Postsynaptic Scaffolding Molecule Research Articles

Development and Application of Technology for Neural Circuit Visualization - Secondary Publication.

The dynamics of neurite extension and synaptic connections are central issues in neural circuit research. The development of technologies for labeling purified cytoskeletal proteins with fluorescent dyes and introducing them into living neurons using microinjection greatly facilitated our understanding of cytoskeletal dynamics in neuronal axons. Imaging data showed that the cytoskeleton repeatedly polymerized and depolymerized within the axon, and elongation was driven by the new cytoskeleton formed at the axon tip. This finding significantly revised previously proposed models that explained slow axonal transport. After the discovery of green fluorescent protein (GFP), its potential application to the live imaging of neurons was recognized in the 1990s, and a new method for visualizing synapses using GFP-tagged postsynaptic scaffolding molecules was established. This method revealed the continuous turnover of synapses during development, which overturned the established theory that synapses are highly stable once they are formed. Live imaging of synapses also demonstrated that the molecular composition of synapses changes rapidly, driven by the rapid replacement of synaptic molecules. Fluorescence measurement of single GFP molecules enabled estimation of the absolute number of postsynaptic molecules in a single synapse. Furthermore, in multiple mouse models of autism spectrum disorders (ASDs), enhanced synapse turnover was detected as a common circuit-level phenotype. This study provides solid experimental evidence that an increase in synapse dynamics underlies the pathophysiology in mouse models of ASDs. The introduction of fluorescence imaging in neurobiology revealed that the neuronal cytoskeleton and synaptic structure are not static but dynamic cellular components. Imaging technology is expected to further advance our understanding of the dynamic properties of neurons and neural circuits.

Convergence of adenosine and GABA signaling for synapse stabilization during development.

During development, neural circuit formation requires the stabilization of active γ-aminobutyric acid–mediated (GABAergic) synapses and the elimination of inactive ones. Here, we demonstrate that, although the activation of postsynaptic GABA type A receptors (GABAARs) and adenosine A2A receptors (A2ARs) stabilizes GABAergic synapses, only A2AR activation is sufficient. Both GABAAR- and A2AR-dependent signaling pathways act synergistically to produce adenosine 3′,5′-monophosphate through the recruitment of the calcium–calmodulin–adenylyl cyclase pathway. Protein kinase A, thus activated, phosphorylates gephyrin on serine residue 303, which is required for GABAAR stabilization. Finally, the stabilization of pre- and postsynaptic GABAergic elements involves the interaction between gephyrin and the synaptogenic membrane protein Slitrk3. We propose that A2ARs act as detectors of active GABAergic synapses releasing GABA, adenosine triphosphate, and adenosine to regulate their fate toward stabilization or elimination.

Differential Scaling of Synaptic Molecules within Functional Zones of an Excitatory Synapse during Homeostatic Plasticity.

Homeostatic scaling is a form of synaptic plasticity where individual synapses scale their strengths to compensate for global suppression or elevation of neuronal activity. This process can be studied by measuring miniature EPSP (mEPSP) amplitudes and frequencies following the regulation of activity in neuronal cultures. Here, we demonstrate a quantitative approach to characterize multiplicative synaptic scaling using immunolabelling of hippocampal neuronal cultures treated with tetrodotoxin (TTX) or bicuculline to extract scaling factors for various synaptic proteins. This approach allowed us to directly examine the scaling of presynaptic and postsynaptic scaffolding molecules along with neurotransmitter receptors in primary cultures from mouse and rat hippocampal neurons. We show robust multiplicative scaling of synaptic scaffolding molecules namely, Shank2, PSD95, Bassoon, and AMPA receptor subunits and quantify their scaling factors. We use super-resolution microscopy to calculate scaling factors of surface expressed GluA2 within functional zones of the synapse and show that there is differential and correlated scaling of GluA2 levels within the spine, the postsynaptic density (PSD), and the perisynaptic regions. Our method opens a novel paradigm to quantify relative molecular changes of synaptic proteins within distinct subsynaptic compartments from a large number of synapses in response to alteration of neuronal activity, providing anatomic insights into the intricacies of variability in strength of individual synapses.

