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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2011 December 17.
Published in final edited form as:
PMCID: PMC3241922
NIHMSID: NIHMS230771

Alternative Splicing of Neuroligin Regulates the Rate of Pre-synaptic Differentiation

Summary

Neuroligins (NLGs) and Neurexins (NRXs) are important adhesion molecules that promote synapse formation. Multiple splice variants of NLG and NRX exist, but their specific functions are unclear. Here we report that a surrogate postsynaptic cell expressing full-length NLG triggers slow presynaptic differentiation in a contacting axon. In contrast, a version of NLG-1, which lacks insert B (NLG-1-ΔB), induces rapid presynaptic differentiation, reaching the rate seen at native neuronal synapses. We show that this acceleration is due to removal of the N-linked glycosylation site within insert B. NLG-1ΔB also increases synaptic density at neuro-neuronal synapses more than does full-length NLG-1. Other postsynaptic adhesion proteins, such as N-cadherin, EphB2 and SynCAM-1, alone or in combination with full-length NLG-1 do not trigger fast differentiation, suggesting that rapid presynaptic differentiation depends on a unique interaction of NLG-1ΔB with axonal proteins. Indeed, we find that NLG-1ΔB recruits more axonal α-NRX. Our results suggest that the engagement of α-NRX is a key to rapid induction of synapses at new sites of axo-dendritic contact.

Introduction

During the development of synapses in the central nervous system, dynamic protrusions from axons and dendrites extend and retract on a time-scale of minutes, probing the environment for targets (Portera-Cailliau et al., 2003). Once appropriate contacts are made, synapse formation must quickly follow to stabilize these transient adhesive connections (Niell et al., 2004; Ruthazer et al., 2006). This requires fast accumulation of adhesion molecules, rapid transport and accurate deposition of synaptic proteins to these locations. On the pre-synaptic side, many synaptic proteins are packaged in complexes for transport. At least two classes of transport packets have been identified: clear circular vesicles about 50 nm in diameter, which likely correspond to synaptic vesicles or their precursors, and 80 nm dense core vesicles, named Piccolo Transport Vesicles (PTVs) because they carry the cytomatrix active zone proteins Piccolo and Bassoon (Zhai et al., 2001; Shapira et al., 2003) as well as other proteins present in the active zone (Garner et al., 2006). Both of these complexes can arrive at new synapses within 20-30 minutes of physical contact between axons and dendrites, leading to the formation of new synapses that release neurotransmitter in an activity dependent manner within one hour of contact (Ahmari et al., 2000; Friedman et al., 2000; Bresler et al., 2004).

Multiple adhesion molecules are present at synapses (Dalva et al., 2007), though single classes of post-synaptic adhesion molecules are sufficient to induce pre-synaptic differentiation at sites of pre-post contact. Five post-synaptic adhesion protein families, Neuroligin (NLG) (Scheiffele et al., 2000; Dean et al., 2003), SynCAM (Biederer et al., 2002), Netrin-G Ligand-2 and 3 (NGL-2, -3) (Kim et al., 2006; Woo et al., 2009), EphB2 (Kayser and Dalva, 2005), and LRRTM2 (de Wit et al., 2009; Ko et al., 2009a) when presented by a non-neuronal cell, trigger presynaptic differentiation. However, the dynamics of the interactions between these adhesion molecules and their presynaptic cognates as well as the subsequent nucleation of the pre-synaptic transmitter release machinery have not been fully determined, although existing data suggests that the interaction between Neuroligin and its ligand β-NRX (Ichtchenko et al., 1995) are fast enough for the fast synapse formation observed in neurons (on the order of minutes) both in non-neuronal S2 and PC12 cells (Nguyen and Sudhof, 1997; Dean et al., 2003) and between β-NRX expressing HEK cells and NLG-bearing supported bilayers (Pautot et al., 2005).

To address these issues, we used an assay in which postsynaptic neurons are replaced by a surrogate cell: a HEK293 cell expressing one or more postsynaptic adhesion proteins (Scheiffele et al., 2000; Biederer and Scheiffele, 2007). This enabled us to define the molecular constituents of the cell-cell interaction and the time and location of contacts. We compared the ability of different post-synaptic adhesion molecules to rapidly (within our hour of contact) induce synapse formation. Strikingly, among NLG-1, NLG-1ΔB (a splice variant of NLG-1 missing an exon of 9 amino acids), SynCAM, EphB2, NGL-2, and N-Cadherin, only NLG-1ΔB was able to recruit Bassoon to new contacts and induce functional presynaptic terminals. We find that α-NRX, an important component for NLG mediated presynaptic differentiation (Ko et al., 2009b), is preferentially concentrated at contacts with NLG-1ΔB expressing cells, consistent with biochemical evidence that absence of insert B enhances α-NRX binding (Boucard et al., 2005). Conversely, a mutation removing an N-linked glycosylation site in insert B known to impair α-NRX binding (Boucard et al., 2005), induced rapid pre-synaptic differentiation. Finally, over-expression of NLG-1ΔB increased synaptic density to a higher degree than NLG-1 (and higher than control vector). Together our data show that NLG-1ΔB is adapted for the fast synapse induction seen at new axo-dendritic contacts. This implies that alternative splicing of NLG plays a role in regulating the rate of synapse formation.

Materials and Methods

All chemicals were purchased from Sigma unless otherwise noted.

DNA Constructs and Antibodies

The expression plasmid for neuroligin-1 was previously described (Scheiffele et al., 2000). Monomeric cyan, green, and yellow fluorescent protein (mCFP, mGFP, mYFP) (Zacharias et al., 2002) or monomeric red fluorescent protein (mRFP) (Campbell et al., 2002) fragments were amplified by PCR and inserted into a unique SalI site in VSV-tagged β–NRX-1 just after the LNS domain. The FP was placed at the same site in FLAG-tagged α-NRX-1 (Boucard et al., 2005) by swapping the ectodomain from FP-tagged β-NRX-1. For NLG-1-mRFP constructs, mRFP was inserted at an engineered SacII site between the AchE-like domain and the transmembrane domain. The NLG-N303S mutation was made by changing AAT to TCT with the QuikChange Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA). SynCAM1, GFP tagged Bassoon (GFP:BSN95-3938), Synaptophysin (SYP-GFP), CASK (GFP-CASK), EphB2, and NGL-2 were provided by Dr. Sudhof, Dr. Gundelfinger (Shapira et al., 2003), Dr. Kaether (Kaether et al., 2000), Dr. Reichardt (Bamji et al., 2003), Dr. Irie, and Dr. Kim (Kim et al., 2006), respectively. SYP-YFP was made by replacing the GFP with YFP. PSD-95-GFP was provided by Dr. Lu Chen, and GFP was replaced with mRFP to make the red version.

