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R620 Dispatch

Synapse formation: If it looks like a duck and quacks like a duck… Isabel Cantallops and Hollis T. Cline

Neuroligin and form an intercellular adhesion Scheiffele et al. [1] used a powerful experimental system complex sufficient to trigger formation of functional to demonstrate that expression of is sufficient presynaptic elements in vitro. This single molecular to trigger the establishment of presynaptic specializations interaction appears to initiate clustering of synaptic in pontine axons. They co-cultured pontine explants with vesicles, assembly of vesicle-release machinery and non-neuronal heterologous cells, HEK cells, which do not morphological changes at the presynaptic membrane. express endogenous neuroligin. The pontine explants extend axons onto the HEK cells and will form presynap- Address: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA. tic junctions if the transfected HEK cells produce neuroli- gin. The presynaptic specializations were recognized at Current Biology 2000, 10:R620–R623 the light microscope level as a clustering of synapsin 1 immunoreactivity, and at the electron microscope level as 0960-9822/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. parallel plasma membranes with electron-dense material and clusters of small clear synaptic vesicles. Scheiffele Synapses are asymmetric junctions between et al. [1] further showed that the presynaptic vesicles through which signals pass from the presynaptic underwent cycles of exocytosis and endocytosis in the to the postsynaptic neuron. The presynaptic axon termi- presence of a depolarizing concentration of K+ ions. These nal is specialized to convert an electrical signal into an observations indicate that the axonal specializations intercellular chemical signal via calcium-dependent exo- induced by neuroligin look like presynaptic terminals and cytosis of neurotransmitter. The postsynaptic element is act like presynaptic terminals. specialized to detect transmitter and convert the chemical signal back into an electrical signal, which is then trans- A structure/function analysis of neuroligin revealed a close mitted through the neuron. In addition, complex signal correlation between the protein’s neurexin-binding and transduction events occur both presynaptically and post- vesicle-clustering activities [1]. The cytoplasmic tail of synaptically, which regulate the formation of synaptic neuroligin was found to be required for targeting to the connections and the strength of synaptic transmission. membrane, but not for either neurexin binding or synaptic The sequence of events that occurs during the establish- vesicle clustering; the extracellular ment of synaptic contacts and the molecular interactions homology domain, however, was required for both that trigger these events in the central nervous system neurexin binding and vesicle clustering. Swapping the remain unclear. acetylcholinesterase homology domain with the catalyti- cally active esterase domain of acetylcholinesterase Recent work by Scheiffele et al. [1] has shed new light on blocked activity. This essential function of the esterase the potential role of the intercellular neuroligin–neurexin homology domain is interesting because the early complex in triggering presynaptic specializations during expression of esterases in the developing central nervous . are heterophilic transmem- system (CNS) has long been a mystery. Recent studies brane cell adhesion molecules that bind β- [2]. [5,6] suggest that a non-catalytic function of acetyl- Both neuroligins and β-neurexins are enriched in synaptic cholinesterase may be required for normal CNS morpho- membranes in adult brain tissue, and in particular neuroligin genesis through an interaction with neurexin. Although localizes to the postsynaptic membrane of glutamatergic the data reported by Scheiffele et al. [1] do not give synapses [3]. β-neurexins are transmembrane proteins with detailed information about the type of interaction an extracellular laminin-like domain and a cytoplasmic tail between the esterase homology domain of neuroligin and with a PDZ-binding domain. Neuroligin is composed of an the binding sites in neurexin, comparison of the conserved extracellular non-catalytic acetylcholinesterase homology and divergent regions of the esterase domains in neuroli- domain, which is required for neurexin binding, and a gin and acetylcholinesterase will provide a starting point cytoplasmic tail with a PDZ-binding domain. Neuroligin for more detailed structure/function studies. has been implicated in mental retardation [4], suggesting a developmental function for neuroligin–neurexin. On The pièce de resistance in this series of experiments was the the basis of neurexin’s synaptic location, heterophilic demonstration that neuroligin–neurexin binding plays a binding properties and interesting binding partners (see role in synaptogenesis between pontine axons and their Figure 1), Scheiffele et al. [1] hypothesized that the inter- normal target, cerebellar granule cells. Using the pontine action between neuroligin and neurexin might be impor- explants co-cultured with HEK cells, Scheiffele et al. [1] tant in synaptogenesis. demonstrated that a soluble neurexin–immunoglobulin Dispatch R621

