Presynaptic Targeting of α4β2 Nicotinic Acetylcholine Receptors Is Regulated by Neurexin-1β*

The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between α4β2 AChRs and the presynaptic cell adhesion molecule, neurexin-1β. In non-neuronal tsA 201 cells, recombinant neurexin-1β and mature α4β2 AChRs form complexes. α4β2 AChRs and neurexin-1β also coimmunoprecipitate from rat brain lysates. When exogenous α4β2 AChRs and neurexin-1β are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1β. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1β lacking the extracellular domain. Additionally, when α4β2 AChRs, α7 AChRs, and neurexin-1β are coexpressed in the same neuron, only the α4β2 AChR colocalizes with neurexin-1β at presynaptic terminals. Collectively, these data suggest that neurexin-1β targets α4β2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on α4β2 AChRs, may contribute to the etiology of these neurological disorders.

The clustering of ion channels or receptors and precise targeting to pre-and postsynaptic specializations in neurons is critical to efficiently regulate synaptic transmission. Within the central nervous system, neuronal nicotinic acetylcholine receptors (AChRs) 5 regulate the release of neurotransmitters at presynaptic sites (1) and mediate fast synaptic transmission at postsynaptic sites of neurons (2). These receptors are part of a family of acetylcholine-gated ion channels that are assembled from various combinations of ␣2-␣10 and ␤2-␤4 subunits (3). AChRs participate in the regulation of locomotion, affect, reward, analgesia, anxiety, learning, and attention (4,5).
The ␣4␤2 subtype is the most abundant AChR receptor expressed in the brain. Multiple lines of evidence support a major role for ␣4␤2 AChRs in nicotine addiction. ␣4␤2 AChRs show high affinity for nicotine (6) and are located on the dopaminergic projections of ventral tegmental area neurons to the medium spiny neurons of the nucleus accumbens (7,8). Furthermore, ␤2 AChR subunit knock-out mice lose their sensitivity to nicotine in passive avoidance tasks (9) and show attenuated self-administration of nicotine (10). ␣4 AChR subunit knock-out mice also exhibit a loss of tonic control of striatal basal dopamine release (11). Finally, experiments with knock-in mice expressing ␣4␤2 AChRs hypersensitive to nicotine demonstrate that ␣4␤2 AChRs indeed mediate the essential features of nicotine addiction including reward, tolerance, and sensitization (12). High resolution ultrastructural studies show that ␣4 subunit-containing AChRs are clustered at dopaminergic axonal terminals (13), and a sequence motif has been identified within the ␣4 AChR subunit cytoplasmic domain that is essential for receptor trafficking to axons (14). However, the mechanisms underlying the targeting and clustering of ␣4␤2 AChRs to presynaptic sites in neurons remain elusive.

EXPERIMENTAL PROCEDURES
Generation of Constructs-All of the constructs were made by PCR using appropriate pairs of forward and reverse synthetic oligonucleotide primers (Invitrogen) and Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA). Rat ␣4, rat ␤2, and chicken ␣7 AChR subunit cDNAs were cloned into the mammalian cell expression vector pEF6/Myc-His A as described previously (28). Mouse neurexin-1␤ lacking the insert at splice site 4 with an extracellular VSV-G epitope tag at the mature N terminus of the protein (NRX) and mouse neuroligin-1 with an extracellular HA epitope tag at the mature N terminus of the protein (NLG) were kind gifts from Dr. Peter Scheiffele (29). The reading frame of full-length mouse NRX (NRX1-447) was amplified by PCR and subcloned between the EcoRI and XbaI sites of pEF6A vector. Truncation mutants were also made by PCR to create NRX⌬C (NRX1-389) lacking the C-terminal cytoplasmic domain and NRX⌬EC (⌬47-360) lacking the entire extracellular domain. Numbering includes the VSV-G tag.
tsA 201 Cell Culture-Human tsA 201 cells, a derivative of the human embryonic kidney cell line 293 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) as previously described (30). tsA 201 cells were transfected using FuGENE 6 (Roche Applied Science).
