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Structural and functional plasticity of the cytoplasmic active zone
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Structural and functional plasticity of the cytoplasmic active zone Stephan J Sigrist1,2,3,4 and Dietmar Schmitz2,3,4 The presynaptic active zone (AZ) membrane is the site where vesicle fusion mediates information transfer between connected neurons. Reaching into the cytoplasm, an electrondense cytomatrix (CAZ) is found to decorate the AZ membranes. CAZ architectures are meant not only to regulate the synaptic vesicle exocycle/endocycle, but also to structurally stabilize the presynaptic site. The CAZ is composed of a set of large scaffold proteins, many of which are evolutionarily conserved. Recently, several signaling factors controlling the developmental assembly of CAZs were found by unbiased genetics in Drosophila and Caenorhabditis elegans. At the same time, post-translational modification of CAZ proteins was implicated in changing the strength of mammalian brain synapses. Studying how processes of structural and functional CAZ plasticity get integrated within circuit remodeling remains an important challenge. Addresses 1 Genetics Institute of Biology, Freie Universita¨t Berlin, Takustr. 6, 14195 Berlin, Germany 2 Cluster of Excellence NeuroCure, Charite´platz 1, 10117 Berlin, Germany 3 Neuroscience Research Center, Charite´ Universita¨tsmedizin Berlin, Charite´platz 1, 10117 Berlin, Germany 4 Bernstein Center for Computational Neuroscience, Philippstr. 13, 10115 Berlin, Germany Corresponding author: Sigrist, Stephan J ([email protected])
Current Opinion in Neurobiology 2011, 21:144–150 This review comes from a themed issue on Developmental neuroscience Edited by Silvia Arber and Graeme Davis Available online 9th September 2010 0959-4388/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2010.08.012
Introduction Chemical synapses are pivotal for information transfer and storage within neuronal circuitry. At the presynaptic site, the active zone (AZ) provides the platform for rapid fusion of neurotransmitter-filled synaptic vesicles (SVs) after Ca2+ influx by a population of clustered Ca2+ channels (Figure 1a). The AZ membrane is decorated by a proteinaceous cytomatrix (cytoplasmic matrix at the active zone, short CAZ), which is characterized by a set of specialized proteins [1,2] often conferring an electron-dense appearance in electron microscopy (dense bodies). While CAZs display variable morphologies at different synapse types, Current Opinion in Neurobiology 2011, 21:144–150
they likely are critical for effective speed and accuracy of the associated SV exocytic/endocytic-cycle [3,4]. The number and the strength of synaptic connections are important for the functional status of a given neuron, during circuit development but equally later in the matured nervous system. Thereby, even rather subtle deficits in defining synaptic connectivity within development and/or later in the matured nervous system might lead to severe diseases. In fact, mutations in Neurexins, AZ-enriched cell adhesion proteins, have been causally linked to autism [5]. Dynamically controlling the molecular architectures of CAZs appears critical for both the structural and functional remodeling of synapses. Here, we highlight recent insights into the assembly of AZ/CAZs, pioneered by fly and worm genetics. Furthermore, we explore acute, CAZ-directed mechanisms for changing synapse function so far dominantly explored in rodent preparations. Likely, both ‘structural and functional plasticity’ co-operate at CAZs during development but equally for information processing and storage in the matured brain. We feel that an integrated view here will be critical for future progress.
Role and molecular composition of the CAZ To allow for the short delay between the influx of Ca2+ through voltage gated Ca2+ channels (localized and enriched at the AZ membrane) and the fusion of SVs, the channels and vesicles at the AZs have to be located in close proximity of each other (Figure 1a) [6,7]. Providing their coupling likely is a major job of the CAZ. Further, the CAZ also might well confer long-term stability (tenacity) to individual synaptic sites [8]. The last years have provided evidence that a rather small but conserved set of large, typically multidomain proteins provide building blocks for the CAZs of both vertebrate and invertebrate synapses. These particularly are proteins of the RIM/ UNC-10, ELKS/CAST/BRP, and Liprin-a/Syd-2 family (Figure 1b). Domain organization and functional analysis of individual CAZ proteins have been reviewed recently [1,9].
Developmental assembly of the CAZ For forming functional circuits, AZs and associated CAZs have to assemble at precise positions with precise timings. The last years have seen the identification of first regulatory proteins important to spatio-temporally define in vivo AZ/CAZ formation (for review see [1]). Thereby, initial assembly steps — that is deciding where and when to start assembly of new AZ/CAZs — might well be differently executed between different synapse types, www.sciencedirect.com
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Figure 1
Proteins and pathways implicated in active zone assembly. (a) A principal outline of CAZ and AZ architecture, (b) domain structure of main AZ protein classes, and (c) pathways controlling CAZ assembly.