All 4 shRNAs were integrated into one pSuper-Retro-GFP plasmid (Oligoengene, Seattle, WA) according to the cloning method described previously (Stove et al., 2006). α-NRX-1 shRNA was directed against residues 2402-2420 (NM 021767) with the sense sequence as follows: 5’-GCTATAACCTCAATGATAA-3’. α-NRX-2 and α-NRX-3 shRNAs were based on the target sequences of siRNAs from Dharmacon: D-096072-1 against rat (Rattus norvegicus) α-NRX-2 (NM 053846); D-096239-03 and D-096239-04 against rat α-NRX-3 (NM 053817).

Anti-PSD-95 antibody (7E3-1B8) and anti-Synapsin-I antibody are from Affinity BioReagents (Golden, CO) and Chemicon International (Temecula, CA) respectively. Anti-Bassoon antibody (SAP7F407) is from Stressgen Bioreagents (San Diego, CA) or Abcam (Cambridge, MA). Anti-FLAG antibody is from Sigma (St. Louis, MO).

Neuronal Cell Culture and Transfection

Rat (Rattus norvegicus) hippocampal neurons were dissociated from embryonic rats of both genders at E18~19 and plated at 500~1000 cells/mm2 on 12 mm coverslips (Carolina Biologicals) in MEM (Invitrogen) supplemented with 20 mM D-glucose, 2% B27, 5% Fetal Bovine Serum, 2 mM L-glutamine, Penicillin/Streptomycin (all from Invitrogen) and Serum Extender (BD Biosciences). Neurons were transfected between day 5 and 10 in culture with a modified method of calcium phosphate precipitation (Xia et al., 1996). In brief, for each 12-mm cover slip, 2 μg DNA was diluted in 15 μL 250 mM CaCl2 solution and mixed with 15 μL HEPES-buffered saline (274 mM NaCl2, 10 mM KCl, 1.4 mM Na2HPO4, 15 mM glucose, and 42 mM HEPES). 30 minutes later, the mixture was combined with 2 mL serum free culture media (MEM with B27, L-glutamine and Penicillin/Streptomycin), added to neuronal cultures in a 35 mm dish and incubated for 30 minutes to 2 hours. The culture was then washed with calcium-free PBS 3 times, and the original culture media replaced. Nucleofection (Lonza Cologne AG, Cologne, Germany) was performed according to the supplier’s manual. In brief, 2 μg of DNA was mixed with 2 × 106 freshly extracted mixed neurons/glia in 100 μL Nucleofector solution and then electroporated with the Nucleofector Program O-003. The mixture was then mixed with 500 μL culture media and incubated for 20 minutes before plating.

Image Acquisition

1 to 2 days after transfection, hippocampal neurons were incubated in modified Tyrode saline solution (In mM, NaCl 115, KCl 2.8, CaCl2 1, MgCl2 1, HEPES 10, Glucose 10, pH 7.3 ~ 7.4). HEK cells were also used 1 to 2 days after transfection and dissociated from the culture plate by enzyme free dissociation media (Specialty Media/Millipore, Billerica, MA). Transfected HEK cells were then manipulated into contact with axons by controlled flow through a glass pipette. Time-lapse images were acquired on a Zeiss LSM510 confocal microscope. The first frame was taken ~1 minute after the HEK293 cell landed on the axon due to the delay of focusing and switching to the time lapse mode. Each frame was an average of two scans and the interval between frames was 60 seconds to 150 seconds depending on the experiments. Laser power, photomultiplier gain and filter sets were selected to minimize bleaching and bleed-through between channels (In nm, CFP: ex 458, em 470-500; GFP: ex 488, em 500-550; YFP: ex 514, em 530-560; RFP: ex 543, em 565-615). Simultaneous imaging of pDisplay or CD8-YFP and FP-tagged NRXs was performed on a Zeiss LSM 5 Live confocal microscope with a similar setting (In nm, CFP: ex 440, em 445-500; YFP: ex 488, em 530-590; RFP ex 533, em 565 long pass).

FM Staining

FM 4-64 (Molecular Probes) was loaded into neurons by a 2-minute high-K (In mM, NaCl 71.5, KCl 50, CaCl2 2, MgCl2 2, HEPES 20, Glucose 20, pH 7.3 ~ 7.4) depolarization in the presence of 15 μM FM 4-64, washed with normal saline solution (In mM, NaCl 119, KCl 2.5, CaCl2 2, MgCl2 2, HEPES 20, Glucose 20, pH 7.3 ~ 7.4) for 15 minute, and unloaded by a 2-minute 90 mM KCl (In mM, NaCl 31.5, KCl 90, CaCl2 2, MgCl2 2, HEPES 20, Glucose 20, pH 7.3 ~ 7.4) depolarization. In some cases, 1 mM Advasep-7 (CyDex, Kansas) was added in the washing solution to facilitate the washout of extracellular FM 4-64. The dye was excited at 514 nm and a 650 nm long-pass filter was used to collect the signal.

Image Analysis

All images are analyzed in Matlab (Mathworks) using custom scripts.

Time-lapse experiments

Offline linescan was acquired by drawing a 10-pixel wide line along the axon, so each pixel in the linescan is an average of 10 pixels perpendicular to the line. Such measure was taken to minimize movement artifacts. Total intensity under the HEK cell was measured by summating the pixel intensity value within regions of interest defined by the contour of the surrogate postsynaptic cells. The average background was then subtracted from this sum. Accumulation plots in Supplemental Fig. 2 were made by setting the initial value at 0% and the final value 100%.

