Transsynaptic channelosomes

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.     

Molecular Mechanism of Active Zone Organization at Vertebrate Neuromuscular Junctions

Organization of presynaptic active zones is essential for development, plasticity, and pathology of the nervous system. Recent studies indicate a trans-synaptic molecular mechanism that organizes the active zones by connecting the pre- and the postsynaptic specialization. The presynaptic component of this trans-synaptic mechanism is comprised of cytosolic active zone proteins bound to the cytosolic domains of voltage-dependent calcium channels (P/Q-, N-, and L-type) on the presynaptic membrane. The postsynaptic component of this mechanism is the synapse organizer (laminin β2) that is expressed by the postsynaptic cell and accumulates specifically on top of the postsynaptic specialization. The pre- and the postsynaptic components interact directly between the extracellular domains of calcium channels and laminin β2 to anchor the presynaptic protein complex in front of the postsynaptic specialization. Hence, the presynaptic calcium channel functions as a scaffolding protein for active zone organization and as an ion-conducting channel for synaptic transmission. In contrast to the requirement of calcium influx for synaptic transmission, the formation of the active zone does not require the calcium influx through the calcium channels. Importantly, the active zones of adult synapses are not stable structures and require maintenance for their integrity. Furthermore, aging or diseases of the central and peripheral nervous system impair the active zones. This review will focus on the molecular mechanisms that organize the presynaptic active zones and summarize recent findings at the neuromuscular junctions and other synapses.     

GABA and neuroligin signaling: linking synaptic activity and adhesion in inhibitory synapse development

GABA-mediated synaptic inhibition is crucial in neural circuit operations. In mammalian brains, the development of inhibitory synapses and innervation patterns is often a prolonged postnatal process, regulated by neural activity. Emerging evidence indicates that gamma-aminobutyric acid (GABA) acts beyond inhibitory transmission and regulates inhibitory synapse development. Indeed, GABA(A) receptors not only function as chloride channels that regulate membrane voltage and conductance but also play structural roles in synapse maturation and stabilization. The link from GABA(A) receptors to postsynaptic and presynaptic adhesion is probably mediated, partly by neuroligin-reurexin interactions, which are potent in promoting GABAergic synapse formation. Therefore, similar to glutamate signaling at excitatory synapse, GABA signaling may coordinate maturation of presynaptic and postsynaptic sites at inhibitory synapses. Defining the many steps from GABA signaling to receptor trafficking/stability and neuroligin function will provide further mechanistic insights into activity-dependent development and possibly plasticity of inhibitory synapses.     

The dynamics of synaptic scaffolds

Complex functions of the central nervous system such as learning and memory are believed to result from the modulation of the synaptic transmission between neurons. The sequence of events leading to the fusion of synaptic vesicles at the presynaptic active zone and the detection of this signal at the postsynaptic density involve the activity of ion channels and neurotransmitter receptors. Their accumulation and dynamic exchange at synapses are dependent on their interaction with synaptic scaffolds. These are synaptic structures composed of adaptor proteins that provide binding sites for receptors and channels as well as other synaptic proteins. While in its entirety the synaptic scaffold is a relatively stable structure, individual adaptor proteins exchange at a fast time scale. These properties of scaffolds help to ensure the stability of synaptic transmission while permitting the modulation of synaptic strength. Here, we review the dynamics of the synaptic scaffold and of adaptor proteins in relation to their roles in the organisation of the synapse as well as in the clustering and trafficking of receptor proteins.     

Cerebellin–neurexin complexes instructing synapse properties

Cerebellins (Cbln1-4) are secreted adaptor proteins that connect presynaptic neurexins (Nrxn1-3) to postsynaptic ligands (GluD1/2 for Cbln1-3 vs. DCC and Neogenin-1 for Cbln4). Classical studies demonstrated that neurexin-Cbln1-GluD2 complexes organize cerebellar parallel-fiber synapses, but the role of cerebellins outside of the cerebellum has only recently been clarified. In synapses of the hippocampal subiculum and prefrontal cortex, Nrxn1-Cbln2-GluD1 complexes strikingly upregulate postsynaptic NMDA-receptors, whereas Nrxn3-Cbln2-GluD1 complexes conversely downregulate postsynaptic AMPA-receptors. At perforant-path synapses in the dentate gyrus, in contrast, neurexin/Cbln4/Neogenin-1 complexes are essential for LTP without affecting basal synaptic transmission or NMDA- or AMPA-receptors. None of these signaling pathways are required for synapse formation. Thus, outside of the cerebellum neurexin/cerebellin complexes regulate synapse properties by activating specific downstream receptors.     

