Synaptic organizer: Slitrks and type IIa receptor protein tyrosine phosphatases.

Slit-like and Trk-like (Slitrk) family members are leucine-rich repeat (LRR)-containing neuronal transmembrane proteins. Slitrks have been highlighted as key synapse organizers at neuronal synapses through interactions with specific members of the presynaptic type IIa receptor protein tyrosine phosphatase (RPTP) family. Recent structural studies on type IIa RPTP/Slitrk1 complexes have unveiled molecular insights into their binding selectivity and have established the role of higher-order receptor clustering in their synaptogenic activity. Here, we will discuss key structural aspects of Slitrk interactions with type IIa RPTP family members, the biological roles of Slitrks in neurons, and our current knowledge of SLITRK mutations in human diseases.

Structural Basis for LAR-RPTP-Mediated Synaptogenesis.

Leukocyte common antigen-related protein tyrosine phosphatases (LAR-RPTPs) are cellular receptors of heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans that regulate neurite outgrowth and neuronal regeneration. LAR-RPTPs have also received particular attention as the major presynaptic hubs for synapse organization through selective binding to numerous postsynaptic adhesion partners. Recent structural studies on LAR-RPTP-mediated trans-synaptic adhesion complexes have provided significant insight into the molecular basis of their specific interactions, the key codes for their selective binding, as well as the higher-order clustering of LAR-RPTPs necessary for synaptogenic activity. In this review, we summarize the structures of LAR-RPTPs in complex with various postsynaptic adhesion partners and discuss the molecular mechanisms underlying LAR-RPTP-mediated synaptogenesis.

LAR-RPTP Clustering Is Modulated by Competitive Binding between Synaptic Adhesion Partners and Heparan Sulfate.

The leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) are cellular receptors of heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans that direct axonal growth and neuronal regeneration. LAR-RPTPs are also synaptic adhesion molecules that form trans-synaptic adhesion complexes by binding to various postsynaptic adhesion ligands, such as Slit- and Trk-like family of proteins (Slitrks), IL-1 receptor accessory protein-like 1 (IL1RAPL1), interleukin-1 receptor accessory protein (IL-1RAcP) and neurotrophin receptor tyrosine kinase C (TrkC), to regulate synaptogenesis. Here, we determined the crystal structure of the human LAR-RPTP/IL1RAPL1 complex and found that lateral interactions between neighboring LAR-RPTP/IL1RAPL1 complexes in crystal lattices are critical for the higher-order assembly and synaptogenic activity of these complexes. Moreover, we found that LAR-RPTP binding to the postsynaptic adhesion ligands, Slitrk3, IL1RAPL1 and IL-1RAcP, but not TrkC, induces reciprocal higher-order clustering of trans-synaptic adhesion complexes. Although LAR-RPTP clustering was induced by either HS or postsynaptic adhesion ligands, the dominant binding of HS to the LAR-RPTP was capable of dismantling pre-established LAR-RPTP-mediated trans-synaptic adhesion complexes. These findings collectively suggest that LAR-RPTP clustering for synaptogenesis is modulated by a complex synapse-organizing protein network.

Structural Insights into Modulation of Neurexin-Neuroligin Trans-synaptic Adhesion by MDGA1/Neuroligin-2 Complex.

Membrane-associated mucin domain-containing glycosylphosphatidylinositol anchor proteins (MDGAs) bind directly to neuroligin-1 (NL1) and neuroligin-2 (NL2), thereby respectively regulating excitatory and inhibitory synapse development. However, the mechanisms by which MDGAs modulate NL activity to specify development of the two synapse types remain unclear. Here, we determined the crystal structures of human NL2/MDGA1 Ig1-3 complex, revealing their stable 2:2 arrangement with three interaction interfaces. Cell-based assays using structure-guided, site-directed MDGA1 mutants showed that all three contact patches were required for the MDGA's negative regulation of NL2-mediated synaptogenic activity. Furthermore, MDGA1 competed with neurexins for NL2 via its Ig1 domain. The binding affinities of both MDGA1 and MDGA2 for NL1 and NL2 were similar, consistent with the structural prediction of similar binding interfaces. However, MDGA1 selectively associated with NL2, but not NL1, in vivo. These findings collectively provide structural insights into the mechanism by which MDGAs negatively modulate synapse development governed by NLs/neurexins.

Slitrk Missense Mutations Associated with Neuropsychiatric Disorders Distinctively Impair Slitrk Trafficking and Synapse Formation.

