RIM Function in Short- and Long-Term Synaptic Plasticity

Cellular Information Processing 1345

RIM function in short- and long-term synaptic plasticity

P.S. Kaeser1 andT.C.Sudhof¨ 1 Center for Basic Neuroscience, Department of Molecular Genetics and Howard Hughes Medical Institute, UT Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390-9111, U.S.A.

Abstract Downloaded from https://portlandpress.com/biochemsoctrans/article-pdf/33/6/1345/540253/bst0331345.pdf by Harvard University user on 02 September 2020 RIM1α (Rab3-interacting molecule 1α) is a large multidomain protein that is localized to presynaptic active zones [Wang, Okamoto, Schmitz, Hofmann and Sudhof¨ (1997) Nature (London) 388, 593–598] and is the founding member of the RIM protein family that also includes RIM2α,2β,2γ ,3γ and 4γ [Wang and Sudhof¨ (2003) Genomics 81, 126–137]. In presynaptic nerve termini, RIM1α interacts with a series of presynaptic proteins, including the synaptic vesicle GTPase Rab3 and the active zone proteins Munc13, liprins and ELKS (a protein rich in glutamate, leucine, lysine and serine). Mouse KOs (knockouts) revealed that, in different types of synapses, RIM1α is essential for different forms of synaptic plasticity. In CA1-region Schaffer- collateral excitatory synapses and in GABAergic synapses (where GABA is γ -aminobutyric acid), RIM1α is required for maintaining normal neurotransmitter release and short-term synaptic plasticity. In contrast, in excitatory CA3-region mossy fibre synapses and cerebellar parallel fibre synapses, RIM1α is necessary for presynaptic long-term, but not short-term, synaptic plasticity. In these synapses, the function of RIM1α in presynaptic long-term plasticity depends, at least in part, on phosphorylation of RIM1α at a single site, suggesting that RIM1α constitutes a ‘phosphoswitch’ that determines synaptic strength. However, in spite of the progress in understanding RIM1α function, the mechanisms by which RIM1α acts remain unknown. For example, how does phosphorylation regulate RIM1α, what is the relationship of the function of RIM1α in basic release to synaptic plasticity and what is the physiological significance of different forms of RIM-dependent plasticity? Moreover, the roles of other RIM isoforms are unclear. Addressing these important questions will contribute to our view of how neurotransmitter release is regulated at the presynaptic active zone.

Structure and protein interactions of RIMs identical with RIM2α.RIM2γ ,3γ and 4γ consist of only the (Rab3-interacting molecules) C-terminal C2 domain with flanking sequences [6]. Active zones are composed of an electron-dense protein RIMs were originally identified as effectors of Rab3 [8]. α α network that is attached to the presynaptic plasma membrane The N-terminal sequence of RIM1 and 2 (that is absent β γ γ γ at the synaptic cleft of the synapse [1,2]. Active zones perform from RIM2 ,2 ,3 and 4 ) binds to two different synaptic a central function in neurotransmitter release because this is proteins: the synaptic vesicle GTPase Rab3 and another active where synaptic vesicles dock and fuse during release [3,4]. zone protein called Munc13 [4,9,10]. This N-terminal region RIM proteins are scaffolding molecules that are composed includes two nested domains that mediate these two binding α of multiple independently folded domains and are thought activities: an -helical Rab3-binding region and a Munc13- to regulate neurotransmitter release by interacting directly or binding zinc-finger domain. Although in vitro binding essays indirectly with multiple other synaptic proteins (Figure 1a) initially suggested that Rab3 and Munc13 compete with each α [4,5]. other for RIM1 binding [9], recent results indicate that The mammalian genome contains four RIM genes (Rims1- they bind simultaneously [10a]. Since Rab3 regulates synaptic 4 [6,7]) that encode six principal RIM forms (RIM1α,2α, vesicle fusion [11,12] and Munc13 is critical for priming α α 2β,2γ ,3γ and 4γ ; Figures 1b and 1c). RIM1α and 2α are synaptic vesicles for fusion [13], RIM1 and 2 probably composed of an N-terminal zinc finger, a central PDZ domain couple these two proteins functionally. In addition to Munc13 and Rab3, RIMs also bind directly to three other active zone and two C-terminal C2 domains [6,8] that are separated by multiple alternatively spliced sequences. RIM2β (which, like proteins and one other synaptic vesicle protein. First, the PDZ α α β RIM2γ , is expressed from an internal promoter in the Rims2 domains of RIM1 ,2 and 2 bind to ELKS proteins [named gene) lacks the N-terminal zinc-finger region but is otherwise after the amino acids glutamate, leucine, lysine and serine in which it is rich, and also referred to as Rab6-interacting