Bilateral processing in chemical synapses with electrical 'ephaptic' feedback: a theoretical model

I have developed a detailed biophysical model of the chemical synapse which hosts voltage-dependent presynaptic ion channels and takes into account the capacitance of synaptic membranes. I find that at synapses with a relatively large cleft resistance (e.g., mossy fiber or giant calyx synapse) the rising postsynaptic current could activate, within the synaptic cleft, electrochemical phenomena that induce rapid widening of the presynaptic action potential (AP). This mechanism could boost fast Ca(2+) entry into the terminal thus increasing the probability of subsequent synaptic releases. The predicted difference in the AP waveforms generated inside and outside the synapse can explain the previously unexplained fast capacitance transient recorded in the postsynaptic cell at the giant calyx synapse. I propose therefore the mechanism of positive ephaptic feedback that acts between the postsynaptic and presynaptic cell contributing to the basal synaptic transmission at large central synapses. This mechanism could also explain the supralinear voltage dependence of EPSCs recorded at hyperpolarizing membrane potentials in low extracellular calcium concentration.     

Orchestrating the synaptic network by tyrosine phosphorylation signalling

The establishment of a functional brain requires coordinated and stereotyped formation of synapses between neurons. For this, trans-synaptic molecular cues (synaptic organizers) are exchanged between a neuron and its target to organize appropriate synapses. The understanding of signalling mechanisms by which such synaptic organizers lead to synapse formation is just being elucidated. However, recent studies revealed that some of these cues act through receptor protein tyrosine kinases (RPTKs) or phosphatases (RPTPs). Synaptogenic RPTKs and RPTPs pattern synaptic network through affecting local protein-protein binding dynamics, changing the phosphorylation state of signalling cascades, or promoting gene expression. Each RPTK or RPTP has distinct roles in synapse formation, serving at different synapses or showing differential synaptogenic effects. Thus, tyrosine phosphorylation signalling plays critical roles in building the orchestrated synaptic circuitry in the brain.     

Mechanisms of excitatory synapse maturation by trans-synaptic organizing complexes

Synapses are specialized cell-cell adhesion contacts that mediate communication within neural networks. During development, excitatory synapses are generated by step-wise recruitment of presynaptic and postsynaptic proteins to sites of contact. Several classes of synaptic organizing complexes have been identified that function during the initial stages of synapse formation. However, mechanisms underlying the later stages of synapse development are less well understood. In recent years, molecules have been discovered that appear to play a role in synapse maturation. In this review, we highlight recent findings that have provided key insights for understanding postsynaptic maturation of developing excitatory synapses with a focus on recruitment of AMPA receptors to developing synapses.     

Agrin and Synaptic Laminin Are Required to Maintain Adult Neuromuscular Junctions

As synapses form and mature the synaptic partners produce organizing molecules that regulate each other’s differentiation and ensure precise apposition of pre- and post-synaptic specializations. At the skeletal neuromuscular junction (NMJ), these molecules include agrin, a nerve-derived organizer of postsynaptic differentiation, and synaptic laminins, muscle-derived organizers of presynaptic differentiation. Both become concentrated in the synaptic cleft as the NMJ develops and are retained in adulthood. Here, we used mutant mice to ask whether these organizers are also required for synaptic maintenance. Deletion of agrin from a subset of adult motor neurons resulted in the loss of acetylcholine receptors and other components of the postsynaptic apparatus and synaptic cleft. Nerve terminals also atrophied and eventually withdrew from muscle fibers. On the other hand, mice lacking the presynaptic organizer laminin-α4 retained most of the synaptic cleft components but exhibited synaptic alterations reminiscent of those observed in aged animals. Although we detected no marked decrease in laminin or agrin levels at aged NMJs, we observed alterations in the distribution and organization of these synaptic cleft components suggesting that such changes could contribute to age-related synaptic disassembly. Together, these results demonstrate that pre- and post-synaptic organizers actively function to maintain the structure and function of adult NMJs.     

Signal regulatory proteins (SIRPS) are secreted presynaptic organizing molecules

Formation of chemical synapses requires exchange of organizing signals between the synaptic partners. Using synaptic vesicle aggregation in cultured neurons as a marker of presynaptic differentiation, we purified candidate presynaptic organizers from mouse brain. A major bioactive species was the extracellular domain of signal regulatory protein alpha (SIRP-alpha), a transmembrane immunoglobulin superfamily member concentrated at synapses. The extracellular domain of SIRP-alpha is cleaved and shed in a developmentally regulated manner. The presynaptic organizing activity of SIRP-alpha is mediated in part by CD47. SIRP-alpha homologues, SIRP-beta and -gamma also have synaptic vesicle clustering activity. The effects of SIRP-alpha are distinct from those of another presynaptic organizer, FGF22: the two proteins induced vesicle clusters of different sizes, differed in their ability to promote neurite branching, and acted through different receptors and signaling pathways. SIRP family proteins may act together with other organizing molecules to pattern synapses.     