Immunostaining

Because individual Bassoon and Synapsin puncta became difficult to resolve at high density, we used coverage as an indicator for number of associated synapses. First, a binary mask was generated based on fluorescence of the HEK cell to cover the whole HEK cell, and a second mask was then made by thresholding the Bassoon or Synapsin fluorescence within the first mask. The coverage percentage was calculated by dividing the area of the second mask by the first one.

Relative density

To calculate the relative density of NRXs at the cell contacts, first a binary mask was generated by thresholding the pDisplay-YFP or CD8-YFP (plasma membrane markers) image. Then within this mask, we calculate the density of NRXs by dividing the intensity of NRXs by the intensity of the plasma membrane marker, which accounts for any heterogeneous membrane protein distribution, assuming the plasma membrane marker is not preferentially distributed to any particular region. Finally, the relative density is the average density at the cell contact divided by the average density elsewhere on the cell.

Density of synapses

Images of transfected neurons were turned into binary images by passing through a threshold. The soma was removed from the binary images, and then skeletonized, with short branches (< 6 μm) removed. The resulting images were the backbones of the dendritic trees where the total lengths can be measured. Particle tracking routines from Blair and Dufresne (http://www.physics.georgetown.edu/matlab/) were used to locate individual Bassoon puncta. In brief, images of Bassoon immunostaing are first filtered spatially to minimize background. Then the filtered images are fitted with a 2D-Gaussian curve to obtain the center coordinates. Any Bassoon punctum within 1 μm of the dendrites is considered to be co-localized.

RNAi interference

Images of transfected neurons co-cultured with HEK cells were analyzed manually. LSM Image Browser (Carl Zeiss) was used to view the images and to identify Bassoon puncta at the contacts between HEK cells and transfected neurons. The lengths of the contacts were traced by hand and measured with the LSM Browser. Synaptic density was calculated by dividing the number of Bassoon puncta by the length of the contact. To confirm the analysis and avoid bias, the analysis was repeated by another experimenter blind to the identity of each experimental group.

Statistical Analysis

All graphs are expressed as average ± SEM. Statistical tests are indicated in the figure legends. Mono-exponential recovery curves are fitted to BSN, SYN, and α-NRX accumulation using the following function:

equation M1

where yi are the coverage at time ti, y is the maximal accumulation amount, τ is the time constant, and εi are the residuals. The fitting was done in Matlab using its Curve Fitting Toolbox with non-linear least square method, starting from (y, τ) = (0.5, 2). The initial rate of accumulation is the derivative of the exponential curve at t = 0, which equals to equation M2.

For analysis of NLG-1 and NLG-1ΔB over-expression in neurons, 2 independent transfections were made and analyzed separately. A total of 39-50 neurons from 4 coverslips per experimental condition were imaged and measured to obtain the synapse density and the total number of synapses in each field of view. We then followed the standard method of analysis of covariance (ANCOVA), in which synaptic densities on transfected neurons are linearly regressed to total number of synapses of each frame and categories, following the function below:

equation M3

where yi is the synapse density of transfected neurons; Xi1 is the total number of synapses in the same visual field;

equation M4

β0, β1, β2, and β3 are fitted coefficients, and εi are the residuals.

The fitted function represents three parallel lines that all have the same slope α, but with different intercepts (CD8-YFP control = β0, NLG-1 = β0 + β2, NLG-1ΔB = β0 + β3). The parallel lines model is then compared to the other restrictive models where two categories are considered the same and the other different, or all three are considered the same. ANOVA on the sum of squared residuals showed that the three-parallel-lines model provides significantly more explanation power than the other restrictive models. The parallel lines model is also compared to the model where each group is linear-regressed separately, or the rotated lines model where slopes differ between each group but the intercept is assumed to be the same. Additional two parameters used in separate regressions did not provide significant explanation power (p = 40.8%), while 3 rotated lines model decreases the explanation power (see table below).

ModelSeparate Regressions3 Parallel Lines3 Rotated LinesNLG-ΔB =NLGNLG=CD8Single line
number of parameters644332
SSE119.51121.25124.52128.40128.44152.70
F0.90--7.437.4816.34
p-value40.8%--0.7%0.7%<0.1%

*abbreviation:

NLG=NLG-ΔB: NLG-1 and NLG-1ΔB transfected neurons are considered drawn from the same population, while CD8-YFP transfected ones are different.

NLG=CD8: NLG-1 and CD8-YFP transfected neurons are considered drawn from the same population while NLG-DB transfected ones are different.

Single line: all three groups belong to the same population.

All F-tests and corresponding p-values are compared to the parallel-lines model, with the listed models as null hypotheses.

Results

Neuroligin-1 ΔB Induces Rapid Synapse Formation

While several classes of post-synaptic adhesion molecules are known to trigger pre-synaptic differentiation, it is unclear whether any of these molecules is sufficient to achieve the rapid synapse formation necessary to stabilize transient contacts (Niell et al., 2004; Ruthazer et al., 2006). We compared the rates of induction of presynaptic terminals in cultured hippocampal neurons at new sites of contact with surrogate postsynaptic cells in the co-culture hemi-synapse formation assay (Scheiffele et al., 2000). The surrogate postsynaptic cells were HEK cells expressing one or more of the six different adhesion molecules. The adhesion molecules included: EphB2 (Kayser and Dalva, 2005), SynCAM (Biederer et al., 2002), NGL-2 (Kim et al., 2006), two alternative splice products of NLG-1, the full-length version, and a version lacking insert B (NLG-1ΔB) (Ichtchenko et al., 1995; Scheiffele et al., 2000) and N-cadherin (Fannon and Colman, 1996; Togashi et al., 2002; Abe et al., 2004). The adhesion molecules were transfected, singly or in pairs, into HEK cells and the HEK cells were dropped onto E18-19 hippocampal neurons that had been developing in vitro for 7 days (7 DIV). Unlike previous studies in which synapse formation on HEK cells was assayed 24 hours or longer after surrogate-neuron contact, our co-cultures were fixed and stained with antibodies against the active zone protein Bassoon one hour after contact. Strikingly, compared to CFP-expressing control HEK cells, only HEK cells expressing NLG-1 lacking insert B induced a significant accumulation of BSN one hour after contact (Fig. 1A, B).