Slit- and Trk-like (Slitrks) are a six-member family of synapse organizers that control excitatory and inhibitory synapse formation by forming trans-synaptic adhesions with LAR receptor protein tyrosine phosphatases (PTPs). Intriguingly, genetic mutations of Slitrks have been associated with a multitude of neuropsychiatric disorders. However, nothing is known about the neuronal and synaptic consequences of these mutations. Here, we report the structural and functional effects on synapses of various rare de novo mutations identified in patients with schizophrenia or Tourette syndrome. A number of single amino acid substitutions in Slitrk1 (N400I or T418S) or Slitrk4 (V206I or I578V) reduced their surface expression levels. These substitutions impaired glycosylation of Slitrks expressed in HEK293T cells, caused retention of Slitrks in the endoplasmic reticulum and cis-Golgi compartment in COS-7 cells and neurons, and abolished Slitrk binding to PTPδ. Furthermore, these substitutions eliminated the synapse-inducing activity of Slitrks, abolishing their functional effects on synapse density in cultured neurons. Strikingly, a valine-to-methionine mutation in Slitrk2 (V89M) compromised synapse formation activity in cultured neuron, without affecting surface transport, expression, or synapse-inducing activity in coculture assays. Similar deleterious effects were observed upon introduction of the corresponding valine-to-methionine mutation into Slitrk1 (V85M), suggesting that this conserved valine residue plays a key role in maintaining the synaptic functions of Slitrks. Collectively, these data indicate that inactivation of distinct cellular mechanisms caused by specific Slitrk dysfunctions may underlie Slitrk-associated neuropsychiatric disorders in humans, and provide a robust cellular readout for the development of knowledge-based therapies.

Neurotrophin-3 Regulates Synapse Development by Modulating TrkC-PTPσ Synaptic Adhesion and Intracellular Signaling Pathways.

UNLABELLED: Neurotrophin-3 (NT-3) is a secreted neurotrophic factor that binds neurotrophin receptor tyrosine kinase C (TrkC), which in turn binds to presynaptic protein tyrosine phosphatase σ (PTPσ) to govern excitatory synapse development. However, whether and how NT-3 cooperates with the TrkC-PTPσ synaptic adhesion pathway and TrkC-mediated intracellular signaling pathways in rat cultured neurons has remained unclear. Here, we report that NT-3 enhances TrkC binding affinity for PTPσ. Strikingly, NT-3 treatment bidirectionally regulates the synaptogenic activity of TrkC: at concentrations of 10-25 ng/ml, NT-3 further enhanced the increase in synapse density induced by TrkC overexpression, whereas at higher concentrations, NT-3 abrogated TrkC-induced increases in synapse density. Semiquantitative immunoblotting and optogenetics-based imaging showed that 25 ng/ml NT-3 or light stimulation at a power that produced a comparable level of NT-3 (6.25 µW) activated only extracellular signal-regulated kinase (ERK) and Akt, whereas 100 ng/ml NT-3 (light intensity, 25 µW) further triggered the activation of phospholipase C-γ1 and CREB independently of PTPσ. Notably, disruption of TrkC intracellular signaling pathways, extracellular ligand binding, or kinase activity by point mutations compromised TrkC-induced increases in synapse density. Furthermore, only sparse, but not global, TrkC knock-down in cultured rat neurons significantly decreased synapse density, suggesting that intercellular differences in TrkC expression level are critical for its synapse-promoting action. Together, our data demonstrate that NT-3 is a key factor in excitatory synapse development that may direct higher-order assembly of the TrkC/PTPσ complex and activate distinct intracellular signaling cascades in a concentration-dependent manner to promote competition-based synapse development processes. SIGNIFICANCE STATEMENT: In this study, we present several lines of experimental evidences to support the conclusion that neurotrophin-3 (NT-3) modulates the synaptic adhesion pathway involving neurotrophin receptor tyrosine kinase C (TrkC) and presynaptic protein tyrosine phosphatase σ (PTPσ) in a bidirectional manner at excitatory synapses. NT-3 acts in concentration-independent manner to facilitate TrkC-mediated presynaptic differentiation, whereas it acts in a concentration-dependent manner to exert differential effects on TrkC-mediated organization of postsynaptic development. We further investigated TrkC extracellular ligand binding, intracellular signaling pathways, and kinase activity in NT-3-induced synapse development. Last, we found that interneuronal differences in TrkC levels regulate the synapse number. Overall, these results suggest that NT-3 functions as a positive modulator of synaptogenesis involving TrkC and PTPσ.