Neuron Article

A Coordinated Local Translational Control Point at the Synapse Involving Relief from Silencing and MOV10 Degradation

Sourav Banerjee,1 Pierre Neveu,1,2 and Kenneth S. Kosik1,* 1Neuroscience Research Institute and Department of Cellular Molecular and Developmental Biology 2Kavli Institute for Theoretical Physics University of California, Santa Barbara, Santa Barbara, CA 93106, USA *Correspondence: [email protected] DOI 10.1016/j.neuron.2009.11.023

SUMMARY 2003; Yi and Ehlers, 2005). These observations suggest a link between localized protein degradation and synthesis in the Persistent changes in synaptic strength are locally regulation of synaptic plasticity. In support of this link, protea- regulated by both protein degradation and synthesis; some blockade can diminish late-phase LTP (L-LTP) induced however, the coordination of these opposing limbs at Schaffer collateral-CA1 synapses, like pharmacological inhibi- is poorly understood. Here, we found that the RISC tion of translation (Fonseca et al., 2006). Interestingly, coapplica- protein MOV10 was present at synapses and was tion of proteasome blockers with translation inhibitors largely rapidly degraded by the proteasome in an NMDA- restores L-LTP, suggesting the need for a crucial balance between protein degradation and synthesis for maintenance of receptor-mediated activity-dependent manner. We L-LTP (Fonseca et al., 2006). Furthermore, activity-dependent designed a translational trap to capture those mRNAs relocation of the proteasome from the dendritic shaft to the whose spatiotemporal translation is regulated by synaptic spine resulted in a spatially precise loss of a degradation MOV10. When MOV10 was suppressed, a set of reporter (Bingol and Schuman, 2006). The relocation of UPS mRNAs—including a-CaMKII, Limk1, and the depal- components is reminiscent of polyribosome redistribution in mitoylating enzyme lysophospholipase1 (Lypla1)— potentiated spines (Ostroff et al., 2002) and suggests local selectively entered the polysome compartment. We remodeling of the spine through coordinated degradation and also observed that Lypla1 mRNA is associated synthesis. A likely target of activity-mediated degradation is with the brain-enriched microRNA miR-138. Using Armitage. In Drosophila, this key RNA-induced silencing a photoconvertible translation reporter, Kaede, we complex (RISC) factor undergoes rapid degradation in response analyzed the activity-dependent protein synthesis to a specific olfactory learning paradigm (Ashraf et al., 2006). The activity-dependent loss of Armitage released translationally driven by Lypla1 and a-CaMKII 30UTRs. We estab- suppressed synaptic mRNAs such as CaMKII, whose translation lished this protein synthesis to be MOV10 and protea- is associated with stable memory (Ashraf et al., 2006). However, some dependent. These results suggest a unifying candidate mRNAs that are regulated by coordinated protein picture of a local translational regulatory mechanism degradation and synthesis within the synaptodendritic compart- during synaptic plasticity. ment in the mammalian central nervous system (CNS) remain elusive. INTRODUCTION Here, we found that MOV10, a homolog of the Drosophila DExD-box protein Armitage (Meister et al., 2005), was present The ubiquitin-proteasome system (UPS) is an important modu- at synapses and was rapidly degraded by the proteasome in an lator of several neuronal functions, including growth cone NMDA-receptor-mediated activity-dependent manner. To iden- guidance, synapse development, and plasticity (Bingol and tify RISC-regulated candidate synaptic mRNAs, we designed Schuman, 2005; DiAntonio et al., 2001; Patrick, 2006; Tai and a translational trap that captured mRNAs in the polyribosome Schuman, 2008; Yi and Ehlers, 2007). Dysregulation of protea- fraction after RNAi knockdown of MOV10. We found, among some-mediated degradation by mutating a specific ubiquitin others, two known RISC-associated synaptic mRNAs–a-CaMKII ligase impaired LTP and contextual learning (Jiang et al., and LimK1 (Ashraf et al., 2006; Schratt et al., 2006)—as well as 1998). The injection of a proteasome inhibitor into hippocampal lypophospholipase 1 (Lypla1), a depalymitoylating enzyme. We CA1 resulted in impairment of some hippocampal-dependent also observed that Lypla1 mRNA is associated with the brain- memory tasks (Lopez-Salon et al., 2001). The proteasome enriched microRNA miR-138. Using a photoconvertible transla- machinery can also regulate activity-dependent remodeling of tion reporter, Kaede, we analyzed the activity-dependent protein the postsynaptic density by incorporating or removing master synthesis driven by Lypla1 and a-CaMKII 30UTRs. We estab- organizing molecules such as scaffolding proteins, glutamate lished this protein synthesis to be MOV10 and proteasome receptors, and signaling enzymes (Ageta et al., 2001; Ehlers, dependent. These results point toward a comprehensive picture

Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. 871 Neuron MOV10 Degradation and Translational Silencing

A Process outline MOV10 PSD95 Merge

B MOV10 PSD95 Merge

CDAP5 - - - + - - - Lactacystin - - + - - - +

Crude SN NMDA - + + kDa - - - - 180 KCl - + + + - - - kDa kDa 180 180 115 MOV10 115 PSD95 115 115 MOV10 82

