BACE1 activity regulates cell surface contactin-2 levels
© Gautam et al.; licensee BioMed Central Ltd. 2014
Received: 18 October 2013
Accepted: 4 December 2013
Published: 9 January 2014
Although BACE1 is a major therapeutic target for Alzheimer’s disease (AD), potential side effects of BACE1 inhibition are not well characterized. BACE1 cleaves over 60 putative substrates, however the majority of these cleavages have not been characterized. Here we investigated BACE1-mediated cleavage of human contactin-2, a GPI-anchored cell adhesion molecule.
Our initial protein sequence analysis showed that contactin-2 harbors a strong putative BACE1 cleavage site close to its GPI membrane linker domain. When we overexpressed BACE1 in CHO cells stably transfected with human contactin-2, we found increased release of soluble contactin-2 in the conditioned media. Conversely, pharmacological inhibition of BACE1 in CHO cells expressing human contactin-2 and mouse primary neurons decreased soluble contactin-2 secretion. The BACE1 cleavage site mutation 1008MM/AA dramatically impaired soluble contactin-2 release. We then asked whether contactin-2 release induced by BACE1 expression would concomitantly decrease cell surface levels of contactin-2. Using immunofluorescence and surface-biotinylation assays, we showed that BACE1 activity tightly regulates contactin-2 surface levels in CHO cells as well as in mouse primary neurons. Finally, contactin-2 levels were decreased in Alzheimer’s disease brain samples correlating inversely with elevated BACE1 levels in the same samples.
Our results clearly demonstrate that mouse and human contactin-2 are physiological substrates for BACE1. BACE1-mediated contactin-2 cleavage tightly regulates the surface expression of contactin-2 in neuronal cells. Given the role of contactin-2 in cell adhesion, neurite outgrowth and axon guidance, our data suggest that BACE1 may play an important role in these physiological processes by regulating contactin-2 surface levels.
Alzheimer’s disease (AD) is the most common neurodegenerative disease that affects millions of people worldwide. Studies strongly suggest that the accumulation of toxic amyloid -β peptides (Aβ) is associated with synaptic dysfunction and neuronal loss in AD . Aβ peptides are generated from sequential cleavages of the amyloid β precursor protein (APP), which are mediated by the β-site APP cleaving enzyme 1 (BACE1) and Presenilin/γ-secretase [2–4]. Therefore, BACE1 and γ-secretase represent two major therapeutic targets for prevention and treatment of AD.
BACE1, also known as memapsin 2 and Asp 2, is a type I transmembrane aspartyl protease that is highly expressed in neuronal tissues [5, 6]. Besides generating pathogenic Aβ, BACE1 also plays crucial roles in numerous physiological processes including neuronal activity, myelination, axonal guidance, presynaptic activity and cognitive behavior in mice [7–14]. These physiological BACE1 functions cast a doubt on the safety of BACE1 inhibition therapy currently being developed to block Aβ generation in AD patients. Currently more than 60 BACE1 substrates have been reported in in vitro and in vivo conditions [15–21]. Therefore characterizing BACE1-mediated cleavage for each substrate may not only contribute to our understanding of how BACE1 regulates crucial physiological processes but also aid in the prevention of potential side effects deriving from BACE1 inhibition therapy.
Contactin-2 (axonin-1 or transient axonal glycoprotein-1 (TAG-1)) is a cell adhesion molecule that belongs to immunoglobulin super family [22, 23]. Contactin-2 is highly expressed at the axon growth cone and plays an important role in regulating axon guidance and path finding [24, 25]. Studies from knockout mice revealed that contactin-2 is also crucial for normal learning and memory functions . While the majority of currently reported BACE1 substrates are type I membrane proteins with transmembrane domains, contactin-2 belongs to the carboxy-terminal glycan phosphatidyl inositol (GPI)-anchored protein family that requires covalent linkage of GPI domains for binding to the plasma membrane. Interestingly, two recent unbiased secretome analyses suggested that BACE1 activity regulates the release of contactin-2 in neurons but the BACE1-mediated contactin-2 cleavage has not been fully characterized [19, 20]. Therefore we decided to characterize BACE1-mediated cleavage of contactin-2 in cellular and neuronal models and explore whether BACE1 cleavage regulates surface expression of contactin-2, potentially affecting the cell adhesion function of the protein.
