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.

IQ Motif and SEC7 Domain-containing Protein 3 (IQSEC3) Interacts with Gephyrin to Promote Inhibitory Synapse Formation.

Gephyrin is a central scaffold protein that mediates development, function, and plasticity of mammalian inhibitory synapses by interacting with various inhibitory synaptic proteins. Here, we show that IQSEC3, a guanine nucleotide exchange factor for ARF6, directly interacts with gephyrin, an interaction that is critical for the inhibitory synapse localization of IQSEC3. Overexpression of IQSEC3 increases inhibitory, but not excitatory, synapse density in a guanine nucleotide exchange factor activity-dependent manner. Conversely, knockdown of IQSEC3 decreases size of gephyrin cluster without altering gephyrin puncta density. Collectively, these data reveal that IQSEC3 acts together with gephyrin to regulate inhibitory synapse development.

LRRTM3 Regulates Excitatory Synapse Development through Alternative Splicing and Neurexin Binding.

The four members of the LRRTM family (LRRTM1-4) are postsynaptic adhesion molecules essential for excitatory synapse development. They have also been implicated in neuropsychiatric diseases. Here, we focus on LRRTM3, showing that two distinct LRRTM3 variants generated by alternative splicing regulate LRRTM3 interaction with PSD-95, but not its excitatory synapse-promoting activity. Overexpression of either LRRTM3 variant increased excitatory synapse density in dentate gyrus (DG) granule neurons, whereas LRRTM3 knockdown decreased it. LRRTM3 also controlled activity-regulated AMPA receptor surface expression in an alternative splicing-dependent manner. Furthermore, Lrrtm3-knockout mice displayed specific alterations in excitatory synapse density, excitatory synaptic transmission and excitability in DG granule neurons but not in CA1 pyramidal neurons. Lastly, LRRTM3 required only specific splice variants of presynaptic neurexins for their synaptogenic activity. Collectively, our data highlight alternative splicing and differential presynaptic ligand utilization in the regulation of LRRTMs, revealing key regulatory mechanisms for excitatory synapse development.

Gephyrin: a central GABAergic synapse organizer.

Gephyrin is a central element that anchors, clusters and stabilizes glycine and γ-aminobutyric acid type A receptors at inhibitory synapses of the mammalian brain. It self-assembles into a hexagonal lattice and interacts with various inhibitory synaptic proteins. Intriguingly, the clustering of gephyrin, which is regulated by multiple posttranslational modifications, is critical for inhibitory synapse formation and function. In this review, we summarize the basic properties of gephyrin and describe recent findings regarding its roles in inhibitory synapse formation, function and plasticity. We will also discuss the implications for the pathophysiology of brain disorders and raise the remaining open questions in this field.

The balancing act of GABAergic synapse organizers.

GABA (γ-aminobutyric acid) is the main neurotransmitter at inhibitory synapses in the mammalian brain. It is essential for maintaining the excitation and inhibition (E/I) ratio, whose imbalance underlies various brain diseases. Emerging information about inhibitory synapse organizers provides a novel molecular framework for understanding E/I balance at the synapse, circuit, and systems levels. This review highlights recent advances in deciphering these components of the inhibitory synapse and their roles in the development, transmission, and circuit properties of inhibitory synapses. We also discuss how their dysfunction may lead to a variety of brain disorders, suggesting new therapeutic strategies based on balancing the E/I ratio.

Structural basis for LAR-RPTP/Slitrk complex-mediated synaptic adhesion.

Synaptic adhesion molecules orchestrate synaptogenesis. The presynaptic leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) regulate synapse development by interacting with postsynaptic Slit- and Trk-like family proteins (Slitrks), which harbour two extracellular leucine-rich repeats (LRR1 and LRR2). Here we identify the minimal regions of the LAR-RPTPs and Slitrks, LAR-RPTPs Ig1-3 and Slitrks LRR1, for their interaction and synaptogenic function. Subsequent crystallographic and structure-guided functional analyses reveal that the splicing inserts in LAR-RPTPs are key molecular determinants for Slitrk binding and synapse formation. Moreover, structural comparison of the two Slitrk1 LRRs reveal that unique properties on the concave surface of Slitrk1 LRR1 render its specific binding to LAR-RPTPs. Finally, we demonstrate that lateral interactions between adjacent trans-synaptic LAR-RPTPs/Slitrks complexes observed in crystal lattices are critical for Slitrk1-induced lateral assembly and synaptogenic activity. Thus, we propose a model in which Slitrks mediate synaptogenic functions through direct binding to LAR-RPTPs and the subsequent lateral assembly of LAR-RPTPs/Slitrks complexes.