Figure 1

Schematic illustration of some components of Synaptic vesicles the glutamatergic synapse in adult brain. Domains Presynaptically, the PDZ-binding domain of Munc18-1 SH3 neurexin binds CASK, which in turn binds Mint CaM PTB kinase and Veli. These proteins affect presynaptic domain GK signal transduction events and indirectly affect PDZ Mint Veli CASK vesicle transport and neurotransmission (LIN-10) (LIN-7) (LIN-2) through an association between Mint-1 and N-cadherin Munc18-1. Postsynaptically, the cytoplasmic Presynaptic L1 membrane Ca2+ channel portion of neuroligin includes a PDZ-binding EphR Synaptic cleft Neuroligin domain through which it binds postsynaptic Neurexin scaffold proteins, including PSD-95 and NMDAR K+ channel AMPAR Postsynaptic S-SCAM. PSD-95 and S-SCAM bind NMDA membrane receptors (NMDAR) and K+ channels, and likely form extensive submembrane scaffolds nRapGEP SynGAP PSD-95 S-SCAM GRIP/ABP that cluster transmembrane proteins and regulate the physical proximity of regulatory Current Biology proteins — such as CaMKII, SynGAP and nRapGEP — with their effectors. Grip/ABP, spectrometric analysis to identify proteins evidence that the formation and maintenance a scaffold protein with seven PDZ domains, associated with the NMDA receptors in the of synaptic contacts includes a combination of binds AMPA receptors (AMPAR) and Ephrin PSD of adult brain, finding that cell adhesion cell adhesion and synaptic signaling receptors (EphR) in a complex that is distinct molecules L1 and N-cadherin are part of the machinery. Symbols for the domains of the from the NMDA receptor complex. Recently NMDA receptors complex, in addition to different proteins are indicated in the upper two groups [20,21] have used mass neuroligin–neurexin. This provides additional right corner.

fusion protein blocked neuroligin-induced synaptic vesicle vesicle protein VAMP, tagged with green fluorescent clustering, presumably by interfering with the binding of protein (GFP), to monitor the establishment of the presy- endogenous neurexin on pontine axons with the exoge- naptic elements in hippocampal cultures. Together, these nous neuroligin in HEK cells. Scheiffele et al. [1] then observations suggest that a heterogeneous population of took advantage of this finding to test whether soluble vesicular bodies, including small clear synaptic vesicles, neurexin–immunoglobulin fusion protein affected cluster- travel within an axon and are ‘captured’ at particular sites ing of synapsin 1 in co-cultures of pontine explants and along the axon. The restricted mobility of the vesicles then granule cells: they found that the soluble fusion protein results in their coalescence at one location, which becomes decreased the number of synapsin 1 clusters per cell by recognizable as a presynaptic specialization. The data of half, indicating that the neuroligin–neurexin interaction Scheiffele et al. [1] now suggest that intercellular neuroli- likely plays a role in synaptogenesis in this system. gin–neurexin signaling is the event that initiates the capture of synaptic vesicles at a particular presynaptic location. Synapse formation requires intimate communication between the presynaptic and postsynaptic cells, so that The capture of synaptic vesicles at the designated presynaptic elements do not develop in the absence of presynaptic site likely involves signaling through a protein postsynaptic specializations and vice versa. Synaptogene- complex that associates with the PDZ-binding domain of sis is therefore likely to be a multistep process, in which the cytoplasmic tail of neurexin. The PDZ-binding domain progression from step to step requires feedback across the of neurexin binds CASK, which in turn associates with pro- synapse (Figure 2). One amazing feature of the observa- teins known as Mint and Veli (Figures 1 and 2). By inter- tions reported by Scheiffele et al. [1] is that neuroligin is acting with these proteins, neurexins can affect signal sufficient to trigger significant presynaptic specializa- transduction, synaptic vesicle transport and exocytosis, tions, apparently without any feedback signals that could thereby providing a direct link between cell adhesion and not be derived from non-neuronal HEK cells. This sug- synaptic function [10]. Furthermore, because these pro- gests that the coalescence of the presynaptic specializa- teins have PDZ domains that are not occupied by neurexin, tion might be triggered simply by the association of they have the potential to recruit other receptors, channels neuroligin with neurexin. and adhesion molecules to the complex. Further studies will certainly yield more detailed information about the A number of recent studies [7,8] have demonstrated that mechanism by which neurexin signaling captures synaptic synaptic vesicles fuse with the presynaptic membrane and vesicles at newly designated presynaptic sites. release transmitter molecules at distributed sites along the developing axon in cell culture. Notably, Smith and If neuroligin expression in heterologous cells can induce colleagues [9] used time-lapse imaging of the synaptic the formation of functional presynaptic specializations, R622 Current Biology Vol 10 No 17

