Elucidation of molecular mechanisms that regulate synapse formation is required for the understanding of neural wiring, higher brain functions, and mental disorders. Despite the wealth of in vitro information, fundamental questions about how glutamatergic synapses are formed in the mammalian brain remain unanswered. Glutamate receptor (GluR) delta2 is essential for cerebellar synapse formation in vivo. Here, we show that the N-terminal domain (NTD) of GluRdelta2 interacts with presynaptic neurexins (NRXNs) through cerebellin 1 precursor protein (Cbln1). The synaptogenic activity of GluRdelta2 is abolished in cerebellar primary cultures from Cbln1 knockout mice and is restored by recombinant Cbln1. Knockdown of NRXNs in cerebellar granule cells also hinders the synaptogenic activity of GluRdelta2. Both the NTD of GluRdelta2 and the extracellular domain of NRXN1beta suppressed the synaptogenic activity of Cbln1 in cerebellar primary cultures and in vivo. These results suggest that GluRdelta2 mediates cerebellar synapse formation by interacting with presynaptic NRXNs through Cbln1.

Mental retardation (MR) and autism are highly heterogeneous neurodevelopmental disorders. IL-1-receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic MR and is associated with autism. Thus, the elucidation of the functional role of IL1RAPL1 will contribute to our understanding of the pathogenesis of these mental disorders. Here, we showed that knockdown of endogenous IL1RAPL1 in cultured cortical neurons suppressed the accumulation of punctate staining signals for active zone protein Bassoon and decreased the number of dendritic protrusions. Consistently, the expression of IL1RAPL1 in cultured neurons stimulated the accumulation of Bassoon and spinogenesis. The extracellular domain (ECD) of IL1RAPL1 was required and sufficient for the presynaptic differentiation-inducing activity, while both the ECD and cytoplasmic domain were essential for the spinogenic activity. Notably, the synaptogenic activity of IL1RAPL1 was specific for excitatory synapses. Furthermore, we identified presynaptic protein tyrosine phosphatase (PTP) ␦ as a major IL1RAPL1-ECD interacting protein by affinity chromatography. IL1RAPL1 interacted selectively with certain forms of PTP␦ splice variants carrying mini-exon peptides in Ig-like domains. The synaptogenic activity of IL1RAPL1 was abolished in primary neurons from PTP␦ knock-out mice. IL1RAPL1 showed robust synaptogenic activity in vivo when transfected into the cortical neurons of wild-type mice but not in PTP␦ knock-out mice. These results suggest that IL1RAPL1 mediates synapse formation through trans-synaptic interaction with PTP␦. Our findings raise an intriguing possibility that the impairment of synapse formation may underlie certain forms of MR and autism as a common pathogenic pathway shared by these mental disorders.

Interleukin-1 receptor accessory protein (IL-1RAcP

Synapse formation is the key step in the development of neuronal networks. Precise synaptic connections between nerve cells in the brain provide the basis of perception, learning, memory, and cognition. Recently, we have shown that the trans-synaptic interaction of postsynaptic glutamate receptor (GluR) d2 and presynaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates excitatory synapse formation in the cerebellum (Uemura et al. 2010). This finding raises a question whether GluRd1 regulates synapse formation by trans-synaptic interaction with NRXNs through Cbln subtypes in the forebrain since GluRd1 shares 56% amino acid identity with GluRd2 (Yamazaki et al. 1992;Araki et al. 1993;Lomeli et al. 1993). Previous studies showed that GluRd1 expressed in human embryonic kidney (HEK) 293T cells induced presynaptic differentiation of cultured cerebellar granule cells AbstractGlutamate receptor (GluR) d1 is widely expressed in the developing forebrain, whereas GluRd2 is selectively expressed in cerebellar Purkinje cells. Recently, we found that trans-synaptic interaction of postsynaptic GluRd2 and presynaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates excitatory synapse formation in the cerebellum. Thus, a question arises whether GluRd1 regulates synapse formation in the forebrain. In this study, we showed that the N-terminal domain of GluRd1 induced inhibitory presynaptic differentiation of some populations of cultured cortical neurons. When Cbln1 or Cbln2 was added to cultures, GluRd1 expressed in HEK293T cells induced preferentially inhibitory presynaptic differentiation of cultured cortical neurons. The synaptogenic activity of GluRd1 was suppressed by the addition of the extracellular domain of NRXN1a or NRXN1b containing splice segment 4. Cbln subtypes directly bound to the N-terminal domain of GluRd1. The synaptogenic activity of GluRd1 in the presence of Cbln subtypes correlated well with their binding affinities. When transfected to cortical neurons, GluRd1 stimulated inhibitory synapse formation in the presence of Cbln1 or Cbln2. These results together with differential interactions of Cbln subtypes with NRXN variants suggest that GluRd1 induces preferentially inhibitory presynaptic differentiation of cortical neurons by interacting with NRXNs containing splice segment 4 through Cbln subtypes.

Synapse formation is triggered by trans-synaptic interactions of cell adhesion molecules, termed synaptic organizers. Three members of type-II receptor protein tyrosine phosphatases (classified as type-IIa RPTPs; PTPδ, PTPσ and LAR) are known as presynaptic organizers. Synaptic adhesion-like molecules (SALMs) have recently emerged as a family of postsynaptic organizers. Although all five SALM isoforms can bind to the type-IIa RPTPs, only SALM3 and SALM5 reportedly have synaptogenic activities depending on their binding. Here, we report the crystal structures of apo-SALM5, and PTPδ–SALM2 and PTPδ–SALM5 complexes. The leucine-rich repeat (LRR) domains of SALMs interact with the second immunoglobulin-like (Ig) domain of PTPδ, whereas the Ig domains of SALMs interact with both the second and third Ig domains of PTPδ. Unexpectedly, the structures exhibit the LRR-mediated 2:2 complex. Our synaptogenic co-culture assay using site-directed SALM5 mutants demonstrates that presynaptic differentiation induced by PTPδ–SALM5 requires the dimeric property of SALM5.