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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell. Author manuscript; available in PMC Mar 7, 2011.
Published in final edited form as:
PMCID: PMC3049808
NIHMSID: NIHMS272642

SCRAPPER-Dependent Ubiquitination of Active Zone Protein RIM1 Regulates Synaptic Vesicle Release

SUMMARY

Little is known about how synaptic activity is modulated in the central nervous system. We have identified SCRAPPER, a synapse-localized E3 ubiquitin ligase, which regulates neural transmission. SCRAPPER directly binds and ubiquitinates RIM1, a modulator of presynaptic plasticity. In neurons from Scrapper-knockout (SCR-KO) mice, RIM1 had a longer half-life with significant reduction in ubiquitination, indicating that SCRAPPER is the predominant ubiquitin ligase that mediates RIM1 degradation. As anticipated in a RIM1 degradation defect mutant, SCR-KO mice displayed altered electrophysiological synaptic activity, i.e., increased frequency of miniature excitatory postsynaptic currents. This phenotype of SCR-KO mice was phenocopied by RIM1 overexpression and could be rescued by re-expression of SCRAPPER or knockdown of RIM1. The acute effects of proteasome inhibitors, such as upregulation of RIM1 and the release probability, were blocked by the impairment of SCRAPPER. Thus, SCRAPPER has an essential function in regulating proteasome-mediated degradation of RIM1 required for synaptic tuning.

INTRODUCTION

During neuronal communication, synaptic vesicles dock and fuse with the plasma membrane of the presynaptic (transmitting) neuron at sites called “active zones.” Subsequently, neurotransmitters released into the extra-cellular synaptic space can bind to cell surface receptors located at sites on the postsynaptic (receiving) cell called “postsynaptic densities.” Both of these specialized intracellular sites contain complexes of scaffolding proteins, neurotransmitter-releasing machinery, receptors, ion channels, and signaling molecules that facilitate synaptic transmission and subsequent signal transduction (Hata and Takai, 1999; Sudhof, 2004; Yao et al., 1999). Modulation of the activity of such protein complexes is important for control of synaptic plasticity. It is not yet fully understood how the activity of these synaptic proteins is regulated, but this sophisticated process includes control at the level of transcription (Bito et al., 1996), translation (Kosik, 2006), and translocation (Ikegami et al., 2007; Matsumoto et al., 2007; Setou et al., 2000, 2002).

Recently, protein degradation has attracted attention as a mechanism to control the level of synaptic proteins. Protein degradation mediated by the ubiquitin-proteasome system (UPS) (Coux et al., 1996; Hershko and Ciechanover, 1998; Varshavsky, 2005) functions in a variety of cellular processes (Pickart, 2001; Varshavsky, 2005). Target proteins are tagged with polyubiquitin via UPS enzymes and then degraded in the proteasome. By controlling the stability, activity, and localization of synaptic proteins, UPS provides an additional mechanism for control of synaptic function. For example, UPS machinery can modulate the level of synaptic proteins such as Vesl-1S/Homer-1a (Ageta et al., 2001), serum-inducible kinase (SNK) (Pak and Sheng, 2003), anaplastic lymphoma kinase (ALK) (Liao et al., 2004), synaptophysin (Wheeler et al., 2002), and syntaxin1 (Chin et al., 2002). Furthermore, it has been suggested that activity-dependent regulation of synaptic function in vivo can be regulated by UPS at both pre- and postsynapses (Ehlers, 2003; Yi and Ehlers, 2005). Indeed, optical analysis of synaptic vesicles indicates that inhibition of proteasome activity triggers a presynaptic modulation in cultured hippocampal neurons (Willeumier et al., 2006). However, the molecular mechanisms whereby UPS regulates synaptic transmission in vivo are unknown.

We have identified SCRAPPER, an ubiquitin ligase found in mammalian CNS synapses and have analyzed its function in synaptic transmission. SCRAPPER directly binds to and ubiquitinates the active zone protein Rab3-interacting molecule 1 (RIM1) in vitro and in vivo. Analysis of mice mutant for SCRAPPER demonstrates that SCRAPPER-dependent UPS contributes to the regulation of synaptic vesicle release probability via RIM1.

RESULTS

SCRAPPER Is a Neural E3 Ubiquitin Ligase Localized on Presynaptic Membrane

Protein ubiquitination involves three classes of enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-protein ligases (E3). Specificity in ubiquitination is often conferred by E3 enzymes due to their high-substrate specificity. We hypothesized that an E3 capable of regulating synaptic function would be membrane bound and would be expressed in neurons. To test this hypothesis we screened the human genome for genes whose coding sequence contained an F box domain (characteristic of E3 ligases), a membrane-targeting sequence, and whose promoter region contained both a neuron-restrictive silencing element and a cAMP-response element (CRE) within 3 kb upstream of exon 1. Only one gene was found with all of these properties. We cloned a full-length cDNA for the mouse ortholog and named the encoded protein “SCRAPPER.” SCRAPPER is a 438 amino acid protein that contains an F box, leucine-rich repeats (LRR), and a CAAX domain. The CAAX domain is a carboxyl-terminal membrane-sorting signal induced by prenylation (Zhang and Casey, 1996).

