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

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.

Synaptogenic Assays Using Neurons Cultured on Micropatterned Substrates.

One of the difficulties for studying the mechanisms of synaptogenesis stems from the spatial unpredictability of contact formation between neurons, and the involvement of many parallel adhesive pathways mediating axon/dendrite recognition. To circumvent these limitations, we describe here a method allowing the investigation of synaptic contacts at controlled locations with high precision and statistics. Specifically, primary neurons are cultured on micropatterned substrates comprising arrays of micron-scale dots coated with purified synaptogenic adhesion molecules. Coating the substrates with the homophilic adhesion molecule SynCAM triggers the formation of functional presynaptic structures in axons, while neurexin elicits postsynapses in dendrites from neurons expressing the counter receptor neuroligin. This assay can be combined with various imaging techniques including immunocytochemistry to screen the accumulation of synaptic components, long-term live cell recordings to probe the kinetics of neurite growth and synapse differentiation, as well as high resolution single molecule tracking.

Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines.

The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin-coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain.

Micropatterned substrates coated with neuronal adhesion molecules for high-content study of synapse formation.

Studying the roles of different proteins and the mechanisms involved in synaptogenesis is hindered by the complexity and heterogeneity of synapse types, and by the spatial and temporal unpredictability of spontaneous synapse formation. Here we demonstrate a robust and high-content method to induce selectively presynaptic or postsynaptic structures at controlled locations. Neurons are cultured on micropatterned substrates comprising arrays of micron-scale dots coated with various synaptogenic adhesion molecules. When plated on neurexin-1ß-coated micropatterns, neurons expressing neuroligin-1 exhibit specific dendritic organization and selective recruitment of the postsynaptic scaffolding molecule PSD-95. Furthermore, functional AMPA receptors are trapped at neurexin-1ß dots, as revealed by live-imaging experiments. In contrast, neurons plated on SynCAM1-coated substrates exhibit strongly patterned axons and selectively assemble functional presynapses. N-cadherin coating, however, is not able to elicit synapses, indicating the specificity of our system. This method opens the way to both fundamental and therapeutic studies of various synaptic systems.

Neurexin-1ß binding to neuroligin-1 triggers the preferential recruitment of PSD-95 versus gephyrin through tyrosine phosphorylation of neuroligin-1.

Adhesion between neurexin-1ß (Nrx1ß) and neuroligin-1 (Nlg1) induces early recruitment of the postsynaptic density protein 95 (PSD-95) scaffold; however, the associated signaling mechanisms are unknown. To dissociate the effects of ligand binding and receptor multimerization, we compared conditions in which Nlg1 in neurons was bound to Nrx1ß or nonactivating HA antibodies. Time-lapse imaging, fluorescence recovery after photobleaching, and single-particle tracking demonstrated that in addition to aggregating Nlg1, Nrx1ß binding stimulates the interaction between Nlg1 and PSD-95. Phosphotyrosine immunoblots and pull-down of gephyrin by Nlg1 peptides in vitro showed that Nlg1 can be phosphorylated at a unique tyrosine (Y782), preventing gephyrin binding. Expression of Nlg1 point mutants in neurons indicated that Y782 phosphorylation controls the preferential binding of Nlg1 to PSD-95 versus gephyrin, and accordingly the formation of inhibitory and excitatory synapses. We propose that ligand-induced changes in the Nlg1 phosphotyrosine level control the balance between excitatory and inhibitory scaffold assembly during synapse formation and stabilization.

Biophysical mechanisms regulating AMPA receptor accumulation at synapses.

Controlling the number of AMPA receptors at synapses is fundamental for fast synaptic transmission as well as for long term adaptations in synaptic strength. In this review, we examine the biophysical mechanisms implicated in regulating AMPAR levels at the cell surface and at synapses. We first describe the structure and function of AMPARs, as well as their interactions with various proteins regulating their traffic and function. Second we review the vesicular trafficking mechanism involving exocytosis and endocytosis, by which AMPARs reach the cell surface and are internalized, respectively. Third, we examine the properties of lateral diffusion of AMPARs and their trapping at post-synaptic densities. Finally, we discuss how these two parallel mechanisms are integrated in time and space to control changes in synaptic AMPAR levels in response to plasticity protocols. This review highlights the important role of the extra-synaptic AMPAR pool, which makes an obligatory link between vesicular trafficking and trapping or release at synapses.

Unified quantitative model of AMPA receptor trafficking at synapses.