Remodeling of synaptic actin induced by photoconductive stimulation.

Use-dependent synapse remodeling is thought to provide a cellular mechanism for encoding durable memories, yet whether activity triggers an actual structural change has remained controversial. We use photoconductive stimulation to demonstrate activity-dependent morphological synaptic plasticity by video imaging of GFP-actin at individual synapses. A single tetanus transiently moves presynaptic actin toward and postsynaptic actin away from the synaptic junction. Repetitive spaced tetani induce glutamate receptor-dependent stable restructuring of synapses. Presynaptic actin redistributes and forms new puncta that label for an active synapse marker FM5-95 within 2 hr. Postsynaptic actin sprouts projections toward the new presynaptic actin puncta, resembling the axon-dendrite interaction during synaptogenesis. Our results indicate that activity-dependent presynaptic structural plasticity facilitates the formation of new active presynaptic terminals.     

Lrp4 is a receptor for Agrin and forms a complex with MuSK

Neuromuscular synapse formation requires a complex exchange of signals between motor neurons and skeletal muscle fibers, leading to the accumulation of postsynaptic proteins, including acetylcholine receptors in the muscle membrane and specialized release sites, or active zones in the presynaptic nerve terminal. MuSK, a receptor tyrosine kinase that is expressed in skeletal muscle, and Agrin, a motor neuron-derived ligand that stimulates MuSK phosphorylation, play critical roles in synaptic differentiation, as synapses do not form in their absence, and mutations in MuSK or downstream effectors are a major cause of a group of neuromuscular disorders, termed congenital myasthenic syndromes (CMS). How Agrin activates MuSK and stimulates synaptic differentiation is not known and remains a fundamental gap in our understanding of signaling at neuromuscular synapses. Here, we report that Lrp4, a member of the LDLR family, is a receptor for Agrin, forms a complex with MuSK, and mediates MuSK activation by Agrin.     

Wnt signaling during synaptic development and plasticity

The formation of synaptic connections requires a dialogue between pre and postsynaptic cells to coordinate the assembly of the presynaptic release machinery and the postsynaptic receptive complexes. Signaling molecules of the Wnt family of proteins are central to this trans-synaptic dialogue. At the neuromuscular junction and central synapses, Wnts promote synaptic assembly by signaling to the developing pre and postsynaptic compartments. In addition, new studies reveal that expression of Wnt proteins and localization of their Fz receptors are regulated by neuronal activity. Importantly, Wnts mediates the synaptic changes induced by patterned neuronal activity or sensory experience in mature neurons. Here we review recent findings into the function of Wnt signaling at the synapse and its link to activity-dependent synaptic growth and function.     

Acute knockdown of AMPA receptors reveals a trans-synaptic signal for presynaptic maturation

Newly formed glutamatergic synapses often lack postsynaptic AMPA-type glutamate receptors (AMPARs). Aside from 'unsilencing' the postsynaptic site, however, the significance of postsynaptic AMPAR insertion during synapse maturation remains unclear. To investigate the role of AMPAR in synapse maturation, we used RNA interference (RNAi) to knockdown AMPARs in cultured hippocampal neurons. Surprisingly, loss of postsynaptic AMPARs increased the occurrence of presynaptically inactive synapses without changing the release probability of the remaining active synapses. Additionally, heterologous synapses formed between axons and AMPAR-expressing HEK cells develop significantly fewer inactive presynaptic terminals. The extracellular domain of the AMPAR subunit GluA2 was sufficient to reproduce this effect at heterologous synapses. Indeed, the retrograde signalling by AMPARs is independent of their channel function as RNAi-resistant AMPARs restore synaptic transmission in neurons lacking AMPARs despite chronic receptor antagonist treatment. Our findings suggest that postsynaptic AMPARs perform an organizational function at synapses that exceeds their standard role as ionotropic receptors by conveying a retrograde trans-synaptic signal that increases the transmission efficacy at a synapse.     

Neurotransmitter release regulated by a MALS-liprin-alpha presynaptic complex

Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-alpha as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS-CASK-liprin-alpha complex recruits components of the synaptic release machinery to adhesive proteins of the active zone.     