PIP3-Phldb2 is crucial for LTP regulating synaptic NMDA and AMPA receptor density and PSD95 turnover

The essential involvement of phosphoinositides in synaptic plasticity is well-established, but incomplete knowledge of the downstream molecular entities prevents us from understanding their signalling cascades completely. Here, we determined that Phldb2, of which pleckstrin-homology domain is highly sensitive to PIP3, functions as a phosphoinositide-signalling mediator for synaptic plasticity. BDNF application caused Phldb2 recruitment toward postsynaptic membrane in dendritic spines, whereas PI3K inhibition resulted in its reduced accumulation. Phldb2 bound to postsynaptic scaffolding molecule PSD-95 and was crucial for localization and turnover of PSD-95 in the spine. Phldb2 also bound to GluA1 and GluA2. Phldb2 was indispensable for the interaction between NMDA receptors and CaMKII, and the synaptic density of AMPA receptors. Therefore, PIP3-responsive Phldb2 is pivotal for induction and maintenance of LTP. Memory formation was impaired in our Phldb2−/− mice.

A Sam68-dependent alternative splicing program shapes postsynaptic protein complexes.

Alternative splicing is one of the key mechanisms to increase the diversity of cellular transcriptomes, thereby expanding the coding capacity of the genome. This diversity is of particular importance in the nervous system with its elaborated cellular networks. Sam68, a member of the Signal Transduction Associated RNA-binding (STAR) family of RNA-binding proteins, is expressed in the developing and mature nervous system but its neuronal functions are poorly understood. Here, we perform genome-wide mapping of the Sam68-dependent alternative splicing program in mice. We find that Sam68 is required for the regulation of a set of alternative splicing events in pre-mRNAs encoding several postsynaptic scaffolding molecules that are central to the function of GABAergic and glutamatergic synapses. These components include Collybistin (Arhgef9), Gephyrin (Gphn), and Densin-180 (Lrrc7). Sam68-regulated Lrrc7 variants engage in differential protein interactions with signalling proteins, thus, highlighting a contribution of the Sam68 splicing program to shaping synaptic complexes. These findings suggest an important role for Sam68-dependent alternative splicing in the regulation of synapses in the central nervous system.

Sequences Flanking the Gephyrin-Binding Site of GlyRβ Tune Receptor Stabilization at Synapses.

The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulation of heteromeric glycine receptors (GlyRs) via the cytoplasmic loop in the β-subunit (β-loop). This binding involves high- and low-affinity interactions, but the molecular mechanism of this bimodal binding and its implication in GlyR stabilization at synapses remain unknown. We have approached this question using a combination of quantitative biochemical tools and high-density single molecule tracking in cultured rat spinal cord neurons. The high-affinity binding site could be identified and was shown to rely on the formation of a 310-helix C-terminal to the β-loop core gephyrin-binding motif. This site plays a structural role in shaping the core motif and represents the major contributor to the synaptic confinement of GlyRs by gephyrin. The N-terminal flanking sequence promotes lower affinity interactions by occupying newly identified binding sites on gephyrin. Despite its low affinity, this binding site plays a modulatory role in tuning the mobility of the receptor. Together, the GlyR β-loop sequences flanking the core-binding site differentially regulate the affinity of the receptor for gephyrin and its trapping at synapses. Our experimental approach thus bridges the gap between thermodynamic aspects of receptor-scaffold interactions and functional receptor stabilization at synapses in living cells.

Dopamine synapse is a neuroligin-2–mediated contact between dopaminergic presynaptic and GABAergic postsynaptic structures

Midbrain dopamine neurons project densely to the striatum and form so-called dopamine synapses on medium spiny neurons (MSNs), principal neurons in the striatum. Because dopamine receptors are widely expressed away from dopamine synapses, it remains unclear how dopamine synapses are involved in dopaminergic transmission. Here we demonstrate that dopamine synapses are contacts formed between dopaminergic presynaptic and GABAergic postsynaptic structures. The presynaptic structure expressed tyrosine hydroxylase, vesicular monoamine transporter-2, and plasmalemmal dopamine transporter, which are essential for dopamine synthesis, vesicular filling, and recycling, but was below the detection threshold for molecules involving GABA synthesis and vesicular filling or for GABA itself. In contrast, the postsynaptic structure of dopamine synapses expressed GABAergic molecules, including postsynaptic adhesion molecule neuroligin-2, postsynaptic scaffolding molecule gephyrin, and GABAA receptor α1, without any specific clustering of dopamine receptors. Of these, neuroligin-2 promoted presynaptic differentiation in axons of midbrain dopamine neurons and striatal GABAergic neurons in culture. After neuroligin-2 knockdown in the striatum, a significant decrease of dopamine synapses coupled with a reciprocal increase of GABAergic synapses was observed on MSN dendrites. This finding suggests that neuroligin-2 controls striatal synapse formation by giving competitive advantage to heterologous dopamine synapses over conventional GABAergic synapses. Considering that MSN dendrites are preferential targets of dopamine synapses and express high levels of dopamine receptors, dopamine synapse formation may serve to increase the specificity and potency of dopaminergic modulation of striatal outputs by anchoring dopamine release sites to dopamine-sensing targets.

