A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation

Abstract

To better understand the molecular mechanisms by which early neuronal connections mature into synapses, we examined the impact of neuroligin-1 (Nlg1) phosphorylation on synapse differentiation, focusing on a unique intracellular tyrosine (Y782), which differentially regulates Nlg1 binding to PSD-95 and gephyrin. By expressing Nlg1 point mutants (Y782A/F) in hippocampal neurons, we show using imaging and electrophysiology that Y782 modulates the recruitment of functional AMPA receptors (AMPARs). Nlg1-Y782F impaired both dendritic spine formation and AMPAR diffusional trapping, but not NMDA receptor recruitment, revealing the assembly of silent synapses. Furthermore, replacing endogenous Nlg1 with either Nlg1-Y782A or -Y782F in CA1 hippocampal neurons impaired long-term potentiation (LTP), demonstrating a critical role of AMPAR synaptic retention. Screening of tyrosine kinases combined with pharmacological inhibitors point to Trk family members as major regulators of endogenous Nlg1 phosphorylation and synaptogenic function. Thus, Nlg1 tyrosine phosphorylation signaling is a critical event in excitatory synapse differentiation and LTP.

Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation

“…Thus, the partial occlusion of LTP observed upon optoFGFR1 stimulation can be explained by a high initial recruitment of synaptic AMPARs, which depletes the extrasynaptic AMPAR reservoir necessary for LTP. Accordingly, we previously reported an almost complete occlusion of LTP upon replacement of endogenous Nlg1 by a Y782A mutant which phenocopies maximally phosphorylated Nlg1 and increases AMPAR-mediated EPSCs by ~4-fold Letellier et al, 2018). Overall, our model predicts a negative correlation between basal synaptic AMPAR number and the ability to respond to LTP (Fig.…”

“…Besides the role of Nlgs in controlling synapse number, there is also a debate about the actual function of Nlgs in regulating basal excitatory synaptic transmission and plasticity. Several studies relying on the expression of Nlg mutants have revealed the potential for Nlg1 to recruit both NMDA receptors (NMDARs) and AMPA receptors (AMPARs) at synapses through extracellular and intracellular interactions, respectively (Budreck et al, 2013;Giannone et al, 2013;Haas et al, 2018;Heine et al, 2008;Letellier et al, 2018;Mondin et al, 2011;Shipman and Nicoll, 2012). However, constitutive or conditional Nlg1/2/3 KO selectively affect basal NMDAR-mediated EPSCs and not AMPARs-EPSCs, and rescue experiments with truncated Nlg1 mutants suggest that the synaptic recruitment of NMDARs requires the intracellular rather than the extracellular domain of Nlg1 (Chanda et al, 2017;Chubykin et al, 2007;Jiang et al, 2017;Wu et al, 2019).…”

“…However, constitutive or conditional Nlg1/2/3 KO selectively affect basal NMDAR-mediated EPSCs and not AMPARs-EPSCs, and rescue experiments with truncated Nlg1 mutants suggest that the synaptic recruitment of NMDARs requires the intracellular rather than the extracellular domain of Nlg1 (Chanda et al, 2017;Chubykin et al, 2007;Jiang et al, 2017;Wu et al, 2019). Finally, while it is generally accepted that NMDAR-dependent long-term potentiation (LTP) is impaired by Nlg1 KD or KO, the issues of which Nlg1 motifs are important in this process and whether the Nlg1-NMDAR interaction is required, are unclear (Jiang et al, 2017;Kim et al, 2008;Letellier et al, 2018;Shipman and Nicoll, 2012;Wu et al, 2019).…”

“…In addition to differences in experimental preparations, these studies relying on the manipulation of the Nlg expression level all have potential biases, including the compensatory expression of proteins in the case of KO (Dang et al, 2018), off-target effects of inhibitory RNAs (Alvarez et al, 2006), and mislocalization of overexpressed Nlgs, e.g. Nlg1 at inhibitory synapses and Nlg2 at excitatory synapses (Chih et al, 2006;Letellier et al, 2018;Nguyen et al, 2016;Tsetsenis et al, 2014). Furthermore, these techniques operate on a long-term basis, i.e.…”

“…To acutely control Nlg1 activity using optogenetics, we aimed at manipulating the phosphotyrosine level of endogenous Nlg1, based on our previous findings that the expression of Nlg1 point mutants in a unique intracellular tyrosine (Y782) affects dendritic spine density and recruitment of PSD-95 and AMPARs Letellier et al, 2018). Here, we show that light stimulation of a photoactivatable receptor tyrosine kinase (optoFGFR1) expressed in hippocampal CA1 neurons increases dendritic spine number, enhances AMPAR-receptor mediated EPSCs, and partially blocks LTP, in a Nlg1 selective fashion, thus demonstrating a major role of the intracellular tyrosine phosphorylation of endogenous Nlg1 in post-synaptic differentiation.…”