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

Shi-Bin Cheng,Stephanie A Amici,Xiao-Qin Ren,Susan B Mckay,Magdalen W Treuil,Jon M Lindstrom,Jayaraman Rao,Rene Anand

doi:10.1074/jbc.m109.017384

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.

Highlights

We developed micro-RNA interference-expressing constructs to silence the expression of neurexin-1␤ and tested their ability to knock down expression of NRX in tsA 201 cells expressing transfected ␣4␤2 acetylcholine receptors (AChRs)
We found that ␣7 does tion or maturation of pre- and post-synaptic specializations not cotarget to terminals with ␣4␤2 AChRs and NRX when in neurons occurs through trans-synaptic, bi-directional sig- expressed in the same neurons
These results indicate that the naling interactions between neurexins and neuroligins

Summary

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). The gel was washed three times with lysis buffer and eluted in sample buffer at 60 °C for 30 min, and ␤-mercaptoethanol was added to the eluted samples prior to analysis by SDS-PAGE. 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. 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 Ca2ϩ and Mg2ϩ), 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. The values obtained are the means Ϯ S.E. and were statistically analyzed by a Student’s t test

RESULTS

Differential splicing of five canonical alternative splice sites in the

Findings

DISCUSSION