Primary Hippocampal Neuron Culture-The cultures were prepared essentially as previously described (31). Briefly, the hippocampi were isolated from embryonic day 18 rat embryos and dissociated by trituration after incubation in 0.25% trypsin/ Hanks' balanced salt solution for 15 min at 37°C (Invitrogen). The cells were plated on poly-L-lysine (Sigma)-coated glass coverslips at 100,000 cells/well and maintained in 500 l of neurobasal media supplemented with B27 and 0.5 mM L-glutamine (Invitrogen). The cells were washed and refed with fresh medium after 16 h. 200 l of medium was exchanged on DIV 3 and 6 -7. The neurons were transfected at DIV 7-10 using the Clontech CalPhos mammalian transfection kit (BD Bioscience, Palo Alto, CA) as described (32).
Bromoacetylcholine (BAC)-conjugated Bead Preparation and Ligand Affinity Capture-Affi-Gel 401 was prepared from Affi-Gel 102 (Bio-Rad). 10 ml of Affi-Gel 102 was washed with 0.5 M NaHCO 3 , followed by incubation with 10% N-acetyl-DL-homocysteine thiolactone, 0.5 M NaHCO 3 , pH 8.5, overnight at 4°C with stirring. The following day, the gel was washed with 0.1 M NaCl and then 0.2 M NaOAc, 0.1 M 2-mercaptoethanol. The gel was slurried with deionized H 2 O, and the pH was adjusted to between 6 and 7. One ml of acetic anhydride was added in five 200-l aliquots at 10-min intervals, adjusting the pH to between 6 and 7 after each addition with 10 M NaOH. Ten min after the last aliquot was added; the pH was adjusted to 9.5 and incubated for 30 min. The gel was rinsed with deionized H 2 O until the pH was under 9.0 and then slurried with 20 ml deionized H 2 O. While stirring, 0.63 g of NaCl, 0.104 g of EDTA, 0.014 g of sodium azide, 0.42 g of Tris, and 55 l of 2-mercaptoethanol were added. The pH was adjusted to 7.75, and the gel was stored at 4°C until use. BAC was coupled to Affi-Gel-401 as described (34). tsA 201 cells were homogenized and incubated in 2% Nonidet P-40 lysis buffer for 2 h at 4°C and then centrifuged at 12,000 ϫ g for 15 min at 4°C. The cleared lysates were incubated with 50 l of BAC-Affi-Gel 401 overnight at 4°C. For the negative control, the lysate was incubated with 10 M nicotine for 15 min prior to the addition of BAC-Affi-Gel 401. The gel was then washed three times with lysis buffer and eluted in sample buffer at 60°C for 30 min, and then ␤-mercaptoethanol was added to the eluted samples prior to analysis by SDS-PAGE.
Pulldowns from Transfected tsA 201 Cells and Rat Brain-Transfected tsA 201 cells were solubilized in 1% Nonidet P-40 and subject to pulldowns using the FLAG M2 beads (Sigma) as previously described (30). For native AChR pulldowns, the Abs were covalently coupled to protein G-Sepharose beads. Briefly, ϳ5 g of affinity purified anti-␤2 (mAb 295) or rat or mouse IgG were incubated with 50 l of a 1:1 slurry of Sepharose beads for 2 h at room temperature in PBS containing 0.1% azide with gentle rotation. After washing with 200 mM borate buffer ϩ 3 M NaCl, pH 9.0, the beads were incubated with 20 M dimethylpimelimidate in the 200 mM borate buffer ϩ 3 M NaCl for 30 min at room temperature. After several washes with 200 mM borate buffer ϩ 3 M NaCl, the unreacted sites on the beads were then blocked using 200 mM ethanolamine, pH 8.0, for 2 h at room temperature. The beads were washed with PBS several times and finally with 200 mM glycine once and then stored at 4°C in PBS containing 0.1% sodium azide. Frozen rat brains were homogenized and solubilized in 1% Nonidet P-40 buffer as previously described (28). Detergent-solubilized brain extracts (typically 1-3 ml) were precleared with 50 l of protein G-Sepharose bead slurry and then incubated with 50 l of mAb covalently cross-linked protein G-Sepharose beads (ϳ5-10 g of antibody/50 l of beads) for 3-4 h at 4°C. The beads were washed and eluted with sample buffer (lacking ␤-mercaptoethanol to avoid reduction of the disulfide linkage of the IgG chains) at 60°C for 30 min, and then ␤-mercaptoethanol was added to the eluted samples prior to analysis by SDS-PAGE.
Immunoblotting-Following separation using SDS-PAGE, the proteins were transferred onto polyvinylidene difluoride membrane and incubated with diluted Abs in PBS containing 5% nonfat milk powder. The binding of the primary Abs to proteins was detected using appropriate secondary Abs as previously described (30).