Figure 2 Development of synaptic contacts New contact Step-wise process of synapse formation in the CNS. Unlike the N-cadherin neuromuscular junction, in the vertebrate CNS most synapses do not L1 form as a result of growth cone arrest and conversion to a presynaptic Presynaptic membrane terminal. Rather they form en passant after the growth cone has Neuroligin navigated past the site of synapse formation [22]. The initial event in Synaptic cleft Neurexin synaptogenesis is likely to be an increase in cell adhesion (top panel) Postsynaptic mediated by classical cell adhesion molecules, including membrane neurexin–neuroligin, N-cadherin and L1. Signaling through the cytoplasmic tail of neurexin presynaptically triggers the insertion and Immature, silent synapse clustering of Ca2+ channels, the coalescence of presynaptic proteins, and the capture of synaptic vesicles. Signaling through the cytoplasmic tail of neuroligin postsynaptically triggers the insertion and clustering of Synaptic vesicles NMDA receptors and K+ channels (middle panel). As synapses mature Munc18-1 (bottom panel), the density of synaptic vesicles increases presynaptically CaM kinase and AMPA receptors are inserted into the postsynaptic membrane to domain render the synapses functional at hyperpolarized potentials. Mint Veli CASK

Ca2+ channel that it could then associate with postsynaptic scaffold proteins and initiate the clustering or insertion of NMDA NMDAR K+ channel receptors at the developing postsynaptic site (Figure 2).

PSD-95 S-SCAM Scheiffele et al. [1] found that the cytoplasmic tail of SynGAP neuroligin is not essential for signaling to the presynaptic axon, using a variety of constructs of neuroligin that AMPAR were altered at the cytoplasmic domain. For instance, a GPI-linked form of neuroligin, which lacks a cytoplasmic Mature, functional synapse tail, was able to induce presynaptic specializations in pontine axons, but there were half as many synapsin 1 clusters as seen with full-length neuroligin. One potential reason for the decreased number of synapsin 1 clusters is that, in the absence of signaling through the cytoplasmic tail to organize postsynaptic elements, positive feedback signals required to proceed to the next step of building a synapse are not generated. Consequently, some presynap- EphR tic specializations do form simply with the GPI-linked AMPAR neuroligin, but the additional coalescence that might normally occur when the cytoplasmic tail of neuroligin is intact did not occur. GRIP/ABP nRapGEP