82 Synapsin I 64 64 64 49 49 Tubulin -CaMKII 49 1.5 64 49 GFAP * 37 ** Histone H1 1 ** 26 0.5

Relative amount 0 E F G AP5 + - - - GFP-UbKO + Lactacystin - - - - + - GFP-Ub : GFP IP : GFP IP KCl - + - - + + + KCl Lactacystin kDa - + + - + 180 kDa KCl + 180 - kDa Poly Ubiquitinated 180 MOV10-PA-GFP 115 MOV10 115 MOV10 115 MOV10-PA-GFP GFP-UbKO 64 26 GFP-Ub 49 TUJ1 WB : GFP 49 Tubulin Poly Ubiquitinated MOV10-PA-GFP * 180 1 ** 115

0.5 WB : Poly Ub Relative amount 0

Figure 1. Synaptic Localization of MOV10 and Its Activity-Dependent Proteasomal Degradation (A) Synaptic localization of endogenous MOV10. DIV 21 neurons were immunostained with MOV10 and PSD95 antibodies. MOV10 shows a punctate pattern along dendrites that colocalized with the synaptic marker PSD95. Neurite processes are shown in outline. Scale bar, 100 mm. (B) High-resolution image from the indicated region of (A). Scale bar, 20 mm. (C) Endogenous MOV10 (130 and 95 kDa) is localized in the synaptodendritic compartment. Synaptoneurosomes (SN) were prepared from 5- to 6-week-old rat hippocampi (Crude: total hippocampal lysate). (D) Proteasomal control of MOV10 degradation is regulated through the NMDA receptor. Hippocampal neurons (DIV 21–23) were stimulated by 60 mM KCl or 20 mM NMDA for 5 min. The block of proteasome activity by lactacystin (10 mM, 10 min pretreatment) or NMDA antagonist AP5 (50 mM, 50 min pretreatment)

872 Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. Neuron MOV10 Degradation and Translational Silencing