Key words: ELKS, long-term potentiation (LPT), mossy-ﬁbre synapse, Rab3A, Rab3-interacting protein or CAST, and also abbreviated as ERC (ELKS/Rab6- molecule function (RIM function), synaptic plasticity. interacting protein/CAST)] [14]. In mammals, two ELKS Abbreviations used: GABA, γ -aminobutyric acid; KO, knockout; LTP, long-term potentiation; genes produce three different ELKS proteins (ELKS1A, 1B NMDA, N-methyl-d-aspartate; PKA, protein kinase A; RIM, Rab3-interacting molecule. 1 Correspondence may be addressed to either of the authors (email pascal.kaeser@ and 2B), of which ELKS1B and 2 are active zone proteins utsouthwestern.edu or [email protected]). [14–16]. Secondly, RIMs bind via their C-terminal C2B

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Figure 1 Domain structure and genetic organization of RIM Figure 2 RIM function in excitatory short-term plasticity proteins (a) Model of the unidirectional hippocampal pathways which were used (a) Domain structure of the RIMα isoform showing RIM’s interaction to analyse several forms of synaptic plasticity in RIM1α KO mice (EC, partners and sites of alternative splicing (SSA, SSB and SSC; splice sites A, entorhinal cortex; PP, perforant path; DG, dentate gyrus; MF, mossy BandC;Zn2+, zinc-finger domain; PxxP, proline-rich region). (b) Domain fibres; AC, associational commissural pathway; SC, Schaffer-collateral structure of RIM isoforms. (c) Genetic representation of RIM proteins. pathway; SB, subiculum). (b, c) Increased paired-pulse facilitation in ERCs, ELKS/Rab6-interacting protein/CAST; RIM-BP, RIM-binding pro- RIM1α and Rab3A KO mice compared with littermate wild-type controls tein; SNAP-25, 25 kDa synaptosome-associated protein. (adapted from [4] with permission. c 2002 Nature Publishing Group; http://www.nature.com). fEPSP, field excitatory post-synaptic potential; ISI, interstimulus internal; WT, wild-type. Downloaded from https://portlandpress.com/biochemsoctrans/article-pdf/33/6/1345/540253/bst0331345.pdf by Harvard University user on 02 September 2020

domain to α-liprins [4]. The functions of mammalian liprins have not been addressed, but in Caenorhabditis elegans and Drosophila, liprins regulate the formation, differentiation and maintenance of synaptic termini [17,18]. Thirdly, RIMs bind via a proline-rich region to the SH3 domain (Src homo- logy 3 domain) protein RIM-BP (RIM-binding protein), which in turn may bind to voltage-gated Ca2+ channels [19].

Fourthly, RIMs interact, via their C-terminal C2B domain, with synaptotagmin 1 in a Ca2+-dependent manner, although the validity of this interaction has not been established. In addition to these interactions, RIMs bind several other pro- synapses in the cerebellum [27,28], corticostriatal synapses teins in vitro, including SNAP-25 (25 kDa synaptosome- [29] and corticothalamic synapses [30] undergo LTP (long- 2+ associated protein) [20], N-type Ca channels [20], cAMP- term potentiation) or LTD (long-term depression) when GEFII (guanine nucleotide-exchange factor II) [21] and presynaptic PKA (protein kinase A) is activated. PKA- 14-3-3 adaptor proteins (P.S. Kaeser and T.C. Sudhof,¨ dependent presynaptic long-term plasticity in mossy fibre unpublished work) [22,23]. However, not all of these inter- synapses is abolished in RIM1α and in Rab3A KO mice actions have been reproducible, and their significance is (Rab3A is one of the Rab3 proteins with which RIM1α unclear at present. Finally, RIMs interact indirectly with other interacts; Figures 3a and 3b). At the same time, the basic synaptic proteins, for example with piccolo and bassoon properties of synaptic transmission, including short-term via ELKS [24], and with receptor-tyrosine phosphatases via synaptic plasticity, appear to be unchanged in these synapses liprins [17,25]. [31–33]. In contrast with synapses capable of PKA-dependent presynaptic LTP,in other excitatory synapses such as the CA3 RIM proteins and synaptic plasticity to CA1 Schaffer-collateral synapses in the hippocampus that RIM1α KO (knockout) mice exhibit severe changes in are incapable of PKA-dependent LTP (Figure 2a), deletion synaptic transmission, whose nature varies dramatically with of RIM1α and of Rab3 proteins caused impairments in basic different types of synapses. A subset of central excita- release properties and in short-term plasticity (Figures 2b tory synapses, such as mossy fibre synapses in the CA3 and 2c) [4,34]. In these synapses, RIM1α KO mice have region of the hippocampus [26] (Figure 2a), parallel fibre a more severe phenotype than Rab3 KO mice even when