CaMKII Current Biology One issue in synaptogenesis that is not resolved by the work of Scheiffele et al. [1] is the relation between cell then what is the evidence for synapse formation being a adhesion and synapse formation. Neuroligin is a cell feedback-dependent multistep process? A hint comes adhesion molecule and so the possibility exists that the from observations following expression of a glycosylphos- formation of presynaptic specializations occurs as a result phate inositol (GPI)-linked form of neuroligin, which is of the cell adhesion properties of neuroligin–neurexin tethered to the extracellular face of the membrane by a complexes, rather than any particular synaptogenic signal- phospholipid link. The cytoplasmic tail of neuroligin ing event, for instance through the cytoplasmic tail of binds several postsynaptic anchor proteins, including neurexin. Scheiffele et al. [1] attempted to address this PSD-95 [11], CIPP [12] and S-SCAM [13]. S-SCAM can issue by testing the synapsin 1-clustering activity of form homo-oligomers, possibly creating large submembrane several other cell adhesion molecules: N-cadherin, ephrin scaffolds. PSD-95, S-SCAM and CIPP can each bind, and B1, agrin and TAG-1. In the examples shown, however, thereby cluster, NMDA receptor molecules. NMDA the numbers of axons contacting the transfected HEK cells receptors mediate synaptic transmission at newly formed appear to be significantly smaller than in the neuroligin- excitatory synapses, so-called ‘silent synapses’ [14,15]. expressing HEK cells. Are there more synapsin 1 clusters The interaction with neurexin could cause a change in the on the neuroligin-expressing cells because the pontine signaling capacity of neuroligin’s cytoplasmic tail, such axons adhere to HEK cells expressing neuroligin or are Dispatch R623

there more axons on these HEK cells because they form 10. Butz S, Okamoto M, Sudhof TC: A tripartite protein complex with the potential to couple synaptic vesicle exocytosis to cell synapses? Clearly axons will not form synapses with cells adhesion in brain. Cell 1998, 94:773-782. that are not an adequate growth substrate. 11. Irie M, Hata Y, Takeuchi M, Ichtchenko K, Toyoda A, Hirao K, Takai Y, Rosahl TW, Sudhof TC: Binding of neuroligins to PSD-95. Science 1997, 277:1511-1515. Expression of neuroligin and the other cell adhesion 12. Kurschner C, Mermelstein PG, Holden WT, Surmeier DJ: CIPP, a molecules tested by Scheiffele et al. [1] appears to cause novel multivalent PDZ domain protein, selectively interacts with morphological changes in HEK cells that are similar to Kir4.0 family members, NMDA receptor subunits, neurexins, and neuroligins. Mol Cell Neurosci 1998, 11:161-172. membrane ruffling and filopodia induced by activated 13. Hirao K, Hata Y, Ide N, Takeuchi M, Irie M, Yao I, Deguchi M, Toyoda forms of the RhoA GTPases [16]. This suggests that A, Sudhof TC, Takai Y: A novel multiple PDZ domain-containing molecule interacting with N-methyl-D-aspartate receptors and neuroligin interacts with endogenous HEK proteins to neuronal cell adhesion proteins. J Biol Chem 1998, affect the actin . Given the known importance 273:21105-21110. of the actin cytoskeleton in structural modifications 14. Liao D, Malinow R: Deficiency in induction but not expression of LTP in hippocampal slices from young rats. Learn Mem 1996, required for synapse formation and plasticity [17–19], 3:138-149. these observations suggest another avenue by which neu- 15. Wu G-Y, Malinow R, Cline HT: Maturation of a central glutamatergic synapse. Science 1996, 274:972-976. roligin may affect synaptic function. 16. Van Aelst L, D’Souza-Schorey C: Rho GTPases and signaling networks. Dev 1997, 11:2295-2322. It is interesting to speculate that the neuroligin–neurexin 17. Dunaevsky A, Tashiro A, Majewska A, Mason C, Yuste R: Developmental regulation of spine motility in the mammalian link may coordinate the development of presynaptic and central nervous system. Proc Natl Acad Sci USA 1999, postsynaptic components at the synapse through a parallel 96:13438-13443. 18. Lee T, Winter C, Marticke SS, Lee A, Luo L: Essential roles of series of interactions with PDZ domain-containing scaffold Drosophila RhoA in the regulation of neuroblast proliferation and proteins on either side of the synapse. Neuroligin appears to dendritic but not axonal morphogenesis. Neuron 2000, trigger the coalescence of the presynaptic specialization 25:307-316. 19. Li Z, Van Aelst L, Cline HT: Rho GTPases regulate distinct aspects through an association with neurexin and communication to of dendritic arbor growth in Xenopus central neurons in vivo. the presynaptic element, as shown by Scheiffele et al. [1]. Nat Neurosci 2000, 3:217-225. Neuroligin could also initiate formation of the postsynaptic 20. Walikonis RS, Jensen ON, Mann M, Provance DW, Jr., Mercer JA, Kennedy MB: Identification of proteins in the element by signaling through the PDZ-binding domain in fraction by mass spectrometry. J Neurosci 2000, 20:4069-4080. its cytoplasmic tail to scaffold proteins such as PSD-95, so as 21. Husi H, Ward MA, Choudhary JS, Blackstock WP, Grant SG: Proteomic analysis of NMDA receptor-adhesion protein signaling to cluster NMDA receptors at newly formed synapses. Such complexes. Nat Neurosci 2000, 3:661-669. an asymmetric signaling mechanism, brought about by het- 22. Jontes JD, Buchanan J, Smith SJ: Growth cone and dendrite erophilic binding partners, could ensure that presynaptic dynamics in zebrafish embryos: early events in synaptogenesis imaged in vivo. Nat Neurosci 2000, 3:231-237. and postsynaptic elements develop opposite one another. This is a fundamental requirement for brain development.