and particularly might make use of different sets of cell adhesion molecules. Along with defining the location where synapses form, interactions between presynaptic and postsynaptic cell adhesion molecules might coordinate the formation of postsynaptic structures, tightly coupled to the ongoing clustering of AZ components. The trans-synaptic complex formed between Neurexins and Neuroligins is a prototypical candidate for mediating such pre-postsynaptic communication. In fact, a Drosophila Neuroligin family member (DNlg1) recently was shown to be important to couple postsynaptic to presynaptic assembly, with dnlg1 mutants forming synaptic terminals where often postsynaptic assembly is lacking opposite to many presynaptic AZs [10]. Downstream of cell adhesion molecules, SYD-2/Liprin-a seems to be a major organizer of AZ/CAZ assembly. In Caenorhabditis elegans syd-2 mutants, the overall synapse numbers appear unchanged, while there is a significant decrease in AZ electron density [11]. Similarly, Drosophila neuromuscular junctions (NMJs) formed less but larger AZs when lacking Liprin-a. At the same time, the membrane tyrosine phosphatase DLar, a binding partner of Liprin-a, was shown to have an essentially identical NMJ phenotype as Liprin-a mutants [12]. The SYD-2/Liprina family is characterized by coiled coil-domains and SAM-domains (Figure 1b), and has been implicated in both presynaptic and postsynaptic assembly by recruiting and interacting with a multitude of synaptic proteins and www.sciencedirect.com
by regulating synaptic cargo transport [13,14]. In fact, Syd-2 recently was shown to be important for effective anterograde axonal transport of SV precursors promoting the formation of transport complexes containing the Kinesin-3 motor UNC-104/KIF1A [15]. The fact that SYD-2/Liprin-a is enriched within the CAZ [16] poses the question of how axonal and AZ-close roles of SYD-2/ Liprin-a get integrated. In addition to SYD-2/Liprin-a, a C2-domain and putative Rho-GAP-domain containing protein named SYD-1 (Figure 1b), enriched within the CAZ of C. elegans and Drosophila, was found to be essential for the assembly of a specific set of AZs in C. elegans (HSNL synapses, [13,17]). There, SYD-1 seems to help the functional recruitment of SYD-2/Liprin-a, since a gain of function allele of syd-2 (a mis-sense mutation in a coiled coil domain of SYD-2, syd2(gf )), allows the suppression of the SYD-1 requirement [17]. Which other CAZ proteins interact functionally with SYD-2/Liprin-a within assembly? Notably, the activity of SYD-2(gf) in C. elegans requires ELKS-1 (Figure 1c), a member of the CAST/ERC family [17,18] (Figure 1b). Moreover, Drosophila Bruchpilot (BRP), whose N-terminal half encodes the Drosophila CAST homolog [19,20], proved to be essential for proper clustering of Ca2+ channels within AZ membranes, as well as for forming the so-called T bar, an electron-dense structure repreCurrent Opinion in Neurobiology 2011, 21:144–150
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senting the CAZ of Drosophila synapses [21]. At the same time, neurotransmitter release is very inefficient at brp mutant synapses [20]. This protein, which in addition to the CAST/ELKS domains contains long additional coiled domains at its C-terminus, seems to provide a large scaffold involved in assembly as well as functional maturation of Drosophila AZ/CAZs. High-resolution light microscopy (<100 nm resolution using stimulated emission depletion microscopy, short STED) together with immuno-electron microscopy showed that BRP is a direct T bar component apparently forming filaments vertically oriented to the AZ membrane center. Thereby, the BRP N terminus faces the AZ membrane while its C-terminus faces away from the AZ membrane [16]. In vivo imaging of intact Drosophila larvae over extended periods [22] showed that AZ assembly at Drosophila NMJs is protracted, with BRP joining the AZ/CAZ assembly late. BRP in this late phase then is responsible for the effective clustering of Ca2+ channels beneath the T bar density, potentially through its direct molecular interaction with intracellular Ca2+ channel domains [16]. As mentioned above, the assembly hierarchy genetically defined for C. elegans HSNL synapses sees ELKS downstream of syd-2 (syd-1 upstream of syd-2 upstream of ELKS, Figure 1c). Drosophila BRP interacts strongly with the Drosophila Syd-1 homolog (DSyd-1), which also is needed for effective addition of AZs [23]. Again, in vivo imaging showed that BRP invariably follows DSyd-1 and Liprin-a (which are closely colocalized) during the assembly of new AZ/CAZs at Drosophila NMJ synapses. Moreover, Liprin-a is unequally distributed over AZs of dsyd-1 mutants. In other words, DSyd-1 seems to ‘stall Liprin-a to AZs’ [23]. Thus, assembly hierarchies defined by means of genetic and imaging analysis increasingly converge in a coherent scenario of AZ/CAZ assembly (Figure 1c).
New pathways steering CAZ assembly It appears likely that SYD-2/Liprin-a together with Syd1 — likely by stabilizing SYD-2/Liprin-a at prospective AZ/CAZs — is needed for effective ‘nucleation’ of AZ/ CAZ assembly at new membrane spots, and that in its absence less native AZ/CAZs can grow over a certain threshold and finally stabilize with mature size [23]. Surprisingly, however, the lack of presynaptic DSyd-1 but not Liprin-a provoked excessively large postsynaptic glutamate receptor fields at Drosophila NMJ synapses [23], potentially indicating that DSyd-1 might stall other substrate proteins (symbolized by X, Figure 1c) to execute a trans-synaptic control over postsynaptic assembly. Thus, our picture of developmental synapse assembly is still far from complete. Recently, unbiased genetics paved the way for the identification of further molecular pathways in this context (also see [21]). Notably, two groups ([24,25]) reported that mutations affecting a serine–arginine protein kinase Current Opinion in Neurobiology 2011, 21:144–150
(SRPK79D) cause very large, electron-dense accumulations of the CAZ component BRP (T bar superassembly) in Drosophila axons [21]. The SRPK79D protein colocalizes with BRP in both the axon (transport aggregates) and synapse (CAZ). Thus, on the one hand SRPK79D activity seemingly protects BRP ‘on its way down the axon’ from ‘precocious oligomerization’ and thus prevents the ectopic formation of CAZ-like material within axons. At ‘prospective AZs’, SRPK79D-dependent repression must then be relieved to facilitate site-specific CAZ assembly. Notably, expression of SRPK79D apparently could ‘dissolve’ BRP from established AZs as well [25]. Thus the role of this kinase seems not restricted to newly forming AZ/CAZs, but SRPK79D might well modulate the AZ/CAZs structure at established synapses as well. While substrate proteins still have to be identified, SRPK79D function depends on its kinase activity. Surprisingly, so far the best-characterized role for SRPKs was in controlling the subcellular localization of SR proteins, thereby regulating their nuclear premRNA splicing activity [26]. Whether control of splicing