Jaewon Ko

LRRTM2 functions as a neurexin ligand in promoting excitatory synapse formation.

Recently, leucine-rich repeat transmembrane proteins (LRRTMs) were found to be synaptic cell-adhesion molecules that, when expressed in nonneuronal cells, induce presynaptic differentiation in contacting axons. We now demonstrate that LRRTM2 induces only excitatory synapses, and that it also acts to induce synapses in transfected neurons similarly to neuroligin-1. Using affinity chromatography, we identified alpha- and beta-neurexins as LRRTM2 ligands, again rendering LRRTM2 similar to neuroligin-1. However, whereas neuroligins bind neurexins containing or lacking an insert in splice site #4, LRRTM2 only binds neurexins lacking an insert in splice site #4. Binding of neurexins to LRRTM2 can produce cell-adhesion junctions, consistent with a trans-interaction regulated by neurexin alternative splicing, and recombinant neurexin-1beta blocks LRRTM2s ability to promote presynaptic differentiation. Thus, our data suggest that two unrelated postsynaptic cell-adhesion molecules, LRRTMs and neuroligins, unexpectedly bind to neurexins as the same presynaptic receptor, but that their binding is subject to distinct regulatory mechanisms.

Association of the Kinesin Motor KIF1A with the Multimodular Protein Liprin-α

Liprin-α/SYD-2 is a multimodular scaffolding protein important for presynaptic differentiation and postsynaptic targeting of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid glutamate receptors. However, the molecular mechanisms underlying these functions remain largely unknown. Here we report that liprin-α interacts with the neuron-specific kinesin motor KIF1A. KIF1A colocalizes with liprin-α in various subcellular regions of neurons. KIF1A coaccumulates with liprin-α in ligated sciatic nerves. KIF1A cofractionates and coimmunopreciptates with liprin-α and various liprin-α-associated membrane, signaling, and scaffolding proteins including α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors, GRIP/ABP, RIM, GIT1, and βPIX. These results suggest that liprin-α functions as a KIF1A receptor, linking KIF1A to various liprin-α-associated proteins for their transport in neurons.

Regulation of Dendritic Spine Morphogenesis by Insulin Receptor Substrate 53, a Downstream Effector of Rac1 and Cdc42 Small GTPases

The small GTPases Rac1 and Cdc42 are key regulators of the morphogenesis of actin-rich dendritic spines in neurons. However, little is known about how activated Rac1/Cdc42 regulates dendritic spines. Insulin receptor substrate 53 (IRSp53), which is highly expressed in the postsynaptic density (PSD), is known to link activated Rac1/Cdc42 to downstream effectors for actin regulation in non-neural cells. Here, we report that IRSp53 interacts with two specific members of the PSD-95 family, PSD-95 and chapsyn-110/PSD-93, in brain. An IRSp53 mutant lacking the C-terminal PSD-95-binding motif shows significant loss of synaptic localization in cultured neurons. Overexpression of IRSp53 in cultured neurons increases the density of dendritic spines but does not affect their length or width. Conversely, short-interfering RNA-mediated knock-down of IRSp53 reduces the density, length, and width of spines. In addition, the density and size of spines are decreased by a dominant-negative IRSp53 with a point mutation in the Src homology 3 (SH3) domain and a dominant-negative proline-rich region of WAVE2 (Wiskott-Aldrich syndrome protein family Verprolin-homologous protein), a downstream effector of IRSp53 that binds to the SH3 domain of IRSp53. These results suggest that PSD-95 interaction is an important determinant of synaptic IRSp53 localization and that the SH3 domain of IRSp53 links activated Rac1/Cdc42 to downstream effectors for the regulation of spine morphogenesis.

SALM Synaptic Cell Adhesion-like Molecules Regulate the Differentiation of Excitatory Synapses

Synaptic cell adhesion molecules (CAMs) are known to play key roles in various aspects of synaptic structures and functions, including early differentiation, maintenance, and plasticity. We herein report the identification of a family of cell adhesion-like molecules termed SALM that interacts with the abundant postsynaptic density (PSD) protein PSD-95. SALM2, a SALM isoform, distributes to excitatory, but not inhibitory, synaptic sites. Overexpression of SALM2 increases the number of excitatory synapses and dendritic spines. Mislocalized expression of SALM2 disrupts excitatory synapses and dendritic spines. Bead-induced direct aggregation of SALM2 results in coclustering of PSD-95 and other postsynaptic proteins, including GKAP and AMPA receptors. Knockdown of SALM2 by RNA interference reduces the number of excitatory synapses and dendritic spines and the frequency, but not amplitude, of miniature excitatory postsynaptic currents. These results suggest that SALM2 is an important regulator of the differentiation of excitatory synapses.