References 1. Scheiffele P, Fan J, Choih J, Fetter R, Serafini T: Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons. Cell 2000, 101:657-669. 2. Ichtchenko K, Hata Y, Nguyen T, Ullrich B, Missler M, Moomaw C, Sudhof TC: Neuroligin 1: a splice site-specific ligand for beta- neurexins. Cell 1995, 81:435-443. 3. Song JY, Ichtchenko K, Sudhof TC, Brose N: Neuroligin 1 is a postsynaptic cell-adhesion molecule of excitatory synapses. Proc Natl Acad Sci USA 1999, 96:1100-1105. 4. Philibert RA, Winfield SL, Sandhu HK, Martin BM, Ginns EI: The structure and expression of the human neuroligin-3 . Gene 2000; 246:303-310. 5. Grifman M, Galyam N, Seidman S, Soreq H: Functional redundancy of acetylcholinesterase and neuroligin in mammalian neuritogenesis. Proc Natl Acad Sci USA 1998, 95:13935-13940. 6. Andres C, Beeri R, Friedman A, Lev-Lehman E, Henis S, Timberg R, Shani M, Soreq H: Acetylcholinesterase-transgenic mice display embryonic modulations in spinal cord choline acetyltransferase and neurexin Ibeta gene expression followed by late-onset neuromotor deterioration. Proc Natl Acad Sci USA 1997, 94:8173-8178. 7. Zakharenko S, Chang S, O’Donoghue M, Popov SV: Neurotransmitter secretion along growing nerve processes: comparison with synaptic vesicle exocytosis. J Cell Biol 1999, 144:507-518. [Erratum: J Cell Biol 1999, 144(4):following 801]. 8. Antonov I, Chang S, Zakharenko S, Popov SV: Distribution of neurotransmitter secretion in growing axons. Neuroscience 1999, 90:975-984. 9. Ahmari SE, Buchanan J, Smith SJ: Assembly of presynaptic active zones from cytoplasmic transport packets. Nat Neurosci 2000, 3:445-451.