Quantitation of Cell Surface ␣4␤2 AChR and Neurexin-Cell surface ␣4␤2 AChRs and NRX were measured using an enzyme-linked immunoassay previously described (28). Briefly, transfected, tsA 201 cells (0.5 ϫ 10 6 cells/well) were blocked with 3% BSA/PBS and incubated for 1 h with anti-␤2 subunit (mAb 295) or anti-VSV-G antibodies in 3% BSA/PBS, washed, fixed with formaldehyde (3%), washed, and blocked again. The cells were incubated with horseradish peroxidase-conjugated goat anti-rat secondary Ab for 1 h in the presence of 3% BSA, washed, and incubated with 300 l of the horseradish peroxidase substrate 3,3Ј,5,5Ј-tetramethylbenzidine (Sigma) for 1 h. The absorbance of the supernatant was then measured at 655 nm in a Beckman spectrophotometer. The values obtained using this assay are the mean Ϯ S.E. and were statistically analyzed using an analysis of variance test. The significance level was set at p Ͻ 0.05. The nonspecific background to nontransfected cells was typically Ͻ0.5% of the total binding observed for transfected cells.
Immunostaining and Imaging-For the mixed neuron/tsA 201 cell assays, the cultures were fixed in 4% paraformaldehyde, 4% sucrose, Hanks' balanced salt solution (with Ca 2ϩ and Mg 2ϩ ), pH 7.3 (15 min at room temperature), blocked with 3% normal goat serum, 3% BSA, Hanks' balanced salt solution with 0.2% Triton X-100 (30 min at room temperature), and incubated with the appropriate primary (overnight at 4°C) and secondary (90 min at room temperature) antibodies. Coverslips were mounted onto slides with ProLong Gold antifade reagent (Invitrogen). The cells were visualized using an Olympus IX81 spinning disc confocal microscope (Tokyo, Japan) with a xenon arc illumination source through a 60ϫ (numerical aperture, 1.42) or 40ϫ (numerical aperture, 1.35) Olympus oil immersion objective. Single-plane fluorescence images were captured using a Hamamatsu EM camera, and the images were processed using the Slide Book version 4.2 software. When the observed fluorescence intensity of antibody staining observed was weak, post acquisition intensities of images were adjusted in the different channels using the gamma function of the slide book software to enhance visibility of axons and terminal in the figures shown. In all cases, the essential features of the original images were not altered. The figures were then processed with Adobe Photoshop CS.
Statistics-Targeting quantification was determined from 29 -71 cells/condition from three independent experiments. Random neuroligin-1-expressing cells were imaged, and the targeting of the constructs was quantified as the number of neurons with targeting/number of neuroligin-1 cells contacted. The values obtained are the means Ϯ S.E. and were statistically analyzed by a Student's t test.

RESULTS
␣4␤2 AChRs in the central nervous system are targeted to presynaptic terminals, but the mechanisms underlying their recruitment remain unclear. We investigated the possibility that ␤-neurexins, which are also highly enriched at axon terminals, have a functional role in the synaptic targeting of ␣4␤2 AChRs. A neurexin-1␤ isoform was tested in subsequent functional studies with recombinant ␣4␤2 AChRs.
Neurexin-1␤ Forms Complexes with Recombinant ␣4␤2 AChRs in tsA 201 Cells-To determine whether NRX forms complexes with recombinant ␣4␤2 AChRs, we coexpressed VSV-G-tagged neurexin-1␤ (NRX) with the ␣4 and the N-terminal FLAG-tagged ␤2 AChR subunits (labeled as ␣4␤2 FLAG AChRs) by transfecting tsA 201 cells with their respective cDNAs. Forty-eight hours post-transfection, 1% Nonidet P-40solubilized cell lysates were incubated with FLAG M2 antibody covalently attached to agarose beads. Proteins eluted from these beads were then fractionated by SDS-PAGE and subjected to immunoblot analyses using Abs recognizing the ␣4 AChR subunit, the ␤2 AChR subunit, and the VSV-G tag. NRX was found in complexes with Nonidet P-40-solubilized ␣4␤2 FLAG AChRs (Fig. 1A). To confirm that the complex formation between NRX and ␣4␤2 AChRs is not an artifact of detergent solubilization, we mixed Nonidet P-40-solubilized extracts from cells expressing NRX alone and cells expressing ␣4␤2 FLAG AChRs alone in a pulldown experiment using FLAG M2 beads (Fig. 1B). No NRX was coimmunoprecipitated with ␣4␤2 FLAG AChRs, indicating that the complex formation between NRX and ␣4␤2 AChRs was not induced by detergent solubilization but instead was due to complex formation within the cell membrane.