We verified that SCRAPPER has these properties in vivo. Cyclic-AMP responsive expression of Scrapper mRNA was observed in primary culture of hippocampal neurons 1 hr following induction by forskolin (a cAMP signaling activator) (Figure S1). Western analysis of levels of SCRAPPER in the mouse brain revealed a gradual increase with age, from midgestation to adult (Figure S2A). In adult mice, highest levels of SCRAPPER were observed in the brain (Figure 1A), where it appeared evenly distributed (Figure S2B). Analysis using in situ hybridization (Figure 1B) and immunohistochemistry (Figure 1C) revealed that Scrapper mRNA and protein were enriched in the CA1, CA3, and dentate gyrus regions of the hippocampus, as well as in the cerebellum and olfactory bulb. Subcellularly, SCRAPPER was enriched in synaptic membrane fractions from the mouse brain (Figure 1D). Immunofluorescence analysis revealed a punctate distribution of SCRAPPER (Figure 1F) that predominantly colocalized with synaptophysin, a known synaptic vesicle protein and presynapse marker. GFP-tagged full-length SCRAPPER (GFP-SCR) was distributed in a punctate manner similar to endogenous SCRAPPER (Figures 1E and 1F). In contrast, GFP-SCR-C435A, which carries a mutation in the CAAX motif, had more diffuse distribution than those observed for wild-type GFP-SCR and -SCR-CAAX. This indicates that the carboxyl-terminal prenylation signal is important for the distinct localization of SCRAPPER. GFP-SCR-LRR, which we expected to have a dominant-negative effect due to presence of the LRR target protein-binding domain, but without the F box or CAAX domains, also displayed a diffuse subcellular distribution (Figure 1F).

Figure 1
SCRAPPER Is a Neuronal E3 Ligase

SCRAPPER Acts as an E3 Ubiquitin Ligase for RIM1 In Vitro

F box-containing proteins are a component of the multisubunit RING-finger type SCF complex (Cardozo and Pagano, 2004), which acts as an E3 enzyme. Similarly, SCRAPPER formed an SCF complex with Skp1 and Cullin1 in mouse brain lysates (Figure 2A). Consistent with a function as a ubiquitin ligase, ubiquitinated proteins coimmunoprecipitated (co-IPed) with FLAG-tagged SCRAPPER (FLAG-SCR) from lysates of MG132-treated (a proteasome inhibitor) cells (Figure 2B). These ubiquitinated proteins may include target proteins and/or autoubiquitinated SCRAPPER. Due to the colocalization of SCRAPPER with synaptophysin (Figure 1F), we screened the SCRAPPER IPs for known presynaptic proteins (Figure 2C). RIM1, a Ca2+-dependent synaptic vesicle-priming factor in the active zone that is required for synaptic plasticity (Wang et al., 1997), co-IPd with SCRAPPER (Figure 2C). SCRAPPER partially colocalized with RIM1 in cultured neurons (Figure 2D). Purified recombinant SCRAPPER and C2B domain of RIM1 directly interacted in an in vitro pull-down assay (Figures 2E and S3). Furthermore, in IPs of native SCRAPPER from the mouse brain, RIM1 was shifted to a higher molecular weight after the addition of a ubiquitination priming mixture (E1, E2, ubiquitin, and a NEDD8 system [Kawakami et al., 2001]) (Figure 2F). Thus, the SCRAPPER complex was sufficient to mediate ubiquitination of RIM1 in vitro.

Figure 2
SCRAPPER Acts as an E3 Ligase for RIM1 In Vitro

SCR-KO Mice Have Deficiency in RIM1 Ubiquitination, Prolonged Half-Life of RIM1, and Increased Steady-State Levels of RIM1

To investigate the physiological function of SCRAPPER, we generated Scrapper-knockout (SCR-KO)- and Scrapper-transgenic (SCR-TG) mice in which the expression of SCRAPPER was either abolished in all tissues or enhanced within the hippocampus, respectively (Figures 3A and 3B). No overt physiological difference was observed between SCR-TG and non-TG mice (Figures 3C and 3D, right). In contrast, the genotypes of offspring from intercross of SCR-KO heterozygous parents did not conform to a Mendelian ratio (29% wild-type [WT], 52% heterozygotes, and 19% homozygotes at birth, p < 0.01, χ2 goodness-of-fit t test; Figure 3C). SCR-KO progeny also died stochastically after birth and had reduced lifespan (Figure 3C) and smaller body size (Figures 3D [left], S4A, and S4B) compared to WT littermates. Necropsy of homozygous SCR-KO mice was unremarkable except for a smaller pancreas (Figure S4D).