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Figure 3 RIM function in excitatory long-term plasticity (a, b) Mossy ﬁbre LTP is abolished in Rab3A and RIM1α KO mice (adapted from [31,32] with permission. c 1997 and 2002 Nature Publishing Group; http://www.nature.com). (c) In mixed cerebellar cultures, the cAMP analogue Sp-8-CPT-cAMP-S [Sp-8-(4-chlorophenylthio)-cAMP-S] triggers a post-synaptic potentiation that is absent from cultured neurons from RIM1α KO animals. This potentiation can be reintroduced by rescuing the KO cultures with wild-type RIM1α. EPSC, excitatory post-synaptic current. Adapted from Cell 115; Lonart, G., Schoch, S., Kaeser, P.S., Larkin, C.J., Sudhof, ¨ T.C. and Linden, D.J. ‘Phos- phorylation of RIM1α by PKA triggers presynaptic long-term potentiation at cerebellar parallel ﬁber synapses.’, pp. 49–60, c 2003 with permission from Elsevier. (d) A current model of how RIM1α integrates Rab3A and PKA signalling by regulating presynaptic LTP. Downloaded from https://portlandpress.com/biochemsoctrans/article-pdf/33/6/1345/540253/bst0331345.pdf by Harvard University user on 02 September 2020

all four Rab3 isoforms are deleted [12], consistent with the either Rab3A or RIM1α (Figure 3c). Direct phosphorylation notion that RIM1α is an effector of Rab3 but performs experiments showed that Rab3A is not a substrate for PKA additional functions beyond acting as a downstream effector [33], but RIM1α is phosphorylated at least at two sites: for Rab3. RIM1α is not required for the induction of classical Ser413, which is in between the zinc-finger domain and NMDA (N-methyl-D-aspartate) receptor-dependent LTP in the PDZ domain, and Ser1548, which is at the very C-ter- Schaffer-collateral synapses, but recently was found to be minus [36]. Identification of these sites allowed testing of essential for ‘late’ NMDA receptor-dependent LTP in these whether phosphorylation of one or both of these sites is synapses [35]. Late LTPis maintained beyond 2 h and depends essential for presynaptic PKA-dependent LTP. Using rescue on PKA activation, similar to presynaptic PKA-dependent experiments in cultured cerebellar RIM1α-deficient neurons, LTP. Thus, although late LTP is initially induced in a post- it was found that serine phosphorylation at Ser413 is essential synaptic NMDA receptor-dependent manner that does not for presynaptic LTP and, moreover, that mutant RIM1α con- require RIM1α, its maintenance may involve presynaptic taining a serine-to-alanine substitution at residue 413 inhibits PKA activation and RIM1α similar to presynaptic LTP. This presynaptic LTP when introduced into wild-type cultured extension of RIM function beyond presynaptically induced cerebellar neurons [36]. These observations suggest that forms of synaptic plasticity suggests that RIM proteins may parallel fibre LTP – and by extension, mossy fibre LTP be involved in more general mechanisms to change the pre- and other long-term forms of PKA-dependent presynaptic synaptic release machinery, probably other forms of long- plasticity – is regulated by the cAMP/PKA pathway at a term plasticity that require post-synaptic induction with single phosphorylation site: Ser413 of RIM1α. It suggests presynaptic feedback mechanisms. a model whereby signalling by Rab3A and PKA converges How does PKA trigger long-term plasticity in synapses on to RIM1α during presynaptic LTP (Figure 3d). such as mossy fibre synapses in the hippocampus or parallel With RIM1α, we now have a molecular handle on PKA- fibre synapses in the cerebellum? A potential avenue to ad- dependent long-term plasticity. However, why does this dressing this question is to test whether PKA might act via form of plasticity not operate in synapses such as