activities or local translation in the axoplasm is linked to T bar assembly via SRPK79D will have to be further addressed. In fact, an SR domain containing protein, RSY-1 (regulator of synaptogenesis 1, [27]) was found to locally inhibit AZ assembly to restrict synapse formation to correct positions during C. elegans development [28]. Collectively, protein interactions and phosphorylation events seem to execute local negative signals controlling CAZ assembly. However, signals promoting CAZ assembly have been identified at the Drosophila NMJ as well. Here, the serine–threonine kinase Unc-51 acts in the presynaptic motoneuron and regulates the localization of BRP opposite to glutamate receptors at individual synapses of fly NMJ [29]. In the absence of Unc-51, many glutamate receptor clusters lack apposed BRP clusters, and ultrastructural analysis demonstrates that fewer AZs contain T bars. Mechanistically, Unc-51 inhibits the activity of the MAP kinase ERK to promote synaptic development. Hence, activated ERK apparently counteracts BRP accumulation at AZ/CAZs. In yet another study, a counterplay of phosphatase PP2A and GSK-3b kinase was recently shown to control BRP levels [30]. Whether BRP or other CAZ proteins are direct substrates of these kinases/phosphatases (SRPK79D, Unc51, PP2A, and GSK-3b, ERK) and how exactly these phosphorylation–dephosphorylation events get integrated into CAZ assembly, remodeling and how finally axonal transport processes are hooked up to AZ-close distribution processes are urgent questions. Apart from regulation by (de)phosphorylation events, very recently the small GTPase Rab3 was implicated in ‘structural’ CAZ dynamics at the fly NMJ [31,32]. At rab3 mutant NMJs, BRP, Ca2+ channels, and electrondense T bars are concentrated at a fraction of the available www.sciencedirect.com
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AZs only, leaving the majority of sites devoid of these AZ/ CAZ components. Late addition of Rab3 to mutant NMJs rapidly reverses this phenotype by recruiting BRP to sites previously lacking the protein, demonstrating that Rab3 can dynamically control the AZ/CAZ composition. Here, physiological analysis (paired pulse measurements) indicated that in the rab3 mutant approximately one-third of AZs have a very high pr, likely reflecting an excess of essential AZ/CAZ components (and thus ultrastructurally often show ‘double T bars’). The remaining sites in contrast lack crucial CAZ proteins, and so might well have a low or even zero pr. It is tempting to speculate that there might be a direct correlation between the amount of CAZ and the pr at individual AZs. However, to further validate this hypothesis it still remains critical to at the Drosophila NMJ directly compare CAZ amount and composition with SV release (e.g. via opto-physiological tools) on the level of individual AZs. In C. elegans, in turn, another small G protein, Arf-like ARL-8, was shown to promote a trafficking identity for presynaptic cargoes (both CAZ and SV components), facilitating their efficient transport by repressing premature self-association [33].
Connecting structural and functional remodeling of CAZs As a result, our mechanistic understanding of how CAZs assemble during initial circuit development is steadily increasing. Clearly, mammalian homologs of factors as Syd-1 or SRPK97 should be studied for similar roles in the development of mammalian brain circuitry. Maybe even more important than sheer developmental roles, it appears likely that these or similar controls operate in the remodeling of AZ/CAZs of already functioning circuits as well. Notably, earlier electron microscopic work already had implied that CAZs of Drosophila photoreceptor neurons are subject to fast (minute range!) assembly and disassembly process when changes in circuit function get executed [34,35]. So far, assembly processes of the CAZ were emphasized in this review. However, these structures apart from a structural clearly also play a functional role [7]. While admittedly we are far from a detailed understanding here, the CAZ likely has roles in tethering and activity-dependent recruitment of SVs to AZ membranes, and might establish physical proximity between exocytic proteins, Ca2+ channels and potentially endocytic machinery to shape release dynamics. Definitively, different CAZ proteins will turn out to have specialized functional and structural roles in this context. Notably, a recent study [36] performed in hippocampal neuron culture provided evidence that the amount of CAZ present at individual AZs can change in the minute time scale. This is in certain contrast to studies emphasizing a tenacity role for CAZ component www.sciencedirect.com
Bassoon [8]. Notably, however, [36] also provided evidence that CAZ amounts were directly correlated with SV release probability. Thus, the functional status of an individual AZ might change in the minutes range as well. Invertebrate synaptic model systems have played out their strengths in identifying proteins relevant for CAZ/ AZ assembly using unbiased genetic strategies. However, detailed functional characterizations of synaptic phenotypes of these factors often are hampered by the small size of invertebrate neurons and are typically restricted to peripheral synapses. In contrast, electrophysiological analysis of vertebrate brain synapses, particularly in rat or mouse, has a long-standing tradition. Here, activity-dependent long-term changes in synaptic transmission strengths, referred to as long-term potentiation (LTP) and long-term depression (LTD), have received long-lasting attention. Unlike postsynaptic long-term plasticity at the Schaffer collateral synapse, the hippocampal mossy fiber synapse [37], the cerebellar parallel fiber synapse [38], and corticothalamic synapses [39] all express presynaptic forms of LTP. For the induction of this form of LTP, presynaptic Ca2+ channels clearly are critical [37,40]. This said, the molecular nature of downstream expression mechanisms is less clear, but undoubtedly involves an increase in transmitter and thus SV release [37]. Both pharmacological and genetic analyses indicate that a rise in presynaptic cAMP is causal for mossy fiber and also parallel fiber LTP. Indeed, pharmacological and genetic analysis demonstrated a role for specific adenylate cyclases and subsequent protein kinase A (PKA) activation in the expression of presynaptic LTP (reviewed in [37]). A simple model would have voltage-dependent Ca2+ entry during intense frequencies of action potentials to directly activate adenylate cyclase. How could a rise in cAMP followed by PKA activation cause a long lasting increase of SV release? Might there be PKA substrate proteins between the CAZ constituents to mediate presynaptic LTP in response to cAMP? In fact, RIM1a was suggested to play such a role. RIM-family proteins (Figure 1b) were recently extensively reviewed [41]. In short, RIMs were implied in CAZ function by unbiased screening in C. elegans, with loss of UNC-10/ RIM, the only RIM isoform in C. elegans, leading to behavioral and physiological deficits [42]. In mice, RIM1a-deficient mice are viable and fertile but exhibit severe deficits in synaptic transmission without showing ultrastructural synaptic deficits [43–45]. However, many Rim-isoforms are present in mammals, and RIM1a RIM2a double knockouts die early, clearly leaving the possibility that important structural roles might have been masked so far due to partial redundancies between RIMfamily members. Coming back to presynaptic LTP, RIM1a was shown to bind the small GTPase Rab3A, Current Opinion in Neurobiology 2011, 21:144–150