of a local translational regulatory mechanism during synaptic was unchanged, p = 0.097, t test) (Figure S2A). Furthermore, plasticity. we observed comparable degradation of ectopically expressed PA-GFP-tagged MOV10 upon neuronal stimulation (only 37% ± 7.1% of MOV10-PA-GFP remained intact, p = 0.002, t test, RESULTS Figure S2B). Thus, like its fly ortholog, MOV10 is degraded by the proteasome in an activity-dependent manner. Synaptic Localization of MOV10 However, depolarization with KCl activates many voltage- and The SDE3 helicase Armitage, member of the RISC, has been ligand-gated channels, including the NMDA-type glutamate shown to regulate synaptic protein synthesis in D. melanogaster receptor, which plays a significant role in synaptic plasticity in (Ashraf et al., 2006). We first showed the synaptic localization of the CNS. Therefore, we tested whether the NMDA receptor the mammalian ortholog of Armitage, MOV10. Rat hippocampal is involved in the proteasomal degradation of MOV10 by neurons (DIV 21) were immunostained for MOV10, and synapses treating neurons with its antagonist, AP5 (D(-)-2-amino-5-phos- were identified with the postsynaptic marker PSD95 (Figures 1A phonovaleric acid) (50 mM, 50 min) (Figure 1D). AP5 pretreatment and 1B). MOV10 pattern was punctate. 62.8% ± 4.0% of PSD95 significantly prevented the KCl-induced loss of MOV10 (73.1% ± puncta colocalized with MOV10 puncta and 25% ± 1.6% of 8.8% of MOV10 was unchanged, p = 0.018, t test, Figure 1D). MOV10 puncta colocalized with PSD95. MOV10 puncta were AP5 treatment without stimulation did not produce significant also present within dendrites (Figure S1C). A biochemical anal- changes in MOV10 levels (81% ± 18% of MOV10 was ysis of synaptoneurosomes prepared from rat hippocampi unchanged, p = 0.30, Figure S2A). Consistent with this observa- confirmed the synaptodendritic localization of endogenous tion, neuronal stimulation by NMDA alone (20 mM, 5 min) was MOV10 (Figure 1C). Similarly, immunostaining of cultured rat sufficient to induce rapid degradation of endogenous MOV10 hippocampal neurons expressing MOV10 tagged with photoac- (only 22% ± 5.6% of MOV10 remained intact, p = 0.002, t test, tivatable GFP (MOV10-PA-GFP) (Patterson and Lippincott- Figure 1D). This NMDA-mediated degradation was rescued Schwartz, 2002)(Figures S1D and S1E) showed a similar by lactacystin (86% ± 7.3% of MOV10 was unchanged, punctate pattern, with 45.6% ± 3.2% of PSD95 puncta colocal- p = 0.002, t test, Figure 1D). These results suggested that ized to MOV10-PA-GFP and 73.2% ± 3.6% of MOV10-PA-GFP NMDA activity can induce activity-dependent proteasomal puncta colocalized to PSD95 (Figures S1D and S1E). Relative regulation of MOV10 in the mammalian CNS. to endogenous MOV10, MOV10-PA-GFP appears to have a rela- If MOV10 degradation regulates synaptic plasticity by tive preference for synapses; however, using antibodies against releasing miRNA-mediated translational repression at synaptic GFP and MOV10, we observed 64.6% ± 1.3% colocalization sites upon neuronal activity, we expect endogenous MOV10 to between endogenous MOV10 puncta and ectopic MOV10-PA- be degraded locally in the synaptodendritic compartment. GFP puncta (Figure S1F), suggesting similar localizations for Biochemical analyses of synaptoneurosomes prepared from both endogenous and ectopically expressed MOV10. Further- stimulated slices (60 mM KCl, 10 min) showed rapid degradation more, ectopic expression of MOV10-PA-GFP did not alter the of MOV10 (only 16% ± 4.6% of MOV10 remained intact, p = protein level of another key RISC component, Argonaute (Nelson 0.0036, t test, Figures 1E and S2F), and this rapid loss of et al., 2007)(Figure S2E). MOV10 was rescued by lactacystin (10 mM, 15 min) (77.4% ± 8.1% of MOV10 was unchanged, p = 0.0028, t test, Figure 1E). Activity-Dependent Degradation of MOV10 Similarly, AP5 (50 mM, 60 min) also prevented the rapid To investigate activity-dependent regulation of MOV10, we synaptic degradation of MOV10 (79.3% ± 9.3% of MOV10 was depolarized hippocampal neurons with KCl (60 mM, 5 min) and unchanged, p = 0.003, t test, Figure 1E). Together, these observed that endogenous MOV10 was rapidly degraded within biochemical observations suggest that activity-regulated pro- 30 min (only 38.5% ± 8.1% of the MOV10-immunoreactive band teasomal degradation of MOV10 can occur locally at synaptic remained intact compared to its basal level, p = 0.0006, t test, sites after NMDA receptor activation. Figure 1D). Pretreatment of neurons with the proteasome inhib- itor lactacystin (10 mM, 10 min) significantly rescued this effect MOV10 Degradation Is Ubiquitin Dependent (75.4% ± 11.2% of MOV10 was unchanged, p = 0.024, t test, Neuronal protein turnover is regulated in both a ubiquitin-depen- Figure 1D). Lactacystin alone without stimulation did not cause dent and -independent manner (Tai and Schuman, 2008). To any significant loss of MOV10 levels (83% ± 8.3% of MOV10 determine whether ubiquitination is required for MOV10 prevented KCl-induced MOV10 degradation. NMDA (20 mM, 5 min) stimulation induced MOV10 degradation that was rescued by lactacystin (10 mM, 10 min pretreatment). Data shown as mean ± SEM (KCl stimulation: n = 8; NMDA stimulation: n = 6), *p < 0.05, t test. (E) Synaptic degradation of endogenous MOV10. Synaptoneurosomal fraction was prepared from hippocampal slices from 5- to 6-week-old rats that were stim- ulated with KCl (60 mM, 10 min). Lactacystin (10 mM, 15 min pretreatment) or NMDA inactivation by AP5 (50 mM, 60 min pretreatment) rescued the KCl-induced degradation. Data shown as mean ± SEM, n = 3, *p < 0.05, t test. (F) Activity-regulated degradation of MOV10 by ubiquitin-dependent 26S proteasome. Hippocampal neurons were transduced with GFP fused to wild-type (GFP-Ub) or mutant (GFP-UbKO) ubiquitin. GFP- Ub and GFP-UbKO expressing neurons (DIV 21) were stimulated with KCl (60 mM, 5 min). The ubiquitin mutant prevented KCl-induced degradation of MOV10. (G) Ubiquitination of MOV10. MOV10-PA-GFP complex was immunoprecipitated from DIV 21–24 neurons (transduced with MOV10-PA-GFP) that were either pretreated with lactacystin (10 mM, 10 min) and stimulated with 60 mM KCl for 5 min or incubated under basal conditions. The immunoprecipitated complexes were then analyzed by western blot using antibodies specific for polyubiquitin conjugates and GFP.

Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. 873 Neuron MOV10 Degradation and Translational Silencing

