Abstract
Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, using LC-MS/MS-based protein analysis, confocal microscopy, RNAscope assays, and electrophysiological recordings, we show that β-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert-positive β-Nrxn variants, but not insert-negative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo Neuron-specific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4-positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits, including Schaffer collateral afferents.
Highlights
Synaptogenic adhesion molecules, a class of synaptic transmembrane proteins that induce synaptic differentiation in vitro [1,2,3], are central to various aspects of synapse development, but their precise roles in synapse assembly, validation, and/or plasticity in vivo are only beginning to be revealed [1, 4, 5]
In assays measuring dimeric ligand binding to mNrxn-expressing cell surfaces, IgClstn3 interacted with both mNrxn1b-splice site 4 (SS4) and mNrxn1b1SS4 with nanomolar affinity (Fig. 1, G and H)
Taken together with our previous findings, the three principal observations of the current study provide plausible explanations for discrepancies surrounding the molecular mechanisms of Clstn3-mediated synapse development
Summary
Our previous cell surface-binding assays used a variety of Nrxn1a deletion variants derived from the bovine Nrxn1a gene or Nrxn1b variants derived from the rat Nrxn1b gene [15]. Expression of Clstn Cad, but not Clstn DCad, restored the decreased VGLUT1 puncta density in the SR layer, but neither Clstn Cad nor Clstn DCad expression exerted a rescue effect in the SO layer of Nestin-Clstn mice (Fig. 8, B–E) These results suggest that Clstn organizes the development of hippocampal CA1 excitatory synapses, and likely acts through cadherin domains-mediated interactions with presynaptic Nrxns to control the specific excitatory synaptic projections involving the SR layer. To complement these anatomical analyses, we performed whole cell electrophysiological recordings of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) in brain slices from Nestin-Clstn and littermate WT mice (Fig. S8). These results suggest that Clstn is not required for basal excitatory synaptic transmission in CA1 hippocampal pyramidal neurons
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