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while studies of knockout mice moreover showed that both Rab3A and RIM1a were required for both mossy fiber LTP [43,46]. Thus, a GTP-dependent interaction between Rab3A and RIM1a, potentially by recruiting SVs to AZs, might be important for cAMP-induced presynaptic LTP expression. However, the fact that these studies found the enhancement by forskolin (increasing cAMP levels) unaltered in RIM1a and Rab3A knockout mice suggests that the role of Rab3A/RIM1a as a mediator of PKA’s effects might be more complex. Interestingly, direct phosphorylation of RIM1a at serine-413 by PKA was suggested to mediate presynaptic LTP at cultivated cerebellar parallel fiber synapses [47]. However, when knockin mice with serine-413 being mutated to nonphosphorylatable alanine were generated, this role of the serine-413 phosphorylation could not be corroborated in vivo [48]. Here, in contrast to [47], three different forms of presynaptic PKA-dependent long-term plasticity were unaffected. Thus, it seems that phosphorylation of serine-413 of RIM1a is not an absolute requirement for PKA-dependent long-term presynaptic plasticity. Similarly, acute in vivo genetic rescue demonstrated that phosphorylation of RIM1a serine-413 is not required for mossy fiber LTP [49]. A parsimonious perspective on these superficially contradictory results might be that phosphorylation of RIM1a at serine-413 per se promotes vesicle release, however, that in vivo a deficit here can be compensated by ‘parallel pathways’. In fact, RIM-family proteins might contribute to protein bridges to anchor the prefusion SVs to the Ca2+ channel protein complexes [50]. In this chapter we so far have been focusing on RIM. How about functional roles for other CAZ proteins at mammalian synapses? Two large scaffolding molecules, Piccolo and Bassoon, were among the first CAZ-specific proteins to be identified. Not conserved in Drosophila or C. elegans, they turned out to be specific to vertebrate synapses. Piccolo seems dispensable for principal AZ assembly at glutamatergic synapses, but influences presynaptic function by negatively regulating SV exocytosis. Piccolo might have a specific role in coupling the mobilization of SVs in reserve pools to events within the AZ [51]. Bassoon is reported to play an important role in the assembly and functioning of various types of synapses, and to be among the first proteins that appear at newly assembling AZs [1,52]. For even another CAZ-protein family, ELKS/CAST (Figure 1b, [18]), a recent study [53] showed that deletion of one ELKS isoform (ELKS2a) in mice caused an increase in inhibitory, but not excitatory, neurotransmitter release, and potentiated the size but not the properties of the readily releasable pool of vesicles at inhibitory synapses. While in ultrastructural terms no severe deficits were found in ELKS2a knockouts, solubility of RIM1a was clearly increased here. We speculate Current Opinion in Neurobiology 2011, 21:144–150
that there might be a graduation from purely functional changes not altering the biochemical composition, compositional biochemical changes associated with robust functional changes and finally ‘structural’ processes involving the full formation/elimination of AZ/CAZs. Notably, following the induction of presynaptic LTP, there might be more than an increase in release probability. Indeed, at hippocampal granule cell autapses, a ‘turning on’ of presynaptically silent synapses [54] could be demonstrated to take place in parallel to increases in release probability. Similarly, optical imaging of postsynaptic Ca2+ transients in slice culture suggested that previously silent presynaptic release sites can be switched on following LTP [55]. Ultimately, a rich combination of cutting edge techniques (e.g. high-resolution microscopy, in vivo imaging, single cell or even single bouton recordings, and modifier screening) will be needed for further progress in our understanding of these delicately controlled architectural modifications of CAZs. An interesting study of such kind [56] recently was conducted at hair cell synapses. Highresolution imaging of these synapses provided insights into presynaptic Ca2+ channel clusters and Ca2+ signals, synaptic CAZs and postsynaptic glutamate receptor clusters, revealing substantial variability for presynaptic Ca2+ signals, even within individual hair cells. Overall, it is also clear that there not one answer but that many answers will have to be derived. This particularly as synapse diversity, often expressed in differences of synaptic short-term plasticity, will also have its grounds in variations of AZ/CAZ organization. Thus, there is still a lot to be done.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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41. Mittelstaedt T, Alvarez-Baron E, Schoch S: RIM proteins and their role in synapse function. Biol Chem 2010, 391:599-606. 42. Koushika SP, Richmond JE, Hadwiger G, Weimer RM, Jorgensen EM, Nonet ML: A post-docking role for active zone protein Rim. Nat Neurosci 2001, 4:997-1005. 43. Castillo PE, Schoch S, Schmitz F, Sudhof TC, Malenka RC: RIM1alpha is required for presynaptic long-term potentiation. Nature 2002, 415:327-330. 44. Schoch S, Castillo PE, Jo T, Mukherjee K, Geppert M, Wang Y, Schmitz F, Malenka RC, Sudhof TC: RIM1alpha forms a protein scaffold for regulating neurotransmitter release at the active zone. Nature 2002, 415:321-326. 45. Calakos N, Schoch S, Sudhof TC, Malenka RC: Multiple roles for the active zone protein RIM1alpha in late stages of neurotransmitter release. Neuron 2004, 42:889-896. 46. Castillo PE, Janz R, Sudhof TC, Tzounopoulos T, Malenka RC, Nicoll RA: Rab3A is essential for mossy fibre long-term potentiation in the hippocampus. Nature 1997, 388:590-593. 47. Lonart G, Schoch S, Kaeser PS, Larkin CJ, Sudhof TC, Linden DJ: Phosphorylation of RIM1alpha by PKA triggers presynaptic long-term potentiation at cerebellar parallel fiber synapses. Cell 2003, 115:49-60. 48. Kaeser PS, Kwon HB, Blundell J, Chevaleyre V, Morishita W, Malenka RC, Powell CM, Castillo PE, Sudhof TC: RIM1alpha phosphorylation at serine-413 by protein kinase A is not required for presynaptic long-term plasticity or learning. Proc Natl Acad Sci U S A 2008, 105:14680-14685. 49. Yang Y, Calakos N: Acute in vivo genetic rescue demonstrates that phosphorylation of RIM1alpha serine 413 is not required for mossy fiber long-term potentiation. J Neurosci 2010, 30:2542-2546.
