The intensity of γ-CTF was measured by densitometric analysis in three independent immunoblots.

The processing of nectin-1 mediated by the PS1/γ-secretase occurs in both constitutive and induced manners at synapses

We also examined the PS1/γ-secretase mediated cleavage of nectin-1 in primary rat hippocampal neurons. Both α-CTFs and γ-CTFs were detected in mature hippocampal neurons, and these bands were further accumulated when neurons were treated with lactacystin, a proteasome inhibitor, for 24 h (Fig. 4A), suggesting that nectin-1 processing occurs constitutively in hippocampal neurons. When γ-secretase activity was selectively blocked by γ-secretase inhibitor, the γ-CTF completely disappeared both in the presence and absence of lactacystin, while the α-CTF was significantly accumulated (Fig. 4A). Interestingly, when neurons were treated with a γ-secretase inhibitor, an additional band at 34 kDa, presumably CTF1, accumulated, suggesting that this band is a product of constitutive ectodomain shedding and rapidly degraded by either ADAM10 and/or PS1/γ-secretase. Interestingly, the size of nectin-1 CTF1 in neurons is slightly lower than that in MEF cells, suggesting that neurons may have different posttranslational modifications on nectin-1.

Figure 4. The PS1/γ-secretase cleavage regulates nectin-1 processing in both a constitutive and regulated manners at synapses.

Figure 4

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A. Rat hippocampal neurons at 28 days in vitro were treated with lactacystin (10 μm) in the presence or absence of γ-secretase inhibitor (1 μm) for 24 h. Cells were collected and analyzed by Western blotting. Treatment with lactacystin caused the accumulation of both α- and γ-CTFs in the absence of γ-secretase inhibitor. The γ-secretase inhibitor treatment increased the α-CTF and CTF1. B. Rat hippocampal neurons were pretreated for 30 min with 100 μM APV or 2 mM EGTA, prior to 50 μM NMDA application for 15 min. The cells were lysed directly in SDS-PAGE sample buffer and analyzed by immunoblotting with nectin-1 and actin antibodies. In the presence of APV (lane 3) and EGTA (lane 4) both α and γ secretase cleavage of nectin-1 was substantially reduced. Beta-actin was used as a loading control for each experiment. C. Oxygenated hippocampal slices were treated with 50 μM Sp-cAMP (a thiophosphate analogue of c-AMP) or a vehicle for 15 min; slices were then transferred to artificial cerebrospinal fluid (aCSF) and harvested after the times indicated. 3-5 slices per condition were used. Tissue samples were lysed in RIPA buffer, 50 μg of protein from each sample were then separated on 12% SDS-PAGE gels and analyzed by immunoblotting with nectin-1 and actin antibodies. D. Adult mouse brains were homogenized and fractionated by different centrifugation and analyzed by immunoblotting for the indicated proteins. 5 μg protein was loaded for all samples.

To investigate whether neuronal activity regulates nectin-1 cleavage mediated by γ-secretase, we treated primary rat hippocampal neurons with NMDA for 15 min and examined the production of CTFs. Activation of NMDA receptors elicited robust accumulation of both α-CTF and γ-CTF and these events were substantially reduced with APV (NMDA receptor antagonist) pretreatment and completely blocked by EGTA, a calcium chelator. However, in this condition, the 34 kDa CTF1 was not detected, clearly indicating that the generation of 34 kDa CTF1 does not depend on synaptic activity. These data demonstrate that nectin-1 ectodomain shedding and intracellular membrane cleavage, generating α-CTF and γ-CTF, can be rapidly induced by NMDA receptor activation and subsequent calcium influx into the neurons.

To investigate if ectodomain shedding and intracellular membrane cleavage of nectin-1 could be elicited in a cellular model of synaptic plasticity and memory formation, we chemically induced LTP in hippocampal slices harvested from adult mice. Both α-CTF and γ-CTF were detected in unstimulated slices (Fig. 4C), indicating that nectin-1 shedding can occur in a constitutive manner in the brain. We observed a pronounced increase in levels of α-CTF and γ-CTF in response to chemical LTP (Fig. 4C). This data suggests that nectin-1 shedding in the adult brain may be a regulated event during conditions of synaptic plasticity.