degradation, we expressed GFP-fused ubiquitin mutant with all ence of the membrane stain FM4-64 (1 mM, 1 min), which tags seven lysines replaced by arginine (GFP-UbKO) or GFP-fused active synapses (Figure 2F). We observed that 34.5% ± 4.7% wild-type ubiquitin (GFP-Ub) (Bergink et al., 2006) in hippo- of MOV10-PA-GFP puncta colocalized with the dye FM4-64. campal neurons. GFP-UbKO has been shown to prevent Moreover, we observed that MOV10 was indeed degraded in ubiquitin-dependent proteasomal degradation by blocking the FM4-64-stained synapses (Figures 2F and 2G). We probed polyubiquitination of the target protein (Bergink et al., 2006). whether endogenous MOV10 could be found in active synapses Then we depolarized neurons expressing GFP-Ub or GFP- (Figure S1G): the 78% ± 2.2% colocalization value with active UbKO. After depolarization, MOV10 degradation was efficiently synapses was much higher than the 25% ± 1.6% colocalization blocked in neurons expressing the ubiquitin mutant, but not value of MOV10 with all synapses stained with PSD95. This wild-type ubiquitin (Figure 1F), thereby suggesting that observation hints at a preferential recruitment of MOV10 to activity-regulated MOV10 degradation occurs in a ubiquitin- active synapses. Taken together, our result suggests a direct dependent manner. link between synaptic activation and MOV10 degradation. To analyze further the ubiquitination of MOV10, we expressed PA-GFP-tagged MOV10 in hippocampal neurons and then NMDA-Dependent Degradation of MOV10 stimulated these neurons after pretreatment with lactacystin Because KCl depolarization affects many voltage- and ligand- (10 mM, 10 min). The MOV10-PA-GFP complex was immunopre- gated channels, we used NMDA to stimulate neurons. With cipitated from neuronal lysates using an antibody against GFP, NMDA, we also derived a mean MOV10 degradation time and the ubiquitination of MOV10 was visualized with an antibody constant of 8.0 ± 2.7 min (mean ± SD) (data not shown); however, that specifically recognizes polyubiquitin conjugates (Fujimuro the Gaussian distribution was sharper than with KCl (p = 0.05, et al., 1994). High molecular weight polyubiquitin labeling was Kolmogorov-Smirnov test), suggesting that the observed observed (Figure 1G), confirming that activity-regulated MOV10 MOV10 degradation events were due to a more restricted mech- degradation occurs through the ubiquitin pathway. anism. We also investigated the effect of NMDA receptor inactivation Single-Puncta Analysis of MOV10 Degradation following by AP5 on the distribution of degradation rates. AP5 treatment KCl Depolarization (50 mM, 50 min) and subsequent neuron depolarization with We imaged rat hippocampal neurons expressing MOV10-PA- KCl induced a significant decrease of the fraction of synaptic GFP. Upon depolarization with KCl (60 mM, 5 min), we observed sites that degraded MOV10 from 55% to 41% (p = 3.107, a rapid loss of fluorescence over the ensuing 45 min (Figures 2A– Kolmogorov-Smirnov test) without altering the degradation time 2D). We extracted the time profiles of the loss in fluorescence constant (Figures 2H and 2I). NMDA receptor activation thus intensity for 1500 puncta (from five neurons) and generated appears to be a triggering factor for MOV10 degradation. The a time constant distribution drawn from individual puncta broad and less specific effects of KCl depolarization may account behavior. From those profiles, degradation appeared to be for the incomplete inhibition of MOV10 degradation by AP5. a first-order reaction. Under basal conditions, i.e., without any stimulation, the loss of fluorescence occurred with an exponen- Proteasomal Control of Activity-Induced MOV10 tial distribution that had a mean time constant of 25 min. This Degradation distribution suggested individual fluorescent loss events were We next sought to determine changes in single MOV10 puncta independent and uncoordinated, i.e., relied on a Poisson degradation rates following pretreatment with the proteasome process (Figure S3). Following KCl treatment, the loss of fluores- inhibitor lactacystin (10 mM, 10 min). Adding lactacystin with cent signal from the puncta appeared to be coordinated, as KCl or NMDA broadened the Gaussian distribution and degradation rates were drawn from a Gaussian distribution. increased the mean degradation time constant to 12 ± 3.7 min We derived a mean MOV10 degradation time constant of 8.0 ± (mean ± SD) for KCl (Figures 2H and 2I) and 12 ± 3.9 min 2.9 min (mean ± SD) (Figures 2D and S3). Analysis of MOV10 (mean ± SD) for NMDA (data not shown). These values signifi- degradation at early time points indicated no measurable time cantly differed from treatment with KCl alone (p = 0.02, t test) lag between the onset of stimulation and the effective loss of or with NMDA alone (p = 0.00017, t test). We could rescale the MOV10-PA-GFP fluorescence (data not shown), suggesting distribution of degradation time constants induced by stimula- that MOV10-PA-GFP was processed for degradation without tion with and without lactacystin in order to yield indistinguish- delay. The activity-dependent loss of MOV10-PA-GFP fluores- able distributions (p = 0.30 for NMDA stimulation, p = 0.56 for cence was not due to diffusion (Supplemental Data). KCl stimulation, Kolmogorov-Smirnov test, see Supplemental The distribution of degradation time constants formed a long Data). Therefore, the MOV10 degradation time constant is tail. 45% of the puncta had estimated time constants >60 min proportional to the proteasome activity or availability. Changes and thus exhibited no effective degradation. This result raised in local proteasome availability (Bingol and Schuman, 2006) the possibility that puncta are differentially degraded depending will thus have a direct effect on the capability of an individual upon their spatial location, perhaps in a distal-proximal gradient. synapse to degrade MOV10. However, this was not true. Puncta degraded in a location-inde- pendent manner (Figure 2E). A Translational Trap Captures mRNAs Regulated To visualize whether MOV10 degradation occurred at active by the RISC synapses, we imaged hippocampal neurons expressing As MOV10 is considered a critical RISC component and involved MOV10-PA-GFP stimulated with KCl (60 mM, 5 min) in the pres- in miRNA-guided translational control (Chendrimada et al.,

874 Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. Neuron MOV10 Degradation and Translational Silencing

