Activity-dependent Protein Dynamics Define Interconnected Cores of Co-regulated Postsynaptic Proteins

Synapses are highly dynamic structures that mediate cell–cell communication in the central nervous system. Their molecular composition is altered in an activity-dependent fashion, which modulates the efficacy of subsequent synaptic transmission events. Whereas activity-dependent trafficking of individual key synaptic proteins into and out of the synapse has been characterized previously, global activity-dependent changes in the synaptic proteome have not been studied.To test the feasibility of carrying out an unbiased large-scale approach, we investigated alterations in the molecular composition of synaptic spines following mass stimulation of the central nervous system induced by pilocarpine. We observed widespread changes in relative synaptic abundances encompassing essentially all proteins, supporting the view that the molecular composition of the postsynaptic density is tightly regulated. In most cases, we observed that members of gene families displayed coordinate regulation even when they were not known to physically interact.Analysis of correlated synaptic localization revealed a tightly co-regulated cluster of proteins, consisting of mainly glutamate receptors and their adaptors. This cluster constitutes a functional core of the postsynaptic machinery, and changes in its size affect synaptic strength and synaptic size. Our data show that the unbiased investigation of activity-dependent signaling of the postsynaptic density proteome can offer valuable new information on synaptic plasticity.Excitatory synaptic transmission is the primary mode of cell–cell communication in the central nervous system. The efficacy of synaptic transmission is highly regulated, and alterations in the strength of synaptic signaling within networks of neurons provide a mechanism for learning and memory storage, as well as for overall network stability. Modulation of synapse efficacy can occur through alterations in the structure and composition of the postsynaptic spine. The synaptic abundance of several molecules has been shown to be regulated in response to activity (1).The levels of individual proteins at postsynaptic spines are regulated through multiple processes. Active transport mechanisms exist and have been well characterized for AMPA-type glutamate receptors (AMPA-Rs)¹ via either insertion into the synapse or tighter association with the postsynaptic density (PSD) following lateral diffusion within the cell membrane (2). In addition to AMPA-Rs, other proteins known to be subject to activity-dependent regulation include calcium calmodulin-dependent protein kinase II alpha and beta, NMDA-type glutamate receptors (NMDA-Rs), and proteosome subunits (3–5). Synaptic protein content is dysregulated in a number of neuropsychiatric and neurodegenerative diseases, including Alzheimer''s disease and fragile X mental retardation (6–8).Most studies reported thus far have focused on a small number of selected molecules in individual experiments using a subset of synapses. Whereas learning and memory rely on the differential response of individual synapses to their specific input patterns, overall network excitability has to be maintained by homeostatic means. This homeostasis is governed by multiple pathways, and very little is known about the principles that regulate synaptic protein content across large numbers of synapses and neurons. The contributions of individual pathways and the interactions among them are largely unknown.In order to explore synaptic dynamics with a global view, we took advantage of a chemically induced mass stimulation protocol to stimulate synapses broadly throughout the central nervous system. We employed mass spectrometry and isotopically encoded isobaric peptide tagging with the iTRAQ reagent to quantify changes in the abundance of 893 proteins (9). We then analyzed changes in the relative abundance of these proteins at 0, 10, 20, and 60 min after the onset of stimulation.We observed evidence of the coordinated activation of synaptic protein groups, thereby identifying functional core complexes within the PSD. We demonstrate that adopting a quantitative systems biology approach provides insight allowing for a new level of analysis of synaptic function.     

Neuronal Ca2+ signaling via caldendrin and calneurons

The calcium sensor protein caldendrin is abundantly expressed in neurons and is thought to play an important role in different aspects of synapto-dendritic Ca2+ signaling. Caldendrin is highly abundant in the postsynaptic density of a subset of excitatory synapses in brain and its distinct localization raises several decisive questions about its function. Previous work suggests that caldendrin is tightly associated with Ca2+ - and Ca2+ release channels and might be involved in different aspects of the organization of the postsynaptic scaffold as well as with synapse-to-nucleus communication. In this report we introduce two new EF-hand calcium sensor proteins termed calneurons that apart from calmodulin represent the closest homologues of caldendrin in brain. Calneurons have a different EF-hand organization than other calcium sensor proteins, are prominently expressed in neurons and will presumably bind Ca2+ with higher affinity than caldendrin. Despite some significant structural differences it is conceivable that they are involved in similar Ca2+ regulated processes like caldendrin and neuronal calcium sensor proteins.     