Figure 1
HEK293 cell expressing NLG-1ΔB induces rapid formation of presynaptic terminal on neurons

We next asked if the newly induced sites of Bassoon accumulation were competent for transmitter release. This was done by determining if membrane depolarization would stimulate the FM dye uptake that is associated with activity-dependent cycling of synaptic vesicles. We found that one hour after contact with NLG-1ΔB expressing HEK cells, axons have significantly more FM 4-64 positive puncta (7.9 ± 0.9 puncta per cell, n = 6) at the sites of the contact than those axons contacting HEK cells expressing NLG-1 (2.8 ± 0.7 puncta per cell, n = 5, p < 0.05, Student’s t-test; Fig. 1C). These observations indicate that NLG-1ΔB has a special ability to induce the rapid recruitment of the presynaptic machinery and the formation of functioning presynaptic nerve terminals.

When the axonal accumulation of synaptic vesicles and PTVs at sites of HEK cell contact was examined over 24 hours by immunostaining for native Synapsin (SYN) and Bassoon (BSN), we found that although the amount of recruitment by NLG-1 and NLG-1ΔB eventually converges, NLG-1ΔB recruits SYN and BSN more rapidly (Fig. 1D). Single exponential fits indicated that the recruitment of SYN and BSN induced by NLG-1ΔB occurred over a similar time course, with a time constant of ~3 hours (τSYN = 2.80 hours, n = 7-10 cells per time point; τBSN = 2.96 hours, n = 7-10 per time point), while the recruitment of BSN by the full-length NLG-1 was more than two-fold slower (τ = 6.52 hours, n = 7-10 per time point) and the recruitment of SYN by the full-length NLG-1 was almost four-fold slower (τ = 11.11 hours, n = 7-10 cells per time point). The experiments were repeated in two separate sets of cell cultures and transfections, yielding an average initial accumulation rates (y/τ) of BSN and SYN, as shown in Fig. 1E. In all cases, NLG-1ΔB always promoted faster pre-synaptic differentiation than did NLG-1. Taken together, the results demonstrate that among all the adhesion molecules tested (NLG-1, NLG-1ΔB, N-cadherin, EphB2, SynCAM, and NGL-2), NLG-1ΔB induces the fastest pre-synaptic differentiation.

We next followed synapse formation in real time using time-lapse microscopy to focus on axons before and after contact with NLG-1-ΔB-expressing HEK cells. To do this, we transfected the neurons at 7-10 DIV with either an N-terminal GFP fusion of Bassoon (GFP-Bassoon) or a C-terminal YFP fusion of the highly restricted synaptic vesicle protein Synaptophysin (Synaptophysin-YFP). One or two days later axons were imaged for a control frame and then HEK cells expressing NLG-1ΔB were positioned into contact with them and imaging was continued. In 25% (5 of 20 experiments) of the axons imaged in this way, contact with the NLG-1ΔB expressing HEK cell triggered the recruitment of GFP-Bassoon puncta within 15-30 minutes (Fig. 2A), comparable to what has been observed at new neuro-neuronal contacts (Friedman et al., 2000). The higher incidence with which we observed the accumulation of native Bassoon in the 1 hour endpoint antibody labeling experiments as opposed to GFP-Bassoon in the time-lapse experiments may reflect the fact that the native Bassoon was usually imaged in multiple axons that came into contact with each HEK cell, while in the time-lapse experiments GFP-Bassoon was followed in only one contacting axon at a time. A second factor is that the time-lapse experiments on GFP-Bassoon were performed at a lower temperature (~25°C) under atmospheric conditions, as compared to the physiological temperature (37°C) and 5% CO2 used in the antibody-labeling experiments.

Figure 2
NLG-1ΔB on HEK cell rapidly clusters axonal PTVs and SVs within first hour after contact

Consistent with what has been described for the dynamics of the synaptic vesicle protein VAMP2 at neuro-neuronal synapses (Ahmari et al., 2000), Synaptophysin-YFP accumulated rapidly in small puncta under the NLG-1-ΔB-expressing HEK cells. As with GFP-Bassoon, the newly recruited Synaptophysin-YFP was seen in some of the contacts (8 out of 15). Also, as with GFP-Bassoon, when Synaptophysin-YFP puncta appeared their fluorescence increased gradually (Fig. 2B). The average intensity of new SYP-YFP puncta at NLG-1ΔB contacts was lower (69 ± 14%, n = 8) than that seen at pre-existing stable puncta. We conclude that both Synaptophysin containing synaptic vesicles and Bassoon containing PTVs arrive rapidly to new presynaptic terminals induced by NLG-1-ΔB.

Neuroligin-1 ΔB Has an Increased Affinity to α-Neurexin in Neurons

Biochemically, the main difference between NLG-1ΔB and full-length NLG-1 is that NLG-1ΔB binds to both α- and β-NRXs, while NLG-1 only binds to β-NRX (Boucard et al., 2005). Moreover, it was shown recently that a long (48 hour) period of contact with a surrogate postsynaptic cell expressing NLG-1ΔB in hemi-synapse formation that primarily depends on α-NRX in the neuron (Ko et al., 2009b). This suggested to us that α-NRX may also play a role in the rapid induction of presynaptic differentiation by NLG-1-ΔB.

