Calsyntenin-3 directly interacts with neurexins to orchestrate excitatory synapse development in the hippocampus

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, 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 insertnegative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo. Neuronspecific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs, and moderate impairment of light-induced anxiety-like behaviors in mice. 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. ResultsClstn3 directly binds β-Nrxns. Our previous cell surface-binding assays used a variety of Nrxn1α deletion variants derived from the bovine Nrxn1α gene or Nrxn1β variants derived from the rat Nrxn1β gene (Um et al., 2014). These vectors have long been used to characterize Nrxn interactions with neuroligins (NLs) and other ligands, including leucine-rich repeat transmembrane neuronal proteins (LRRTMs), neurexophilins, and latrophilin-1 (Boucard et al., 2012;Ko et al., 2009a;Missler et al., 1998). However, using Nrxn vectors constructed from mouse Nrxn genes, Craig and colleagues reported that Nrxn1α, but not Nrxn1β, binds to Clstn3 (Pettem et al., 2013). To resolve this discrepancy, we performed affinity chromatography of solubilized mouse synaptosomes using immobilized recombinant Ig-Clstn3 followed by mass spectrometry (MS). Intriguingly, among the captured proteins was a tryptic peptide unique to Nrxn1β (in addition to Nrxn1α peptides) ( Figures 1A-D; see Table 1). To confirm binding between Clstn3 and Nrxn1β, we engineered mouse Nrxn1β expression constructs and performed cell surface-binding assays. We found robust binding of Ig-Clstn3 to HEK293T cells expressing C-terminally FLAG-tagged mNrxn1β lacking (mNrxn1β -SS4 -FLAG) or containing (mNrxn1β +SS4 -FLAG) the SS4 insert (Figures 1E and 1F). In assays measuring dimeric ligand binding to mNrxn-expressing cell surfaces, Ig-Clstn3 interacted with both mNrxn1β -SS4 and mNrxn1β +SS4 with nanomolar affinity (Figures 1G and 1H). Kd values calculated by Scatchard analyses were 51.39 ± 5.26 nM for Nrxn1β +SS4 and 105.94 ± 7.89 nM for Nrxn1β -SS4 (Figures 1G and 1H). These findings indicate that Clstn3 binds Nrxn1β with high affinity and exhibits a slight preference for SS4 insert-positive splice variants. To investigate differences between bovine and rat Nrxn1 plasmids (bNrxn1α and rNrxn1β) used in our previous studies and t...

Activation of the CaMKII-Sarm1-ASK1-p38 MAP kinase pathway protects against axon degeneration caused by loss of mitochondria

“…Calsyntenins are type I transmembrane (TM) proteins and are members of the cadherin superfamily ( Hill et al, 2001 ). Their extracellular region contains two cadherin domains and an LG/LNS (laminin G-like) domain and has been shown to regulate synapse development through interactions with neurexin ( Kim et al, 2020a ; Kim et al, 2020b ; Pettem et al, 2013 ). Their intracellular region is characterized by two kinesin-binding sites (KBS), which are important for its anterograde trafficking ( Araki et al, 2003 ; Konecna et al, 2006 ).…”