Figure 3
SCRAPPER-Dependent UPS Ubiquitinates RIM1 In Vivo

We used SCR-KO mice to investigate SCRAPPER-dependent RIM1 ubiquitination in vivo. In hippocampal acute slices prepared from WT mice, the steady-state level of RIM1 was increased by treatment (50 µM, 1 hr) with MG132 (Figure 3E). Consistent with a role for SCRAPPER in facilitating degradation of RIM1, untreated brains of SCR-KO mice displayed increased levels of RIM1, which increased further following treatment with MG132, although the relative increase was smaller than that observed after MG132 treatment in WT mice (Figures 3E and 3F). In addition, in MG132-treated IPs from WT mice, RIM1 was shifted to a higher molecular weight, whereas this was not detected in similarly treated IPs from SCR-KO mice (see “RIM1 IP” in Figure 3E). These results indicate that SCRAPPER is the main E3 ligase for RIM1 ubiquitination in a short time window in vivo. Consistent with these findings, the half-life of RIM1 was 0.7 ± 0.1 hr in WT neurons and 5.0 ± 0.1 hr in neurons from SCR-KO mice (Figures 3G, 3H, and S5), confirming that SCRAPPER enhances the rate of turnover of RIM1. In contrast, there was no significant difference in the half-life of β-catenin, an additional synaptic protein (5.4 ± 0.3 hr, WT; 5.0 ± 0.1 hr SCR-KO; Figure S5).

SCRAPPER Regulates the Synaptic Level of RIM1 In Vivo

As the steady-state level of RIM1 was increased in SCR-KO mice, we investigated whether a supraphysiological level of SCRAPPER was sufficient to mediate a decrease in levels of RIM1 in vivo. To do so we performed western analyses of brain lysates from each SCR-TG mouse line (TG-22, TG-26, and TG-31), SCR-KO, and WT mice. Indeed, an increased steady-state level of SCRAPPER produced a reduction in the level of RIM1 as well as several presynaptic proteins, including synaptophysin and synapsin IIa in vivo (Figure 4A). The level of mRNA for presynaptic proteins such as RIM1, synaptotagmin, and SNAP-25 were unchanged in SCR-TG, SCR-KO, and WT mice (Figure S6). In conventional two-dimensional polyacrylamide gel electrophoresis (2D) analyses, almost all brain proteins in SCR-KO and in SCR-TG (data not shown) were unchanged compared with those of WT animals. Thus, the changes of RIM1 protein level in the SCR-KO brain were specific.

Figure 4
Inverse Relationship between Steady-State Levels of SCRAPPER and a Subset of Presynaptic Proteins In Vivo

Immunofluorescence analyses of SCR-KO hippocampi revealed that the increased level of RIM1 occurred in the synaptic region, not in the cell body (Figures 4B, 4C, and S7). Conversely, the intensity of RIM-specific fluorescence in the synaptic region was reduced in SCR-TG hippocampi relative to that of WT (Figures 4B, 4C, and S7). In parallel, we assessed the distribution and number of synapses of the SCR-KO mice. No significant difference was observed in the number of synapses per neurite length (Figure 4G) between WT and SCR-KO neurons. Analysis of the hippocampal CA1 region by transmission electron microscopy (TEM) revealed an increased local density and fewer docked synaptic vesicles in SCR-KO neurons (Figures 4D–4F), although the total number of synaptic vesicles was unchanged (Figure 4F). The number of synapses and the sizes of the active zones were also similar in WT and SCR-KO mice (Figure 4F).

SCRAPPER Is a Regulator of Presynaptic Vesicle Release

As RIM1 is known to regulate synaptic transmission (Wang et al., 1997), we investigated whether altered levels of RIM1 and morphological changes of synaptic vesicles in SCR-KO mice was associated with altered neural transmission. We analyzed AMPA-receptor-mediated miniature excitatory postsynaptic currents (mEPSC) from SCR-KO hippocampal primary culture (Inoue et al., 2006). The mEPSC frequency was increased in neurons from SCR-KO mice compared to WT littermates, and the increment was corrected by exogenous re-expression of SCRAPPER (WT; 0.91 ± 0.23 Hz, n = 15, SCR-KO; 3.05 ± 1.12 Hz, n = 14, SCR-KO-rescue; 0.98 ± 0.27 Hz, n = 10) (Figures 5A and 5B). Thus, SCRAPPER plays a significant role in regulation of neurotransmitter release.