A BCDE

FGHI

Figure 2. Neuronal Stimulation Triggers Proteasome-Mediated Localized Degradation of MOV10 (A) Time-lapse imaging of hippocampal neurons (DIV 21–24) expressing MOV10-PA-GFP under basal conditions or stimulated with KCl (60 mM, 5 min) and subse- quent degradation of GFP-labeled MOV10. Scale bar, 10 mm. (B and C) Temporal variation in the relative fluorescence intensity of randomly chosen puncta from KCl-stimulated neuron: the locations of the puncta were iden- tified with a circle (B) of the same color as the fluorescence trace (C). The white rectangle in (B) corresponds to the dendrite shown in (A). MOV10-PA-GFP degra- dation occurred on the 10 min timescale. Scale bar, 20 mm. (D) Temporal variation in the total relative fluorescence intensity of 1500 puncta with (solid blue line) or without (dotted blue line) KCl stimulation. The distribution of degradation time constants over all puncta could be adjusted (red lines) to a population of puncta degrading with a rate drawn from an exponential distribution (absence of stimulation) or from a Gaussian distribution (KCl stimulation). See Figure S3 for details. Shades represent mean ± SEM. (E) Median degradation time constant (red line) plotted against the distance of individual puncta from an arbitrary point. Time constants of individual puncta are shown as blue dots. MOV10 degradation did not depend on the puncta location and was much faster than the relatively rare MOV10 degradation events under basal conditions (black line: median degradation time constant). (F and G) Simultaneous imaging of synaptic activity and MOV10-PA-GFP degradation upon KCl stimulation. (F) Neurons expressing MOV10-PA-GFP were stim- ulated with KCl (60 mM, 5 min) in the presence FM4-64 (1 mM, 1 min). Active synapses showed 34.5% colocalization with MOV10-PA-GFP. (G) Temporal vari- ations of the relative fluorescence intensity of MOV10-PA-GFP puncta shown in panel (F). No degradation was observed in puncta indicated with green arrows (puncta showing no FM4-64 staining). MOV10-PA-GFP was degraded in puncta indicated with yellow arrows (puncta exhibiting FM4-64 staining). Scale bar, 20 mm. (H) Proteasome block by lactacystin (10 mM, 10 min) or inhibition of the NMDA receptor by AP5 (50 mM, 50 min) inhibited MOV10-PA-GFP degradation. Scale bar, 10 mm. (I) Distribution of time constants of individual puncta with KCl stimulation. Proteasome inhibition with lactacystin slowed the degradation. Inhibition of NMDA receptors with AP5 induced a decrease in the number of MOV10-degrading puncta. Time constants <2 min cannot be measured with our imaging frequency. Error bars indicate statistical error.

2007), like its fly ortholog Armitage (Cook et al., 2004; Tomari increased polysomal fraction (Figures 3A and 3B). We then iden- et al., 2004), the loss of MOV10 could relieve translational tified the translationally upregulated mRNAs enriched in the silencing. We hypothesized that with neuronal activation, a set polyribosomal fraction by quantitative PCR. We selected 54 of mRNAs silenced by the RISC would be released into an candidates based on their synaptodendritic localization (Poon actively translating polysome pool in the synaptodendritic et al., 2006; Zhong et al., 2006), the presence of a conserved compartment and elsewhere. We designed a translational trap miRNA seed match in their UTR, and a context score value method to screen mRNAs that are actively translated after the from Targetscan 4.0. Those candidate mRNAs enriched in the loss of MOV10. We disrupted RISC function with RNAi polyribosomal fraction following MOV10 knockdown by RNAi constructs against MOV10 and then fractionated the actively points to a pool of mRNAs that are regulated by miRNAs at translating mRNAs using density gradient centrifugation. The the level of translation initiation. 24% of the tested candidate fractionation profile showed a reduced monosomal peak and mRNAs showed a 2-fold enrichment in the actively translating

Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. 875 Neuron MOV10 Degradation and Translational Silencing

ACB M Candidate Mean fold M change P P Limk1 5.47 Grm1 3.73 Elavl2 3.56 Gria3 3.23 Camk2a 3.01 Grin2a 2.96 Universal Control MOV10 RNAi Zdhhc2 2.92 D Universal MOV10 Universal MOV10 Sdc2 2.86 Control RNAi Control RNAi Zdhhc17 2.49 kDa kDa Lypla1 180 180 2.35 Zdhhc15 2.34 Gria1 2.20 Gria2 2.16 115 115 MOV10 Cpeb1 1.42 Ppp1r9b 1.35 64 Eif3s9 64 1.14 Eif3s10 0.91 49 49 -CaMKII Actb 0.87 E 26 26 Lypla1 1.5 * *

19 19 1 64 64 49 49 Tubulin RNAi RNAi MOV10 0.5 MOV10 Construct # 1 Construct # 2 * Relative amount Universal Control Universal Control Universal Control RNAi 0 MOV10 MOV10 -CaMKII Lypla1