Lateral thinking: CaMKII uncouples kainate receptors from mossy fibre synapses

EMBO J (2013) 32: 496–510 doi:10.1038/emboj.2012.334; published online January042013Alteration of the efficacy of excitatory synaptic transmission between neurons is a critical element in the processes of learning, memory, and behaviour. Despite decades of research aimed at elucidating basic cellular mechanisms underlying synaptic plasticity, new pathways and permutations continue to be discovered. Carta et al (2013) now show that activation of the calcium/calmodulin dependent kinase II (CaMKII) induces an unusual postsynaptic form of long-term depression (LTD) at the hippocampal mossy fibre synapse by promoting lateral diffusion of kainate receptors (KARs), a family of ionotropic glutamate receptors (iGluRs) that influence pyramidal neuron excitability. This report therefore reveals a new and mechanistically unique way of fine-tuning synaptic plasticity at this central synapse in the hippocampus.Information transfer within the nervous system is regulated at the synaptic level by diverse cellular mechanisms. Synaptic efficacy is not static (i.e., it is ‘plastic''), and the capacity to adjust the strength of communication between neurons in a network has been shown to be a critical component of diverse aspects of brain function that include many forms of behavioural learning (Martin et al, 2000). The complex means by which neurons adjust their synaptic properties in response to changes in local and global activity in the central nervous system has been the subject of intensive investigation spanning multiple decades (Malenka and Bear, 2004; Feldman, 2009). Nonetheless, new mechanisms underlying plasticity of excitatory and inhibitory synaptic transmission continue to be elucidated; these can vary depending on the experimental parameters for induction of plasticity, the particular type of synapse under investigation, and even the prior history of activation at the synapse. Long-term potentiation (LTP) and LTD of excitatory synaptic transmission are two well-known phenomena in which efficacy is increased or decreased, respectively, and at many synapses in the CNS occur through concomitant alterations in the number of postsynaptic iGluRs. The movement of excitatory receptors in and out of synapses, and more generally to and from the neuronal plasma membrane, is dictated by their association with a wide variety of scaffolding and chaperone proteins, whose interactions are often controlled by various protein kinases (Anggono and Huganir, 2012).It is generally appreciated now that long-term synaptic plasticity can be elicited by a variety of mechanisms even within a single type of synaptic connection. In addition to postsynaptic alterations in receptor content, for example, synaptic efficacy can also be tuned by regulated alterations in the probability of vesicular release of the neurotransmitter. Until recently, this presynaptic form of plasticity was thought to be the exclusive mechanism for altering excitatory synaptic strength at a morphologically unusual synapse in the hippocampus formed between large bouton-like presynaptic terminals arising from granule cell axons, or mossy fibres, and proximal dendrites on CA3 pyramidal neurons (Nicoll and Schmitz, 2005). These synaptic connections allow for single dentate granule cells to profoundly influence the likelihood of action potential firing in CA3 pyramidal neurons in a frequency-dependent manner, and for that reason have been referred to as ‘conditional detonator'' synapses (Henze et al, 2002). The precise mechanisms that lead to increased vesicular release probability following LTP-inducing stimulation of mossy fibre axons, including a potential role for retrograde signalling, remain the subject of debate, although there is general consensus that activation of presynaptic protein kinase A (PKA) is a key step in this form of synaptic plasticity (Figure 1A). Enhancing release probability impacts signalling through all three types of iGluRs present at mossy fibre synapses—AMPA, NMDA, and KARs. Recently, however, novel postsynaptic forms of mossy fibre plasticity were discovered in which induction protocols specifically increased the number of NMDA receptors (Kwon and Castillo, 2008; Rebola et al, 2008) or decreased the number of KARs (Selak et al, 2009), expanding the mechanistic repertoire at this historical site of focus of research on presynaptic LTP. Alterations in the synaptic content of particular iGluRs could serve as an additional means to fine-tune synaptic integration at the mossy fibre—CA3 synapse and therefore have important consequences for hippocampal network excitability.Open in a separate windowFigure 1Kainate receptor-dependent plasticity mechanisms at the hippocampal mossy fibre–CA3 synapse. (A) Activation of presynaptic receptors enhances glutamate release from the mossy fibre terminals. (B) A spike-timing-dependent plasticity protocol known to activate postsynaptic CaMKII results in long-term synaptic depression. CaMKII phosphorylates the GluK5 kainate receptor subunit, which uncouples the receptor from PSD-95 in the postsynaptic density. This leads to an increase in receptor mobility and diffusion away from the synapse. (C) Low-frequency stimulation of mossy fibres and activation of postsynaptic group 1 mGluRs leads to activation of PKC, which promotes the association of SNAP-25 to the GluK5 kainate receptor subunit and the subsequent endocytosis of synaptic receptors.In this issue, Carta et al (2013) identify a new postsynaptic mechanism for shaping mossy fibre plasticity that is specific to synaptic KARs, which serve to influence temporal integration of synaptic input as well as pyramidal neuron excitability through modulation of intrinsic ion channels. The authors paired postsynaptic depolarization of CA3 pyramidal neurons with a precisely timed presynaptic release of glutamate in a pattern that is known to produce LTP at many central synapses (Feldman, 2012). At mossy fibre synapses, however, this form of spike-timing-dependent plasticity (STDP) instead caused LTD of KAR-mediated excitatory synaptic potentials (KAR-LTD) while leaving AMPA receptor function unaltered (Figure 1B) (Carta et al, 2013). Using a series of genetic and pharmacological manipulations, Carta et al (2013) found that KAR-LTD was dependent upon the activation of postsynaptic KARs themselves, a rise in postsynaptic Ca²⁺, and CaMKII phosphorylation of a specific protein component of synaptic KARs, the GluK5 subunit. Unlike other mechanisms of postsynaptic mossy fibre plasticity, KAR-LTD was independent of NMDA or metabotropic glutamate receptor activation. Most surprisingly, KAR-LTD did not require receptor endocytosis from the plasma membrane, as is the case with most other forms of postsynaptic depression of excitatory transmission, including a distinct form of KAR-LTD reported previously (Selak et al, 2009) (Figure 1C). Instead, CaMKII-mediated phosphorylation of GluK5 subunits likely uncoupled receptors from the postsynaptic scaffolding protein PSD-95, which then led to enhanced lateral diffusion of KARs out of mossy fibre synapses. As KAR endocytosis was not altered in mossy fibre STDP, the activity-dependent reduction in KAR signalling was effectively limited to those receptors in the synapse. A molecular replacement strategy was employed using biolistic-based expression of mutant KARs in cultured hippocampal slices prepared from KAR knockout mice, which allowed Carta et al (2013) to corroborate their detailed biochemical studies by showing that reconstituted KAR currents in CA3 neurons expressing recombinant GluK5 phosphorylation site substitutions were unable to express KAR-LTD. In summary, KAR-mediated activation of CaMKII leads to phosphorylation of the GluK5 subunit and subsequent KAR-LTD through enhanced lateral mobility of synaptic receptors (Figure 1B).These findings are intriguing for several reasons. Most notably, they stand in stark contrast to studies in which CaMKII activation primarily triggers potentiation, rather than depression, of excitatory synaptic transmission at other synapses (Lisman et al, 2012). CaMKII recently was shown to cause diffusional trapping of AMPA receptor complexes within the postsynaptic density following phosphorylation of a closely associated auxiliary subunit, stargazin (Opazo et al, 2010), which is precisely the opposite of the effects of activation of the enzyme on KAR mobility at mossy fibre synapses. Further, these divergent consequences are both dependent upon carboxy-terminal PDZ interactions with scaffolding proteins, although in each case further research is needed to dissect out the relevant binding partners that control lateral mobility. It is of interest that KAR-LTD required synaptic activation of KARs to initiate signalling via CaMKII, which implies a tight coupling exists between KARs and the holoenzyme in the mossy fibre postsynaptic density. This observation also raises the possibility that activated CaMKII could phosphorylate other targets to effect other, yet-to-be-discovered, changes in synaptic function. Finally, the report by Carta et al expands our understanding of how excitatory synaptic transmission is fine-tuned at an important central synapse and underscores the fact that even well-trod ground (or synapses) continue to yield surprises that inform our understanding of the remarkable mechanistic diversity underlying synaptic plasticity in the CNS.     