To further verify that NRX forms complexes with assembled ␣4␤2 AChRs, tsA 201 cells were cotransfected with untagged ␣4␤2 AChRs and NRX and processed as in Fig. 1A. However, in this case (Fig. 1C), the ␣4␤2 AChRs and their associated proteins were captured with a ligand that has a high affinity for ␣4␤2 AChRs (BAC-conjugated to Affi-Gel 401 resin) (35). When ␣4␤2 AChRs and NRX were coexpressed, BAC-conjugated beads captured both ␣4 and ␤2 AChR subunits, as well as NRX (Fig. 1C, BAC affinity capture, first lane). As a control for BAC capture specificity, lysates were incubated with 10 M nicotine for 15 min prior to the addition of the BAC-coupled beads. Pretreatment with nicotine blocked the binding of BAC to the receptor complex (Fig. 1C, BAC, second lane). Additionally, BAC failed to capture the ␣4 AChR subunit if it was not coexpressed with the ␤2 AChR subunit (data not shown), indicating that only fully formed pentamers are affinity-purified. When the N-terminal HA-tagged neuroligin-1 (NLG), a trans-synaptic binding partner of NRX, was coexpressed with ␣4␤2 AChRs and incubated with BAC, the anti-HA antibody did not detect NLG in the pulldown (Fig. 1C, BAC, third lane), suggesting that the complex formation between NRX and ␣4␤2 AChRs is specific.
Neurexin-1␤ Forms Complexes with Native ␣4␤2 AChRs Isolated from Rat Brain-To determine whether neurexin-1␤ forms complexes with native ␣4␤2 AChRs, as it does with recombinant ␣4␤2 AChRs, 1% Nonidet P-40-solubilized rat brain membrane extracts were incubated with a ␤2 AChR sub-unit-specific mAb (mAb 295 or 270) or nonspecific rat IgGs (as a control), and the eluates were fractionated by SDS-PAGE and immunoblotted using Abs to the ␣4 AChR subunit, the ␤2 AChR subunit, and neurexin-1 (that was reported to cross-react with both the 1␣ and 1␤ isoforms). Both ␣4 and ␤2 AChR subunits are captured by the anti-␤2 antibody. Note that the endogeneous levels of ␣4␤2 AChRs in the lysates lanes of the blot are below the threshold for detection by the anti-␣4 and anti-␤2 AChR antibodies (Fig. 2A). The anti-neurexin antibody (P-15) detects a ϳ66-kDa band in both the lysate lane and the ␤2 immunoprecipitation lane ( Fig. 2A, IP, NRX). No band of this size was observed in the pulldown using the nonspecific IgG as a control. Furthermore, no bands corresponding to neurexin-1␣ isoforms expected at ϳ165 kDa were observed in either the lysates or the pulldowns (Fig. 2A). Hence, we were unable to experimentally determine whether other neurexin-1␣ isoforms also form complexes with the ␣4␤2 AChRs. When the immunoblots were probed with an anti-neuroligin-1 Ab, a strong band of the expected size (ϳ110 kDa) was detected in the lysate but was absent in the precipitate captured with the anti-␤2 AChR antibody (Fig. 2B). Additionally, an antibody against N-cadherin, a protein expressed in both pre-and postsynaptic membranes, did not detect this protein in the pulldown. These data suggest that neurexin-1␤ and the ␣4␤2 AChRs are present in specific complexes in vivo.
Because the evidence that neurexins form complexes with native ␣4␤2 AChRs relied on the use of a commercially generated anti-neurexin goat polyclonal antiserum (P-15, sc-1334; Santa Cruz) that has not been extensively characterized by other investigators, we additionally verified that this antiserum could recognize recombinant NRX expressed in tsA 201 cells (Fig. 2C). Equal amounts of samples from two eluates of pulldown experiments, one from cells coexpressing ␣4␤2 FLAG AChRs and VSV-G-tagged NRX and another from cells coexpressing ␣4␤2 FLAG AChRs and VSV-G-tagged NRX lacking its C terminus (NRX⌬C), were loaded in parallel, and two sets of blots were probed with neurexin I or VSV-G antiserum. The results show that the neurexin I antiserum recognized both NRX and NRX⌬C and that this antiserum weakly binds fulllength NRX but is specific in its binding. Stronger reactivity of the Abs with the NRX⌬C compared with the full-length NRX is observed possibly because truncation of the C terminus in the NRX⌬C construct facilitates increased Ab access to highly antigenic terminal residues of the peptide epitope originally used to raise this antiserum. It is possible that our inability to detect the neurexin-1␣ isoforms may also be due to conformational masking of this epitope by the extracellular domains of the neurexin-1␣ isoforms.