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50. Wong FK, Stanley EF: Rab3a interacting molecule (RIM) and the tethering of pre-synaptic transmitter release site-associated CaV2.2 calcium channels. J Neurochem 2010, 112:463-473. 51. Leal-Ortiz S, Waites CL, Terry-Lorenzo R, Zamorano P, Gundelfinger ED, Garner CC: Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis. J Cell Biol 2008, 181:831-846. 52. Regus-Leidig H, Tom Dieck S, Specht D, Meyer L, Brandstatter JH: Early steps in the assembly of photoreceptor ribbon synapses in the mouse retina: the involvement of precursor spheres. J Comp Neurol 2009, 512:814-824. 53. Kaeser PS, Deng L, Chavez AE, Liu X, Castillo PE, Sudhof TC: ELKS2alpha/CAST deletion selectively increases neurotransmitter release at inhibitory synapses. Neuron 2009, 64:227-239. Surprisingly, an ELKS family member in mice plays a functional role in suppressing SV release specifically at GABAergic synapses. 54. Tong G, Malenka RC, Nicoll RA: Long-term potentiation in cultures of single hippocampal granule cells: a presynaptic form of plasticity. Neuron 1996, 16:1147-1157. 55. Reid CA, Dixon DB, Takahashi M, Bliss TV, Fine A: Optical quantal analysis indicates that long-term potentiation at single hippocampal mossy fiber synapses is expressed through increased release probability, recruitment of new release sites, and activation of silent synapses. J Neurosci 2004, 24:3618-3626. 56. Meyer AC, Frank T, Khimich D, Hoch G, Riedel D, Chapochnikov NM, Yarin YM, Harke B, Hell SW, Egner A et al.: Tuning of synapse number, structure and function in the cochlea. Nat Neurosci 2009, 12:444-453. State-of-the-art study correlating AZ organization, local Ca2+ dynamics and SV release on single active zone level.
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Structural and functional plasticity of the cytoplasmic active zone Stephan J Sigrist1,2,3,4 and Dietmar Schmitz2,3,4 The presynaptic active zone (AZ) membrane is the site where vesicle fusion mediates information transfer between connected neurons. Reaching into the cytoplasm, an electrondense cytomatrix (CAZ) is found to decorate the AZ membranes. CAZ architectures are meant not only to regulate the synaptic vesicle exocycle/endocycle, but also to structurally stabilize the presynaptic site. The CAZ is composed of a set of large scaffold proteins, many of which are evolutionarily conserved. Recently, several signaling factors controlling the developmental assembly of CAZs were found by unbiased genetics in Drosophila and Caenorhabditis elegans. At the same time, post-translational modification of CAZ proteins was implicated in changing the strength of mammalian brain synapses. Studying how processes of structural and functional CAZ plasticity get integrated within circuit remodeling remains an important challenge. Addresses 1 Genetics Institute of Biology, Freie Universita¨t Berlin, Takustr. 6, 14195 Berlin, Germany 2 Cluster of Excellence NeuroCure, Charite´platz 1, 10117 Berlin, Germany 3 Neuroscience Research Center, Charite´ Universita¨tsmedizin Berlin, Charite´platz 1, 10117 Berlin, Germany 4 Bernstein Center for Computational Neuroscience, Philippstr. 13, 10115 Berlin, Germany Corresponding author: Sigrist, Stephan J ([email protected])
Current Opinion in Neurobiology 2011, 21:144–150 This review comes from a themed issue on Developmental neuroscience Edited by Silvia Arber and Graeme Davis Available online 9th September 2010 0959-4388/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2010.08.012
Introduction Chemical synapses are pivotal for information transfer and storage within neuronal circuitry. At the presynaptic site, the active zone (AZ) provides the platform for rapid fusion of neurotransmitter-filled synaptic vesicles (SVs) after Ca2+ influx by a population of clustered Ca2+ channels (Figure 1a). The AZ membrane is decorated by a proteinaceous cytomatrix (cytoplasmic matrix at the active zone, short CAZ), which is characterized by a set of specialized proteins [1,2] often conferring an electron-dense appearance in electron microscopy (dense bodies). While CAZs display variable morphologies at different synapse types, Current Opinion in Neurobiology 2011, 21:144–150
they likely are critical for effective speed and accuracy of the associated SV exocytic/endocytic-cycle [3,4]. The number and the strength of synaptic connections are important for the functional status of a given neuron, during circuit development but equally later in the matured nervous system. Thereby, even rather subtle deficits in defining synaptic connectivity within development and/or later in the matured nervous system might lead to severe diseases. In fact, mutations in Neurexins, AZ-enriched cell adhesion proteins, have been causally linked to autism [5]. Dynamically controlling the molecular architectures of CAZs appears critical for both the structural and functional remodeling of synapses. Here, we highlight recent insights into the assembly of AZ/CAZs, pioneered by fly and worm genetics. Furthermore, we explore acute, CAZ-directed mechanisms for changing synapse function so far dominantly explored in rodent preparations. Likely, both ‘structural and functional plasticity’ co-operate at CAZs during development but equally for information processing and storage in the matured brain. We feel that an integrated view here will be critical for future progress.
Role and molecular composition of the CAZ To allow for the short delay between the influx of Ca2+ through voltage gated Ca2+ channels (localized and enriched at the AZ membrane) and the fusion of SVs, the channels and vesicles at the AZs have to be located in close proximity of each other (Figure 1a) [6,7]. Providing their coupling likely is a major job of the CAZ. Further, the CAZ also might well confer long-term stability (tenacity) to individual synaptic sites [8]. The last years have provided evidence that a rather small but conserved set of large, typically multidomain proteins provide building blocks for the CAZs of both vertebrate and invertebrate synapses. These particularly are proteins of the RIM/ UNC-10, ELKS/CAST/BRP, and Liprin-a/Syd-2 family (Figure 1b). Domain organization and functional analysis of individual CAZ proteins have been reviewed recently [1,9].