Protein components of post‐synaptic density lattice,a backbone structure for type I excitatory synapses

It is essential to study the molecular architecture of post‐synaptic density (PSD ) to understand the molecular mechanism underlying the dynamic nature of PSD , one of the bases of synaptic plasticity. A well‐known model for the architecture of PSD of type I excitatory synapses basically comprises of several scaffolding proteins (scaffold protein model). On the contrary, ‘PSD lattice’ observed through electron microscopy has been considered a basic backbone of type I PSD s. However, major constituents of the PSD lattice and the relationship between the PSD lattice and the scaffold protein model, remain unknown. We purified a PSD lattice fraction from the synaptic plasma membrane of rat forebrain. Protein components of the PSD lattice were examined through immuno‐gold negative staining electron microscopy. The results indicated that tubulin, actin, α‐internexin, and Ca²⁺/calmodulin‐dependent kinase II are major constituents of the PSD lattice, whereas scaffold proteins such as PSD ‐95, SAP 102, GKAP , Shank1, and Homer, were rather minor components. A similar structure was also purified from the synaptic plasma membrane of forebrains from 7‐day‐old rats. On the basis of this study, we propose a ‘PSD lattice‐based dynamic nanocolumn’ model for PSD molecular architecture, in which the scaffold protein model and the PSD lattice model are combined and an idea of dynamic nanocolumn PSD subdomain is also included. In the model, cytoskeletal proteins, in particular, tubulin, actin, and α‐internexin, may play major roles in the construction of the PSD backbone and provide linker sites for various PSD scaffold protein complexes/subdomains.

     

Morphine administration alters the profile of hippocampal postsynaptic density-associated proteins: a proteomics study focusing on endocytic proteins

Numerous studies have shown that drugs of abuse induce changes in protein expression in the brain that are thought to play a role in synaptic plasticity. Drug-induced plasticity can be mediated by changes at the synapse and more specifically at the postsynaptic density (PSD), which receives and transduces synaptic information. To date, the majority of studies examining synaptic protein profiles have focused on identifying the synaptic proteome. Only a handful of studies have examined the changes in synaptic profile by drug administration. We applied a quantitative proteomics analysis technique with the cleavable ICAT reagent to quantitate relative changes in protein levels of the hippocampal PSD in response to morphine administration. We identified a total of 102 proteins in the mouse hippocampal PSD. The majority of these were signaling, trafficking, and cytoskeletal proteins involved in synaptic plasticity, learning, and memory. Among the proteins whose levels were found to be altered by morphine administration, clathrin levels were increased to the largest extent. Immunoblotting and electron microscopy studies showed that this increase was localized to the PSD. Morphine treatment was also found to lead to a local increase in two other components of the endocytic machinery, dynamin and AP-2, suggesting a critical involvement of the endocytic machinery in the modulatory effects of morphine. Because alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are thought to undergo clathrin-mediated endocytosis, we examined the effect of morphine administration on the association of the AMPA receptor subunit, GluR1, with clathrin. We found a substantial decrease in the levels of GluR1 associated with clathrin. Taken together, these results suggest that, by causing a redistribution of endocytic proteins at the synapse, morphine modulates synaptic plasticity at hippocampal glutamatergic synapses.     

Ca2+ Permeable AMPA Receptor Induced Long-Term Potentiation Requires PI3/MAP Kinases but Not Ca/CaM-Dependent Kinase II

Ca²⁺ influx via GluR2-lacking Ca²⁺-permeable AMPA glutamate receptors (CP-AMPARs) can trigger changes in synaptic efficacy in both interneurons and principle neurons, but the underlying mechanisms remain unknown. We took advantage of genetically altered mice with no or reduced GluR2, thus allowing the expression of synaptic CP-AMPARs, to investigate the molecular signaling process during CP-AMPAR-induced synaptic plasticity at CA1 synapses in the hippocampus. Utilizing electrophysiological techniques, we demonstrated that these receptors were capable of inducing numerous forms of long-term potentiation (referred to as CP-AMPAR dependent LTP) through a number of different induction protocols, including high-frequency stimulation (HFS) and theta-burst stimulation (TBS). This included a previously undemonstrated form of protein-synthesis dependent late-LTP (L-LTP) at CA1 synapses that is NMDA-receptor independent. This form of plasticity was completely blocked by the selective CP-AMPAR inhibitor IEM-1460, and found to be dependent on postsynaptic Ca²⁺ ions through calcium chelator (BAPTA) studies. Surprisingly, Ca/CaM-dependent kinase II (CaMKII), the key protein kinase that is indispensable for NMDA-receptor dependent LTP at CA1 synapses appeared to be not required for the induction of CP-AMPAR dependent LTP due to the lack of effect of two separate pharmacological inhibitors (KN-62 and staurosporine) on this form of potentiation. Both KN-62 and staurosporine strongly inhibited NMDA-receptor dependent LTP in control studies. In contrast, inhibitors for PI3-kinase ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin) or the MAPK cascade (PD98059 and U0126) significantly attenuated this CP-AMPAR-dependent LTP. Similarly, postsynaptic infusion of tetanus toxin (TeTx) light chain, an inhibitor of exocytosis, also had a significant inhibitory effect on this form of LTP. These results suggest that distinct synaptic signaling underlies GluR2-lacking CP-AMPAR-dependent LTP, and reinforces the recent notions that CP-AMPARs are important facilitators of synaptic plasticity in the brain.     

