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.     

Synapses are regulated by the cytoplasmic tyrosine kinase Fer in a pathway mediated by p120catenin,Fer, SHP-2, and β-catenin

Localization of presynaptic components to synaptic sites is critical for hippocampal synapse formation. Cell adhesion–regulated signaling is important for synaptic development and function, but little is known about differentiation of the presynaptic compartment. In this study, we describe a pathway that promotes presynaptic development involving p120catenin (p120ctn), the cytoplasmic tyrosine kinase Fer, the protein phosphatase SHP-2, and β-catenin. Presynaptic Fer depletion prevents localization of active zone constituents and synaptic vesicles and inhibits excitatory synapse formation and synaptic transmission. Depletion of p120ctn or SHP-2 similarly disrupts synaptic vesicle localization with active SHP-2, restoring synapse formation in the absence of Fer. Fer or SHP-2 depletion results in elevated tyrosine phosphorylation of β-catenin. β-Catenin overexpression restores normal synaptic vesicle localization in the absence of Fer or SHP-2. Our results indicate that a presynaptic signaling pathway through p120ctn, Fer, SHP-2, and β-catenin promotes excitatory synapse development and function.     

Sensory Regulation of Neuroligins and Neurexin I in the Honeybee Brain

Background

Neurexins and neuroligins, which have recently been associated with neurological disorders such as autism in humans, are highly conserved adhesive proteins found on synaptic membranes of neurons. These binding partners produce a trans-synaptic bridge that facilitates maturation and specification of synapses. It is believed that there exists an optimal spatio-temporal code of neurexin and neuroligin interactions that guide synapse formation in the postnatal developing brain. Therefore, we investigated whether neuroligins and neurexin are differentially regulated by sensory input using a behavioural model system with an advanced capacity for sensory processing, learning and memory, the honeybee.

Methodology/Principal Findings

Whole brain expression levels of neuroligin 1–5 (NLG1–5) and neurexin I (NrxI) were estimated by qRT-PCR analysis in three different behavioural paradigms: sensory deprivation, associative scent learning, and lateralised sensory input. Sensory deprived bees had a lower level of NLG1 expression, but a generally increased level of NLG2–5 and NrxI expression compared to hive bees. Bees that had undergone associative scent training had significantly increased levels of NrxI, NLG1 and NLG3 expression compared to untrained control bees. Bees that had lateralised sensory input after antennal amputation showed a specific increase in NLG1 expression compared to control bees, which only happened over time.

Conclusions/Significance

Our results suggest that (1) there is a lack of synaptic pruning during sensory deprivation; (2) NLG1 expression increases with sensory stimulation; (3) concomitant changes in gene expression suggests NrxI interacts with all neuroligins; (4) there is evidence for synaptic compensation after lateralised injury.     

DNA binding and synapsis by the large C-terminal domain of ϕC31 integrase

The integrase (Int) from phage ϕC31 acts on the phage and host-attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. Excision (attL × attR recombination) is prevented, in the absence of accessory factors, by a putative coiled-coil motif in the C-terminal domain (CTD). Int has a serine recombinase N-terminal domain, required for synapsis of recombination substrates and catalysis. We show here that the coiled-coil motif mediates protein–protein interactions between CTDs, but only when bound to DNA. Although the histidine-tagged CTD (hCTD) was monomeric in solution, hCTD bound cooperatively to three of the recombination substrates (attB, attL and attR). Furthermore, when provided with attP and attB, hCTD brought these substrates together in a synaptic complex. Substitutions in the coiled-coil motif that greatly reduce Int integration activity, L460P and Y475H, prevented CTD–CTD interactions and led to defective DNA binding and no detectable DNA synapsis. A substitution, E449K, in full length Int confers the ability to perform excision in addition to integration as it has gained the ability to synapse attL × attR. hCTD^E449K was similar to hCTD in DNA binding but unable to form the CTD synapse suggesting that the CTD synapse is not essential but could be part of the mechanism that controls directionality.     

Transsynaptic Coordination of Synaptic Growth,Function, and Stability by the L1-Type CAM Neuroglian

The precise control of synaptic connectivity is essential for the development and function of neuronal circuits. While there have been significant advances in our understanding how cell adhesion molecules mediate axon guidance and synapse formation, the mechanisms controlling synapse maintenance or plasticity in vivo remain largely uncharacterized. In an unbiased RNAi screen we identified the Drosophila L1-type CAM Neuroglian (Nrg) as a central coordinator of synapse growth, function, and stability. We demonstrate that the extracellular Ig-domains and the intracellular Ankyrin-interaction motif are essential for synapse development and stability. Nrg binds to Ankyrin2 in vivo and mutations reducing the binding affinities to Ankyrin2 cause an increase in Nrg mobility in motoneurons. We then demonstrate that the Nrg–Ank2 interaction controls the balance of synapse growth and stability at the neuromuscular junction. In contrast, at a central synapse, transsynaptic interactions of pre- and postsynaptic Nrg require a dynamic, temporal and spatial, regulation of the intracellular Ankyrin-binding motif to coordinate pre- and postsynaptic development. Our study at two complementary model synapses identifies the regulation of the interaction between the L1-type CAM and Ankyrin as an important novel module enabling local control of synaptic connectivity and function while maintaining general neuronal circuit architecture.     