Developmental assembly of the CAZ For forming functional circuits, AZs and associated CAZs have to assemble at precise positions with precise timings. The last years have seen the identification of first regulatory proteins important to spatio-temporally define in vivo AZ/CAZ formation (for review see [1]). Thereby, initial assembly steps — that is deciding where and when to start assembly of new AZ/CAZs — might well be differently executed between different synapse types, www.sciencedirect.com
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Figure 1
Proteins and pathways implicated in active zone assembly. (a) A principal outline of CAZ and AZ architecture, (b) domain structure of main AZ protein classes, and (c) pathways controlling CAZ assembly.
and particularly might make use of different sets of cell adhesion molecules. Along with defining the location where synapses form, interactions between presynaptic and postsynaptic cell adhesion molecules might coordinate the formation of postsynaptic structures, tightly coupled to the ongoing clustering of AZ components. The trans-synaptic complex formed between Neurexins and Neuroligins is a prototypical candidate for mediating such pre-postsynaptic communication. In fact, a Drosophila Neuroligin family member (DNlg1) recently was shown to be important to couple postsynaptic to presynaptic assembly, with dnlg1 mutants forming synaptic terminals where often postsynaptic assembly is lacking opposite to many presynaptic AZs [10]. Downstream of cell adhesion molecules, SYD-2/Liprin-a seems to be a major organizer of AZ/CAZ assembly. In Caenorhabditis elegans syd-2 mutants, the overall synapse numbers appear unchanged, while there is a significant decrease in AZ electron density [11]. Similarly, Drosophila neuromuscular junctions (NMJs) formed less but larger AZs when lacking Liprin-a. At the same time, the membrane tyrosine phosphatase DLar, a binding partner of Liprin-a, was shown to have an essentially identical NMJ phenotype as Liprin-a mutants [12]. The SYD-2/Liprina family is characterized by coiled coil-domains and SAM-domains (Figure 1b), and has been implicated in both presynaptic and postsynaptic assembly by recruiting and interacting with a multitude of synaptic proteins and www.sciencedirect.com
by regulating synaptic cargo transport [13,14]. In fact, Syd-2 recently was shown to be important for effective anterograde axonal transport of SV precursors promoting the formation of transport complexes containing the Kinesin-3 motor UNC-104/KIF1A [15]. The fact that SYD-2/Liprin-a is enriched within the CAZ [16] poses the question of how axonal and AZ-close roles of SYD-2/ Liprin-a get integrated. In addition to SYD-2/Liprin-a, a C2-domain and putative Rho-GAP-domain containing protein named SYD-1 (Figure 1b), enriched within the CAZ of C. elegans and Drosophila, was found to be essential for the assembly of a specific set of AZs in C. elegans (HSNL synapses, [13,17]). There, SYD-1 seems to help the functional recruitment of SYD-2/Liprin-a, since a gain of function allele of syd-2 (a mis-sense mutation in a coiled coil domain of SYD-2, syd2(gf )), allows the suppression of the SYD-1 requirement [17]. Which other CAZ proteins interact functionally with SYD-2/Liprin-a within assembly? Notably, the activity of SYD-2(gf) in C. elegans requires ELKS-1 (Figure 1c), a member of the CAST/ERC family [17,18] (Figure 1b). Moreover, Drosophila Bruchpilot (BRP), whose N-terminal half encodes the Drosophila CAST homolog [19,20], proved to be essential for proper clustering of Ca2+ channels within AZ membranes, as well as for forming the so-called T bar, an electron-dense structure repreCurrent Opinion in Neurobiology 2011, 21:144–150
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senting the CAZ of Drosophila synapses [21]. At the same time, neurotransmitter release is very inefficient at brp mutant synapses [20]. This protein, which in addition to the CAST/ELKS domains contains long additional coiled domains at its C-terminus, seems to provide a large scaffold involved in assembly as well as functional maturation of Drosophila AZ/CAZs. High-resolution light microscopy (<100 nm resolution using stimulated emission depletion microscopy, short STED) together with immuno-electron microscopy showed that BRP is a direct T bar component apparently forming filaments vertically oriented to the AZ membrane center. Thereby, the BRP N terminus faces the AZ membrane while its C-terminus faces away from the AZ membrane [16]. In vivo imaging of intact Drosophila larvae over extended periods [22] showed that AZ assembly at Drosophila NMJs is protracted, with BRP joining the AZ/CAZ assembly late. BRP in this late phase then is responsible for the effective clustering of Ca2+ channels beneath the T bar density, potentially through its direct molecular interaction with intracellular Ca2+ channel domains [16]. As mentioned above, the assembly hierarchy genetically defined for C. elegans HSNL synapses sees ELKS downstream of syd-2 (syd-1 upstream of syd-2 upstream of ELKS, Figure 1c). Drosophila BRP interacts strongly with the Drosophila Syd-1 homolog (DSyd-1), which also is needed for effective addition of AZs [23]. Again, in vivo imaging showed that BRP invariably follows DSyd-1 and Liprin-a (which are closely colocalized) during the assembly of new AZ/CAZs at Drosophila NMJ synapses. Moreover, Liprin-a is unequally distributed over AZs of dsyd-1 mutants. In other words, DSyd-1 seems to ‘stall Liprin-a to AZs’ [23]. Thus, assembly hierarchies defined by means of genetic and imaging analysis increasingly converge in a coherent scenario of AZ/CAZ assembly (Figure 1c).