Figure 5
SCRAPPER-Mediated UPS Functions in Presynaptic Transmission

To further investigate the functions of SCRAPPER in synaptic transmission via RIM1 ubiquitination, we expressed various forms of GFP-SCRAPPER (Figure 1E) or nontagged red fluorescent protein (RFP) in primary hippocampal neurons (Figure 5C). To determine if SCRAPPER was acting at the pre- or postsynaptic site, we cocultured neurons expressing the various GFP-SCR constructs with neurons expressing only nontagged RFP and recorded mEPSCs. Neurons transfected with GFP-SCR exhibited significant decrease in frequency but not amplitude of mEPSC, whereas the expression of either GFP-SCR-C435A (CAAX mutation) or GFP-SCR-CAAX, which lacks the RIM1 binding domain, had no significant effect on the frequency and amplitude of mEPSC (Figures 5D and 5E). In contrast, neurons transfected with GFP-SCR-LRR, in which we expected a dominant-negative effect caused by binding of the LRR to RIM1, exhibited increased mEPSC frequency but not amplitude (Figures 5D and 5E). Recording of mEPSC from RFP (i.e., non-SCR)-transfected cells in the mixed culture showed the same significant change in mEPSC, demonstrating that the SCRAPPER-dependent effect on neurotransmitter release was generated at the presynaptic site.

Because RIM1 is a known component of a Ca2+ sensor (Sudhof, 2004), we tested the Ca2+ sensitivity of neurons expressing altered forms of SCRAPPER. The effect of expression of SCR forms on modulating mEPSC frequency was observed in the presence of 5 mM but not 10 mM or 20 mM extracellular Ca2+ (Figure 5F). Similar results were also observed in the case of evoked-field EPSP (fEPSP) at the hippocampal acute slice preparations (Figures S8A–S8C). We verified that the intracellular Ca2+ level of SCR-KO neurons was within the normal range in assays by using Fura2 or a FRET-based Ca2+ indicator (Miyawaki et al., 1999) (Figures S8D and S8E). These results indicate that SCRAPPER can regulate the Ca2+ sensitivity in presynaptic machinery.

SCRAPPER Regulates Synaptic Vesicle Release via RIM1 and Proteasome Activity

To determine if the altered mEPSC frequency in SCR-KO neurons is mediated specifically via RIM1, we knocked down expression of RIM1 in SCR-KO neurons and analyzed if this was sufficient to rescue the SCR-KO phenotype. Indeed, reduction of RIM1 in SCR-KO neurons was sufficient to rescue the increased frequency of mEPSC (Figures 6A–6C). These results demonstrate that mEPSC frequency can be regulated via SCRAPPER-mediated RIM1 degradation. Overexpression of RIM1 promoted neurotransmitter release (Figures 6D and 6E), which mimicked the SCR-KO phenotype, indicating that the increased RIM1 in SCR-KO mice is sufficient to account for the mEPSC phenotype.

Figure 6
SCRAPPER Tunes Synaptic Transmission via Regulation of RIM1

To evaluate the relative contribution of SCRAPPER to UPS-mediated degradation of proteins in neurons, we recorded mEPSC from SCR-KO in the hippocampal CA1 pyramidal neurons in acute slices and analyzed the effect of treatment with MG132 or epoxomicin, another proteasome inhibitor. Significantly, the effect of proteasome inhibitors on mEPSC frequency in acute slices prepared from WT mice was mostly abolished in samples from SCR-KO mice (2.1-fold in WT to 1.2-fold in SCR-KO [MG132 treatment]; 1.9-fold in WT to 1.2-fold in SCR-KO [epoxomicin treatment]; Figure 6H). In contrast, MG132 had no effect on amplitude of mEPSC between SCR-KO and WT mice (Figures 6F–6H).

We investigated the SCRAPPER-proteasome effect on mEPSC not only in acute slices but also in primary cultures of dissociated neurons. When we applied MG132 (50 µM) to primary cultured hippocampal neurons, the frequency and amplitude of mEPSC was increased within 60 min (Figure S9). In contrast, neither amplitude nor frequency of mEPSC was altered following treatment of neurons with the calpain inhibitor ALLM (Figure S9). The mEPSC upregulated by MG132 was completely suppressed under extracellular Ca2+-free conditions and was diminished at higher Ca2+ conditions (Figures 6I and 6J). The increase in mEPSC frequency by proteasome inhibitor and the poor response to MG132 in SCR-KO neurons were also demonstrated in the dissociation cultures (7.2-fold in WT to 2.1-fold in SCR-KO, Figure S10).