Identification of substrates of palmitoyl protein thioesterase 1 highlights roles of depalmitoylation in disulfide bond formation and synaptic function

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein–associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.

Unbiased proteomics with acyl resin-assisted capture reveals diverse novel substrates of the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) at the synapse, with potential implications for the pathogenesis of neuronal ceroid lipofuscinosis, disulfide bond formation, synaptic adhesion and additional critical synaptic functions.     

Neuroligins mediate excitatory and inhibitory synapse formation: involvement of PSD-95 and neurexin-1beta in neuroligin-induced synaptic specificity

The balance between excitatory and inhibitory synapses is a tightly regulated process that requires differential recruitment of proteins that dictate the specificity of newly formed contacts. However, factors that control this process remain unidentified. Here we show that members of the neuroligin (NLG) family, including NLG1, NLG2, and NLG3, drive the formation of both excitatory and inhibitory presynaptic contacts. The enrichment of endogenous NLG1 at excitatory contacts and NLG2 at inhibitory synapses supports an important in vivo role for these proteins in the development of both types of contacts. Immunocytochemical and electrophysiological analysis showed that the effects on excitatory and inhibitory synapses can be blocked by treatment with a fusion protein containing the extracellular domain of neurexin-1beta. We also found that overexpression of PSD-95, a postsynaptic binding partner of NLGs, resulted in a shift in the distribution of NLG2 from inhibitory to excitatory synapses. These findings reveal a critical role for NLGs and their synaptic partners in controlling the number of inhibitory and excitatory synapses. Furthermore, relative levels of PSD-95 alter the ratio of excitatory to inhibitory synaptic contacts by sequestering members of the NLG family to excitatory synapses.     

Targeting of the Synaptic Vesicle Protein Synaptobrevin in the Axon of Cultured Hippocampal Neurons: Evidence for Two Distinct Sorting Steps

Synaptic vesicles are concentrated in the distal axon, far from the site of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins travel en route to the synaptic vesicle, we have used a defective herpes virus amplicon expression system to study the targeting of a synaptobrevin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neurons. Addition of the cytoplasmic domain of synaptobrevin onto human transferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was instead found in an organelle with biochemical and functional characteristics of an endosome. The chimera recycled in parallel with synaptic vesicle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequence that controls targeting to the presynaptic endosome was not localized to a single, 10– amino acid region of the molecule, indicating that this targeting signal may be encoded by a more distributed structural conformation. However, the chimera could be shifted to synaptic vesicles by deletion of amino acids 61–70 in synaptobrevin, suggesting that separate signals encode the localization of synaptobrevin to the synapse and to the synaptic vesicle.     

The 13-kD FK506 Binding Protein, FKBP13, Interacts with a Novel Homologue of the Erythrocyte Membrane Cytoskeletal Protein 4.1

We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G–CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677– 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705–708). We report the specific association of FKBP13 with 4.1G–CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G–CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G–CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G–CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds.     

Presynaptic CK2 promotes synapse organization and stability by targeting Ankyrin2

The precise regulation of synapse maintenance is critical to the development and function of neuronal circuits. Using an in vivo RNAi screen targeting the Drosophila kinome and phosphatome, we identify 11 kinases and phosphatases controlling synapse stability by regulating cytoskeletal, phospholipid, or metabolic signaling. We focus on casein kinase 2 (CK2) and demonstrate that the regulatory (β) and catalytic (α) subunits of CK2 are essential for synapse maintenance. CK2α kinase activity is required in the presynaptic motoneuron, and its interaction with CK2β, mediated cooperatively by two N-terminal residues of CK2α, is essential for CK2 holoenzyme complex stability and function in vivo. Using genetic and biochemical approaches we identify Ankyrin2 as a key presynaptic target of CK2 to maintain synapse stability. In addition, CK2 activity controls the subcellular organization of individual synaptic release sites within the presynaptic nerve terminal. Our study identifies phosphorylation of structural synaptic components as a compelling mechanism to actively control the development and longevity of synaptic connections.     

ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

Insights into mechanisms coordinating membrane remodeling, local actin nucleation, and postsynaptic scaffolding during postsynapse formation are important for understanding vertebrate brain function. Gene knockout and RNAi in individual neurons reveal that the F-BAR protein syndapin I is a crucial postsynaptic coordinator in formation of excitatory synapses. Syndapin I deficiency caused significant reductions of synapse and dendritic spine densities. These syndapin I functions reflected direct, SH3 domain–mediated associations and functional interactions with ProSAP1/Shank2. They furthermore required F-BAR domain-mediated membrane binding. Ultra-high-resolution imaging of specifically membrane-associated, endogenous syndapin I at membranes of freeze-fractured neurons revealed that membrane-bound syndapin I preferentially occurred in spines and formed clusters at distinct postsynaptic membrane subareas. Postsynaptic syndapin I deficiency led to reduced frequencies of miniature excitatory postsynaptic currents, i.e., to defects in synaptic transmission phenocopying ProSAP1/Shank2 knockout, and impairments in proper synaptic ProSAP1/Shank2 distribution. Syndapin I–enriched membrane nanodomains thus seem to be important spatial cues and organizing platforms, shaping dendritic membrane areas into synaptic compartments.     

Scribble Interacts with β-Catenin to Localize Synaptic Vesicles to Synapses

An understanding of how synaptic vesicles are recruited to and maintained at presynaptic compartments is required to discern the molecular mechanisms underlying presynaptic assembly and plasticity. We have previously demonstrated that cadherin–β-catenin complexes cluster synaptic vesicles at presynaptic sites. Here we show that scribble interacts with the cadherin–β-catenin complex to coordinate vesicle localization. Scribble and β-catenin are colocalized at synapses and can be coimmunoprecipitated from neuronal lysates, indicating an interaction between scribble and β-catenin at the synapse. Using an RNA interference approach, we demonstrate that scribble is important for the clustering of synaptic vesicles at synapses. Indeed, in scribble knockdown cells, there is a diffuse distribution of synaptic vesicles along the axon, and a deficit in vesicle recycling. Despite this, synapse number and the distribution of the presynaptic active zone protein, bassoon, remain unchanged. These effects largely phenocopy those observed after ablation of β-catenin. In addition, we show that loss of β-catenin disrupts scribble localization in primary neurons but that the localization of β-catenin is not dependent on scribble. Our data supports a model by which scribble functions downstream of β-catenin to cluster synaptic vesicles at developing synapses.     

Liprinalpha1 degradation by calcium/calmodulin-dependent protein kinase II regulates LAR receptor tyrosine phosphatase distribution and dendrite development

Neural activity regulates dendrite and synapse development, but the underlying molecular mechanisms are unclear. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an important sensor of synaptic activity, and the scaffold protein liprinalpha1 is involved in pre- and postsynaptic maturation. Here we show that synaptic activity can suppress liprinalpha1 protein level by two pathways: CaMKII-mediated degradation and the ubiquitin-proteasome system. In hippocampal neurons, liprinalpha1 mutants that are immune to CaMKII degradation impair dendrite arborization, reduce spine and synapse number, and inhibit dendritic targeting of receptor tyrosine phosphatase LAR, which is important for dendrite development. Thus, regulated degradation of liprinalpha1 is important for proper LAR receptor distribution, and could provide a mechanism for localized control of dendrite and synapse morphogenesis by activity and CaMKII.     

LIM Kinase1 controls synaptic stability downstream of the type II BMP receptor

Here, we demonstrate that the BMP receptor Wishful Thinking (Wit) is required for synapse stabilization. In the absence of BMP signaling, synapse disassembly and retraction ensue. Remarkably, downstream Smad-mediated signaling cannot fully account for the stabilizing activity of the BMP receptor. We identify LIM Kinase1 (DLIMK1)-dependent signaling as a second, parallel pathway that confers the added synapse-stabilizing activity of the BMP receptor. We show that DLIMK1 binds a region of the Wit receptor that is necessary for synaptic stability but is dispensable for Smad-mediated synaptic growth. A genetic analysis demonstrates that DLIMK1 is necessary, presynaptically, for synapse stabilization, but is not necessary for normal synaptic growth or function. Furthermore, presynaptic expression of DLIMK1 in a wit or mad mutant significantly rescues synaptic stability, growth, and function. DLIMK1 localizes near synaptic microtubules and functions independently of ADF/cofilin, highlighting a novel requirement for DLIMK1 during synapse stabilization rather than actin-dependent axon outgrowth.     