New pathways steering CAZ assembly It appears likely that SYD-2/Liprin-a together with Syd1 — likely by stabilizing SYD-2/Liprin-a at prospective AZ/CAZs — is needed for effective ‘nucleation’ of AZ/ CAZ assembly at new membrane spots, and that in its absence less native AZ/CAZs can grow over a certain threshold and finally stabilize with mature size [23]. Surprisingly, however, the lack of presynaptic DSyd-1 but not Liprin-a provoked excessively large postsynaptic glutamate receptor fields at Drosophila NMJ synapses [23], potentially indicating that DSyd-1 might stall other substrate proteins (symbolized by X, Figure 1c) to execute a trans-synaptic control over postsynaptic assembly. Thus, our picture of developmental synapse assembly is still far from complete. Recently, unbiased genetics paved the way for the identification of further molecular pathways in this context (also see [21]). Notably, two groups ([24,25]) reported that mutations affecting a serine–arginine protein kinase Current Opinion in Neurobiology 2011, 21:144–150
(SRPK79D) cause very large, electron-dense accumulations of the CAZ component BRP (T bar superassembly) in Drosophila axons [21]. The SRPK79D protein colocalizes with BRP in both the axon (transport aggregates) and synapse (CAZ). Thus, on the one hand SRPK79D activity seemingly protects BRP ‘on its way down the axon’ from ‘precocious oligomerization’ and thus prevents the ectopic formation of CAZ-like material within axons. At ‘prospective AZs’, SRPK79D-dependent repression must then be relieved to facilitate site-specific CAZ assembly. Notably, expression of SRPK79D apparently could ‘dissolve’ BRP from established AZs as well [25]. Thus the role of this kinase seems not restricted to newly forming AZ/CAZs, but SRPK79D might well modulate the AZ/CAZs structure at established synapses as well. While substrate proteins still have to be identified, SRPK79D function depends on its kinase activity. Surprisingly, so far the best-characterized role for SRPKs was in controlling the subcellular localization of SR proteins, thereby regulating their nuclear premRNA splicing activity [26]. Whether control of splicing activities or local translation in the axoplasm is linked to T bar assembly via SRPK79D will have to be further addressed. In fact, an SR domain containing protein, RSY-1 (regulator of synaptogenesis 1, [27]) was found to locally inhibit AZ assembly to restrict synapse formation to correct positions during C. elegans development [28]. Collectively, protein interactions and phosphorylation events seem to execute local negative signals controlling CAZ assembly. However, signals promoting CAZ assembly have been identified at the Drosophila NMJ as well. Here, the serine–threonine kinase Unc-51 acts in the presynaptic motoneuron and regulates the localization of BRP opposite to glutamate receptors at individual synapses of fly NMJ [29]. In the absence of Unc-51, many glutamate receptor clusters lack apposed BRP clusters, and ultrastructural analysis demonstrates that fewer AZs contain T bars. Mechanistically, Unc-51 inhibits the activity of the MAP kinase ERK to promote synaptic development. Hence, activated ERK apparently counteracts BRP accumulation at AZ/CAZs. In yet another study, a counterplay of phosphatase PP2A and GSK-3b kinase was recently shown to control BRP levels [30]. Whether BRP or other CAZ proteins are direct substrates of these kinases/phosphatases (SRPK79D, Unc51, PP2A, and GSK-3b, ERK) and how exactly these phosphorylation–dephosphorylation events get integrated into CAZ assembly, remodeling and how finally axonal transport processes are hooked up to AZ-close distribution processes are urgent questions. Apart from regulation by (de)phosphorylation events, very recently the small GTPase Rab3 was implicated in ‘structural’ CAZ dynamics at the fly NMJ [31,32]. At rab3 mutant NMJs, BRP, Ca2+ channels, and electrondense T bars are concentrated at a fraction of the available www.sciencedirect.com
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AZs only, leaving the majority of sites devoid of these AZ/ CAZ components. Late addition of Rab3 to mutant NMJs rapidly reverses this phenotype by recruiting BRP to sites previously lacking the protein, demonstrating that Rab3 can dynamically control the AZ/CAZ composition. Here, physiological analysis (paired pulse measurements) indicated that in the rab3 mutant approximately one-third of AZs have a very high pr, likely reflecting an excess of essential AZ/CAZ components (and thus ultrastructurally often show ‘double T bars’). The remaining sites in contrast lack crucial CAZ proteins, and so might well have a low or even zero pr. It is tempting to speculate that there might be a direct correlation between the amount of CAZ and the pr at individual AZs. However, to further validate this hypothesis it still remains critical to at the Drosophila NMJ directly compare CAZ amount and composition with SV release (e.g. via opto-physiological tools) on the level of individual AZs. In C. elegans, in turn, another small G protein, Arf-like ARL-8, was shown to promote a trafficking identity for presynaptic cargoes (both CAZ and SV components), facilitating their efficient transport by repressing premature self-association [33].
Connecting structural and functional remodeling of CAZs As a result, our mechanistic understanding of how CAZs assemble during initial circuit development is steadily increasing. Clearly, mammalian homologs of factors as Syd-1 or SRPK97 should be studied for similar roles in the development of mammalian brain circuitry. Maybe even more important than sheer developmental roles, it appears likely that these or similar controls operate in the remodeling of AZ/CAZs of already functioning circuits as well. Notably, earlier electron microscopic work already had implied that CAZs of Drosophila photoreceptor neurons are subject to fast (minute range!) assembly and disassembly process when changes in circuit function get executed [34,35]. So far, assembly processes of the CAZ were emphasized in this review. However, these structures apart from a structural clearly also play a functional role [7]. While admittedly we are far from a detailed understanding here, the CAZ likely has roles in tethering and activity-dependent recruitment of SVs to AZ membranes, and might establish physical proximity between exocytic proteins, Ca2+ channels and potentially endocytic machinery to shape release dynamics. Definitively, different CAZ proteins will turn out to have specialized functional and structural roles in this context. Notably, a recent study [36] performed in hippocampal neuron culture provided evidence that the amount of CAZ present at individual AZs can change in the minute time scale. This is in certain contrast to studies emphasizing a tenacity role for CAZ component www.sciencedirect.com