Altered Short-Term Synaptic Plasticity in SCR-KO Mice

RIM1 mutant mice have increased paired-pulse facilitation (PPF) (Schoch et al., 2002), which is a form of short-term synaptic plasticity (STP) (Katz and Miledi, 1968). Thus, we predicted the PPF in SCR-KO would be affected. We analyzed PPF from the CA3-CA1 synapse of the hippocampal acute slice preparation (Figure 7). The PPF ratio was significantly reduced in SCR-KO mice at every stimulation interval (50, 100, 200, 300, 400, and 500 ms) tested (Figures 7A and 7B). Furthermore, a gradual increase in fEPSP slope and decrease in PPF ratio was observed during treatment of neurons with 50 µM of MG132 for 20 min, and this effect became saturated after 1 hr in both WT and SCR-KO mice (Figures 7C–7E). The effect of exposure to MG132 on fEPSP slope was significantly smaller in SCR-KO mice than in WT mice (normalized fEPSP slope after 1 hr MG132 treatment; 1.31 ± 0.05 in WT, n = 7, versus 1.14 ± 0.04 in KO, n = 7). As with alteration of the mEPSC frequency by MG132 in SCR-KO neurons, the effect of MG132 on the PPF ratio was significantly smaller in SCR-KO mice (i.e., a change from 1.61 ± 0.06 at −10 to 0 min before the application of MG132 to 1.36 ± 0.08 after 1 hr of MG132 treatment, n = 7) compared to WT mice (1.94 ± 0.05 at −10 to 0 min before the application of MG132 to 1.41 ± 0.10 after 1 hr of MG132 treatment, n = 7) (Figures 7D and 7E). These results demonstrate that SCRAPPER can regulate presynaptic STP.

Figure 7
SCRAPPER Functions in Short-Term Synaptic Plasticity

DISCUSSION

SCRAPPER Is an E3 Ligase on Synaptic Membranes

We used bioinformatics to identify SCRAPPER, a neuronal and membranous ubiquitin ligase. SCRAPPER was the only protein identified by our strategy to screen for F box containing proteins that could be membrane localized and whose expression is predicted in neurons. Among the 68 F box protein-coding genes in the human genome (Jin et al., 2004; Winston et al., 1999), SCRAPPER is one of six independent genes that have orthologs in C. elegans, D. melanogaster, and mammals (data not shown), which suggests that it might function as an important membrane-localized E3 ligase in various species. SCRAPPER is broadly expressed within the mouse CNS and is abundant at the presynaptic membrane. Many E3s have been identified whose activities are localized to specific subcellular compartments such as nuclei or cytoplasm, for the regulation of transcription or cell cycles (Coux et al., 1996; Hershko and Ciechanover, 1998).

RIM1 Is a Target of SCRAPPER

Experimentally, SCRAPPER behaves as an F box type E3 ligase and RIM1 is a target of SCRAPPER in the mouse brain. Under normal circumstances, UPS-targeted multiubiquitinated RIM1 are rarely detected due to their rapid turnover. This may account for the relatively weak signal observed of coIPed RIM1. Consistent with this prediction, the RIM1-specific signal was shifted upward after in vitro ubiquitination. At present, we cannot exclude the existence of additional SCRAPPER targets in the synapses. Indeed, many E3 enzymes recognize several substrates as a target (Hatanaka et al., 2006b; Ingham et al., 2004).

RIM1 plays an important role in the vesicle priming step in the active zone of the presynapse (Betz et al., 2001; Kaeser and Sudhof, 2005). Recently, we reported that SAD kinase, which can phosphorylate RIM1, is expressed at presynapses and can regulate synaptic transmission (Inoue et al., 2006). Among the F box protein family, the binding of some LRR-type F box proteins to substrates can be influenced by phosphorylation (Hsiung et al., 2001). Because SCRAPPER may recognize the phosphorylation of the substrate as predicted from the leucine-rich sequence, it is possible that SCRAPPER and SAD cooperatively regulate synaptic transmission and plasticity via modulation of RIM1.

Reduction of RIM1 Ubiquitination and Increased Levels of RIM1 in Presynapses in SCR-KO Mice

Analysis of SCR-KO mice revealed that SCRAPPER regulates steady-state level of RIM1. RIM1 degradation can also be controlled via SCRAPPER-independent mechanisms as treatment with MG132, a proteasome inhibitor, produced a further increase in levels of RIM1 in SCR-KO mice. However, the majority of RIM1 degradation appears to be SCRAPPER dependent. This conclusion is supported by the fact that the lifetime of RIM1 was seven times greater in neurons from SCR-KO compared to WT mice. Interestingly, the levels of several presynaptic-localized proteins such as synaptotagmin were also inversely proportional to steady-state level of SCRAPPER in vivo, although the mRNA levels of these presynaptic proteins were unchanged. A 2D analysis indicated that relatively few proteins were affected by the absence of SCRAPPER. This suggests that stabilization of RIM complex proteins in SCR-KO mice impacts relatively few proteins.