We asked if α-NRX in the neuron is preferentially delivered to sites of contact with NLG-1ΔB expressing HEK cells. To do this, we generated FP-tagged versions of α- and β-NRXs by inserting a monomeric fluorescent protein between the last LG/LNS domain and transmembrane domain (Supplement Fig. 1A). The tagged proteins were shown to retain the ability of untagged NRXs to undergo heterophilic adhesion with NLG-1 and NLG-1ΔB in the following experiments. First, when α-NRX-mYFP or β-NRX-mCFP expressing HEK cells were brought into contact with NLG-1 were expressed in two separate HEK cells and the cells were brought into contact, α-NRX-mYFP and β-NRX-mCFP concentrated at cell-cell junctions (as shown for β-NRX-mCFP in Supplement Fig. 1B and for α-NRX-mYFP in Fig. 3B). To quantify the recruitment, we measured the relative density of NRXs at the cell-cell junction (i.e. the average density at the junction divided by the average density elsewhere on the cell). pDisplay-YFP, a plasma membrane marker, was used to control for any unrelated heterogeneous membrane protein distribution, so the density of NRXs was normalized to the density of pDisplay-YFP. As expected, both NLG-1 and NLG-1ΔB recruited β-NRX equally well, with the relative density of β-NRX-mCFP at the junction being approximately 2 (i.e. the normalized fluorescence of β-NRX-mCFP at the junction was two-times higher than elsewhere on the cell) (Fig. 3A). In contrast, the relative density of α-NRX-mCFP at the NLG-1ΔB junctions was 2.08 ± 0.22 (n = 10), significantly higher than at the NLG-1 junctions (1.19 ± 0.08, n = 10), indicating that there is a preference of α-NRX-mCFP for NLG-1-ΔB. To test whether such a preference remains even when both types of FP-tagged NRXs are present in the same cell, as is the case in neurons, we co-expressed α-NRX-mYFP and β-NRX-mCFP in the same HEK cell and brought this cell into contact with another HEK cell bearing either NLG-1 or NLG-1ΔB (Fig. 3B). The preference could be visualized by plotting the fraction of α-NRX-mYFP at the junction versus that of β-NRX-mCFP (Fig. 3C). NLG-1ΔB recruited both α- and β-NRX (thus the magenta points in Fig. 3C roughly follow the 1:1 ratio diagonal line), whereas NLG-1 recruited mostly β-NRX (thus the blue points in Fig. 3C deviate toward the ordinate). These results confirmed that insertion of fluorescent protein in α- and β-NRXs did not modify their affinities for NLG-1 and NLG-1ΔB.

Figure 3
Preferential recruitment of α-NRX to NLG-1ΔB at HEK-HEK contacts

Having found that NLG-1ΔB recruits α-NRX more potently than does full-length NLG-1 at HEK-HEK contacts, we next asked if the same would be true at contacts between an NLG-1 presenting HEK cell serving as a postsynaptic surrogate and an axon expressing the panoply of other neuronal proteins. We co-expressed α-NRX-mCFP in neurons along with CD8-YFP (a plasma membrane marker) as a reference and examined sites of contact with HEK cells expressing NLG-1ΔB or NLG-1 over 24 hours. We observed a stronger α-NRX-mCFP accumulation at sites of contact with HEK cells expressing NLG-1ΔB than with HEK cells expressing NLG-1. The difference was evident at 1 hour (relative density for NLG-1ΔB/NLG-1 = 3.33 ± 0.43), 10 hours (relative density = 3.69 ± 0.48) and 24 hours (relative density = 3.75 ± 0.81) after contact (Fig. 4A). While α-NRX-mCFP was also recruited to sites of contact with NLG-1 expressing HEK cells, its concentration was significantly lower (relative density = 1.38 ± 0.18 at 1 hour, 1.41 ± 0.15 at 10 hours, and 2.00 ± 0.31 at 24 hours) (Fig. 4B, p < 0.05). In contrast, the concentration of β-NRX-mCFP did not differ between neurons contacting NLG-1 and those contacting NLG-1ΔB, although no further increase was observed after 1 hr as well (Fig. 4C). Notice that no further increase of α-NRX-mCFP was observed beyond an hour after contact, indicating its accumulation reaches maximum within one hour, preceding the recruitments of BSN and SYN (Fig. 4D).

Figure 4
Preferential recruitment of α-NRX to NLG-1ΔB at HEK-neuron contacts

Our observations at HEK-HEK and neuron-HEK contacts generally agree with earlier work on the interaction of the soluble ectodomains of NRX and NLG (Ichtchenko et al., 1995; Boucard et al., 2005). Although these previous studies suggested that α-NRX and full-length NLG-1 do not interact, our results indicate that they have a weak interaction when they are presented on cell membranes.

We next asked if the unique potency of NLG-1ΔB could be due to a more rapid recruitment of axonal α-NRX than β-NRX to the contact site. To address this we carried out time-lapse imaging of both FP-tagged α-NRX and β-NRX in axons before and after contact with HEK cells expressing NLG-1 or NLG-1ΔB. When such neurons were monitored for an hour after contacting HEK cells expressing NLG-1, the accumulation of α-NRX-mCFP was always less than that of β-NRX-mYFP (α/β = 0.81 ± 0.05, n = 5, Fig. 5A & B). However, when the transfected neurons were in contact with HEK cells expressing NLG-1-ΔB, the accumulation of α- and β-NRX was similar (α/β = 0.96 ± 0.10, n = 5, Fig. 5A & B), and significantly different from the case of NLG-1 (paired t-test, p < 0.05, Fig. 5B). These data indicated that the removal of insert B in NLG-1 increases α-NRX concentration at HEK/neuron contacts, but has no effect on β-NRX concentration.

Figure 5
Time-lapse of axonal NRX recruitment to NLG-1ΔB contacts

So far, we have shown that contact with a cell presenting NLG-1ΔB induces a more potent local accumulation of active zone proteins in an axon than does contact with a cell presenting other adhesion proteins and the full-length NLG-1 (Fig. 1A-C). We have also shown that synaptic vesicles and active zone proteins can appear within tens of minutes of contact (Fig. 2) and that, within an hour, the site has activity-dependent vesicle cycling that is characteristic of transmitter release (Fig. 1C). In contrast, the induction is delayed when full-length NLG-1 is the trigger for differentiation (Fig. 1D, E). This led us to extend earlier studies on soluble ectodomains, and much longer (24-48 hr.) interaction times between the full length proteins presented on the surfaces of contacting cells, to show that NLG-1ΔB is able to concentrate more α-NRX in HEK cells (Fig. 3) and in neurons (Fig. 4). In addition, time-lapse experiment in neurons validated that NLG-1ΔB shows no preference between α-NRX and β-NRX (Fig. 5), while NLG-1 prefers β-NRX over α-NRX. We further found that an associated scaffolding protein, CASK, an intracellular partner for both α- and β-NRXs (Hata et al., 1996), accumulates equally fast at NLG-1 and NLG-1ΔB contacts (Supplement Fig. 2).