Bassoon [8]. Notably, however, [36] also provided evidence that CAZ amounts were directly correlated with SV release probability. Thus, the functional status of an individual AZ might change in the minutes range as well. Invertebrate synaptic model systems have played out their strengths in identifying proteins relevant for CAZ/ AZ assembly using unbiased genetic strategies. However, detailed functional characterizations of synaptic phenotypes of these factors often are hampered by the small size of invertebrate neurons and are typically restricted to peripheral synapses. In contrast, electrophysiological analysis of vertebrate brain synapses, particularly in rat or mouse, has a long-standing tradition. Here, activity-dependent long-term changes in synaptic transmission strengths, referred to as long-term potentiation (LTP) and long-term depression (LTD), have received long-lasting attention. Unlike postsynaptic long-term plasticity at the Schaffer collateral synapse, the hippocampal mossy fiber synapse [37], the cerebellar parallel fiber synapse [38], and corticothalamic synapses [39] all express presynaptic forms of LTP. For the induction of this form of LTP, presynaptic Ca2+ channels clearly are critical [37,40]. This said, the molecular nature of downstream expression mechanisms is less clear, but undoubtedly involves an increase in transmitter and thus SV release [37]. Both pharmacological and genetic analyses indicate that a rise in presynaptic cAMP is causal for mossy fiber and also parallel fiber LTP. Indeed, pharmacological and genetic analysis demonstrated a role for specific adenylate cyclases and subsequent protein kinase A (PKA) activation in the expression of presynaptic LTP (reviewed in [37]). A simple model would have voltage-dependent Ca2+ entry during intense frequencies of action potentials to directly activate adenylate cyclase. How could a rise in cAMP followed by PKA activation cause a long lasting increase of SV release? Might there be PKA substrate proteins between the CAZ constituents to mediate presynaptic LTP in response to cAMP? In fact, RIM1a was suggested to play such a role. RIM-family proteins (Figure 1b) were recently extensively reviewed [41]. In short, RIMs were implied in CAZ function by unbiased screening in C. elegans, with loss of UNC-10/ RIM, the only RIM isoform in C. elegans, leading to behavioral and physiological deficits [42]. In mice, RIM1a-deficient mice are viable and fertile but exhibit severe deficits in synaptic transmission without showing ultrastructural synaptic deficits [43–45]. However, many Rim-isoforms are present in mammals, and RIM1a RIM2a double knockouts die early, clearly leaving the possibility that important structural roles might have been masked so far due to partial redundancies between RIMfamily members. Coming back to presynaptic LTP, RIM1a was shown to bind the small GTPase Rab3A, Current Opinion in Neurobiology 2011, 21:144–150
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while studies of knockout mice moreover showed that both Rab3A and RIM1a were required for both mossy fiber LTP [43,46]. Thus, a GTP-dependent interaction between Rab3A and RIM1a, potentially by recruiting SVs to AZs, might be important for cAMP-induced presynaptic LTP expression. However, the fact that these studies found the enhancement by forskolin (increasing cAMP levels) unaltered in RIM1a and Rab3A knockout mice suggests that the role of Rab3A/RIM1a as a mediator of PKA’s effects might be more complex. Interestingly, direct phosphorylation of RIM1a at serine-413 by PKA was suggested to mediate presynaptic LTP at cultivated cerebellar parallel fiber synapses [47]. However, when knockin mice with serine-413 being mutated to nonphosphorylatable alanine were generated, this role of the serine-413 phosphorylation could not be corroborated in vivo [48]. Here, in contrast to [47], three different forms of presynaptic PKA-dependent long-term plasticity were unaffected. Thus, it seems that phosphorylation of serine-413 of RIM1a is not an absolute requirement for PKA-dependent long-term presynaptic plasticity. Similarly, acute in vivo genetic rescue demonstrated that phosphorylation of RIM1a serine-413 is not required for mossy fiber LTP [49]. A parsimonious perspective on these superficially contradictory results might be that phosphorylation of RIM1a at serine-413 per se promotes vesicle release, however, that in vivo a deficit here can be compensated by ‘parallel pathways’. In fact, RIM-family proteins might contribute to protein bridges to anchor the prefusion SVs to the Ca2+ channel protein complexes [50]. In this chapter we so far have been focusing on RIM. How about functional roles for other CAZ proteins at mammalian synapses? Two large scaffolding molecules, Piccolo and Bassoon, were among the first CAZ-specific proteins to be identified. Not conserved in Drosophila or C. elegans, they turned out to be specific to vertebrate synapses. Piccolo seems dispensable for principal AZ assembly at glutamatergic synapses, but influences presynaptic function by negatively regulating SV exocytosis. Piccolo might have a specific role in coupling the mobilization of SVs in reserve pools to events within the AZ [51]. Bassoon is reported to play an important role in the assembly and functioning of various types of synapses, and to be among the first proteins that appear at newly assembling AZs [1,52]. For even another CAZ-protein family, ELKS/CAST (Figure 1b, [18]), a recent study [53] showed that deletion of one ELKS isoform (ELKS2a) in mice caused an increase in inhibitory, but not excitatory, neurotransmitter release, and potentiated the size but not the properties of the readily releasable pool of vesicles at inhibitory synapses. While in ultrastructural terms no severe deficits were found in ELKS2a knockouts, solubility of RIM1a was clearly increased here. We speculate Current Opinion in Neurobiology 2011, 21:144–150
that there might be a graduation from purely functional changes not altering the biochemical composition, compositional biochemical changes associated with robust functional changes and finally ‘structural’ processes involving the full formation/elimination of AZ/CAZs. Notably, following the induction of presynaptic LTP, there might be more than an increase in release probability. Indeed, at hippocampal granule cell autapses, a ‘turning on’ of presynaptically silent synapses [54] could be demonstrated to take place in parallel to increases in release probability. Similarly, optical imaging of postsynaptic Ca2+ transients in slice culture suggested that previously silent presynaptic release sites can be switched on following LTP [55]. Ultimately, a rich combination of cutting edge techniques (e.g. high-resolution microscopy, in vivo imaging, single cell or even single bouton recordings, and modifier screening) will be needed for further progress in our understanding of these delicately controlled architectural modifications of CAZs. An interesting study of such kind [56] recently was conducted at hair cell synapses. Highresolution imaging of these synapses provided insights into presynaptic Ca2+ channel clusters and Ca2+ signals, synaptic CAZs and postsynaptic glutamate receptor clusters, revealing substantial variability for presynaptic Ca2+ signals, even within individual hair cells. Overall, it is also clear that there not one answer but that many answers will have to be derived. This particularly as synapse diversity, often expressed in differences of synaptic short-term plasticity, will also have its grounds in variations of AZ/CAZ organization. Thus, there is still a lot to be done.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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