Although the sizes of the active zones were unchanged in SCR-KO mice, we found several presynaptic morphological phenotypes such as an increased density of synaptic vesicles and reduced number of docked vesicles. How increased RIM1 generates these morphological phenotypes is not immediately apparent as the synaptic morphology in RIM1-mutant mice does not involve alteration in localization or density of synaptic vesicles (Schoch et al., 2002). The multidomain structure of RIM1 complicates reconciliation of phenotypes in gain- and loss-of-function mice. It is possible that altered expression of presynaptic proteins other than RIM1 contribute to the altered synaptic morphology in SCR KO.

We found no difference in the number and basic structure of synapses in either cultured neurons or hippocampal CA1 region in SCR-KO and WT mice, suggesting that SCRAPPER had no overt effect on synapse development. In contrast, other neural E3 ligase such as Drosophila Highwire, the C. elegans homolog RPM-1, and the mammalian proteins Phr1 and Pam, constitute a conserved family of proteins, all of which influence synapse development (DiAntonio et al., 2001; McCabe et al., 2004). Additional, as yet unknown E3 ligases may exist that can regulate synaptic development in mammals.

SCRAPPER Is an Important Regulator of Synaptic Transmission

Electrophysiological analyses verified that SCRAPPER can regulate synaptic transmission, especially neurotransmitter release. Recording of mEPSC frequency in mixtures of neurons expressing either nontagged RFP or GFP displayed no significant difference between cells. In contrast, changes observed when recording from nontagged RFP-positive cells in the presence of GFP-positive neurons transfected with different GFP-SCR constructs were interpreted as an effect generated in the presynaptic (green) cell. Use of this strategy (suggested by a reviewer) enabled us to clarify the importance of SCRAPPER function at the presynaptic site. The synapses of neurons expressing elevated levels of SCRAPPER displayed a lower mEPSC frequency via lower Ca2+ sensitivity. In contrast, neurons expressing SCR-LRR (RIM1-binding domain) showed a higher Ca2+ sensitivity, most likely as a consequence of the dominant-negative effect of this protein. This indicates that SCRAPPER can regulate neurotransmitter release in a LRR domain-dependent manner. This effect was not significantly observed in cells overexpressing SCR-CAAX. Cells expressing SCR-C435A, where the cysteine in the canonical CAAX prenylation motif is replaced by alanine, displayed an intermediate reduction in the frequency of mEPSC, though this was a trend and not statistically significant. One possible explanation why mutation of the cysteine residue diminished SCR activity only moderately is this sequence was able to target SCR to membranes with reduced efficiency. Further experiments are required to discriminate between this and other possibilities. It is possible that the increased mEPSC frequency reduced the number of docked vesicles observed by electron microscopy, and the rate of supply of newly synthesized synaptic vesicles to active zones is unable to support the consumption of synaptic vesicles at the increased mEPSC frequency, although other explanations are possible.

The Ca2+ sensitivity curve suggests that the targets of the SCRAPPER-proteasome axis should include molecules that regulate Ca2+-dependent neurotransmitter release at the presynapse and that these targets are not molecules such as Ca2+ channels, which directly regulate the Ca2+ influx, but are likely due to the modulation of the Ca2+ sensitivity of presynaptic machinery.

SCRAPPER Regulates Synaptic Transmission via RIM1

Are the effects of loss or gain of function of SCRAPPER on synaptic function mediated specifically through RIM1? RNAi-mediated knockdown of RIM1 in neurons from SCR-KO mice was able to reverse the change in mEPSC, which supports that SCRAPPER does indeed function via RIM1. Moreover, overexpression of RIM1 in WT neurons was sufficient to phenocopy the increased mEPSC frequency in SCR-KO mice. Thus, alteration in steady-state level of RIM1 is sufficient to modulate mEPSC and upstream signaling pathways that function through SCRAPPER can in turn regulate this process. At present we cannot exclude the possibility that monoubiquitination of RIM1 by SCRAPPER may also regulate RIM1 before RIM1 is polyubiquitinated and degraded by the proteasome. Mutation of SCRAPPER abolished the increase in mEPSC observed following treatment of WT neurons with proteasome inhibitors, indicating that SCRAPPER is the major mediator of ongoing proteasomal regulation of mEPSC frequency. The rapidity of MG132 action suggests that the effects are independent of transcriptional regulation. These results also indicate that there is robust proteasome activity for the regulation of Ca2+-dependent synaptic vesicle release.

SCRAPPER Contributes to Regulation of Synaptic Plasticity

Synaptic plasticity is thought to be an important basis for learning and memory (Brown et al., 1990). PPF is one form of STP, the fundamental basis of synaptic plasticity. Ataxin-1 knockout mice show learning deficits and decreased hippocampal PPF, despite having normal LTP induction (Matilla et al., 1998). This indicates that PPF is related to learning. We showed that SCRAPPER functions as a regulator of PPF (i.e., synaptic plasticity), at presynapses.