Neurexin-1␤ Does Not Affect the Expression Levels of ␣4␤2 AChRs-To investigate the functional significance of the interaction of NRX with the ␣4␤2 AChRs, we first determined whether it affected the steady state levels of recombinant ␣4 or ␤2 AChR subunits. Either the pEF6A vector (as a control) or NRX was coexpressed in tsA 201 cells with ␣4␤2 AChRs, and 48 h after transfection, the cells were lysed, separated by SDS-PAGE, and subjected to immunoblot analyses using Abs to the ␣4 and ␤2 AChR subunits. No significant change in the steady state levels of the ␣4 or ␤2 AChR subunits was observed, sug- gesting that NRX does not play a role in the early events that regulate AChR subunit stability (Fig. 3A).
Next, we assessed whether coexpression of NRX with ␣4␤2 AChRs altered the steady state levels of either the ␣4␤2 AChRs or NRX itself on cell surface membranes. Surface expression of the ␣4␤2 AChRs was measured using an Ab to the extracellular domain of the ␤2 AChR subunit (mAb 295) in conjunction with a previously described enzyme-linked immunoassay (28). Similarly, the surface expression level of the NRX was measured with this same assay but using an Ab to the VSV-G tag. The coexpression of ␣4␤2 AChRs with NRX did not significantly change their surface expression levels compared with when they were expressed alone (Fig. 3, B and C). The results suggest that NRX does not affect the trafficking of ␣4␤2 AChRs to the cell surface membrane and vice versa and thus their rates of exo-or endocytosis to cell surface membranes of tsA 201 cells.
Neurexin-1␤ Induces Presynaptic Targeting of ␣4␤2 AChRs in Rat Hippocampal Neurons-To test whether the presynaptic maturation functions of neurexins included recruitment of ␣4␤2 AChRs to synaptic terminals, we transfected rat hippocampal neurons with ␣4 and ␤2 AChR subunits and NRX cDNAs and cocultured them with tsA 201 cells transfected with neuroligin-1-HA (NLG). A similar in vitro assay for neurexinneuroligin signaling has been used extensively by other investi-gators (33). Neurons expressing ␣4␤2 AChRs, in the absence of exogenous NRX, exhibited a low level (ϳ11.1 Ϯ 7.4%, n ϭ 71) of AChR accumulation at contact sites formed with tsA 201 cells expressing NLG (Fig. 4A). It is important to note that this low level accumulation was qualitatively very different from those observed when NRX was coexpressed in neurons. These boutons were quite small and frequently did not recruit NLG to the contact sites (Fig. 4A, arrowhead), suggesting they were most likely immature synaptic boutons.
To determine whether the hemi-synapses that formed between NRX-expressing neurons and NLG-expressing tsA 201 cells could recruit presynaptic vesicle markers, cocultures were costained with Abs to the ␤2 AChR subunit, VSV-G, and synapsin-1, a synaptic vesicle protein. Nearly all the contact sites at which ␣4␤2 AChRs and NRX were enriched were also positive for synapsin-1, indicating that these were indeed mature presynaptic terminals (Fig. 5, A-D, arrows). Synapsin-1 staining was also observed at the small number of vestigial synapses formed (as shown in Fig. 4) between neurons expressing ␣4␤2 AChR alone and NLG-expressing tsA 201 cells (Fig. 5, E-G). Similarly, synapsin-1 staining was also observed at synaptic contacts sites between neurons expressing NRX alone and NLG-expressing tsA 201 cells (Fig. 5, H-J).