Breaking down to build up: Neuroligin’s C-terminal domain strengthens the synapse

The mechanisms by which neuroligin adhesion molecules modulate synaptic plasticity remain unclear. In this issue, Liu et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509023) demonstrate that neuroligin 1 promotes actin assembly associated with synaptic strengthening independent of adhesion, suggesting additional ways for neuroligins to contribute to neuronal development and disease pathology.Spines are actin-enriched dendritic protrusions that serve as the major site of excitatory neurotransmission, underlying learning and memory formation (Lynch et al., 2007). Spines associate with presynaptic axon terminals through diverse adhesion molecules to form synapses (Siddiqui and Craig, 2011). Dynamic rearrangements of these synaptic adhesions and of the underlying actin cytoskeleton lead to either strengthening or weakening of particular synaptic connections. Synaptic strengthening, or long-term potentiation (LTP), is initiated by excitation of glutamate N-methyl-d-aspartate (NMDA) receptors, which promotes cleavage of synaptic adhesion molecules and disassembly of actin filaments (Lynch et al., 2007). Actin disassembly is mediated in part by recruitment of the actin-severing protein cofilin into the spine (Bosch et al., 2014). After the breakdown of the existing synaptic architecture, the actin cytoskeleton is stabilized again via Rac1-driven actin polymerization (Rex et al., 2009) and phosphorylation-mediated cofilin inactivation (Bosch et al., 2014). In parallel, recruitment and anchoring of synaptic adhesion molecules, including neuroligin 1 (NLG1; Schapitz et al., 2010) and glutamate receptors, increases the size of the postsynaptic signaling scaffold (PSD) across from the presynaptic terminal. In the final stage of LTP, the changes in synaptic morphology are consolidated by stabilization of actin filaments through actin capping and cross-linking together with the insertion of newly synthesized synaptic proteins (Lynch et al., 2007). Although the different steps of LTP shaping spine morphology and stability are generally understood, the signaling events that coordinate the initial disassembly of the existing synaptic architecture with reassembly of a stronger synaptic connection remain unclear.Neuroligins (NLGs) are a family of four transmembrane postsynaptic adhesion molecules (NLGs 1–4) that form heterotypic adhesions with presynaptic neurexins via an extracellular acetylcholinesterase-like domain (Südhof, 2008). Of the four NLG family members, NLG1 localizes predominantly to excitatory glutamatergic synapses (Song et al., 1999). Both in vitro and in vivo evidence demonstrate that the NLG–neurexin binding interaction is sufficient to promote synapse formation (Südhof, 2008; Chen et al., 2010). However, NLG knockout mice exhibit normal spine density but impaired synaptic transmission, suggesting that NLGs may regulate synaptic function independent of adhesion (Südhof, 2008). In addition to trans-synaptic adhesion mediated by the extracellular domain of NLGs, their short intracellular C-terminal domain (CTD) contains a PDZ binding domain (PBD) that facilitates binding and recruitment of postsynaptic density scaffold proteins, such as PSD95 (Irie et al., 1997; Dresbach et al., 2004). NLG1 is cleaved in an activity-dependent manner, leading to the release of an extracellular fragment that destabilizes synaptic adhesion and of the intracellular CTD (Suzuki et al., 2012).In this issue, Liu et al. focused on how activity-dependent cleavage of NLG1 and the subsequent release of its CTD affect actin organization and spine stability at excitatory synapses. They first observed that NLG1 knockout mouse brains, as well as cultured neurons infected with an shRNA targeting NLG1, exhibit decreased cofilin-S3 phosphorylation when compared with wild-type levels. Cofilin-S3 phosphorylation functions as a marker of mature dendritic spines, as cofilin inactivation results in F-actin assembly and is associated with the later stages of LTP (Calabrese et al., 2014). In addition, the absence of NLG1 prevented dynamic regulation of cofilin phosphorylation in response to KCl-induced neuronal excitation of brain slices, suggesting that cofilin phosphorylation depends on NLG1 both basally and in an activity-dependent manner. Remarkably, incubation with recombinant NLG1-CTD increased spine-associated cofilin phosphorylation in cultured neurons and rescued cofilin phosphorylation in NLG1 knockout mouse brain slices. Using full-length or truncated NLG1 constructs with a wild-type or mutated PDB sequence, Liu et al. (2016) demonstrated that NLG1-induced cofilin phosphorylation depends on both NLG1 cleavage and an intact PBD sequence within the released CTD. As the NLG1-CTD alone induced spine-associated cofilin phosphorylation, the researchers investigated its impact on actin assembly associated with synapse formation and function. In cultured neurons, recombinant NLG1-CTD increased F-actin levels together with spine and synapse formation. Similarly, intravenous injection of NLG1-CTD increased spine density in the CA1 region of the mouse hippocampus. This increased spine and synapse formation resulted in a corresponding increase in the frequency of excitatory postsynaptic currents, which was inhibited by a peptide that blocked cofilin phosphorylation. Together, these results establish that the NLG1-CTD requires cofilin phosphorylation to strengthen synaptic connections, prompting Liu et al. (2016) to investigate the mechanism underlying NLG1-induced cofilin phosphorylation.SPAR is a known regulator of the actin cytoskeleton that is hypothesized to bind to NLG1 (Craig and Kang, 2007). Using brain lysates and HEK293 cells expressing both NLG1 and SPAR, Liu et al. (2016) demonstrated that SPAR interacts with NLG1-CTD via its PBD domain. In brain slices, KCl-mediated excitation, which induces proteolytic cleavage of endogenous NLG1, increased the association of NLG1 and SPAR, suggesting that the interaction occurs in response to activity-dependent release of an intracellular CTD. To test whether this interaction regulates cofilin phosphorylation, Liu et al. (2016) expressed SPAR in HEK293 cells, where it decreased cofilin-S3 phosphorylation. However, incubation with a recombinant NLG1-CTD containing an intact PBD restored cofilin phosphorylation, demonstrating that this interaction alleviates SPAR-mediated repression of cofilin phosphorylation. In neurons, NLG1-CTD reduced the levels of synaptic SPAR, as assessed by both immunofluorescence and Western blotting of purified synaptosomes. SPAR is known to negatively regulate Rap1 signaling, and Rap1 signaling is important for Rac1 activation and spine morphogenesis (Pak et al., 2001; Maillet et al., 2003). In cultured neurons, a Rap1 inhibitor prevented NLG1-CTD–induced cofilin phosphorylation, whereas treatment with recombinant NLG1-CTD without Rap1 inhibition activated Rac1 signaling, leading to phosphorylation of its downstream targets, LIMK1 and cofilin. The results demonstrate that the CTD of NLG1 binds and displaces SPAR from the spine, alleviating its inhibition on Rap1 signaling. In turn, increased Rap1 signaling promotes Rac1 activation, leading to LIMK-1 and cofilin phosphorylation (Fig. 1). Lastly, these NLG-driven changes in actin assembly were found to simultaneously inhibit long-term depression, an activity-dependent reduction in the efficacy of synapses, and facilitate LTP, as determined by whole-cell patch clamping of brain slices incubated with NLG1-CTD.Open in a separate windowFigure 1.NLG’s CTD strengthens the synapse from within through dynamic actin remodeling. Excitatory activation of NMDA receptors (NMDAR) results in sequential cleavage of NLG1 (Suzuki et al., 2012). Liu et al. (2016) describe how the CTD of NLG1 interacts with SPAR, a negative regulator of Rap GTPase activity. This activity-dependent interaction displaces SPAR and alleviates the local inhibition of Rap activity within the dendritic spine. Rap drives a corresponding increase in Rac activation and phosphorylation of its downstream target, the actin regulator cofilin, thereby increasing F-actin filament assembly within spines. These changes in actin organization ultimately result in increased spine density and promote LTP.This work provides important insights into the mechanism by which NLG1 impacts synapse development and function by highlighting a critical role for SPAR in the regulation of actin assembly mediating synaptic strengthening. Interestingly, the temporal delay between the release of the NLG1-CTD and the subsequent sequestration of SPAR from the PSD could serve to distinguish an early disassembly phase following excitatory stimulation from later LTP consolidation, which is known to rely on both Rac1 activation (Rex et al., 2009) and cofilin phosphorylation (Bosch et al., 2014). Furthermore, it will be of interest to determine whether NLG1’s CTD affects the localization of other proteins known to bind its PBD, such as PSD95 (Irie et al., 1997), and whether these dynamic rearrangements at the postsynaptic scaffold also serve to simultaneously promote actin assembly while alleviating SPAR-mediated negative regulation of actin remodeling. For example, NLG1 has been shown to interact with Kalirin-7 (Owczarek et al., 2015), an activator of Rac1 that binds to PSD95 at the synapse; however, binding to PSD95 reduces Kalirin-7–mediated activation of Rac1 (Penzes et al., 2001). It is therefore attractive to speculate that the activity-dependent release of protein fragments, such as the CTD of NLG1, might alter postsynaptic density interactions that further promote localized Rac1-driven F-actin assembly. Consistent with this hypothesis, adhesion disassembly triggered by the extracellular domain of NLG1’s binding partner (β-neurexin) increases Rac1 activation (Owczarek et al., 2015). Ultimately, more work is necessary to determine how the strengthening effects of the intracellular CTD compete with the destabilizing effects of the extracellular domain (Suzuki et al., 2012). Recent research demonstrates that CAMKII phosphorylates and increases NLG1 surface expression in response to NMDA receptor activation (Bemben et al., 2014). If this phosphorylation event protects NLG1 from cleavage, it could serve to stabilize an adhesive pool of NLG1 while allowing for the release of the CTD from an unprotected population. Alternatively, this phosphorylation event could serve to recruit new NLG1 proteins to the synapse later in the LTP process when adhesions are reestablished. Further research is needed to understand how the adhesive and intracellular signaling capabilities of NLG1 are balanced at discrete stages of synaptic plasticity, and in particular how phosphorylation of NLG1 regulates both its surface expression as well as its cleavage.Consistent with the multiple roles of NLGs in modulating synaptic architecture, it is not surprising that NLG mutations have been implicated in diverse cognitive and neurodevelopmental disorders, such as Alzheimer’s disease and autism (Südhof, 2008; Tristán-Clavijo et al., 2015). In light of this study, it will be interesting to determine how disease-associated NLG mutations contribute to both synaptic adhesion as well as stabilization of the actin cytoskeleton that supports synaptic strengthening. This is particularly important because both Alzheimer’s disease and autism-associated NLG mutant proteins exhibit decreased surface expression (Chubykin et al., 2005; Tristán-Clavijo et al., 2015), although the autism-associated mutant NLG proteins present at the cell surface still promote synapse formation (Chubykin et al., 2005). However, the decreased postsynaptic NLG pool could impair subsequent activity-dependent synaptic strengthening. Likewise, understanding whether binding of the postsynaptic scaffolding protein Shank3 to the CTD of NLG1 (Arons et al., 2012) affects NLG1 cleavage could provide insights into the mechanism by which Shank3 affects activity-dependent synaptic remodeling in autism pathogenesis. The work by Liu et al. (2016), demonstrating that adhesion disassembly coordinates subsequent actin assembly underlying synaptic strengthening, takes an important step toward shedding light on the altered synaptic plasticity underlying both complex neurodevelopmental and neurodegenerative pathologies.     