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Schaffer-collateral synapses? And why does the RIM1α the same time, tests of motor co-ordination using the rotorod deletion so dramatically alter synaptic strength and short- assay did not uncover an abnormality, indicating that the term synaptic plasticity in Schaffer-collateral synapses, but RIM1α KO mice did not simply show incapacitated nervous not in synapses competent for PKA-dependent presynaptic system function. Moreover, although some of the behavioural LTP? RIM1α is clearly present, and also phosphorylated on changes in RIM1α KO mice are compatible with a deficit of Ser413, in Schaffer-collateral synapses, indicating that PKA PKA-dependent LTP, this loss of synaptic plasticity by itself may be active in these termini. Several hypotheses may be probably does not explain these behavioural changes because advanced that answer these questions. First, it is possible Rab3A KO mice that also lack presynaptic PKA-dependent that, in Schaffer-collateral synapses, RIM1α is constitutively LTP (at least in hippocampal mossy fibre synapses) did not phosphorylated on Ser413, maybe via other kinases, such exhibit any behavioural change. Thus, at present, it is clear

that RIM1α is always ‘turned on’. This would explain that RIM1α function has a profound role in animal behaviour, Downloaded from https://portlandpress.com/biochemsoctrans/article-pdf/33/6/1345/540253/bst0331345.pdf by Harvard University user on 02 September 2020 why the RIM1α KO diminishes synaptic strength and why but the precise role of different facets of RIM1α function in changing PKA activity does not lead to changes in synaptic behaviour remains to be explored. strength. Alternatively, it is also possible that the downstream effectors for phospho-RIM1α are different in these synapses, Summary and outlook 413 resulting in a situation whereby phosphorylation of Ser is The currently available data establish that RIM1α is an regulatory in all synapses, but the endpoints of regulation active zone protein with a central function in regulating are different. A third possibility is based on the fact that, up neurotransmitter release and furthermore suggest that other to now, the functions of other RIM isoforms have not been RIM isoforms may also act in regulating neurotransmitter α considered. It is thus possible that RIM1 is co-expressed release. Together with work on other active zone proteins with distinct additional RIM isoforms in the various such as Munc13 and bassoon [13,24,39–41], a picture emerges synapses and that the pattern of RIM expression deter- whereby the protein network that makes up the active α mines the contribution of RIM1 to overall synaptic strength zone performs two basic functions: first, it organizes the 413 and plasticity. In this regard, it is noteworthy that the Ser docking and priming of synaptic vesicles for exocytosis. α α phosphorylation site of RIM1 is conserved in RIM2 Secondly, it mediates use-dependent changes in release during β and 2 , suggesting that PKA might operate in synaptic short- and long-term synaptic plasticity. RIMs are clearly plasticity on multiple RIMs depending on which RIMs are central components of this protein network and may in expressed. To differentiate between these possibilities, it fact be like the proverbial spider in the net in that RIMs will be necessary to test the effect of deleting other RIM interact with most other active zone proteins directly or isoforms on synaptic transmission and of mutating the phos- indirectly. However, it is also clear that research on RIMs α phorylation site of RIM1 in vivo. Moreover, it will be has only started; major questions remain to be addressed important to understand the biochemical properties of differ- before even a rudimentary understanding can be claimed. ent RIM isoforms and, in particular, the molecular con- These questions can be grouped into two classes. First, what sequences of RIM phosphorylation. are the fundamental mechanisms by which RIMs act? For α Finally, RIM1 is not only involved in excitatory but example, how does RIM1α mediate presynaptic LTP and how also in inhibitory synapses. Whereas regulation of synaptic does it work in short-term plasticity? Secondly, what is the release at excitatory synapses has been intensively studied, physiological significance of RIM functions? For example, markedly less is known about GABAergic inhibitory what is the importance of RIM1α’s role in presynaptic LTP γ synapses (where GABA is -aminobutyric acid). Generally, and what do other RIM isoforms do? Addressing these ques- inhibitory synapses have a high release probability and tions will provide a fundamental insight into how synapses express paired-pulse depression instead of paired-pulse work and how their use-dependent plasticity shapes syn- facilitation when two action potentials are applied with a aptic networks. short interpulse interval [37]. Unexpectedly, RIM1α KO mice exhibit increased paired-pulse depression [4], indicating that References the RIM1α deletion may increase the release probability at 1 Akert, K. and Tokunaga, A. (1979) Zh. Evol. Biokhim. Fiziol. 15, GABAergic synapses, opposite to the decrease in release 263–267 2 Harlow, M.L., Ress, D., Stoschek, A., Marshall, R.M. and McMahan, U.J. probability at excitatory synapses. 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