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-1β-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-1β 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.

Postsynaptic scaffolding molecules modulate the localization of neuroligins

Previous work has shown an important role for neuroligins in promoting the formation of synaptic connections in cultured cells. Although neuroligins enhance both excitatory and inhibitory synapse formation, individual neuroligin isoforms have been shown to preferentially localize to either glutamatergic or GABAergic synapses. Current evidence points to an important role for both the extracellular and intracellular domains of neuroligins in their synaptic localization. Although postsynaptic density protein 95 (PSD-95) has been shown to be involved in the recruitment of neuroligin 1 to excitatory synapses, the localization of neuroligin 2 (NL2) and neuroligin 3 (NL3) to excitatory and inhibitory synapses is less well defined. We assessed the roles of gephyrin and PSD-95, postsynaptic scaffolding molecules exclusively localized to inhibitory and excitatory synapses, respectively, in localizing NL2 and NL3 in primary neuronal cultures. We demonstrate that knockdown of gephyrin results in a significant shift of NL2 from inhibitory to excitatory synaptic contacts, while knockdown of PSD-95 leads to a partial shift of NL2 and NL3 from excitatory to inhibitory contacts. Furthermore, analysis of specific domain deletions within the C-terminal, intracellular domain of NL2 reveals that the region between amino acids 716 and 782 is required for the normal synaptic clustering of this protein. Together, these data suggest that intracellular mechanisms are involved in the targeting of different neuroligin family members to synapses (216).

An Essential Role for PICK1 in NMDA Receptor-Dependent Bidirectional Synaptic Plasticity

PICK1 is a calcium-sensing, PDZ domain-containing protein that interacts with GluR2 and GluR3 AMPA receptor (AMPAR) subunits and regulates their trafficking. Although PICK1 has been principally implicated in long-term depression (LTD), PICK1 overexpression in CA1 pyramidal neurons causes a CaMK- and PKC-dependent potentiation of AMPAR-mediated transmission and an increase in synaptic GluR2-lacking AMPARs, mechanisms associated with NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here, we directly tested whether PICK1 participates in both hippocampal NMDAR-dependent LTP and LTD. We show that the PICK1 potentiation of AMPAR-mediated transmission is NMDAR dependent and fully occludes LTP. Conversely, blockade of PICK1 PDZ interactions or lack of PICK1 prevents LTP. These observations demonstrate an important role for PICK1 in LTP. In addition, deletion of PICK1 or blockade of PICK1 PDZ binding prevented NMDAR-dependent LTD. Thus, PICK1 plays a critical role in bidirectional NMDAR-dependent long-term synaptic plasticity in the hippocampus.

Ubiquitin and Protein Turnover in Synapse Function

Enduring modification of synapses is central to long-lasting neural circuit plasticity. Such adaptations include rapid posttranslational modification of existing synaptic proteins over periods of minutes and persisting changes in the abundance of synaptic proteins over hours to days. Recently, ubiquitination and protein degradation have emerged as additional mechanisms for modifying the function and molecular composition of synapses. These recent findings raise intriguing questions as to how enduring changes at synapses are accomplished in the face of robust, ongoing molecular turnover.

Polo-like kinases in the nervous system

Polo like kinases (Plks) are key regulators of the cell cycle, but little is known about their functions in postmitotic cells such as neurons. Recent findings indicate that Plk2 and Plk3 are dynamically regulated in neurons by synaptic activity at the mRNA and protein levels. In COS cells, Plk2 and Plk3 interact with spine-associated Rap guanosine triphosphatase-activating protein (SPAR), a regulator of actin dynamics and dendritic spine morphology, leading to its degradation through the ubiquitin-proteasome system. Induction of Plk2 in hippocampal neurons eliminates SPAR protein, depletes a core postsynaptic scaffolding molecule (PSD-95), and causes loss of mature dendritic spines and synapses. These findings implicate neuronal Plks as mediators of activity-dependent change in molecular composition and morphology of synapses. Induction of Plks might provide a homeostatic mechanism for global dampening of synaptic strength following heightened neuronal activity ('synaptic scaling'). Synapse-specific actions of induced Plks are also possible, particularly in light of the discovery of phosphoserine/threonine peptide motifs as binding targets of the polo box domain, which could allow for 'priming' phosphorylation by upstream kinases that could 'tag' Plk substrates only in specific synapses.