Heterogeneous nuclear ribonucleoprotein k interacts with Abi-1 at postsynaptic sites and modulates dendritic spine morphology

Background

Abelson-interacting protein 1 (Abi-1) plays an important role for dendritic branching and synapse formation in the central nervous system. It is localized at the postsynaptic density (PSD) and rapidly translocates to the nucleus upon synaptic stimulation. At PSDs Abi-1 is in a complex with several other proteins including WASP/WAVE or cortactin thereby regulating the actin cytoskeleton via the Arp 2/3 complex.

Principal Findings

We identified heterogeneous nuclear ribonucleoprotein K (hnRNPK), a 65 kDa ssDNA/RNA-binding-protein that is involved in multiple intracellular signaling cascades, as a binding partner of Abi-1 at postsynaptic sites. The interaction with the Abi-1 SH3 domain is mediated by the hnRNPK-interaction (KI) domain. We further show that during brain development, hnRNPK expression becomes more and more restricted to granule cells of the cerebellum and hippocampal neurons where it localizes in the cell nucleus as well as in the spine/dendritic compartment. The downregulation of hnRNPK in cultured hippocampal neurons by RNAi results in an enlarged dendritic tree and a significant increase in filopodia formation. This is accompanied by a decrease in the number of mature synapses. Both effects therefore mimic the neuronal morphology after downregulation of Abi-1 mRNA in neurons.

Conclusions

Our findings demonstrate a novel interplay between hnRNPK and Abi-1 in the nucleus and at synaptic sites and show obvious similarities regarding both protein knockdown phenotypes. This indicates that hnRNPK and Abi-1 act synergistic in a multiprotein complex that regulates the crucial balance between filopodia formation and synaptic maturation in neurons.     