Materials and methods
Antibodies and reagents
Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA). The rabbit monoclonal anti-human contactin-2 antibody was from Abcam (Cambridge, USA) while rabbit monoclonal BACE1 antibody was from Cell Signaling Inc. (Boston, USA). Anti-mouse V5 antibody was from Life Technologies (Grand Island, USA). Rabbit anti-APP was described earlier . sAPP (22C11), N-cadherin and NrCAM antibodies were from EMD Millipore (Billerica, USA). BACE1 Inhibitor IV was also purchased from EMD Millipore.
Human contactin-2 plasmid (MGC:157722) was obtained from Harvard Plasmid DNA Resource Core (Harvard Medical School, Boston). Contactin-2 cDNA coding region was amplified using the following primers: forward, CACCATGGGGACAGCCACCAGG AGG; reverse, TCAGAGCTCCAGGGAGCCTATGAGG. The amplified fragments were subcloned into pcDNA6.1 vector (directed TOPO system, Life Technologies). For generation of the soluble form of contactin-2, a V5-tag was C-terminally added to inactivate the GPI-anchor domain. The putative BACE1 cleavage site was mutated in the contactin-2 cDNA construct (CNTN2-MM1008AA) with the help of Quick Change site-directed mutagenesis kit from Agilent Technologies (Santa Clara, USA) according to the manufacturer’s protocol using the following primers: forward, GAGGAATGGAGGCACAAGCGCGGCGGTGGAGAACATGGCAGTC; reverse, GACTGCCATGTTCTCCACCGCCGCGCTTGTGCCTCCATTCCTC. All the constructs were sequenced and verified at the MGH DNA sequencing core facility (Boston, USA).
Generation of contactin-2 stable cell lines
Single cell stable lines were generated for both GPI-anchored contactin-2 and soluble contactin-2 (V5-tagged) in CHO cells. Briefly, 4 μg of GPI-anchored contactin-2 and soluble contactin-2 cDNA were transfected in CHO cells with the help of Effectene transfection reagent (Qiagen, Valencia, USA) according to the manufacture’s protocol. 48 h after transfection, cells were trypsinized and replated in the presence of 10 μg/μl of Blasticidin selection marker. After 2 weeks of selection, Blasticidin resistant cells were further plated in serial dilutions in a 96-well plate in order to get single cell colonies. Later, different single cell clones were picked and analyzed by Western blotting for the optimal expression of contactin-2 with the help of specific antibodies.
Primary neuronal cultures
Primary neuronal cultures were prepared from 16-day old pregnant female mice (CD-1). Mice were purchased from Charles River Laboratories, Cambridge, and the animal protocol was approved by the MGH Institutional Animal Committee. In brief, hippocampi and frontal cortices were dissected and isolated from pups at embryonic day 16 (E-16). Dissected tissue was further triturated using fine pasture pipette and later plated on poly-D-lysine/laminin coated 6-well tissue culture plate using Neurobasal media containing 2% B-27 serum supplement (Life Technologies). Cultures were maintained in a humidified environment at 37°C with 5% CO2 and 50% of the media were replaced every 3rd day.
Lentiviral generation and infection
BACE1 and the control mCherry lentiviral particles were generated at the MGH Vector Core facility (Charlestown, USA). Contactin-2 expressing CHO cells at 40% confluency were infected with 1 × 106 lentiviral particles. 24 h after infection, media was replaced and the cells were allowed to grow for a total of 5 days before extraction. In case of primary neurons, cultures were infected at DIV5 with 1 × 106 lentiviral particles. In order to reduce the lentivirus-mediated toxicity, 50% of the culture media was replaced 12 h after infection. Cultures were allowed to grow for 6 additional days after infection.
BACE1 inhibitor treatment
CHO cells stably expressing GPI-anchored contactin-2 were plated on 60 mm tissue culture plate and treated with either 4 μM of BACE1 Inhibitor IV (EMD Millipore) or the same volume of DMSO control vehicles. The treated cells were allowed to grow for 48 h and the media was replaced with fresh media containing BACE1 Inhibitor IV. 48 h after conditioning, both the media and the cells were collected, processed and analyzed by Western blot. For primary neuronal cultures, the neurons were treated with 1 μM BACE1 Inhibitor IV for 48 h, replaced with fresh media containing 1 μM of BACE1 Inhibitor IV, and then incubated for additional 48 h before collecting the media and the cells.