Knock-down of α-Neurexin-1, 2, and 3 Delays Neuroligin-1ΔB Induced Synapse Formation

Our above results suggest that NLG-1ΔB is uniquely capable of inducing the fast formation of presynaptic nerve terminals because of its interaction with α-NRXs. If this is correct, then we would expect that a reduction of expression levels of α-NRX in the neuron would delay this process. To test this, we used RNA interference to knock down α-NRXs protein levels in cultural neurons. Rats have three α-NRXs, all of which are known to bind NLG-1ΔB (Boucard et al., 2005) and to be present in the hippocampus (Puschel and Betz, 1995). Four shRNA sequences were designed to knock down expression of these three α-NRXs by targeting unique α-NRXs sequences not present in the β form (see Materials and Methods). Each shRNA was driven by its own H1 RNA promoter and the four were incorporated in tandem into a single pSuper-GFP vector (Stove et al., 2006) to ensure that the four α-NRXs shRNAs would be transcribed in the same cell. Co-expression of this quadruple shRNA vector in HEK cells along with each of the FLAG-α-NRX-1, 2, or 3 resulted in a strong reduction in expression, but had no effect on NLG-1-ΔB (Fig. 6A). Having demonstrated the efficacy of the shRNA vector, we next transfected it into neurons by Nucleofection, achieving a transfection rate of 10-30%, based on GFP expression. HEK cells co-expressing NLG-1ΔB and mRFP were co-cultured with the shRNA-transfected neurons and the cultures were stained for endogenous BSN at 1, 3, and 6 hours after HEK-neuron contact. We found that α-NRXs knock down reduced the number of synapses induced by NLG-1-ΔB on transfected neurons both at 3 and 6 hours after contact, indicating that the rapid synapse formation induced by NLG-1ΔB was delayed (Fig. 6B & C). This result supports the interpretation that α-NRX is a key mediator of rapid synapse induction by NLG-1 splice variants.

Figure 6
α-NRX-1, 2, 3 triple knock-down reduces the number of synapses induced by NLG-1-ΔB

The Glycosylation Site in Insert B Reduces Affinity for α-Neurexin and Triggers a Slow Synapse Formation

If the affinity of NLG-1ΔB for α-NRX makes it uniquely capable of inducing fast synapse formation then we would predict that the manipulation of the affinity of NLG-1 for α-NRX should change the rate of synapse induction by NLG-1. Insert B of NLG-1 is known to lower its affinity for the soluble domain of α-NRX due to an N-linked glycosylation site (N303) located within insert B (Boucard et al., 2005). We wondered if mutation of this site would increase NLG-1 affinity to α-NRX in cells. To test this, we performed a HEK cell binding assay with pDisplay-YFP as a membrane marker. We found that the relative density of α-NRX at the junction with NLG-1-N303S expressing cells was 1.84 ± 0.22, similar to what was seen at junctions with cells expressing NLG-1ΔB (2.09 ± 0.22), and significantly higher than what was seen at contacts with NLG-1 expressing cells (1.20 ± 0.08) (Fig. 3A, n = 10 for each group). In contrast, the recruitment of β-NRX was the same for NLG-1, NLG-1ΔB and NLG-1-N303S (Fig. 3A, n = 10 for each group).

Having observed that the apparent affinity of NLG-1-N303S for α-NRX was similar to that of NLG-1ΔB, we asked whether NLG-1-N303S would also induce new presynaptic terminals with the greater speed of NLG-1-ΔB. We found that within an hour of contact, native Bassoon accumulated significantly around HEK cells transfected with NLG-1-N303S (NLG-1: 1.2% ± 0.7%; NLG-1-N303S: 10.4% ± 3.8%, n = 6 and 10 respectively; p < 0.05, Mann-Whitney Ranksum test), as found with NLG-1-ΔB, but not NLG-1 (Fig. 1). This finding supports the idea that the accelerated rate of synapse induction correlates with the recruitment of α-NRX in the axon at the site of contact with the NLG-1 presenting cell.

Intracellular Clustering of NLG-1 by PSD-95 Accelerates Bassoon Recruitment

If the affinity of NLG-1 for α-NRX determined the rate with which it induces pre-synaptic differentiation, as proposed above, then clustering of NLG-1 in the membrane would be predicted to increase binding and accelerate pre-synaptic differentiation. NLG-1 forms oligomers on its own (Scheiffele et al., 2000; Comoletti et al., 2007), but it also binds the multimerizing scaffolding protein PSD-95 (Hsueh et al., 1997; Hsueh and Sheng, 1999), suggesting that PSD-95 might increase NLG-1 clustering and, consequently, increase α-NRX binding and favor synapse induction. We tested this notion by comparing the recruitment of active zone proteins in hippocampal axons at contacts with HEK cells that expressed either NLG-1 alone or NLG-1 along with PSD-95-mRFP. Adding PSD-95-mRFP was found to increase the recruitment of Bassoon (Fig. 7). Co-expression of PSD-95-mRFP also boosted Bassoon recruitment to NLG-1ΔB contacts (Fig. 7), indicating that Bassoon recruitment by NLG-1ΔB can be further enhanced.

Figure 7
PSD-95 co-expression with NLG in HEK cell accelerates recruitment of presynaptic machinery to contact site with axon

Neuroligin-1ΔB Exhibits an Enhanced Ability to Increase Synaptic Density in Neurons

Over-expression of NLG-1 in neurons is known to increase synaptic density (Dean et al., 2003; Boucard et al., 2005; Sara et al., 2005; Ko et al., 2009b). We asked whether over-expression of NLG-1ΔB in neurons, with its ability to accelerate presynaptic differentiation, would further increase the density of synapses at neuro-neuronal contacts at later time points. DIV 9 Neurons were grown for 16 hours following transfection with CD8-YFP alone, as a control, or CD8-YFP along with either NLG-1-ΔB or NLG-1. The cultures were then stained with anti-BSN antibodies (Fig. 8A). Because the density of synapses on a CD8-YFP transfected neuron was highly correlated with the average synaptic density within the field of view (R2 = 0.77, Fig. 8B), we evaluated the data with an analysis of covariance (ANCOVA) by fitting the data to a linear additive parallel lines model, where the slope is taken to be an invariant function of the number of processes in the field of view and greater potency of synapse induction by the adhesion protein results in higher values along the y-axis (see Materials and Methods). We found that on average, NLG-1ΔB increased Bassoon puncta density over the CD8-YFP control by 1.2 ± 0.42 synapses/10 μm length of dendrite, whereas NLG-1 increased Bassoon puncta density by only half that amount, 0.6 ± 0.44 synapses/10 μm (Fig. 8C). Thus, not only is NLG-1ΔB more potent in inducing the recruitment of presynaptic machinery than full-length NLG-1 at contacts with surrogate postsynaptic cells, but it is also more potent at axo-dendritic contacts.