Figure 3. Translational Regulation of Dendritic mRNAs by the RISC

(A and B) A254 profile of ribosomal fractionation after RNAi knockdown of MOV10. Cortical neurons were transduced with lentiviral RNAi construct against MOV10 or a nontargeting control construct (Universal Control) at DIV 7–8. Cytosolic extracts (from neurons at DIV 18–19) were fractionated with continuous monitoring at 254 nm. Untranslated mRNAs accumulated in the lighter monosomal fraction (M), whereas translating mRNAs were captured in the heavier polysomal fraction (P). (C) RNAi against MOV10 upregulated the translation of a group of selected dendritic mRNAs. These candidates were enriched in the polysomal fraction compared to the universal control. (D) RNAi against MOV10 upregulated a-CaMKII and Lypla1 protein levels. (E) Quantification of the experiments shown in (D) (n = 2). Data shown as mean ± SEM, *p < 0.05, t test. polyribosomal fraction after RNAi against MOV10 (Figure 3C). translational modification that has recently been implicated in Among the top changing mRNAs, our screen identified two synaptic plasticity (el-Husseini and Bredt, 2002) and synaptic known mRNAs whose translation is known to be regulated by development (Kang et al., 2008; Mukai et al., 2008). In addition the RISC: Limk1 and a-CaMKII (5.5- and 3-fold change, respec- to the translational trap result, we confirmed the increase of tively, Figure 3C). On the other hand, several dendritic mRNAs Lypla1 and a-CaMKII protein levels after knocking down such as b-actin (ActB), translation initiation factors Eif3S9 and MOV10 (Figures 3D and 3E). Taken together, these observations Eif3S10, protein phosphatase 1 (Ppp1r9b), and others did not further support our hypothesis that activity-regulated proteaso- change (less than 1.5 fold) after RNAi knockdown of MOV10 mal control of MOV10 degradation may lead to localized transla- (Figure 3C). Thus, our screen specifically identified candidate tion of these mRNAs. mRNAs that could be potentially regulated by the RISC. Among the top changing mRNAs was Lypla1, with a 2-fold Lypla1 and miR-138 Are a Regulated Duplex in Dendrites change. Lypla1, also known as acyl protein thioesterase 1 We tested our hypothesis that activity-regulated local proteaso- (APT1), removes palmitate groups from proteins, including mal control of MOV10 degradation leads to localized trans- endothelial nitric-oxide synthase (Yeh et al., 1999), Ga, and p21 lation of mRNAs with Lypla1 as a potential candidate. The (Duncan and Gilman, 1998). Palmitoylation is a reversible post- 30UTR of Lypla1 contains a conserved 8-mer seed match of

876 Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. Neuron MOV10 Degradation and Translational Silencing

A B CD 2 * * Control LNA miR-138 LNA 2 25 nM 50 nM 100 nM 25 nM 50 nM 100 nM kDa 1.2 1.5 26 * Lypla1 1.5

1 37 GAPDH 1 1 4 0.5 0.5 * 3 0.8 Relative mRNA abundance

Relative luciferase intensity 2

Relative luciferase intensity 0 0 1 25 nM 50 nM 25 nM 50 nM abundance 100 nM 100 nM 25 nM 50 nM100 nM 25 nM 50 nM100 nM

mutant Relative protein Control LNA mir-138 LNA wild type 0 Control LNA mir-138 LNA SynapsinI Merge E miR-138 Lypla1