Dynamin-dependent Membrane Drift Recruits AMPA Receptors to Dendritic Spines

The surface expression and localization of AMPA receptors (AMPARs) at dendritic spines are tightly controlled to regulate synaptic transmission. Here we show that de novo exocytosis of the GluR2 AMPAR subunit occurs at the dendritic shaft and that new AMPARs diffuse into spines by lateral diffusion in the membrane. However, membrane topology restricts this lateral diffusion. We therefore investigated which mechanisms recruit AMPARs to spines from the shaft and demonstrated that inhibition of dynamin GTPase activity reduced lateral diffusion of membrane-anchored green fluorescent protein and super-ecliptic pHluorin (SEP)-GluR2 into spines. In addition, the activation of synaptic N-methyl-d-aspartate (NMDA) receptors enhanced lateral diffusion of SEP-GluR2 and increased the number of endogenous AMPARs in spines. The NMDA-invoked effects were prevented by dynamin inhibition, suggesting that activity-dependent dynamin-mediated endocytosis within spines generates a net inward membrane drift that overrides lateral diffusion barriers to enhance membrane protein delivery into spines. These results provide a novel mechanistic explanation of how AMPARs and other membrane proteins are recruited to spines by synaptic activity.AMPA³ receptors (AMPARs) are of fundamental importance because they mediate the majority of fast excitatory synaptic transmission in the mammalian central nervous system (1). Most excitatory synapses are characterized morphologically by dendritic spines that contain an electron-dense postsynaptic density (PSD) at their head (2, 3). PSD is highly enriched in AMPARs and associated proteins equired for synaptic transmission and signal transduction (4-6). Activity-evoked changes in functional postsynaptic AMPARs mediate the two main forms of synaptic plasticity believed to underlie learning and memory in the hippocampus (7). Long term potentiation involves the activity-dependent recruitment of AMPARs to the postsynaptic membrane and a concurrent increase in AMPA-mediated transmission, whereas long term depression is a decrease in synaptic AMPAR function (8).The number and subunit composition of synaptic AMPARs are stringently regulated, but despite intense investigation, the processes by which AMPARs are delivered to and retained at the PSD remain controversial. Using photoreactive antagonists and electrophysiology, it has been proposed that AMPARs are only inserted in the plasma membrane at the cell body and laterally diffuse long distances to synapses (9). In direct contrast, approaches using real-time imaging have suggested that AMPARs are inserted in the plasma membrane of the dendritic shaft close to, but not at, dendritic spines (10). It has also been suggested that AMPARs could be inserted directly into the plasma membrane of the PSD (11).Independent of the route of delivery for new AMPARs to synapses, it is well established that lateral diffusion in the plasma membrane allows the exchange of receptors in and out of the PSD (12-14). Using palmitoylated membrane-anchored GFP (mGFP), which partitions to the inner leaflet of the plasma membrane, it has also been reported that diffusion is significantly retarded within spines compared with the shaft and that AMPAR activation increases the rates of mGFP diffusion in spines (15). In addition, we have shown previously that membrane protein movement into and out of spines is slow compared with lateral diffusion on non-spiny membrane (16), and modeling studies have predicted that spine length is a major determinant of the time a protein takes to reach the PSD (17). More recently, it has been proposed that endocytosis at specialized endocytic zones close to the PSD within spines is required to maintain the steady state complement of synaptic AMPARs (18).Taken together these findings suggest that endocytosis and exocytosis as well as lateral diffusion and membrane topology may all play important roles in regulating membrane protein mobility in spines. The interrelationships between these processes, however, remain unclear. Here we used FRAP (fluorescence recovery after photobleaching) and multisite FLIP (fluorescence loss in photobleaching) to visualize super-ecliptic pHluorin-tagged GluR2 surface expression and AMPAR movement in real time. We examined how lateral diffusion is regulated in spines both by blocking dynamin GTPase activity and stimulating NMDARs. Combined with Monte Carlo simulations on lattices fitting theoretical spines, our data indicate that the membrane topology of spines alone is sufficient to constrain lateral diffusion. NMDAR activation facilitates AMPAR recruitment to spines by a process that involves the recruitment of plasma membrane, together with the constituent membrane proteins, from adjacent regions of the dendritic shaft being drawn into the spine to replace membrane that is internalized during endocytosis. In other words, our results suggest a mode of lateral diffusion that is neither free nor anomalous. Rather, we show the directional diffusion of membrane-embedded proteins toward the postsynapse driven by the endocytosis within the spine. These results provide a new mechanistic explanation of how synaptic activity can overcome topology-induced diffusion barriers to recruit new membrane proteins to the spine.     

Transsynaptic channelosomes: non-conducting roles of ion channels in synapse formation

Recent findings demonstrate that synaptic channels are directly involved in the formation and maintenance of synapses by interacting with synapse organizers. The synaptic channels on the pre- and postsynaptic membranes possess non-conducting roles in addition to their functional roles as ion-conducting channels required for synaptic transmission. For example, presynaptic voltage-dependent calcium channels link the target-derived synapse organizer laminin β2 to cytomatrix of the active zone and function as scaffolding proteins to organize the presynaptic active zones. Furthermore, postsynaptic δ2-type glutamate receptors organize the synapses by forming transsynaptic protein complexes with presynaptic neurexins through synapse organizer cerebellin 1 precursor proteins. Interestingly, the synaptic clustering of AMPA receptors is regulated by neuronal activity-regulated pentraxins, while postsynaptic differentiation is induced by the interaction of postsynaptic calcium channels and thrombospondins. This review will focus on the non-conducting functions of ion-channels that contribute to the synapse formation in concert with synapse organizers and active-zone-specific proteins.     

Modeling synaptic transmission of the tripartite synapse

The tripartite synapse denotes the junction of a pre- and postsynaptic neuron modulated by a synaptic astrocyte. Enhanced transmission probability and frequency of the postsynaptic current-events are among the significant effects of the astrocyte on the synapse as experimentally characterized by several groups. In this paper we provide a mathematical framework for the relevant synaptic interactions between neurons and astrocytes that can account quantitatively for both the astrocytic effects on the synaptic transmission and the spontaneous postsynaptic events. Inferred from experiments, the model assumes that glutamate released by the astrocytes in response to synaptic activity regulates store-operated calcium in the presynaptic terminal. This source of calcium is distinct from voltage-gated calcium influx and accounts for the long timescale of facilitation at the synapse seen in correlation with calcium activity in the astrocytes. Our model predicts the inter-event interval distribution of spontaneous current activity mediated by a synaptic astrocyte and provides an additional insight into a novel mechanism for plasticity in which a low fidelity synapse gets transformed into a high fidelity synapse via astrocytic coupling.     

