Presynaptic targeting of alpha4beta 2 nicotinic acetylcholine receptors is regulated by neurexin-1beta

Abstract

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 alpha4beta2 AChRs and the presynaptic cell adhesion molecule, neurexin-1beta. In non-neuronal tsA 201 cells, recombinant neurexin-1beta and mature alpha4beta2 AChRs form complexes. alpha4beta2 AChRs and neurexin-1beta also coimmunoprecipitate from rat brain lysates. When exogenous alpha4beta2 AChRs and neurexin-1beta 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-1beta. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1beta lacking the extracellular domain. Additionally, when alpha4beta2 AChRs, alpha7 AChRs, and neurexin-1beta are coexpressed in the same neuron, only the alpha4beta2 AChR colocalizes with neurexin-1beta at presynaptic terminals. Collectively, these data suggest that neurexin-1beta targets alpha4beta2 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 alpha4beta2 AChRs, may contribute to the etiology of these neurological disorders.

FIGURE 1.

NRX forms complexes with α4β2 AChRs in vitro. A, coimmunoprecipitation of NRX with recombinant α4β2 AChRs from tsA 201 cells. tsA 201 cells were transfected with VSV-G-tagged NRX and untagged or FLAG-tagged α4β2 AChRs, as indicated along the bottoms of the blots, and were then lysed and immunoprecipitated (IP) with FLAG M2 beads. Lysates (input) and immunoprecipitates were immunoblotted for α4 (mAb 299), NRX (rabbit polyclonal anti-VSV-G), and β2 (goat polyclonal anti-β2), indicated along the right side of the image. Detergent-solubilized extract from the cells coexpressing VSV-G-tagged NRX and α4β2 ^FLAG AChRs was incubated with IgG-coupled beads as an additional control (IgG immunoprecipitates). B, NRX does not coimmunoprecipitate with α4β2^FLAG AChRs when they are transfected separately, and the lysates are combined. Detergent-solubilized extracts from the tsA201 cells expressing α4β2^FLAG AChRs alone and tsA 201 cells expressing VSV-G-tagged NRX alone were mixed and pulled down using FLAG M2 beads. The eluates were subject to SDS-PAGE, followed by Western blotting with the same antibodies as in A. No band for NRX was observed in the IP lane. C, copurification of NRX with assembled and mature recombinant α4β2 AChRs from tsA 201 cells using BAC affinity purification. Protein complexes captured with BAC-conjugated beads show the presence of the α4, β2 subunits, and NRX (BAC capture, lane 1). Pretreatment with 10 μm nicotine blocked binding of BAC to the mature α4β2 AChRs complexes (BAC capture, lane 2). N-terminal HA-tagged neuroligin-1, the trans-synaptic partner of NRX, was not detected (BAC capture, lane 3), suggesting that the complex formation between NRX and mature α4β2 AChRs is specific. A faint nonspecific band is detected in the cell lysates with the VSV-G antibody (B, α4β2^FLAG input lane; C, α4β2+NLG-HA input lane; empty vector-transfected lysates (data not shown)).

FIGURE 2.

Neurexin-1β forms complexes with α4β2 AChRs in vivo. A, coimmunoprecipitation of NRX and α4β2 AChRs from whole rat brain lysates. Rat brains were homogenized, solubilized in 1% Nonidet P-40 lysis buffer, and incubated with beads conjugated to rat IgGs or rat anti-β2 (mAb 295). Lysate and immunoprecipitates (IP) were immunoblotted with rat anti-α4 (mAb 299), goat polyclonal anti-neurexin-1 (P-15), or goat polyclonal anti-β2 (C-20) antibodies. B, neuroligin 1 does not coimmunoprecipitate with α4β2 AChRs. Rat brain lysate and immunoprecipitates were incubated with rat anti-β2 (mAb 270) and immunoblotted with rat anti-α4 (mAb 299) and a mouse monoclonal anti-neuroligin 1 antibody. C, neurexin I antiserum cross-reacts with recombinant neurexin-1β. Samples were from the two eluates of pulldown experiments, one from cells coexpressing α4β2 ^FLAG AChRs and VSV-G-tagged NRX and another from cells coexpressing α4β2 ^FLAG AChRs and VSV-G-tagged NRXΔC. The same amounts of samples were loaded side by side, and two sets of blots were probed with neurexin I or VSV-G antiserum. Neurexin I antiserum is also capable of recognizing the recombinant mouse VSV-G-tagged NRX and VSV-G-tagged NRXΔC.

