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. 2012 Nov 20;109(47):19432-7.
doi: 10.1073/pnas.1217633109. Epub 2012 Nov 5.

Dimerization of postsynaptic neuroligin drives synaptic assembly via transsynaptic clustering of neurexin

Seth L Shipman  1 , Roger A Nicoll
Affiliations

Dimerization of postsynaptic neuroligin drives synaptic assembly via transsynaptic clustering of neurexin

Seth L Shipman et al. Proc Natl Acad Sci U S A. .

Abstract

The transsynaptic complex of neuroligin (NLGN) and neurexin forms a physical connection between pre- and postsynaptic neurons that occurs early in the course of new synapse assembly. Both neuroligin and neurexin have, indeed, been proposed to exhibit active, instructive roles in the formation of synapses. However, the process by which these instructive roles play out during synaptogenesis is not well understood. Here, we examine one aspect of postsynaptic neuroligin with regard to its synaptogenic properties: its basal state as a constitutive dimer. We show that dimerization is required for the synaptogenic properties of neuroligin and likely serves to induce presynaptic differentiation via a transsynaptic clustering of neurexin. Further, we introduce chemically inducible, exogenous dimerization domains to the neuroligin molecule, effectively bestowing chemical control of neuroligin dimerization. This allows us to identify the acute requirements of neuroligin dimerization by chemically manipulating the monomeric-to-dimeric conversion of neuroligin. Based on the results of the inducible dimerization experiments, we propose a model in which dimerized neuroligin induces the mechanical clustering of presynaptic molecules as part of a requisite step in the coordinated assembly of a chemical synapse.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Mutations of neuroligin affecting dimerization abolish the synaptogenic effects of postsynaptic expression. (A) Structure of the neuroligin 1/neurexin 1β extracellular domains viewed side-on, looking through the synaptic cleft. (Left) Schematic (neuroligin in blue, neurexin in purple) indicates the viewing angle. (Right) Two neuroligin molecules, shown in dark and light blue, form a dimer with each neuroligin molecule bound to neurexin, shown in purple. Calcium ions at the neuroligin/neurexin interface are shown in gray. Locations of the dimerization-inhibiting mutations are indicated in orange. Structure from Araç et al. (10). (B) As in A, but viewed from the presynaptic side of the synapse, looking toward the postsynaptic side. (C) Postsynaptic expression of wild-type NLGN1 results in increased AMPAR-mediated EPSCs compared with control (P < 0.01, n = 11), whereas the expression of dimerization-null mutants do not (NLGN1DM1 P > 0.05, n = 15; NLGN1DM2 P > 0.05, n = 10; NLGN1DM3 P > 0.05, n = 8). Open circles represent individual pairs; closed circles indicate mean ± SEM. (D) Postsynaptic expression of wild-type NLGN3 also results in increased AMPAR-mediated EPSCs compared with control (P < 0.001, n = 12), whereas expression of dimerization-null mutants results in decreased AMPAR-mediated EPSCs (NLGN3DM1 P < 0.005, n = 9; NLGN3DM2 P < 0.05, n = 11; NLGN3DM3 P < 0.05, n = 9). As in C, open circles represent individual pairs; closed circles indicate mean ± SEM. Expression of wild-type NLGN1 and NLGN3, previously shown in Shipman et al. (25) are repeated here for clarity. Sample traces in C and D show individual paired recordings with control AMPAR-mediated currents in black and experimental in green (scale bar, 20 pA/20 ms.)
Fig. 2.
Fig. 2.
Dimerization-null neuroligin mutant retains the ability to enhance the postsynaptic site in the absence of presynaptic enhancements. (A) Postsynaptic expression of NLGN3D-N results in an increased spine density along the apical dendrite of CA1 pyramidal neurons in organotypic hippocampal cultures compared with control neurons [P < 0.0001, n = 8 control (ctrl), n = 8 experimental). Circles represent individual cells; horizontal bars indicate mean ± SEM. Sample images show individual sections of dendrite (Scale bar, 5 µm.) (B) Postsynaptic expression of either wild-type or dimerization-null NLGN3 increases PSD-95 immunofluorescence intensity in dissociated hippocampal neurons (NLGN3 P < 0.001, n = 12; NLGN3D-N P < 0.05, n = 12; ctrl n = 13). Circles represent individual cells; horizontal bars indicate mean ± SEM. In sample images, dendrites are shown using mCherry, red (Left), whereas PSD-95 staining is in green (Right). (Scale bar, 5 µm.) (C) Postsynaptic expression of wild-type NLGN3 increases VGLUT1 staining onto the postsynaptic cell (P < 0.005, n = 7; ctrl n = 7), whereas expression of NLGN3D-N decreases VGLUT1 staining onto the postsynaptic cell (P < 0.05, n = 9). Circles represent individual cells; horizontal bars indicate mean ± SEM. In sample images, dendrites are shown using mCherry, red (Left), whereas VGLUT1 staining is in green (Right). (Scale bar, 5 µm.)
Fig. 3.
Fig. 3.
Chemically induced dimerization and monomerization can acutely alter the synaptic phenotype of neuroligin expression. (A) Schematic of induced-dimerization experiments. Neuroligin monomers are acutely dimerized via an exogenous, inducible dimerization domain (blue) in the presence of the small molecule B/B homodimerizer (yellow). (B) Acute dimerization rescues the synaptogenic phenotype of the dimerization-null neuroligin mutant. In the absence of the homodimerizing compound, expression of the NLGN3D-N-iDmr construct (n = 9) has an inhibitory effect on evoked AMPAR-mediated EPSCs compared with wild-type NLGN3 expression (n = 12, P < 0.005). 48 h of exposure to the B/B Homodimerizer (n = 10) results in an increase in the AMPAR-mediated current amplitudes compared with the no drug condition (P < 0.005), rescuing the phenotype to a wild-type level (48 h condition versus wild-type expression, P > 0.05; 24 h homodimerizer n = 10). Closed circles indicate mean AMPAR-mediated current amplitudes expressed as percent of control ± SEM. Example traces illustrate individual pairs, with control cells shown in black and experimental cells shown in green. (Scale bar, 20 pA/20 ms.) (C) Schematic of the induced-monomerization experiments. Dimerization-null mutant neuroligin is artificially dimerized via an exogenous domain (light blue), which, in the presence of the small molecule D/D solubilizer (yellow), is converted to a monomeric state. (D) Artificial dimerization via the exogenous dimerization domain rescues basal synaptogenic properties of neuroligin as evidenced by an increase in AMPAR-mediated synaptic currents upon expression of NLGN3D-N–revDmr (n = 12) compared with NLGN3D-N (n = 10) in the absence of drug (P < 0.005). NLGN3D-N expression, originally shown in Fig. 1D, is repeated here for clarity. Monomerization of NLGN3D-N–revDmr by the addition of the D/D solubilizer for 24 h (n = 10) results in elimination of the synaptogenic phenotype compared with the no-drug condition (P < 0.005), returning the synaptic phenotype to the dimerization-null condition (24-h condition versus NLGN3D-N expression, P > 0.05; 5 h solubilizer n = 8). Graphs and sample traces as in B. (Scale bar, 20 pA/20 ms.) (E) Model for neuroligin-induced synaptogenesis whereby postsynaptic, dimerizered neuroligin binds to and clusters presynaptic neurexin, leading to the differentiation of a presynaptic terminal.
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