Western blot analysis
Cells were lysed in 1X GTIP buffer containing 10 mM Tris-HCl (pH 6.8), 2 mM EDTA (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.25% Nonidet P-40, and a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA). The lysates were centrifuged at 16,000 × g in order to remove insoluble materials and the protein concentration was measured using a BCA protein assay kit (Pierce Biotechnology, Rockford, USA). 25-75 μg protein samples were separated either on 3-8% Tris-Acetate gels, 4-12% gradient Bis-Tris gels, or 12% Bis-Tris gels (Life Technologies) and transferred on PVDF membrane. Blots were then blocked either with 5% skimmed milk or with 5% BSA (Sigma, St. Louis, MO, USA) for overnight at 4°C. Primary antibodies were used at the following dilutions: human contactin-2 (1:200), mouse contactin-2 (1:200), anti-V5 (1:3000), anti-APP C-66 (1:1000), anti-BACE1 (1:1000), anti-sAPPβ (1:200), anti-N-cadherin (1:1000), anti-NrCAM (1:1000) and anti-GAPDH (1:2000). Blots were developed by chemiluminescence using Biomax light film (Kodak, Rochester, USA) or Versa Doc imaging system and quantified using Quantity One software (Biorad).
CHO cells stably expressing GPI-anchored contactin-2 were plated on glass coverslips in a 6-well tissue culture plate. Cells were treated with 4 μM BACE1 Inhibitor IV or DMSO for 48 h, fixed and then stained with anti-human contactin-2 antibody (1:200) without permeabilization for overnight at 4°C. After incubating with Alexa Fluor488-conjugated secondary antibody, cover slips were mounted on the glass slides with the help of mounting media containing DAPI (Life Technologies). Images were taken on Olympus IX 70 microscope with the same exposure settings and later processed by IPLab software.
Cell surface biotinylation
Cell surface biotinylation experiments were performed on primary neuronal cultures at day 15 (DIV15). Cultures grown in 6-well plate were washed three times with ice cold Hank’s balanced salt solution (HBSS) and incubated in dark for 1 h with 2 ml of ice cold HBSS containing 0.5 mg/mL Sulfo-NHS-Biotin (Pierce). Five-minute incubation with 100 μM lysine solution was used to quench the reaction followed by three washes with cold HBSS. Cells were then extracted directly in an extraction buffer containing 10 mM Tris-HCl (pH 6.8), 2 mM EDTA (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% Sodium Deoxycolate, 0.2% SDS, 1 mM PMSF, 20 μM ALLN and a protease inhibitor cocktail (Roche Molecular Biochemicals). The insoluble fractions were removed by centrifugation at 16,000 × g and the protein concentrations were determined using the BCA protein assay kit (Pierce). 200-600 μg of protein was immunoprecipitated using NeutrAvidin beads (Pierce) overnight at 4°C. Next day, samples were washed 3 times with the extraction buffer, eluted with the LDS sample loading buffer (Life Technologies) supplemented with 2% (v/v) β-mercaptoethanol and separated on 4-12% Bis Tris-Acetate NuPage gel (Life Technologies) followed by Western blot analysis using various primary antibodies.
Analysis of AD brain samples
Brain samples from 9 AD and 8 Non-AD patients (age-matched, temporal lobe region) were obtained from Dr. Yong Shen (Roskamp Institute, Sarasota, FL). The same set of samples were previously used to analyze altered sodium channel metabolism . Frozen tissue samples were lysed with extraction buffer containing 10 mM Tris-HCl (pH 6.8), 1 mM EDTA, 150 mM NaCl, 0.25% Nonidet P-40, 1% Triton X-100, 0.2% SDS and a protease inhibitor cocktail (Roche Molecular Biochemicals). 75 μg of protein was resolved on 12% Bis/Tris NuPage gels or 3-8% Tris/Acetate gels. BACE1 levels were determined in our previous study  while amyloid plaque density information for individual samples were provided by Dr. Shen’s laboratory.
Results and discussion
Contactin-2 is a substrate for BACE1
To test whether contactin-2 is a BACE1 substrate in cells, we first generated expression constructs with full-length human contactin-2 (Figure 1B, GPI-anchored CNTN2) or secreted contactin-2 where the GPI-anchor domain was inactivated by the addition of a V5-epitope tag (Figure 1B, sCNTN2). These constructs were transfected into Chinese Hamster Ovary (CHO) cells and stable CHO cell clones with high expression of GPI-anchored CNTN2 or sCNTN2, were selected for the experiments (Additional file 1: Figure S1). As expected, Western blot analysis of the conditioned media using a human contactin-2 antibody revealed a strong increase in the secreted form of contactin-2 in CHO cells with sCNTN2 due to the lack of active GPI-anchor domain (Additional file 1: Figure S1A). Interestingly, we also found a small but consistent release of contactin-2 into cell culture media of CHO cells with GPI-anchored contactin-2 (Additional file 1: Figure S1B). To explore whether BACE1-mediated cleavage regulates contactin-2 release into cell culture media, we coexpressed human BACE1 in CHO cells with contactin-2 and analyzed contactin-2 levels in total cell lysates and conditioned cell culture media (Figure 1C and D). BACE1 overexpression increased secreted contactin-2 levels by ~2 fold in CHO cells with GPI-anchored contactin-2 (Figure 1C and E). As a negative control, we co-expressed BACE1 in CHO cells expressing sCNTN2 with the inactive GPI anchor domain (Figure 1D and F). As expected, BACE1 expression did not induce significant changes in soluble contactin-2 levels both in the conditioned media and in the total cell lysate (Figure 1D and F). These data confirm that BACE1 cleaves GPI-anchored contactin-2 and therefore regulates the release of contactin-2 ectodomain.