FIGURE 3.

NRX does not affect steady state expression of α4β2 AChRs. A, NRX does not affect steady state levels of α4β2 AChRs. Whole cell lysates of tsA 201 cells expressing α4β2 and pEF6A vector only or coexpressing α4β2 and NRX were separated by SDS-PAGE and Western blotted with rat anti-α4 (mAb 299), goat polyclonal anti-β2 (C-20), or goat polyclonal anti-neurexin-1 (P-15). Duplicates of each condition are shown. B, NRX does not affect the surface expression of recombinant α4β2 AChRs in tsA 201 cells. tsA 201 cells were transfected with α4β2, α4β2+pEF6A vector, and α4β2+NRX. Cell surface expression of α4β2 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 α4β2 AChRs does not affect surface NRX expression. tsA 201 cells were transfected with NRX, NRX+pEF6A vector, and NRX+α4β2, 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 ± S.E., and analyzed using analysis of variance test. The differences are not significant (p > 0.05).

FIGURE 4.

α4β2 AChRs target with NRX to presynaptic terminals in hippocampal neurons. Single plane images of neurons that were transfected at 8 DIV, coplated with tsA 201 cells at 12 DIV and fixed, permeabilized, and immunostained at 14 DIV. A, neurons expressing α4β2 AChRs were coplated with tsA 201 cells expressing NLG. B, neurons expressing α4β2 AChRs and NRX were coplated with tsA 201 cells expressing NLG. α4β2 AChRs and NRX-expressing cultures exhibit enhanced targeting of α4β2 AChRs to synapses (arrows) compared with α4β2 AChRs alone (A, arrowhead). C, neurons expressing α4β2 AChRs and NRX lacking its extracellular domain (NRXΔEC) were coplated with tsA 201 cells expressing NLG. D, quantification of results shown in A–C. *, p < 0.05, expressed as the means ± S.E. and analyzed using Student's t test. Antibody combinations: A–C, anti-β2 AChR (mAb 295, red), anti-VSV-G (green), and anti-HA (blue) antibodies. Scale bar, 10 μm.

FIGURE 5.

Synaptic vesicles are present at α4β2 AChR/NRX-positive presynaptic terminals. A–D, neurons expressing α4β2 AChRs (red) and NRX (green) were coplated with tsA 201 cells expressing NLG and stained as described in the legend to Fig. 4, except that cells were alternatively costained for synapsin-1 (blue) instead of HA to detect synaptic vesicles at neuron-tsA 201 cell contact sites. Enhanced synapsin-1 clustering is observed at contact sites with NLG-expressing tsA 201 cells (arrows). Robust staining of presynaptic boutons with synapsin-1 Ab indicates presynaptic terminal maturation. The tsA201 cell is indicated by the dotted line. E–G, neurons expressing α4β2 AChRs (red) alone were coplated with tsA 201 cells expressing NLG (not immunostained). A vestigial synapse is shown (arrows). H–J, neurons expressing NRX (green) alone were coplated with tsA 201 cells expressing NLG (not immunostained). Single plane confocal images are shown. Scale bar, 10 μm.

FIGURE 6.

α4β2 AChRs and α7 AChRs exhibit differential targeting when coexpressed with NRX. A–D, neurons expressing α4β2 AChRs (red), α7 AChRs (green), and NRX (blue) were coplated with tsA 201 cells expressing NLG (not immunostained). The cultures were immunostained with antibodies against β2 (rat mAb 295), α7 (mouse mAb 306), and VSV-G (rabbit polyclonal). The arrows point to areas of colocalization between α4β2 and NRX that do not include α7 AChRs. A1–D1, these panels represent the upper boxes magnified four times. The dotted lines indicate cell outlines. Antibodies recognizing the α4β2 AChR and NRX, but not the α7 AChR, show accumulation on a cell soma. A2–D2, these panels represent the lower boxes magnified four times. The axon is immunolabeled with antibodies recognizing the α4β2 AChR and NRX, but not the α7 AChR. Scale bars, A–D, 20 μm; A1–D1 and A2–D2, 10 μm.