Figure 8
Over-expression of NLG-1 or NLG-1ΔB in neurons elevates synaptic density to different levels

Discussion

A number of post-synaptic adhesion molecules have been implicated in synapse formation. We examined several of these and asked which is able to induce presynaptic differentiation at a rate comparable to the one observed at new neuro-neuronal contacts (Ahmari et al., 2000; Friedman et al., 2000; Bresler et al., 2004; Garner et al., 2006). We found that, between the full-length NLG-1 and its alternative splice product NLG-1-ΔB, SynCAM, EphB2, NGL-2 and N-Cadherin, only NLG-1ΔB induces fast presynaptic differentiation (Fig. 1A, B).

The unique ability of NLG-1ΔB to rapidly induce functional presynaptic terminals was mimicked by removing the N-linked glycosylation site (N303) within Insert B from the full length NLG-1. As shown earlier in isolated ecto-domains mixed in solution (Boucard et al., 2005), we found that absence of insert B or its glycosylation site makes NLG-1 bind more strongly to α-NRX (Fig. 3A). The results suggest that the fast presynaptic differentiation results from the recruitment of α-NRX to the site of contact with the NLG-presenting cell. Indeed, mutants of NLG-1 that are unable to bind α-NRXs have been shown earlier to fail in synapse induction (Ko et al., 2009b), and LRRTM2, which binds both α- and β-NRX, was found to be more potent than full length NLG-1 (Siddiqui et al., 2010). Consistent with this, we found that NLG-1ΔB recruits more α-NRX than does the full-length NLG-1 in HEK cells (Fig. 3) and neurons (Fig. 4). Moreover, we found that knockdown of all three α-NRXs delays the rate of synapse formation induced by NLG-1ΔB (Fig. 6), demonstrating that NLG-1ΔB/α-NRX interaction is vital to rapid pre-synaptic differentiation.

The knock-down of α-NRXs results in fewer induced synapses at 3 and 6 hours after contact (Fig. 6B). No difference was observed at 1 hour, but this likely reflects the fact that the biggest divergence between pre-synaptic differentiation induced by NLG-1 and NLG-1ΔB occurs between 2-12 hours (Fig. 1D), while the difference at 1 hour is small and close to the detection limit (Fig. 1B, D).

While affinity chromatography on soluble ecto-domains suggests that NLG-1 exclusively interacts with β-NRX (Ichtchenko et al., 1995; Boucard et al., 2005), we found that full-length NLG-1 presented on a contacting cell is capable of recruiting α-NRX. This may explain why, although delayed in comparison to NLG-1ΔB, NLG-1 eventually induces presynaptic terminals capable of activity-dependent vesicle release. It is possible that clustering in the membrane and/or high expression in HEK cells partially overcomes the relatively low affinity of NLG-1 and recruits sufficient α-NRX to induce new synapses. Consistent with this, we find that PSD-95, a postsynaptic scaffolding protein, which clusters post-synaptic proteins (including NLG-1) into density arrays (Hsueh et al., 1997; Hsueh and Sheng, 1999), accelerates the pre-synaptic differentiation triggered by NLG-1. In a similar vein, it was suggested recently that glial factor Thrombospondin-1 accelerates pre-synaptic differentiation in neurons by clustering NLG-1 (Xu et al., 2010).

Over-expression of NLG-1 in neurons was shown earlier to increase the density of synapses in hippocampal cultures, as assayed by staining for pre-synaptic proteins (Dean et al., 2003; Boucard et al., 2005; Sara et al., 2005; Ko et al., 2009b). We found that over-expression of NLG-1ΔB increases synaptic density even more. While this is in contrast to previous reports (Boucard et al., 2005; Ko et al., 2009b), which showed that NLG/α-NRX interaction influences synaptic size rather than density, two possible explanations may account for the discrepancy. First, in our study the fluorescent protein tag was inserted into the extracellular domain of NLG-1, whereas in the earlier studies (Boucard et al., 2005; Ko et al., 2009b) it was inserted into the intracellular domain, where it might influence protein trafficking or signaling. Second, our analysis took into account the synaptic density within the field of view. All in all, our results support the idea that in addition to the role of NLG/α-NRX interaction in the determination of excitatory vs. inhibitory synaptic connections (Chih et al., 2005) this interaction is a key to rapid synapse formation.

With fluorescent imaging and immunocytochemistry over 24 hours, we were able to define the time course of pre-synaptic differentiation induced by NLG-1 and NLG-1ΔB. The initial adhesion between α-NRX and NLG-1, or its splice variants, occurs quickly: within a few minutes (Fig. 5A), reaching a maximum within an hour (Fig. 4B), consistent with a role in triggering synapse formation. Downstream components such as active zone proteins and synaptic vesicles (marked by BSN and SYN, respectively) arrive at the new contact sites within tens of minutes to hours, depending on the level of α-NRX (Fig. 1D & 4D). Interestingly, at the site of contact with the NLG-expressing HEK cells there is a high degree of co-localization of active zone proteins (including Bassoon) and synaptic vesicles (containing Synapsin) and this was seen at all time points after contact (Figure 1E), indicating that their recruitments are coordinated.