Figure 4. Lypla1 Is a Target of Brain-Enriched microRNA, miR-138 (A) Luciferase activity was measured from hippocampal neurons (DIV 7) transfected with Lypla1 30UTR fused luciferase reporter construct in the presence of lock nucleic acid (LNA) inhibitor of miR-138 or a nontargeting inhibitor (25 nM, 50 nM, and 100 nM). A luciferase assay was performed after 48 hr. Data shown as mean ± SEM, n = 3, *p < 0.05, t test. (B) The luciferase assay was performed with wild-type and mutant Lypla1 30UTR to monitor the effect of miR-138 binding site on reporter expression. Wild-type or mutant Lypla1 30UTR was transfected into hippocampal neurons (DIV 7), and a reporter assay was performed 48 hr after transfection. Data shown as mean ± SEM, n = 9, *p < 0.05, t test. (C) LNA miR-138 enhances endogenous protein levels of Lypla1. LNA inhibitor of miR-138 and control LNA (25 nM, 50 nM, and 100 nM) were transfected into hippocampal neurons (DIV 7). Neuronal lysates were prepared from LNA-treated samples after 48 hr. Data shown as mean ± SEM, n = 3, *p < 0.05, t test. (D) Inhibition of miR-138 has no effect on the stability of Lypla1 mRNA as measured by quantitative PCR. LNA inhibitor of miR-138 and control LNA (25 nM, 50 nM, and 100 nM) were transfected into hippocampal neurons (DIV 7). Transcript levels were measured after 48 hr of LNA treatment. Data shown as mean ± SEM, n = 3, *p < 0.05, t test. (E) Both miR-138 and Lypla1 mRNA are localized at synapses. Double in situ hybridization of hippocampal neurons (DIV 21) using LNA probes for miR-138 and Lypla1 mRNA and immunostained for the synaptic marker synapsin I. 32% of the synapses possessed both miR-138 and Lypla1 mRNA. Scale bar, 10 mm. a brain-enriched microRNA, miR-138 (Lagos-Quintana et al., lated inactivation of a functional RISC through proteasomal 2002). Inhibition of endogenous miR-138 by lock nucleic acid degradation of MOV10 will subsequently release the silenced (LNA) in hippocampal neurons led to a significant increase of Ly- miRNA-bound Lypla1 mRNA for translation at dendritic spines pla1 30UTR fused luciferase reporter expression (80% ± 2% with spatiotemporal precision. increase, p = 0.0003, t test) as well as endogenous protein level (90% ± 23% increase, p = 0.05, t test) compared to a control Single-Puncta Analysis of Local Dendritic LNA inhibitor (Figures 4A and 4C, respectively). Mutation in the Protein Synthesis seed region of the predicted miR-138 binding site in Lypla1 To investigate whether activity-dependent proteasomal control 30UTR also increased the luciferase reporter expression (2-fold of the RISC is linked to new protein synthesis at synaptic sites, increase; p < 0.05, t test, Figure 4B). LNA-mediated inhibition we expressed the a-CaMKII and Lypla1 30UTRs fused to the of miR-138 did not change the endogenous Lypla1 mRNA level photoconvertible translation reporter, Kaede (Leung et al., (Figure 4D), suggesting that miR-138 regulates Lypla1 expres- 2006; Raab-Graham et al., 2006), in hippocampal neurons. sion at the translational level rather than at the level of mRNA Kaede had a punctate pattern when its expression levels were decay. low and a more uniform pattern once expression levels became We next examined whether miR-138 and Lypla1 mRNA could high (data not shown). We studied diffusion properties of Kaede be found at the same synapses using double in situ hybridization by FRAP (Supplemental Data) and found that Kaede behavior combined with immunostaining with scrambled probes as was governed by an unbinding reaction and not by free diffusion. controls (Figures S4A–S4D). 32% of synapsin I-positive puncta These features were probably due to Kaede’s tetrameric nature were labeled with a miR-138 and Lypla1 mRNA probes (Figures that leads to aggregates even at low concentration (Ando et al., 4E and S4E). In separate colocalization studies, we observed 2002; Dittrich et al., 2005). The special geometric properties of that MOV10 colocalized with Lypla1 mRNA (60%) (Figure S4F). a synapse, i.e., a restricted space, could also contribute to the Together these observations led us to reason that activity-regu- aggregate formation.

Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. 877 Neuron MOV10 Degradation and Translational Silencing

A BC

D E F

Figure 5. Proteasomal Control of Localized Synthesis of a-CaMKII and Lypla1 mRNA (A and B) Neuronal activity induced a-CaMKII 30UTR-mediated local synthesis of the translation reporter Kaede. Hippocampal neurons (DIV 14) were transduced with a lentiviral construct containing Kaede fused to the 30UTR of a-CaMKII. Kaede was photoconverted, and then the neurons (DIV 21–24) were stimulated with NMDA (20 mM, 5 min). Time-lapse images were collected to track new protein synthesis. Upon stimulation, puncta were either synthesizing new proteins (green arrows) or not (white arrows). Scale bar, 10 mm. Neuronal processes are shown as outline. The temporal variations of the relative fluorescence intensity for randomly chosen puncta (encircled in A) are shown in (B). (C) Median synthesis time constant (red line) plotted against the puncta distance from an arbitrary point. Time constants of individual puncta are shown as blue dots. a-CaMKII 30UTR-driven Kaede synthesis did not depend on puncta location. (D) Synaptic activation induced localized new synthesis of Lypla1 30UTR-driven translation reporter. Hippocampal DIV 21–24 neurons transduced with lentiviral construct (at DIV 16–18) containing Kaede fused to the 30UTR of Lypla1 were stimulated with glutamate (20 mM for 5min) after photoconversion of the Kaede. Time-lapse images were collected to track new protein synthesis. Upon stimulation, puncta were synthesizing new proteins (green arrows) or not (white arrows). Neuronal processes are shown as outline. Scale bar, 10 mm. (E) Temporal variation of the total relative fluorescence intensity of 1500 puncta under basal conditions (black line) or upon glutamate stimulation (dark blue line). Anisomycin (green line) (40 mM, 20 min) and lactacystin (red line) (10 mM, 10 min) completely blocked new protein synthesis. New synthesis was not affected by actinomycin D (light blue line) (5 mM, 10 min). The variation could be adjusted (dotted lines) to a population of synapses synthesizing Kaede with a time constant drawn from a Gaussian distribution with the same mean as a-CaMKII 30UTR (Figure S5B). Shades represent mean ± SEM. (F) Activity-dependent proteasomal degradation of MOV10 is critical for localized protein synthesis of RISC-regulated candidate mRNA. After RNAi knockdown of MOV10, hippocampal neurons expressing Lypla1 30UTR-fused Kaede were imaged for 20 min to record the level of basal Kaede fluorescence and then stimu- lated with glutamate (20 mM, 5 min). After stimulation, neurons were further imaged for 20 min. A temporal profile of the total relative fluorescence intensity under basal conditions as well as upon glutamate stimulation obtained from neurons after RNAi knockdown of MOV10 (dark blue line) showed no activity-dependent increase of localized translation as compared to neurons without RNAi knockdown of MOV10 (red dotted line). Shades represent mean ± SEM.