Neurexins Physically and Functionally Interact with GABAA Receptors

Neurexins are presynaptic cell-adhesion molecules that form trans-synaptic complexes with postsynaptic neuroligins. When overexpressed in nonneuronal cells, neurexins induce formation of postsynaptic specializations in cocultured neurons, suggesting that neurexins are synaptogenic. However, we find that when overexpressed in neurons, neurexins do not increase synapse density, but instead selectively suppressed GABAergic synaptic transmission without decreasing GABAergic synapse numbers. This suppression was mediated by all subtypes of neurexins tested, in a cell-autonomous and neuroligin-independent manner. Strikingly, addition of recombinant neurexin to cultured neurons at submicromolar concentrations induced the same suppression of GABAergic synaptic transmission as neurexin overexpression. Moreover, experiments with native brain proteins and purified recombinant proteins revealed that neurexins directly and stoichiometrically bind to GABA(A) receptors, suggesting that they decrease GABAergic synaptic responses by interacting with GABA(A) receptors. Our findings suggest that besides their other well-documented interactions, presynaptic neurexins directly act on postsynaptic GABA(A) receptors, which may contribute to regulate the excitatory/inhibitory balance in brain.

Phosphorylation of Stargazin by Protein Kinase A Regulates Its Interaction with PSD-95

Stargazin is the first transmembrane protein known to associate with AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate) glutamate receptors (AMPARs) and regulate their synaptic targeting by two distinct mechanisms, specifically via delivery of AMPARs to the surface membrane and synaptic targeting of these receptors by binding to PSD-95/SAP-90 and related PDZ proteins. However, it is not known whether and how this stargazin-mediated synaptic targeting of AMPARs is regulated. Stargazin interacts with the PDZ domains of PSD-95 through the C-terminal PDZ-binding motif. The stargazin C terminus contains a consensus sequence for phosphorylation by cAMP-dependent protein kinase A (PKA). Phosphorylation site-specific stargazin antibodies reveal that the stargazin C terminus is phosphorylated at the Thr-321 residue in heterologous cells and in vivo. Stargazin phosphorylation is enhanced by the catalytic subunit of PKA. Mutations mimicking stargazin phosphorylation (T321E and T321D) lead to elimination of yeast two-hybrid interactions, in vitrocoimmunoprecipitation, and coclustering between stargazin and PSD-95. Phosphorylated stargazin shows a selective loss of coimmunoprecipitation with PSD-95 in heterologous cells and limited enrichment in postsynaptic density fractions of rat brain. These results suggest that phosphorylation of the stargazin C terminus by PKA regulates its interaction with PSD-95 and synaptic targeting of AMPARs.

An autism-associated point mutation in the neuroligin cytoplasmic tail selectively impairs AMPA receptor-mediated synaptic transmission in hippocampus

Neuroligins are evolutionarily conserved postsynaptic cell‐adhesion molecules that function, at least in part, by forming trans‐synaptic complexes with presynaptic neurexins. Different neuroligin isoforms perform diverse functions and exhibit distinct intracellular localizations, but contain similar cytoplasmic sequences whose role remains largely unknown. Here, we analysed the effect of a single amino‐acid substitution (R704C) that targets a conserved arginine residue in the cytoplasmic sequence of all neuroligins, and that was associated with autism in neuroligin‐4. We introduced the R704C mutation into mouse neuroligin‐3 by homologous recombination, and examined its effect on synapses in vitro and in vivo. Electrophysiological and morphological studies revealed that the neuroligin‐3 R704C mutation did not significantly alter synapse formation, but dramatically impaired synapse function. Specifically, the R704C mutation caused a major and selective decrease in AMPA receptor‐mediated synaptic transmission in pyramidal neurons of the hippocampus, without similarly changing NMDA or GABA receptor‐mediated synaptic transmission, and without detectably altering presynaptic neurotransmitter release. Our results suggest that the cytoplasmic tail of neuroligin‐3 has a central role in synaptic transmission by modulating the recruitment of AMPA receptors to postsynaptic sites at excitatory synapses.

