Abstract
Beta-neurexin and neuroligin cell adhesion molecules contribute to synapse development in the brain. The longer alpha-neurexins function at both glutamate and gamma-aminobutyric acid (GABA) synapses in coupling to presynaptic calcium channels. Binding of alpha-neurexins to neuroligins was recently reported, but the role of the alpha-neurexins in synapse development has not been well studied. Here we report that in COS cell neuron coculture assays, all three alpha-neurexins induce clustering of the GABAergic postsynaptic scaffolding protein gephyrin and neuroligin 2 but not of the glutamatergic postsynaptic scaffolding protein PSD-95 or neuroligin 1/3/4. alpha-Neurexins also induce clustering of the GABA(A) receptor gamma2 subunit. This synapse promoting activity of alpha-neurexins is mediated by the sixth LNS (laminin neurexin sex hormone-binding protein) domain and negatively modulated by upstream sequences. Although inserts at splice site 4 (S4) in beta-neurexins promote greater clustering activity for GABA than glutamate proteins in coculture assay, alpha-neurexin-specific sequences confer complete specificity for GABA proteins. We further report a developmental increase in the ratio of -S4 to +S4 forms of neurexins correlating with an increase in glutamate relative to GABA synaptogenesis and activity regulation of splicing at S4. Thus, +S4 beta-neurexins and, even more selectively, alpha-neurexins may be mediators of GABAergic synaptic protein recruitment and stabilization.
Highlights
Synaptic connections in the brain require precise alignment of neurotransmitter receptors on dendrites opposite transmitter release sites on axons
We further report a developmental increase in the ratio of ؊site 4 (S4) to ؉S4 forms of neurexins correlating with an increase in glutamate relative to glutamate and ␥-aminobutyric acid (GABA) synaptogenesis and activity regulation of splicing at S4
For comparison, we assayed in the same experiments the sixth LNS domain of neurexin 1␣, 2␣, and 3␣ each in the context of the neurexin 1-flanking sequences, corresponding to neurexin 1 itself, and constructs termed neurexin 2Ј and neurexin 3Ј
Summary
Primary Neuronal Culture and COS Cell Coculture—Dissociated primary hippocampal neuronal cultures were prepared from embryonic day 18 (E18) rats as previously described [37, 38]. M. Fritschy [40]) or ␥2 antibodies for 30 min in the neuronal medium plus 50 mM HEPES, pH 7.4, at room temperature, washed extensively, fixed, permeabilized, and incubated with anti-synapsin followed by secondary antibodies. The area associated with each chosen COS cell was visually scored as either positive or negative, positive indicating the presence of any clusters of postsynaptic protein (PSD-95, gephyrin, or neuroligin 2) that lacked associated staining for synapsin. For RNA extraction from hippocampal cultures, roughly 1 ϫ 106 cells that were either untreated or chronically treated with 100 M APV were harvested by briefly washing in cold phosphate-buffered saline and either scraping directly in Trizol reagent or by using the RNeasy mini kit (Qiagen) according to the manufacturer’s protocol. For neurexins 1 and 2, a closely spaced doublet PCR product was obtained for the ϩS4 PCR product even using cloned ϩS4 cDNA template; both bands were included in quantitation
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