We further investigated whether endogenous neurexin-1␤ had a role in the development of the vestigial synaptic terminals observed at ϳ11% of contact sites in cocultures of neurons expressing only ␣4␤2 AChRs and NLG-expressing tsA 201 cells. We developed micro-RNA interference-expressing constructs (miRNAs) to silence the expression of neurexin-1␤ and tested their ability to specifically knock down expression of NRX in tsA 201 cells expressing transfected ␣4␤2 AChRs. At least one miRNA was able to silence the expression of NRX, as compared with the negative control miRNA (supplemental Fig. S2A). However, when the miRNA constructs were coexpressed with ␣4␤2 AChRs in neurons cocultured with tsA 201 cells expressing NLG, we did not observe significant changes in the size of the synaptic boutons formed or in the extent to which low level targeting of ␣4␤2 AChRs occurs at these contact sites on tsA cells expressing NLG (supplemental Fig. S2B). There was a trend toward an increase in puncta, but it did not reach significance. Overall, the base-line targeting of ␣4␤2 AChRs observed was not significantly altered. This lack of phenotype could be due to multiple compensatory mechanisms, including functional redundancies among other the neurexin isoforms or other presynaptic cell adhesion molecules or because endogenous neurexin-1␤ does not mediate the low level of synaptic targeting observed with ␣4␤2 AChRs expressed alone. . Cell surface expression of ␣4␤2 was measured using an enzyme-linked immunoassay in which tsA 201 cells were washed, blocked, and then incubated with mAb (mAb 295). The cells were blocked again, fixed, and incubated with horseradish peroxidase-conjugated secondary Abs followed by incubation with the horseradish peroxidase substrate. The absorbance of the supernatant was then measured at 655 nm in a Beckman spectrophotometer. C, coexpression of NRX with ␣4␤2 AChRs does not affect surface NRX expression. tsA 201 cells were transfected with NRX, NRXϩpEF6A vector, and NRXϩ␣4␤2, and cell surface expression of NRX was measured using an enzyme-linked immunoassay using anti-VSV-G. The values in B and C are each from three separate experiments, expressed as the means Ϯ S.E., and analyzed using analysis of variance test. The differences are not significant (p Ͼ 0.05).
kinase was shown to phosphorylate neurexin-1 (45), so it is possible that neurexin-1 binds different proteins depending on its phosphorylation state. Future experiments are necessary to sort out the full repertoire of neurexin isoforms involved in the synaptic targeting of the different AChR subtypes.
Our finding that neurexin-1␤ is involved in the targeting of ␣4␤2 AChRs may have significant implications for the role of neurexins in the etiology of different neurological diseases typically associated with pathophysiological functions of AChRs. In this regard, it is significant that a recent high density genome-wide association study for nicotine dependencelinked single nucleotide polymorphisms in the neurexin-1 gene to the development of nicotine dependence and thus smoking behavior (46), and this association was replicated in an independent study (47). The ␣4␤2 AChRs play a significant role in mediating the essential features of nicotine addiction including reward, tolerance, and sensitization (13). Thus, changes in the expression level of neurexin-1␤ could be expected to affect functions mediated by ␣4␤2 AChRs. Little is known about how the neurexin-1␣ and -1␤ splicing is regulated to generate the predicted hundreds of neurexin-1 isoforms. Hence, it is possible that a regulatory single nucleotide polymorphism, linked to nicotine dependence, in an intron of neurexin-1␣ could modulate neurexin-1␤ levels. Alternatively, a specific neurexin-1␣ isoform may also influence AChR functions. Future, more challenging studies analyzing whether any of the hundreds of neurexin-1␣ isoforms also perform similar targeting functions are necessary to elucidate the linkage between neurexin-1 gene variants, ␣4␤2 AChR synaptic targeting, and nicotine dependence. Nevertheless, our results provide support for a possible mechanism by which changes in neurexin-1 function could contribute to nicotine dependence.
The interactions between neurexin-1␤ and ␣4␤2 AChRs may also shed light on the association between deficits in AChR, neurexin and neuroligin functions, and autism spectrum disorders (ASD). ␤2-Containing AChRs have been shown to regulate executive and social behaviors in ␤2 AChR subunit knock-out mice, and some of these affected behaviors have been reported to resemble behavioral deficits characteristic of ASD (48). Additionally, postmortem analyses of autistic patient brains show an extremely significant reduction in the expression levels of ␣4␤2 AChRs in the cerebellar cortex (49,50) and the parietal cortex (51). In addition, multiple recent linkage analysis studies (52,53) and an analysis of structural variants in the ␤-neurexin genes (54) implicate neurexin-1 dysfunctions in ASD. Our results complement these studies and suggest that some behavioral deficits characteristic of ASD are highly likely to be due to defects in ␣4␤2 AChR-mediated functions caused by neurexin or neuroligin dysfunctions.