The neurexin ligands, neuroligins and leucine-rich repeat transmembrane proteins, perform convergent and divergent synaptic functions in vivo

Synaptic cell adhesion molecules, including the neurexin ligands, neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs), are thought to organize synapse assembly and specify synapse function. To test the synaptic role of these molecules in vivo, we performed lentivirally mediated knockdown of NL3, LRRTM1, and LRRTM2 in CA1 pyramidal cells of WT and NL1 KO mice at postnatal day (P)0 (when synapses are forming) and P21 (when synapses are largely mature). P0 knockdown of NL3 in WT or NL1 KO neurons did not affect excitatory synaptic transmission, whereas P0 knockdown of LRRTM1 and LRRTM2 selectively reduced AMPA receptor-mediated synaptic currents. P0 triple knockdown of NL3 and both LRRTMs in NL1 KO mice yielded greater reductions in AMPA and NMDA receptor-mediated currents, suggesting functional redundancy between NLs and LRRTMs during early synapse development. In contrast, P21 knockdown of LRRTMs did not alter excitatory transmission, whereas NL manipulations supported a role for NL1 in maintaining NMDA receptor-mediated transmission. These results show that neurexin ligands in vivo form a dynamic synaptic cell adhesion network, with compensation between NLs and LRRTMs during early synapse development and functional divergence upon synapse maturation.

Neuroligin‐1 performs neurexin‐dependent and neurexin‐independent functions in synapse validation

Postsynaptic neuroligins are thought to perform essential functions in synapse validation and synaptic transmission by binding to, and dimerizing, presynaptic α‐ and β‐neurexins. To test this hypothesis, we examined the functional effects of neuroligin‐1 mutations that impair only α‐neurexin binding, block both α‐ and β‐neurexin binding, or abolish neuroligin‐1 dimerization. Abolishing α‐neurexin binding abrogated neuroligin‐induced generation of neuronal synapses onto transfected non‐neuronal cells in the so‐called artificial synapse‐formation assay, even though β‐neurexin binding was retained. Thus, in this assay, neuroligin‐1 induces apparent synapse formation by binding to presynaptic α‐neurexins. In transfected neurons, however, neither α‐ nor β‐neurexin binding was essential for the ability of postsynaptic neuroligin‐1 to dramatically increase synapse density, suggesting a neurexin‐independent mechanism of synapse formation. Moreover, neuroligin‐1 dimerization was not required for either the non‐neuronal or the neuronal synapse‐formation assay. Nevertheless, both α‐neurexin binding and neuroligin‐1 dimerization were essential for the increase in apparent synapse size that is induced by neuroligin‐1 in transfected neurons. Thus, neuroligin‐1 performs diverse synaptic functions by mechanisms that include as essential components of α‐neurexin binding and neuroligin dimerization, but extend beyond these activities.

Slitrks control excitatory and inhibitory synapse formation with LAR receptor protein tyrosine phosphatases

The balance between excitatory and inhibitory synaptic inputs, which is governed by multiple synapse organizers, controls neural circuit functions and behaviors. Slit- and Trk-like proteins (Slitrks) are a family of synapse organizers, whose emerging synaptic roles are incompletely understood. Here, we report that Slitrks are enriched in postsynaptic densities in rat brains. Overexpression of Slitrks promoted synapse formation, whereas RNAi-mediated knockdown of Slitrks decreased synapse density. Intriguingly, Slitrks were required for both excitatory and inhibitory synapse formation in an isoform-dependent manner. Moreover, Slitrks required distinct members of the leukocyte antigen-related receptor protein tyrosine phosphatase (LAR-RPTP) family to trigger synapse formation. Protein tyrosine phosphatase σ (PTPσ), in particular, was specifically required for excitatory synaptic differentiation by Slitrks, whereas PTPδ was necessary for inhibitory synapse differentiation. Taken together, these data suggest that combinatorial interactions of Slitrks with LAR-RPTP family members maintain synapse formation to coordinate excitatory–inhibitory balance.