Calsyntenin-3 interacts with both α- and β-neurexins in the regulation of excitatory synaptic innervation in specific Schaffer collateral pathways

Abstract

<jats:p>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 <jats:italic>in vivo</jats:italic>. 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.</jats:p>

Calsyntenin-3, an atypical cadherin, suppresses inhibitory basket- and stellate-cell synapses but boosts excitatory parallel-fiber synapses in cerebellum

“…Third, what trans-synaptic interactions mediate the functions of Clstn3? Several papers describe binding of calsyntenins to neurexins (Kim et al, 2020;Pettem et al, 2013). However, our data uncover a phenotype that is different from that observed with deletions of neurexins or neurexin ligands, suggesting that Clstn3 does not function exclusively by binding to neurexins.…”

“…The parallel-fiber synapse increase is likely not a homeostatic response to the loss of inhibitory synapses because such a response, which would aim to maintain the correct excitatory/inhibitory balance, should involve a decrease, and not an increase, in parallel-fiber synapses. The increase in parallel-fiber synapse numbers is also unexpected given previous results showing that in hippocampal CA1 neurons, the Clstn3 KO decreases excitatory synapse numbers (Kim et al, 2020;Pettem et al, 2013). To independently confirm this increase, we analyzed the dendritic spine density in Purkinje cells.…”

“…In the CA1 region, Clstn1 and Clstn3 levels were highest, in the CA3 region all three calsyntenins were similarly abundant, and in the dentate gyrus, Clstn1 and Clstn2 were most strongly present (Figure 1A). These results suggest that most hippocampal neurons co-express multiple calsyntenin isoforms, which may account for the modest phenotypes observed with Clstn1, Clstn2, and Clstn3 KO mice (Kim et al, 2020;Lipina et al, 2016;Pettem et al, 2013;Ster et al, 2014).…”

“…Moreover, calsyntenins induce presynaptic specializations in heterologous synapse formation assays when expressed in non-neuronal cells (Pettem et al, 2013). Most importantly, knockout (KO) mice of all three calsyntenins exhibit synaptic impairments (Kim et al, 2020; Lipina et al, 2016; Pettem et al, 2013; Ster et al, 2014). Careful analyses revealed that Clstn3 KO mice exhibit a 20-30% decrease in excitatory synapse density in the CA1 region of the hippocampus (Kim et al, 2020; Pettem et al, 2013).…”

Neurexins regulate presynaptic GABAB-receptors at central synapses

“…Such an impact of FMR1 on the presynaptic GABA Breceptor signaling was not found in hippocampal inhibitory synapses 43 . Interestingly, calsyntenin-3, one of the integrated GABA B -receptor macromolecule complexes 26 , has been shown to interact with both α-neurexin and β-neurexin extracellularly 51,52 and to mediate GABAergic and glutamatergic synapse formation 52 . Neurexophilin, another α-neurexin ligand with restricted expression in subpopulations of inhibitory neurons, has also been reported to support presynaptic GABA B -receptor function 53 .…”