Endogenous BACE1 activity regulates contactin-2 cleavage
BACE1 cleaves contactin-2 at Met1008-Met1009
BACE1 regulates cell surface contactin-2 levels in mouse primary neurons
Decreased contactin-2 levels in AD brain samples
In this study, we showed that BACE1 activity tightly regulates cell surface contactin-2 levels in CHO cells and cultured mouse primary neurons by selectively cleaving cell surface contactin-2. Recent studies indicated that BACE1-null neurons display axon guidance defects but the underlying molecular mechanisms are not fully elucidated [12–14]. While the biological activity of the released contactin-2 remains unknown, lack of proper BACE1-mediated cleavage of surface neural adhesion molecules such as contactin-2 may provide an explanation for axon guidance defects observed in BACE1-null mice [13, 20]. Since contactin-2 regulates axon guidance through homophilic and heterophilic interactions with other neural adhesion molecules [25, 26, 33–38], abnormal accumulation of surface contactin-2 by BACE1 knock-down may interfere the proper axon guidance in vivo. In mice, premature early overexpression of contactin-1 (F3/contactin) leads to the reduction in the cerebellar size, granule cell numbers and Purkinje cell maturation , which suggests the importance of the precise contactin expression in early brain development. Similarly, Hitt et al. recently proposed that decreased BACE1-mediated shedding of CHL1 may also contribute to axon guidance deficits in BACE1-null neurons . Our findings, together with those of Hitt et al., suggest that lack of proper BACE1-mediated shedding of neural adhesion molecules may produce the final phenotype of axon guidance deficits found in BACE1-null neurons. It will be interesting to identify all major neural adhesion molecules mediating the effect of BACE1 on axonal guidance in neural tissues.
Our data in Figure 6 also suggest that elevated BACE1 in brains of AD patients may also contribute to AD pathogenesis by decreasing contactin-2 levels and possibly its surface expression. Mice lacking contactin-2 show deficits in neuronal migration (for a subset of cerebella neurons) , neurogenesis , learning and memory  and ion channel clustering [26, 42]. Therefore, decreased contactin-2 levels in brains of AD patients may contribute the neuronal deficits through multiple mechanisms. However, further studies will be required to fully characterize the functional consequences of BACE1-mediated contactin-2 cleavage in AD pathogenesis as well as its potential side-effects during BACE1 inhibitor therapies currently in clinical trials.
While we were investigating human contactin-2 cleavage in our cellular model systems, two unbiased secretome analyses have been published showing that TAG-1 (mouse contactin-2) ectodomain release is regulated by BACE1 activity in mouse primary neuronal cultures [19, 20]. Kuhn et al. also confirmed that TAG-1 ectodomain release was significantly decreased in brains of BACE1-null mice . Consistent with these findings, we confirmed that BACE1 activity tightly regulates the release of contactin-2 in mouse primary neuronal cultures (Figure 3). Moreover, we characterized human contactin-2 cleavage by BACE1 and identified the cleavage site for the first time (Figures 1, 2 and 4). Our surface biotinylation studies also demonstrated that BACE1 activity tightly regulates cell surface contactin-2 levels in cultured mouse primary neurons (Figure 5). Together, our data show that human and mouse contactin-2 are endogenous substrates for BACE1 and that BACE1-mediated cleavage modulates the surface expression of contactin-2.
We would like to thank Dr. Yong Shen (Roskamp Institute, Sarasota, FL) for providing AD and control age-matched brain samples. We also would like to thank Ms. Carmilla Peach, Carolyn C. Sachse and Mr. Manuel T. Gersbacher (Massachusetts General Hospital, Boston, MA) for technical help in this study. This work is supported by grants from the NIA to DMK and DYK.
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