To examine the location of nectin-1 shedding in the brain, we performed subcellular fractionations of brain homogenates by different centrifugations of adult mice (P60-90). The enrichment of PSD95 in the PSD (postsynaptic density) fraction and synaptophysin in the LP2 (synaptic vesicle-enriched fraction) fraction confirmed the effectiveness of the fractionation procedure (Fig. 4D). In the adult cerebral cortex, full-length nectin-1 was detected in membrane fractions P2 (crude synaptosome fraction), PSD, LP1 (lysed synaptosomal membranes) and LP2 (Fig. 4D). The intensity of full-length nectin-1 in the LP2 fraction was seven fold greater than that in PSD. This data indicates that nectin-1 is localized to postsynaptic membranes but more preferentially to presynaptic membranes in the cortex. The α-CTF was more abundant than the CTF1 in these brain fractionations, and exhibited a similar distribution as full-length nectin-1 (Fig. 4D), indicating that the α-CTF is associated with synaptic membranes and is a main byproduct of ectodomain shedding. The CTF1 was detected in membrane fractions P2 and LP1, but rarely detected in PSD and LP2, presumably due to low abundance level. The γ-CTF was rarely detected due to low abundance but longer film exposure exhibited the γ-CTF in every fraction with the highest accumulation in LS2 (presynaptic soluble cellular components) (Fig. 4D), indicating that γ-CTF is a soluble fragment. These studies indicate that nectin-1 localized to postsynaptic and preferentially presynaptic membranes, and that ectodomain shedding and intramembrane cleavage occurs in both compartments.

Point mutants identify critical amino acids involved in nectin-1 processing within the transmembrane (TM) domain

Since γ-secretase cleavage occurs within TM domains of substrates, we addressed which amino acid residues are involved in the cleavage of nectin-1. We used site-directed alanine scanning mutagenesis to change each amino acid to alanine, with the exception of the residues at 352, 358 and 374 (Fig. 5), which were mutated to isoleucine. Each construct was expressed in HEK 293 cells and harvested 48 h post-transfection. We found that seven amino acids, when mutated, affect nectin-1 processing (Fig. 5A and B). Substitutions G355A, G356A, A358I, G359A, and S360A consistently altered molecular weights of α-CTF and CTF1 to a greater apparent mass than those of wild-type (Fig. 5A). Mutation of residue 357 from valine to alanine reduced the formation of CTF1 and split α-CTF into two CTFs, generating an additional band at 40 kDa (Fig. 5A). However, all of these point mutants still generated a γ-secretase product of ~24 kDa without changing molecular mass.

Figure 5. Point mutants identify critical amino acid residues involved in nectin-1 processing within the TM domain.

Figure 5

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HEK 293 cells were transfected with each mutant. After 24 h, cells were lysed in reducing sample buffer and analyzed on 12% SDS-PAGE. Samples were transferred to nitrocellulose, and the blot was probed with anti-nectin-1 antibody. To visualize the γ-CTF more clearly, longer exposure of the same film is shown at the bottom of the figure. A. Immunoblot analysis of amino acids 352 to 363. B. Immunoblot analysis of amino acids 364 to 375. C. Molecular structure of nectin-1. The residues that affect the cleavage events within the transmembrane domain are depicted with the colors. The residue that blocks the formation of α- and γ-CTFs is indicated by an arrow.

Conversely, the mutation G369A reduced the formation of α-CTF, and completely abolished the production of the 24 kDa band (Fig. 5B). This point mutation analysis identified residues that are important for the generation of α-CTF and CTF1 within the TM region. The remainder of the point mutants exhibited normal nectin-1 processing identical to wild-type nectin-1 (Fig. 5A and B). All these mutants exhibited the correct subcellular distribution to cell-cell contact sites as similar to that of wild-type (Supplemental Fig. 2), suggesting that the alternative cleavages by mutations were not due to the endoplasmic reticulum retention or misfolding. These results show that six amino acids, located proximate to the outer cell surface (Fig. 5C), are important for determining the correct positioning for the action of enzymes involved in the generation of α-CTF and CTF1, and that one amino acid may play a major role in α- and γ-secretase cleavages within the TM region.

Nectin-3 is also a γ-secretase substrate

In the CNS, nectin-1 and -3 localize at the pre- and postsynaptic sides of synaptic junctions formed in the CA3 pyramidal region of adult mouse hippocampi (Mizoguchi et al. 2002). Nectin-3 forms hetero-trans-dimerization with nectin-1, an interaction stronger than nectin-1 homo-trans-dimerization (Satoh-Horikawa et al. 2000). This is different from cadherins, whose extracellular regions only homophilically interact in trans with each other (Takeichi 1995). This trans-dimerization between nectin-1 and nectin-3 has been shown to play an important role in sustaining the normal associations between axons and dendrites (Togashi et al. 2006).