Trafficking of AMPA receptors (AMPARs) plays a key role in synaptic transmission. However, a general framework integrating the two major mechanisms regulating AMPAR delivery at postsynapses (i.e., surface diffusion and internal recycling) is lacking. To this aim, we built a model based on numerical trajectories of individual AMPARs, including free diffusion in the extrasynaptic space, confinement in the synapse, and trapping at the postsynaptic density (PSD) through reversible interactions with scaffold proteins. The AMPAR/scaffold kinetic rates were adjusted by comparing computer simulations to single-particle tracking and fluorescence recovery after photobleaching experiments in primary neurons, in different conditions of synapse density and maturation. The model predicts that the steady-state AMPAR number at synapses is bidirectionally controlled by AMPAR/scaffold binding affinity and PSD size. To reveal the impact of recycling processes in basal conditions and upon synaptic potentiation or depression, spatially and temporally defined exocytic and endocytic events were introduced. The model predicts that local recycling of AMPARs close to the PSD, coupled to short-range surface diffusion, provides rapid control of AMPAR number at synapses. In contrast, because of long-range diffusion limitations, extrasynaptic recycling is intrinsically slower and less synapse-specific. Thus, by discriminating the relative contributions of AMPAR diffusion, trapping, and recycling events on spatial and temporal bases, this model provides unique insights on the dynamic regulation of synaptic strength.

Protein kinase D controls the integrity of Golgi apparatus and the maintenance of dendritic arborization in hippocampal neurons.

Protein kinase D (PKD) is known to participate in various cellular functions, including secretory vesicle fission from the Golgi and plasma membrane-directed transport. Here, we report on expression and function of PKD in hippocampal neurons. Expression of an enhanced green fluorescent protein (EGFP)-tagged PKD activity reporter in mouse embryonal hippocampal neurons revealed high endogenous PKD activity at the Golgi complex and in the dendrites, whereas PKD activity was excluded from the axon in parallel with axonal maturation. Expression of fluorescently tagged wild-type PKD1 and constitutively active PKD1(S738/742E) (caPKD1) in neurons revealed that both proteins were slightly enriched at the trans-Golgi network (TGN) and did not interfere with its thread-like morphology. By contrast, expression of dominant-negative kinase inactive PKD1(K612W) (kdPKD1) led to the disruption of the neuronal Golgi complex, with kdPKD1 strongly localized to the TGN fragments. Similar findings were obtained from transgenic mice with inducible, neuron-specific expression of kdPKD1-EGFP. As a prominent consequence of kdPKD1 expression, the dendritic tree of transfected neurons was reduced, whereas caPKD1 increased dendritic arborization. Our results thus provide direct evidence that PKD activity is selectively involved in the maintenance of dendritic arborization and Golgi structure of hippocampal neurons.

NMDA receptor NR2B subunit over-expression increases cerebellar granule cell migratory activity.

Glutamate acting on NMDA receptors (NMDARs) is known to influence cerebellar granule cell migration. Subunit composition of NMDARs in granule cells changes characteristically during development: NR2B subunit containing receptors are abundant during migration towards the internal granule cell layer but are gradually replaced by NR2A and/or NR2C subunits once the final position is reached. Cerebellar granule cell migration was investigated using mutant mouse lines either with a deletion of the NR2C gene (NR2C(-/-) mice) or expressing NR2B instead of the NR2C subunit (NR2C-2B mice). BrdU-labeling revealed that over-expression of NR2B increased granule cell translocation in vivo, while the lack of NR2C subunit did not have any detectable effects on cell migration. Cellular composition of wild-type and mutant dissociated cerebellar granule cell cultures isolated from 10-day-old cerebella were similar, but NR2C-2B cultures had elevated level of NR2B subunits and intracellular Ca2+ imaging revealed higher sensitivity towards the addition of NR2B-selective antagonist in vitro. Time-lapse videomicroscopic observations revealed that average migratory velocity and the proportion of translocating cell bodies were significantly higher in NR2C-2B than in wild-type cultures. Our results provide evidence that NR2B-containing NMDARs can have specialized roles during granule cell migration and can increase migratory speed.

Cerebellar granule cells show age-dependent migratory differences in vitro.

Developmental differences between cerebellar granule cells during their migratory period were revealed using dissociated granule cell cultures isolated from 4, 7, or 10 days old (P4, P7, P10) mice. Under all culture conditions, the great majority of cultivated cell populations consisted of those granule cells that had not reach their final destination in the internal granule cell layer (IGL) by the age of isolation. In vitro morphological development and the expression of migratory markers (TAG-1, astrotactin, or EphB2) showed similar characteristics between the cultures. The migration of 1008 granule cells isolated from P4, P7, and P10 cerebella and cultivated under identical conditions were analyzed using statistical methods. In vitro time-lapse videomicroscopy revealed that P4 cells possessed the fastest migratory speed while P10 granule cells retained their migratory activity for the longest time in culture. Cultures obtained from younger postnatal ages showed more random migratory trajectories than P10 cultures. Our observations indicate that despite similar morphological and molecular properties, migratory differences exist in granule cell cultures isolated from different postnatal ages. Therefore, the age of investigation can substantially influence experimental results on the regulation of cell migration.