SCR-KO mice displayed decreased PPF ratio. In addition, MG132-dependent upregulation of fEPSP was suppressed in SCR-KO animals. Our findings demonstrate that UPS-dependent regulation of PPF and mEPSC is mainly controlled by SCRAPPER. Based on analysis of RIM1 knockout mice in which the PPF value was increased at the hippocampal CA1 synapse (Schoch et al., 2002), together with the increased steady-state level of RIM1 in SCR-KO mice, we propose that increased RIM1 is a major contributor to the decreased PPF observed in SCR-KO mice. Thus, SCRAPPER appears to induce degradation of RIM1 complex via the proteasome, which regulates the PPF ratio. We note that overexpression of Munc13 also increases mEPSC (Betz et al., 1998) and that synaptotagmin I increased the probability of vesicle fusion (Kreft et al., 2003). Hence, it is formally possible that upregulation of Munc13 and synaptotagmin could also contribute to the changes of PPF and mEPSC in SCR-KO mice.

Neural activity-dependent alteration of Scrapper expression directly implicates SCRAPPER as being involved in neural activity-dependent protein degradation, which in turn alters synaptic transmission efficiency. Regulation of RIM1 protein complex level is likely to cause both short- and long-term changes in the PPF ratio. That SCRAPPER can facilitate a long-term change in synaptic efficacy suggests that SCRAPPER could influence regulation of “the plasticity of plasticity” at the presynapse—i.e., the metaplasticity (Abraham and Tate, 1997). We have focused our investigation on presynaptic functions. However, as SCRAPPER is also localized to dendrites, there may be additional effects of SCRAPPER disruption on neuronal function and postsynaptic function. Finally, we speculate that the activation of certain neural networks does not occur in SCR-KO, because SCRAPPER may also serve as E3 for an unknown substrate in inhibitory neurons, which in turn may give rise to the inactivation of a certain population of excitatory neurons. In summary, we have identified a physiological role of SCRAPPER-dependent RIM1 ubiquitination for proteasomal degradation in presynaptic function. Many kinds of neuronal disorder/disease are caused by excessive neurotransmitter release. An attractive possibility is that SCRAPPER can be a potential target of new drug designs for the treatment of neuronal diseases.

EXPERIMENTAL PROCEDURES

Identification of SCRAPPER and Cloning of Scrapper Gene

Eleven hundred thirty genes with both neuron-restrictive silencing element and cAMP-response element were found using the Celera Discovery System (a search program that is no longer available). The F box domain was identified by the Pfam program. Cloning of the Scrapper gene was performed by RT-PCR, using cDNA from newborn mouse brain and Scrapper-specific primers. The NCBI accession number of Scrapper is EF649694.

Animals

Care and experiments with animals were in accordance with institutional guidelines and those of the National Institute of Health and the Animal Care and Use Committee (Mitsubishi Kagaku Institute of Life Science). C57BL/6 mice and Wistar SD rats were used.

Antibodies

Rabbit anti-SCRAPPER antibody was raised against amino residues 321–380 of mouse SCRAPPER expressed in bacteria. Other antibodies used are described in the Supplemental Data.

Miscellaneous Procedures

In situ hybridization for Scrapper mRNA was performed as described (Ikegami et al., 2006). Details are described in the Supplemental Data. Plasmid construction, cell culture, and Southern and western blotting were performed by conventional methods (Hatanaka et al., 2006c). Subcellular fractionation of mouse brain was performed as described (Yao et al., 1999). Fractions were analyzed by western blotting. Stability of RIM1 was measured by the treatment of 20 µg/ml cycloheximide (Hatanaka et al., 2006a) to cerebral cortical primary culture from WT or SCR-KO mice. RIM1 was knockdowned by the miR-RNAi system (Invitrogen).

Neuron Culture and Immunostaining

Hippocampal neurons were prepared (Kato et al., 2001) with minor modifications described in the Supplemental Data. Cultured cells were transfected with 1 µg DNA with Lipoofectamine2000 (Invitrogen). Primary cultured neurons from SCR-KO mice were prepared with P1–P3 mice. Immunofluorescence and immunohistochemical studies were performed as described (Yao et al., 2002) and imaged and quantified using confocal microscopy operated under manual control (Zeiss LSM5 PASCAL, Olympus FV-1000). For mixed culture, neurons were incubated separately with each DNA mixture, subsequently washed to remove any adherent DNA before plating then cocultured. The cells transfected independently with a vector expressing either nontagged RFP (DsRed2, Clontech), or different GFP-SCR constructs were plated together with other group neurons and used at 12–15 days in vitro for electrophysiological recordings. The mEPSC frequency recorded form neurons expressing either nontagged RFP or nontagged GFP displayed no significant difference between cells. The changes observed when recording from Red cells in the presence of green neurons transfected with different GFP-SCR constructs were interpreted as an effect generated in the presynaptic (green) cell.