We examined whether nectin-3 also undergoes a PS1/γ-secretase cleavage event. Due to a lack of antibodies recognizing nectin-3 CTFs efficiently, we looked at the level of full-length mouse nectin-3 in PS1 +/+, +/-, and -/- MEF cells. Full-length nectin-3 was highly accumulated in PS1 -/- cells compared to that of either homozygous or heterozygous cells, which can be visualized by Western blot (Fig. 6A) and the densitometry analysis (Fig. 6B). Immunostaining of nectin-3 in PS1 -/- cells further supported the accumulation of nectin-3 molecules at the cell-cell adhesion sites (Fig. 6C). These observations suggested that nectin-3 is a potential substrate of PS1/γ-secretase.

Figure 6. Nectin-3α is a substrate of PS1/γ-secretase.

Figure 6

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A. PS1 +/+, +/-, and -/- MEF cells were lysed in lysis buffer and 50 μg cell lysates were analyzed on a 12% SDS-PAGE gel. Samples were transferred to nitrocellulose, and the blot was probed with anti-nectin-3 antibody and beta-actin. Compared with wild-type MEFs, a significant accumulation of full-length nectin-3 (100 kDa) was observed in both PS1 +/- and deficient fibroblasts. B. Relative ratio of full-length nectin-3. The intensity of full-length nectin-3 was measured by densitometric analysis in three independent immunoblots. C. PS1 +/- and -/- cells were plated on poly-l-lysine coated coverslips, fixed in methanol, and stained for nectin-3 three days later. Scale bar = 5 μm. D. PS1 +/+ and -/- MEF cells were infected with a recombinant adenovirus expressing nectin-3α-v5 at an MOI of 1000. Twenty-four h after infection, cells were treated with 1 μM γ-secretase inhibitor for 24 h. Cells were then collected and analyzed by immunoblot. The 19 kDa band shown in PS1 +/+ MEF cells disappeared in the presence of γ-secretase inhibitor and was missing in PS1 -/- cells. E. Primary hippocampal neurons at 7 DIV were infected with recombinant adenovirus expressing nectin-3α-v5 at an MOI of 200. Twenty-four h after infection, neurons were treated with 1 μM γ-secretase inhibitor for 24 h. Neurons were collected in reducing sample buffer and analyzed by immunoblot. The blot was probed with anti-v5 antibody. Several CTFs were detected and the 19 kDa band, indicated by an arrow, disappeared when treated with γ-secretase inhibitor in the long exposure blot. Beta-actin was used as a loading control.

Next, we addressed whether nectin-3 is cleaved by PS1/γ-secretase activity by utilizing an overexpression system. MEF cells were infected with recombinant adenovirus expressing mouse nectin-3α tagged with v5 epitope at its C-terminus. Immunoblot exhibited two CTFs at 19 and 23 kDa (Fig. 6D), indicating that nectin-3 also undergoes ectodomain shedding mediated by unidentified sheddases. When MEF cells were treated with γ-secretase inhibitor, the 19 kDa band disappeared and the 23 kDa was highly accumulated (Fig. 6D; left panel, lane 2), suggesting that the 19 kDa band is likely the product of γ-secretase activity, and the 23 kDa band is likely the substrate. We also infected PS1 -/- cells with recombinant adenovirus expressing nectin-3α tagged with v5 epitope at its C-terminus. In PS1 -/- cells, the 23 kDa band was highly accumulated, but the 19 kDa band was not seen and the addition of γ-secretase inhibitor had no effect on the expression level of the 23 kDa band (Fig. 6D; right panel), indicating that the 19 kDa band is the product of γ-secretase activity.

To further address whether nectin-3 is cleaved by PS1/γ-secretase in neurons, we infected primary rat hippocampal neurons with recombinant adenovirus expressing mouse nectin-3α. The 19 kDa band was clearly detected in rat hippocampal neurons, whereas this band was disappeared when neurons were treated with γ-secretase inhibitor. This data indicates that nectin-3α is a substrate of γ-secretase in neurons (Fig. 6E).