Synapse reorganization—a new partnership revealed

Changes in synaptic function require both qualitative and quantitative reorganization of the synaptic components. Ca²⁺ plays a central role in this process, but the mechanism has not been fully elucidated. Zhang et al report a novel mechanism whereby Ca²⁺/calmodulin (CaM) regulates the stability of the postsynaptic scaffold. Ca²⁺/CaM interacts with PSD-95, a core protein in the postsynaptic density (PSD) that supports synaptic signaling and structural components. Ca²⁺/CaM interferes with the palmitoylation of PSD-95, resulting in the dissociation of PSD-95 from the postsynaptic membrane. This process may explain the reduction of surface glutamate receptor observed during synaptic depression and homeostatic regulation of the synaptic response after prolonged neuronal activity.The adaptation of synaptic strength is a fundamental process for learning and memory. This form of synaptic plasticity is triggered by influx of Ca²⁺ from NMDA-type glutamate receptor (NMDAR), leading to the synaptic insertion or removal of AMPA-type glutamate receptor (AMPAR) and determines the strength of synaptic transmission. This process is mediated by the gross reorganization of the postsynaptic composition in a qualitative and quantitative fashion (Bosch et al, 2014). Importantly, the number of synaptic AMPAR is regulated by the number and affinity of postsynaptic ‘slots’, a hypothetical receptor binding site within a synapse, which is regulated during synaptic plasticity processes.PSD-95, a scaffolding protein at excitatory synapses, has been considered as a major candidate for the slot. It interacts with AMPAR through the TARP/stargazin protein family and modulates the synaptic localization of the receptor as well as the strength of synaptic transmission (El-Husseini et al, 2000). In turn, the localization of PSD-95 at the synapse is regulated by a constant cycle of palmitoylation by protein palmitoyl acyltransferases (PAT) at cysteines 3 and 5, which is required for efficient synaptic targeting of the protein, and depalmitoylation by palmitoyl protein thioesterases (PPT) (El-Husseini et al, 2002; Noritake et al, 2009). Activation of glutamate receptors increases depalmitoylated PSD-95 and releases it from the postsynaptic site (El-Husseini et al, 2002; Sturgill et al, 2009), whereas blockage of neuronal activity by TTX increases palmitoylated PSD-95 and targets it to the synapse (Noritake et al, 2009). However, it remains unclear how neuronal activity controls the palmitoylation/depalmitoylation cycle and the subsequent trafficking of PSD-95 to and from the synapse.In this issue of The EMBO Journal, using a combination of structural biological, biochemical, and cell biological approaches, Zhang et al (2014) revealed a novel mechanism by which Ca²⁺ regulates the synaptic localization of PSD-95. The authors found that Ca²⁺ complexed to CaM can bind PSD-95 within the first 13 residues—the exact location where palmitoylation takes place. Ca²⁺/CaM binds preferentially to unmodified PSD-95, thereby blocking the accessibility of the PAT to the palmitoylation sites. In contrast, Ca²⁺/CaM does not bind to palmitoylated PSD-95, and therefore, palmitoylated PSD-95 is subject to depalymitoylation by PPT. Overall, the net effect of Ca²⁺/CaM is a reduction of PSD-95 bound to the synaptic membrane (Fig​(Fig1).1). Consistent with this, a PSD-95 mutant that was unable to bind Ca²⁺/CaM does not leave the synapse following glutamate/glycine treatment. Furthermore, the mutant PSD-95 showed an increased in synaptic accumulation, indicating that the treatment also increases palmitoylation activity but it is normally dominated by the depalmitoylating action of Ca²⁺/CaM binding (Noritake et al, 2009).Open in a separate windowFigure 1Calcium influx-induced release of PSD-95 and glutamate receptor from the synapseAt a synapse, cysteine residues of PSD-95 (C3 and C5) are under a continuous cycle of palmitoylation by protein palmitoyl acyltransferase (PAT), and depalmitoylation by palmitoyl protein thioesterase (PPT). A. Palmitoylated PSD-95 associates with the synaptic membrane and CDKL5 and serves as a ‘slot’ for AMPAR at the synapse through the interaction with TARP/stargazin. B. Upon glutamate stimulation, Ca²⁺ influx through NMDARs induces binding of Ca²⁺/CaM to PSD-95. Ca²⁺/CaM blocks the accessibility of PAT, thereby facilitating the depalmitoylation of PSD-95, which subsequently allows PSD-95 to dissociate from the synaptic membrane and CDKL5. C. The dissociation of PSD-95 from the synaptic membrane reduces the number of available ‘slots’ for AMPAR on the postsynaptic membrane, leading to a reduction of AMPAR-mediated synaptic transmission.The beauty of the work by Zhang et al (2014) is that the structure fully explains the biology. However, several important questions remain. Above all, it is still unclear when this mechanism would operate. Generally, the palmitoylation/depalmitoylation cycle is considered to be in the order of minutes. Therefore, for Ca²⁺/CaM to effectively reduce the palmitoylation of PSD-95, a prolonged influx of Ca²⁺ is required. In contrast, the rise in intracellular Ca²⁺ induced by a single excitatory postsynaptic current (EPSC) or dendritic action potential is in the order of milliseconds to seconds. A slow repetitive stimulation protocol, such as that used to induce long-term depression (LTD) (1 Hz, 15 min), may be effective in increasing Ca²⁺/CaM for a sufficiently long period of time. Homeostatic scaling induced by a prolonged increase in network activity (for example, via the pharmacological blockade of inhibitory synaptic transmission) may also be a possible mechanism for the Ca²⁺/CaM-mediated removal of synaptic PSD-95. In contrast, stimulation used to induce long-term potentiation (LTP) (a brief high-frequency stimulation such as 100 Hz, 1 s) may not be effective. Indeed, Bosch et al (2014) found that the induction of LTP at single dendritic spines in hippocampal CA1 pyramidal neurons does not decrease or increase PSD-95 during the first hour after induction even if the dendritic spine enlarges during this period.In this context, it is important to understand what impact Ca²⁺/CaM-mediated removal of synaptic PSD-95 has on synaptic transmission. If PSD-95 is indeed the slot for AMPAR, the Ca²⁺/CaM-mediated removal of synaptic PSD-95 is expected to reduce the synaptic transmission. The stimulation protocol used here by Zhang et al (bath application of glutamate/glycine) is similar to previously reported approaches to induce ‘chemical’ LTD and hence can provide an explanation for the decrease in PSD-95 from the synapse for 10–15 min after stimulation. The PSD-95 mutant that is unable to bind Ca²⁺/CaM will be useful to further analyze the link between the observed PSD-95-Ca²⁺/CaM interaction and synaptic plasticity.PSD-95 is also known to interact with the cyclin-dependent protein kinase-like kinase 5 (CDKL5) at the first 19 residues in a palmitoylation-dependent manner (Zhu et al, 2013). As expected, Ca²⁺/CaM binding also regulates CDKL5 association with PSD-95. The treatment of neurons with NMDA reduces the palmitoylation of PSD-95 and, concomitantly, the association with CDKL5. Mutations of CDKL5 and netrin-G1 gene have been reported in patients with an atypical form of Rett syndrome. Netrin-G1 ligand (NGL1) has been identified as an interaction partner and substrate of CDKL5 (Ricciardi et al, 2012). CDKL5 phosphorylates NGL1, and this phosphorylation stabilizes the interaction of NGL1 with PSD-95. Given that the Ca²⁺ signal only lasts a few milliseconds to seconds, it is important to investigate the spatiotemporal interaction between CaM, PSD-95, CDKL5, and NGL1 in dendritic spines during physiological and pathological conditions. In addition, CDKL5 can function as an upstream modulator of Rac signaling during development (Chen et al, 2010). Together with the fact that Rac also plays an important role in long-term potentiation, the PSD-95/CDKL5 complex might regulate Rac activity in the vicinity of the PSD.Other neuronal proteins including AMPAR subunit GluR1/2, glutamate receptor interacting protein (GRIP), G-protein-coupled receptors, δ-catenin, and small and trimeric G-proteins can also undergo palmitoylation, suggesting that this process plays an essential role in subcellular targeting and that these proteins can be regulated by activity (Kang et al, 2008). The question of whether Ca²⁺/CaM can regulate the palmitoylation of these proteins remains. Interestingly, the Ca²⁺/CaM interaction site of PSD-95 does not conform to a canonical IQ-motif, a CaM binding motif found in many other proteins (Zhang et al, 2014). Therefore, a bioinformatic approach is not possible at this point in time. This opens further directions of research.     