Although the amount of α-NRX recruitment and the rate of formation of new synapses are greater for NLG-1ΔB, the number of NLG-1 triggered synapses catches up after a few hours when the postsynaptic element is a HEK cell. This suggests that the assembly of pre-synaptic components is rate limiting, but once pre-synaptic assembly has occurred the synapses are stable. While the HEK cell is a powerful minimal model for the dendrite, permitting us to define the molecular interactions and the time and place of contact with the axon, however, it lacks the dynamic nature of the dendrite’s morphology. In the natural case of axo-dendritic contacts, where the presentation of the postsynaptic adhesion protein is made by the dynamic dendritic filopodia, the pre-post contact is transient and, as a consequence, the speed of the presynaptic adhesion complex formation and the release machinery recruitment will likely make a significant difference to the number of synapses that form. This would be expected to favor NLG-1ΔB for its more efficient recruitment of α-NRX. Indeed, over-expression of NLG-1ΔB results in a larger number of neuro-neuronal synapses than does NLG-1 (Fig. 8C).

An important objective for future studies will be to determine why α-NRX induces quicker presynaptic differentiation. α-NRX and β-NRX have a common cytoplasmic domain, but differ in their extracellular domains. Since the recruitment of β-NRX is insufficient for fast maturation, acceleration of synapse formation appears likely to require the part of the protein that is unique to α-NRX, namely the extracellular domain that only exists in α-NRX (Ushkaryov et al., 1992; Ushkaryov and Sudhof, 1993). This raises the possibility that extracellular α-NRX binding partners may be involved in rapid synapse induction. These include Neurexophilin (Petrenko et al., 1996) and dystroglycans (Sugita et al., 2001). However, Neurexophilin-1 is only found in a subset of inhibitory neurons (Petrenko et al., 1996), Neurexophilin-3 is absent from hippocampus (Beglopoulos et al., 2005), and dystroglycans are only found at inhibitory synapses (Lévi et al., 2002), making them unlikely candidates to participate in accelerating synapse formation at all synapses. In addition, although synaptic N-type Ca2+ channels are significantly reduced in α-NRX triple knock-out mice, they do not interact with α-NRX directly (Missler et al., 2003), suggesting there may well be other, as yet unidentified, binding partners.

In conclusion, while multiple synaptogenic adhesion molecules are able to induce pre-synaptic terminal differentiation, we showed that the induction rates by several of these molecules is much slower than what is seen during the formation of synapses at new sites of axo-dendritic contact. The exceptions were an alternative splice product of NLG-1 that was missing its Insert B and a mutant of NLG-1 missing just the N-linked glycosylation site that is situated within Insert B. Using a combination of immunostaining and time-lapse microscopy, we found that the fast presynaptic induction by the NLG-1ΔB alternative splice product is associated with its stronger affinity to α-NRXs. This provides a potential mechanism for neurons to regulate their synapse formation rates during development to alter the connectivity of the network.

Supplementary Material

Supp1

Supplemental Figure 1. Construction of FP-tagged NLG-1 and β-NRX-1:

(A) Schematic diagrams of FP-tagged Neuroligin (NLG-1) and β-Neurexin (β-NRX-1). The monomeric FP is inserted between the transmembrane domain and the extracellular region that mediates adhesion. (B) NLG-1-mRFP (red) in one HEK cell and β-NRX-mCFP (cyan) in another HEK cell accumulate at the HEK-HEK contact. Merged fluorescence image superimposed on DIC image shows NLG-1 expressing cell (asterisk) and β-NRX expressing cell (diamond). (C) NLG-1-mCFP and NLG-1-mRFP retain synaptogenic activity when expressed in HEK cells co-cultured with neurons for 24 hours. Strong immunoreactivity of presynaptic vesicle marker Synapsin (green on left panel and red on right panel) surrounds the NLG-1-FP transfected cell.

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Supp2

Supplemental Figure 2. Rapid recruitment of axonal CASK to contact with HEK cell expressing NLG-1 or NLG-1ΔB:

(A) Time-lapse imaging of GFP-CASK in the axons of transfected neurons. HEK cells (white circles) co-expressing NLG-1ΔB and mRFP were positioned in contact with the axon of a transfected neuron and imaged for 1 hour. Accumulation of GFP-CASK is seen minutes after contact, beginning with one or two initial spots, depending on the geometry of the cell-axon contact. Within 1 hour the initial spots expand laterally with strong GFP-CASK fluorescence. Images were acquired every 60 seconds. The first frame was taken ~1 minute after the HEK cell landed on the axon. Linescans along the axon between yellow arrowheads are shown on right. (B) Quantification of GFP-CASK accumulation (n = 4) overlaying on that of α-NRX-mCFP and β-NRX-mYFP (n = 4 from Figure 5B) in axons under HEK cells expressing NLG-1ΔB + mRFP shows that NRXs and CASK accumulate at a similar rate. (C) Time-lapse imaging of GFP-CASK in neurons contacting HEK cells transfected with NLG-1-mRFP for 1 hour where images are acquired every 150 seconds. (D) Quantification of GFP-CASK accumulation (n = 4) overlaying on that of β-NRX-mYFP (n = 8) under HEK cells expressing NLG-1-mRFP shows that β-NRX and CASK accumulate at a similar rate. In both (B) and (D), the curves were normalized such that the initial value is 0% and final value at one hour is 100%

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Supp3

Acknowledgments

We thank T. Sudhof for SynCAM1; L. Reichardt for GFP-CASK; E. D. Gundelfinger for Bassoon-GFP; R. Tsien for mRFP; C. Kaether for Synaptophysin-GFP; F. Irie for EphB2; E. Kim for NGL-2. We also warmly thank S. DeMaria for initiating the project, S. Wiese for general technical assistance; I. Hafez for assistance with FM staining; H. Aaron and T. Machen for help with confocal imaging and microscopy equipment; S. Pautot for helpful discussion; G. Agarwal for aid in image analysis programming; O. Tulyathan for the blind analysis of Bassoon immunostaining for the shRNA knock-down experiment; and countless rats for their sacrifice. This work was supported by US National Health Institute (NIH) grants to E.I (RO1NS050833) and the Nanomedicine Development Center for the Optical Control of Biological Function (5PN2EY018241). H.L. was supported by a US National Science Foundation (NSF) IGERT grant (0437079), and C.D. by an NIH training grant (T32 GM007048).

Footnotes

Competing Interest Statement The authors declare that they have no competing financial interests.

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