Taken all together, nectin-3α also undergoes ectodomain shedding, followed by intramembranous cleavage mediated by PS1/γ-secretase, consequently releasing the nectin-3 intracellular domain. The coordinated cleavages of nectin-1 and nectin-3 by PS1/ γ-secretase activity may be significant for biological functions such as synapse formation and synaptic remodeling in neurons.

DISCUSSION

Elucidating roles of PS1/γ-secretase in regulation of nectin function, we might be able to understand the mechanism underlying the control of synaptic function, in addition to that of synapse loss, which is an early event seen in Alzheimer's disease. In this study, we have shown that the loss of PS1/γ-secretase activity caused the accumulation of full-length and α-CTF of nectin-1 (Fig. 1A) as well as full-length nectin-3 under basal conditions (Fig. 6A), indicating that PS1/γ-secretase activity regulates the processing of both nectin-1 and nectin-3. In primary hippocampal neurons, both nectin-1 and nectin-3 undergo ectodomain shedding, followed by PS1/ γ-secretase cleavage (Fig. 3 and Fig. 6E respectively). Pronounced increases in levels of α- and γ-CTFs of nectin-1 in response to chemical LTP was observed in mouse brain slices. The mouse brain synaptosomal fractionation study showed that nectin-1 localized to postsynaptic and, preferentially, presynaptic membrane fractions, and that ectodomain shedding and intramembrane cleavage of nectin-1 occur in both compartments. These data suggest that nectin ectodomain shedding and PS-dependent intramembrane cleavage occur at synapses and is a regulated event during conditions of synaptic plasticity in the brain. These data also suggest that secretase cleavage of synaptic adhesion molecules such as nectin-1 may allow the activated synapse to undergo both an increase and decrease in synaptic density observed during induction of LTP and LTD.

Our data showed that the enhanced nectin expression at cell-cell contact sites caused by the loss of PS1 was directly correlated with a high l-afadin level at the cell-cell contact sites (Fig. 1E and F), although the increased nectins did not affect the overall expression level of l-afadin (Fig. 1D and E). Afadin functions as a molecular scaffold that integrates signals related to cell adhesion and cytoskeletal reorganization. Afadin is known to interact with a number of proteins through its multiple domains (Hock et al. 1998, Kuriyama et al. 1996, Boettner et al. 2000, Linnemann et al. 1999, Su et al. 2003, Yamamoto et al. 1999, Radziwill et al. 2003). The loss of afadin results in embryonic lethality by 10.5 days post cotium, apparently due to a loss of neuronal polarity (Zhadanov et al. 1999), demonstrating the important role of afadin in the generation and maintenance of cell-cell junctions. The ectodomain shedding and intramembrane cleavage of nectins are likely major mechanisms in the destabilization of intercellular junctions by dispatching afadin and its associated proteins from shed nectins at the intercellular junctions including synaptic junctions.

Interestingly, the overexpression of PS2 in PS1-/- MEF cells induced the generation of the 24 kDa γ-CTF under basal conditions (Fig. 3B), indicating that PS2 can compensate for the loss of PS1 function in nectin-1 processing, further confirming that PS1 and PS2 share some functional redundancy (Lai et al. 2003). However, PS1 -/- MEF cells express endogenous PS2 and neither stimulating PS1 -/- cells with IM or overexpressing nectin-1 in PS1 -/- cells could produce the γ-CTF, suggesting that PS2 is less efficiently involved in γ-secretase activity or unlikely involved in nectin-1 processing in normal conditions. This question should be addressed in future studies.