Immunoprecipitation and In Vitro Pull-Down Assay

Immunoprecipitation of the mouse brain was performed as described (Yao et al., 1999). HEK293T cells were lysed 48 hr after transfection with a solution containing 50 mM Tris-Cl (pH 7.4), 100 mM NaCl, 1% (v/v) Triton X-100, and a cocktail of protease inhibitors (Complete EDTA-free, Roche). Cell lysates were incubated with 5 µg of antibody in 10 µl of protein G-sepharose beads (Amersham) for 4 hr at 4°C. Immunoprecipitates were washed four times with ice-cold lysis buffer and were blotted with appropriate antibodies. In vitro binding using GST fusion proteins and cell extract was performed as described (Yao et al., 1999).

In Vitro Immunoprecipitation and Ubiquitination Assays

Recombinant Uba1 (E1), GST-UbcH5 (E2), Hisx6-ubiquitin, APP-BP1/Uba3, GST-UbcH12, and NEDD8 were purchased from Medical and Biological laboratories Company, Limited. The NEDD8 system containing APPBP1/Uba3, UbcH12, and NEDD8 was added simultaneously to the mixture. SCRAPPER was immunoprecipitated with anti-SCRAPPER conjugated to Protein G sepharose, and denatured after being washed three times. The ubiquitination assay was carried out as described (Kawakami et al., 2001).

SCRAPPER Knockout and Transgenic Mice

We used homologous recombination in ES cells to mutate Scrapper. Exon 3 (which encodes the region including the F box domain) and a part of exon 4 (a recombination that generates a frameshift) were replaced by a Neo selection cassette. Analysis of SCR-KO mice was performed on littermates derived from mating heterozygous mutant mice on a hybrid 129Sv/C57BL6 background and was confirmed with several independent litters derived from independent generations of heterozygous breeding. We established three independent mouse lines overexpressing GFP-fused SCRAPPER (TG-22, TG-26, and TG-31). The GFP-SCRAPPER transgene contains a CaMKII promoter, an EGFP tag, a Scrapper-coding region, and a polyadenylation signal. Details are described in the Supplemental Data. Two-dimensional analyses of the brain homogenate of Scrapper knockout mice were performed as described (Omori et al., 2002).

Transmission Electron Microscopy

Transmission electron microscopy (TEM) was performed as described (Ikegami et al., 2006) and in the Supplemental Data. Quantitative analysis of TEM micrographs was performed as described (Altrock et al., 2003; Schoch et al., 2002).

Recording of mEPSC

mEPSC was recorded as described (Inoue et al., 2006), see the Supplemental Data. Cells for whole-cell recording configuration were selected on the status of RFP or GFP expression. The average frequency (Hz), amplitude (pA), rise time (ms), and decay time (ms) from each neuron were then averaged to give a value for the entire population. Statistical significance was determined using the two-tailed, paired Student’s t test. *p < 0.05 was considered to be statistically significant.

Electrophysiology of Hippocampal Slices

Hippocampal 300-µm-thickness slices were prepared from ether-anesthetized 3- to 4-week-old SCR-KO mice as described in the Supplemental Data. PPF was measured by using two-paired 50, 100, 200, 300, and 400-ms interpulse interval stimuli. The PPF ratio (2nd/1st fEPSP slope) was evaluated in each interpulse interval (IPI).

Supplementary Material

01

ACKNOWLEDGMENTS

We are grateful to K. Nakamura, R. Migishima, and T. Hino, Mouse Genome Technology Center at MITILS, for generating SCR-KO and SCR-TG mice. We also wish to thank T. Sekiya, S. Song, A. Omori, S. Kamijyo, and K. Nagayama and M. Arai, Y. F.-Tsukamoto, Y. Hinohara, and other members of the Setou group and MITILS. We thank Profs. Mayford and Kida for the CaMKII promoter vector, Dr. Seino for the RIM1 constructs, and Ms. Takamura for critical reading of this manuscript. We thank three anonymous reviewers for constructive criticism and superb suggestions for experiments to strengthen the study. This work was supported by Research Grants for PRESTO and SENTAN, from JST and a Grant-In-Aid for Young Scientists A to M.S., by a Grant-In-Aid for Young Scientists B to I.Y., by NANO-001 (N.M.), NIBIO 05-32 (S.Y.) to N.M., and in part by a grant from company, MITILS.

Footnotes

Supplemental Data

Supplemental Data include Supplemental Experimental Procedures, Supplemental References, and ten figures and can be found with this article online at http://www.cell.com/cgi/content/full/130/5/943/DC1/.

A portion of this study was presented at 46th Annual Meeting of the ASCB.

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