The presynaptic active zone protein bassoon is essential for photoreceptor ribbon synapse formation in the retina

The photoreceptor ribbon synapse is a highly specialized glutamatergic synapse designed for the continuous flow of synaptic vesicles to the neurotransmitter release site. The molecular mechanisms underlying ribbon synapse formation are poorly understood. We have investigated the role of the presynaptic cytomatrix protein Bassoon, a major component of the photoreceptor ribbon, in a mouse retina deficient of functional Bassoon protein. Photoreceptor ribbons lacking Bassoon are not anchored to the presynaptic active zones. This results in an impaired photoreceptor synaptic transmission, an abnormal dendritic branching of neurons postsynaptic to photoreceptors, and the formation of ectopic synapses. These findings suggest a critical role of Bassoon in the formation and the function of photoreceptor ribbon synapses of the mammalian retina.     

Synaptogenesis of hippocampal neurons in primary cell culture

Hippocampal neurons in dissociated cell culture are one of the most extensively used model systems in the field of molecular and cellular neurobiology. Only limited data are however available on the normal time frame of synaptogenesis, synapse number and ultrastructure of excitatory synapses during early development in culture. Therefore, we analyzed the synaptic ultrastructure and morphology and the localization of presynaptic (Bassoon) and postsynaptic (ProSAP1/Shank2) marker proteins in cultures established from rat embryos at embryonic day 19, after 3, 7, 10, 14, and 21 days in culture. First excitatory synapses were identified at day 7 with a clearly defined postsynaptic density and presynaptically localized synaptic vesicles. Mature synapses on dendritic spines were seen from day 10 onward, and the number of synapses steeply increased in the third week. Fenestrated or multiple synapses were found after 14 or 21 days, respectively. So-called dense-core vesicles, responsible for the transport of proteins to the active zone of the presynaptic specialization, were seen on cultivation day 3 and 7 and could be detected in axons and especially in the presynaptic subcompartments. The expression and localization of the presynaptic protein Bassoon and of the postsynaptic molecule ProSAP1/Shank2 was found to correlate nicely with the ultrastructural results. This regular pattern of development and maturation of excitatory synapses in hippocampal culture starting from day 7 in culture should ease the comparison of synapse number and morphology of synaptic contacts in this widely used model system.     

Signaling at the vertebrate synapse: new roles for embryonic morphogens?

The formation of synapses is critical for functional neuronal connectivity. The coordinated assembly at both sides of the synapse is fundamental for the proper apposition of the neurotransmitter release machinery on the presynaptic neuron and the clustering of neurotransmitter receptors and ion channels on the receptive postsynaptic cell. This process requires bidirectional communication between the presynaptic neuron and its postsynaptic target, another neuron, or muscle fiber. Extracellular signals such as WNT, TGF-beta, and FGF factors are emerging as key target-derived signals required for the initial stages of synaptic assembly. Studies in invertebrates are also providing new insights into the function of these signals in synaptic growth and homeostasis. During early embryonic patterning, WNT, TGF-beta, and FGF factors function as typical morphogens in a concentration-dependent manner to regulate cell fate decisions. This mode of action raises the provocative idea that these same morphogens might also provide a coordinate system for axons to establish the distance to their targets during axon guidance and synapse formation.     

Neurobeachin regulates neurotransmitter receptor trafficking to synapses

The surface density of neurotransmitter receptors at synapses is a key determinant of synaptic efficacy. Synaptic receptor accumulation is regulated by the transport, postsynaptic anchoring, and turnover of receptors, involving multiple trafficking, sorting, motor, and scaffold proteins. We found that neurons lacking the BEACH (beige-Chediak/Higashi) domain protein Neurobeachin (Nbea) had strongly reduced synaptic responses caused by a reduction in surface levels of glutamate and GABA_A receptors. In the absence of Nbea, immature AMPA receptors accumulated early in the biosynthetic pathway, and mature N-methyl-d-aspartate, kainate, and GABA_A receptors did not reach the synapse, whereas maturation and surface expression of other membrane proteins, synapse formation, and presynaptic function were unaffected. These data show that Nbea regulates synaptic transmission under basal conditions by targeting neurotransmitter receptors to synapses.     

PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7

Both postsynaptic density and presynaptic active zone are structural matrix containing scaffolding proteins that are involved in the organization of the synapse. Little is known about the functional role of these proteins in the signaling of presynaptic receptors. Here we show that the interaction of the presynaptic metabotropic glutamate (mGlu) receptor subtype, mGlu7a, with the postsynaptic density-95 disc-large zona occludens 1 (PDZ) domain-containing protein, PICK1, is required for specific inhibition of P/Q-type Ca(2+) channels, in cultured cerebellar granule neurons. Furthermore, we show that activation of the presynaptic mGlu7a receptor inhibits synaptic transmission and this effect also requires the presence of PICK1. These results indicate that the scaffolding protein, PICK1, plays an essential role in the control of synaptic transmission by the mGlu7a receptor complex.