Structural and functional analysis of the MutS C-terminal tetramerization domain

The Escherichia coli DNA mismatch repair (MMR) protein MutS is essential for the correction of DNA replication errors. In vitro, MutS exists in a dimer/tetramer equilibrium that is converted into a monomer/dimer equilibrium upon deletion of the C-terminal 53 amino acids. In vivo and in vitro data have shown that this C-terminal domain (CTD, residues 801–853) is critical for tetramerization and the function of MutS in MMR and anti-recombination. We report the expression, purification and analysis of the E.coli MutS-CTD. Secondary structure prediction and circular dichroism suggest that the CTD is folded, with an α-helical content of 30%. Based on sedimentation equilibrium and velocity analyses, MutS-CTD forms a tetramer of asymmetric shape. A single point mutation (D835R) abolishes tetramerization but not dimerization of both MutS-CTD and full-length MutS. Interestingly, the in vivo and in vitro MMR activity of MutS^CF/D835R is diminished to a similar extent as a truncated MutS variant (MutS800, residues 1–800), which lacks the CTD. Moreover, the dimer-forming MutS^CF/D835R has comparable DNA binding affinity with the tetramer-forming MutS, but is impaired in mismatch-dependent activation of MutH. Our data support the hypothesis that tetramerization of MutS is important but not essential for MutS function in MMR.     

Munc13 controls the location and efficiency of dense-core vesicle release in neurons

Neuronal dense-core vesicles (DCVs) contain diverse cargo crucial for brain development and function, but the mechanisms that control their release are largely unknown. We quantified activity-dependent DCV release in hippocampal neurons at single vesicle resolution. DCVs fused preferentially at synaptic terminals. DCVs also fused at extrasynaptic sites but only after prolonged stimulation. In munc13-1/2–null mutant neurons, synaptic DCV release was reduced but not abolished, and synaptic preference was lost. The remaining fusion required prolonged stimulation, similar to extrasynaptic fusion in wild-type neurons. Conversely, Munc13-1 overexpression (M13OE) promoted extrasynaptic DCV release, also without prolonged stimulation. Thus, Munc13-1/2 facilitate DCV fusion but, unlike for synaptic vesicles, are not essential for DCV release, and M13OE is sufficient to produce efficient DCV release extrasynaptically.     

Lateral assembly of the immunoglobulin protein SynCAM 1 controls its adhesive function and instructs synapse formation

Synapses are specialized adhesion sites between neurons that are connected by protein complexes spanning the synaptic cleft. These trans-synaptic interactions can organize synapse formation, but their macromolecular properties and effects on synaptic morphology remain incompletely understood. Here, we demonstrate that the synaptic cell adhesion molecule SynCAM 1 self-assembles laterally via its extracellular, membrane-proximal immunoglobulin (Ig) domains 2 and 3. This cis oligomerization generates SynCAM oligomers with increased adhesive capacity and instructs the interactions of this molecule across the nascent and mature synaptic cleft. In immature neurons, cis assembly promotes the adhesive clustering of SynCAM 1 at new axo-dendritic contacts. Interfering with the lateral self-assembly of SynCAM 1 in differentiating neurons strongly impairs its synaptogenic activity. At later stages, the lateral oligomerization of SynCAM 1 restricts synaptic size, indicating that this adhesion molecule contributes to the structural organization of synapses. These results support that lateral interactions assemble SynCAM complexes within the synaptic cleft to promote synapse induction and modulate their structure. These findings provide novel insights into synapse development and the adhesive mechanisms of Ig superfamily members.     

Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD

Long-term depression (LTD) is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR)–dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs) via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.     

beta-Amyloid-induced dynamin 1 degradation is mediated by N-methyl-D-aspartate receptors in hippocampal neurons

Alzheimer disease (AD) is a progressive, neurodegenerative disorder that leads to debilitating cognitive deficits. Although little is known about the early functional or ultrastructural changes associated with AD, it has been proposed that a stage of synaptic dysfunction might precede neurodegeneration in the development of this disease. Unfortunately, the molecular mechanisms that underlie such synaptic dysfunction remain largely unknown. Recently we have shown that beta-amyloid (Abeta), the main component of senile plaques, induced a significant decrease in dynamin 1, a protein that plays a critical role in synaptic vesicle recycling, and hence, in the signaling properties of the synapse. We report here that this dynamin 1 degradation was the result of calpain activation induced by the sustained calcium influx mediated by N-methyl-D-aspartate receptors in hippocampal neurons. In addition, our results showed that soluble oligomeric Abeta, and not fibrillar Abeta, was responsible for this sustained calcium influx, calpain activation, and dynamin 1 degradation. Considering the importance of dynamin 1 to synaptic function, these data suggest that Abeta soluble oligomers might catalyze a stage of synaptic dysfunction that precedes synapse loss and neurodegeneration. These data also highlight the calpain system as a novel therapeutic target for early stage AD intervention.