We have previously shown that ADAM10 is responsible for the generation of nectin-1 α-CTF, and that this cleavage event is dependent on postsynaptic NMDA receptor activity (Kim et al. 2010). N-cadherin and E-cadherin, which are colocalized with nectins at synapses and adherens junctions respectively, also undergo ectodomain shedding mediated mainly by ADAM10 (Reiss et al. 2005, Maretzky et al. 2005a, Uemura et al. 2006a). The ADAM10 conditional KO mice die perinatally with a disrupted neocortex, severely reduced ganglionic eminence, and a disrupted organization of the cortical region (Jorissen et al. 2010). Although these phenotypes of ADAM10 cKO mice are highly due to the role of ADAM10 in Notch-1 signaling, it is possible that some of these defects may be due to a reduced ectodomain shedding of cell adhesion molecules including nectins and cadherins. Analogous with N- and E-cadherin processing, nectin-1 and nectin-3 also undergo γ-secretase cleavage of the membrane-anchored CTF after ectodomain shedding (Fig. 2, 3, 4, and 6) (Kim et al. 2010, Kim et al. 2002). The nectin/afadin adhesion system colocalizes with components of the cadherin/catenin system at synapses (Mizoguchi et al. 2002, Nishioka et al. 2000, Lim et al. 2008). PS1 forms complexes with N-cadherin (Georgakopoulos et al. 1999), and these complexes are present at the synapses as well (Georgakopoulos et al. 1999, Uchida et al. 1996). These data imply that the nectin/afadin system also forms complexes with PS1 at synapses. This juxtaposition of enzymatic machinery with nectins appears poised to affect a rapid remodeling of spine structure in response to synaptic transmission. Our work now leads to the proposal that nectins participate in synaptic remodeling by the orchestrated regulation of sheddases including ADAM10 and γ-secretase. This change may lead to long-term changes in synaptic function, which are required for higher order processes in the brain such as learning and memory (Shiosaka 2004, Lee et al. 2008, Zhang et al. 2005).

It has been shown that the PS1-dependent ε-cleavage product of N-cadherin functions as a potent repressor of CBP/CREB-mediated transcription (Marambaud et al. 2003). Furthermore, N-cadherin cleavage resulted in a dramatic redistribution of β-catenin from the cell surface to the cytoplasmic pool, which influences the expression of β-catenin target genes (Reiss et al. 2005). Therefore, it is also possible that the intracellular domain of nectin-1 released by gamma-secretase activity may elicit an intracellular signal through unidentified cytoplasmic binding partners that are involved in gene regulation, which may influence the long-term synaptic plasticity.

Mutagenesis analysis in the TM domain of nectin-1 suggests that the TM domain plays a significant role in the association with ectodomain sheddases. Six residues within the TM domain are critical for the determination of cleavage positions by ectodomain sheddases since changes in any of these residues shifts the molecular weight of both α-CTF and CTF1 (Fig. 5). These residues may play an important role in interactions with the transmembrane domains of ectodomain sheddases, including ADAM10, or participate in determining the right position for the action of the sheddases. It is also possible that ectodomain sheddases compete for these residues, and that this determines which form of CTF will be produced. However, these mutations, except G369A, did not impair the γ-secretase cleavage event and did not change the size of γ-CTF, suggesting that these residues are less likely play a role in the interaction with γ-secretase subunits. Mutation of V357A generated different molecular weight CTFs of nectin-1 (Fig. 5B), implying that this residue plays a key role in positioning the cleavage site, or alternatively, its mutation causes a conformational change of nectin-1, allowing a new sheddase to access the cleavage site. Mutation of G369A dramatically impaired the generation of both α-CTF and γ-CTF, suggesting that this residue is absolutely necessary for nectin-1 to have the conformation necessary to allow for proteolytic processing by ADAM10 and subsequently PS1/γ-secretase. However, our study does not provide the exact cleavage sites mediated by ectodomain sheddases and PS1/ γ-secretase. Further study is required to identify the exact cleavage sites.

In summary we have shown that nectin ectodomain shedding and PS-dependent intramembrane cleavage occur at synapses and is a regulated event during conditions of synaptic plasticity in the brain. Future work will be focused on whether FAD mutations in PS directly perturb synaptic activity by aberrantly modulating nectins and if this altered processing could ultimately lead to synaptic dysfunction, which is the earliest detectable abnormality in AD.

Supplementary Material

Supp Fig S1-S2

ACKNOWLEDGEMENT

The authors thank Dr. Phillip Wang for Presenilin KO cell lines and comments on the manuscript and Dr. Katherine Conant for providing comments on the manuscript. Funding for this project was provided by NIH-RO1 AG027233-01.

Abbreviations used

AD

Alzheimer's disease

AJ

adherens junction

CAM

cell adhesion molecule

CTF

C-terminal fragment

CNS

central nervous system

FAD

familiar Alzheimer's disease

IM

ionomycin

LTD

long term depression

LTP

long-term potentiation

MEF

mouse embryonic fibroblast

NMDA

N-Methyl-D-aspartic acid

NE1-ICD

nectin-1 intracellular domain

PAJ

puncta adherens junction

PS1

Presenilin-1

PS2

presenilin-2

TM

transmembrane

Footnotes

The authors have no conflicts of interest to declare.

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Supp Fig S1-S2
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