NESH regulates dendritic spine morphology and synapse formation

Background

Dendritic spines are small membranous protrusions on the neuronal dendrites that receive synaptic input from axon terminals. Despite their importance for integrating the enormous information flow in the brain, the molecular mechanisms regulating spine morphogenesis are not well understood. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family, and its overexpression is known to reduce cell motility and tumor metastasis. NESH is prominently expressed in the brain, but its function there remains unknown.

Methodology/Principal Findings

NESH was strongly expressed in the hippocampus and moderately expressed in the cerebral cortex, cerebellum and striatum, where it co-localized with the postsynaptic proteins PSD95, SPIN90 and F-actin in dendritic spines. Overexpression of NESH reduced numbers of mushroom-type spines and synapse density but increased thin, filopodia-like spines and had no effect on spine density. siRNA knockdown of NESH also reduced mushroom spine numbers and inhibited synapse formation but it increased spine density. The N-terminal region of NESH co-sedimented with filamentous actin (F-actin), which is an essential component of dendritic spines, suggesting this interaction is important for the maturation of dendritic spines.

Conclusions/Significance

NESH is a novel F-actin binding protein that likely plays important roles in the regulation of dendritic spine morphogenesis and synapse formation.     

Perisynaptic Schwann Cells at the Neuromuscular Synapse: Adaptable,Multitasking Glial Cells

The neuromuscular junction (NMJ) is engineered to be a highly reliable synapse to carry the control of the motor commands of the nervous system over the muscles. Its development, organization, and synaptic properties are highly structured and regulated to support such reliability and efficacy. Yet, the NMJ is also highly plastic, able to react to injury and adapt to changes. This balance between structural stability and synaptic efficacy on one hand and structural plasticity and repair on another hand is made possible by the intricate regulation of perisynaptic Schwann cells, glial cells at this synapse. They regulate both the efficacy and structural plasticity of the NMJ in a dynamic, bidirectional manner owing to their ability to decode synaptic transmission and by their interactions via trophic-related factors.The vertebrate neuromuscular junction (NMJ), arguably the best characterized synapse in the peripheral nervous system (PNS), is composed of three closely associated cellular components: the presynaptic nerve terminal, the postsynaptic specialization, and nonmyelinating Schwann cells. These synapse-associated glial cells are called perisynaptic Schwann cells (PSCs), or terminal Schwann cells (see reviews by Todd and Robitaille 2006; Feng and Ko 2007; Griffin and Thompson 2008; Sugiura and Lin 2011). Multiple roles of PSCs have gained great appreciation since the 1990s and, along with the novel roles of astrocytes in central synapses, have led to the concept of the “tripartite” synapse (Araque et al. 1999, 2014; Volterra et al. 2002; Auld and Robitaille 2003; Kettenmann and Ransom 2013).Thus, to fully understand synaptic formation and function, it is critical to also consider the active and essential roles of synapse-associated glial cells. We will discuss evidence supporting the existence of a synapse–glia–synapse regulatory loop that helps maintain and restore synaptic efficacy at the NMJ. We will also explore the multiple functions that PSCs exert, functions that are adapted to a given situation at the NMJ (e.g., synapse formation, stability, and reinnervation). This will highlight the great adaptability and plasticity of the morphological and functional properties of PSCs.In this review, we will focus on the multiple roles PSCs play in synaptic formation, maintenance, remodeling, and regeneration, as well as synaptic function and plasticity. Based on the evidence presented, we propose a model in which PSCs, through specific receptor activation, play a prominent role in a continuum of synaptic efficacy, stability, and plasticity at the NMJ. These synaptic-regulated functions allow PSCs to orchestrate the stability and plasticity of the NMJ and, hence, are important for maintaining and adapting synaptic efficacy.