Data for Alternative Splicing of Adhesion-GPCR Latrophilin-3 Controls Synapse Formation

How synapses are assembled and specified in brain is incompletely understood. Latrophilin-3, a postsynaptic adhesion-GPCR, mediates Schaffer-collateral synapse formation in the hippocampus but the mechanisms involved remained unclear. Here we show that Latrophilin-3 organizes synapses by a convergent dual-pathway mechanism by which Latrophilin-3 simultaneously activates GaS/cAMP-signaling and recruits phase-separated postsynaptic protein scaffolds. We found that cell type-specific alternative splicing of Latrophilin-3 controls its G protein coupling mode, resulting in Latrophilin-3 variants that predominantly signal via Gas and cAMP or via Gα12/13. A CRISPR-mediated genetic switch of Latrophilin-3 alternative splicing from a GaS- to a Gα12/13-coupled mode impaired synaptic connectivity similar to the overall deletion of Latrophilin-3, suggesting that GaS/cAMP-signaling by Latrophilin-3 splice variants mediates synapse formation. Moreover, GaS- but not Gα12/13-coupled splice variants of Latrophilin-3 recruit phase-transitioned postsynaptic protein scaffolds that are clustered by binding of presynaptic Latrophilin-3 ligands. Strikingly, neuronal activity promotes alternative splicing of the synaptogenic variant of Latrophilin-3, thereby enhancing synaptic connectivity. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways, GaS/cAMP signaling and the phase separation of postsynaptic protein scaffolds. Two experiments included in this submission: (1) RNA sequencing in primary hippocampal neurons cultured from Cas9-expressing mice (JAX ##024858), to study the transcriptomic alterations after Lphn3 is deleted (Lphn3 KO), or the Exon31 of Lphn3 is disrupted (Lphn3 Exon31 KO). In this experiment, neurons were infected with lentiviruses expressing gRNA which either: has no target as negative control (control group, 3 replicates), or targets Lphn3 constitutive exon7 to induce KO of all Lphn3 (Lphn3 KO group, 3 replicates), or targets the splice acceptor of Exon31 of Lphn3 in order to disrupt the splicing of Exon31 (Lphn3 Exon31 KO group, 3 replicates). (2) Amplicon sequencing of PDZ-binding motif (PBM) region of Lphn3 in primary hippocampus neurons cultured from Cas9-expressing mice (JAX ##024858), to assess the efficiency of PBM KO in all Lphn3 mRNAs. In this experiment, neurons were infected with lentiviruses expressing gRNA which either: has no target as negative control (control group, 2 replicates), or targets the upstream of PBM of Lphn3 to induce KO of PBM (PBM KO group, 2 replicates).