As NMDA receptor activation was sufficient to trigger each punctum and a single arbitrary point. We found that the MOV10 degradation, we examined whether its activation could median synthesis time constant was uniform regardless of puncta induce local protein synthesis by recording the temporal varia- location with a variation of 15% (11.4 ± 1.1 min [median ± SD] for tions of green Kaede fluorescence for 60 min in 1500 individual a-CaMKII UTR and NMDA stimulation, Figure 5C). puncta. After NMDA activation (20 mM, 5 min), the a-CaMKII Similar protein synthesis events upon NMDA stimulation were 30UTR-driven Kaede reporter indicated that new protein observed with the Lypla1 30UTR-driven Kaede reporter (data synthesis, as monitored by the appearance of green Kaede fluo- not shown). Glutamate (20 mM, 5 min) stimulation of neurons rescence, occurred with a time constant of 11 min, i.e., after 11 expressing Lypla1 30UTR fused Kaede also induced a 20% local min the increasing fluorescence intensity began to plateau increase in the relative amount of the translation reporter (Figures 5A and 5B). To ensure that the signal increase was not compared to the basal level within 11 min of synaptic activation due to an enlargement in spine volume, we imaged the temporal (Figures 5D, 5E, and S5C). The timescale and the amount of variation of fluorescence of the photoconverted Kaede proteins protein synthesis were in good agreement with previously in the red channel before stimulation and for 30 min after stimula- reported values in the literature, with different translation tion: we observed that the red signal remained constant over time reporters such as membrane-anchored GFP with a 1 hr half- (data not shown); this could be explained by the lack of diffusion life (Aakalu et al., 2001) or Kaede (Leung et al., 2006). We found of Kaede due to its propensity to aggregate. that the protein synthesis kinetics and amount did not depend Two populations of puncta were apparent: one in which activity- significantly on the stimulation method (KCl, 60 mM, 5 min; gluta- dependent synthesis occurred and one in which Kaede fluores- mate, 20 mM, 5 min; or NMDA, 20 mM, 5 min) or the UTR reporter cence was unchanged (Figure 5B). Those two populations were used (a-CaMKII or Lypla1). not regionally localized within the neuron, as neighboring puncta Pretreatment with anisomycin (40 mM, 20 min), a protein- could belong to either population (Figures 5A and 5B). Like for synthesis inhibitor, completely blocked on-site reporter syn- MOV10 degradation, we studied whether local protein synthesis thesis (Figures 5E, S5B, and S5D). Pretreatment with actino- occurred in a spatially regulated way. We plotted the median mycin D (5 mM, 10 min), a transcription inhibitor, did not affect synthesis time constant as a function of the distance between new synthesis of the translation reporter (Figure 5E). It suggested

878 Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. Neuron MOV10 Degradation and Translational Silencing

MOV10 degrading/ MOV10 degrading/ ACB MOV10 degrading puncta D Kaede synthesizing puncta Kaede synthesizing puncta MOV10-CFP Kaede Merge 0.14 60 no synthesis 0.12 Kaede synthesis 1 50 0.1 40

0.08 (min) 30 0.06 0.5

probability 20 0.04 synthesis 0.02 10

0 synthesis amplitude (a.u.) 0 0 0 0.2 0.40.6 0.8 1 0 0.5 1 0102030405060 degradation amplitude (a.u.) degradation amplitude (a.u.) degradation (min)

Figure 6. Degradative Control of RISC Regulates Localized Synthesis of Lypla1 30UTR Fused Translation Reporter (A) Localization of ectopically expressed MOV10 and Kaede fused with Lypla1 30UTR. Hippocampal neurons (DIV 14) were transduced with MOV10-CFP and Kaede-Lypla1 30UTR and imaged (DIV 21) before stimulation. MOV10-CFP colocalized with Kaede reporter fused with Lypla1 30UTR. Scale bar, 20 mm. (B) Distribution of degradation amplitudes in puncta synthesizing Kaede (black line) or not (gray line). Puncta that were synthesizing Kaede exhibited a stronger overall MOV10 degradation (p = 3 3 1011, Kolmogorov-Smirnov test). (C) Comparison between Kaede synthesis amplitude and MOV10 degradation amplitude in individual puncta. The red line shows the median synthesis amplitude. Those quantities were correlated (r = 0.5). A greater extent of MOV10 degradation tended to foster new protein synthesis.

(D) Comparison between Kaede synthesis time constants tsynthesis and MOV10 degradation time constants tdegradation in individual puncta. Synthesis time constants depended only weakly on degradation time constants (red line shows the median synthesis time constant). See Supplemental Data for details (Figure S6). that local translation at the synapse was not dependent on the fused with CFP and the Kaede-Lypla1 30UTR translation reporter longer timescale of transcription. in hippocampal neurons. Both proteins, MOV10-CFP and the Kaede-Lypla1 reporter, colocalized (Figure 6A). We stimulated Activity-Dependent Protein Synthesis Is MOV10 neurons with KCl (60 mM, 5 min) and imaged MOV10 degrada- and Proteasome Dependent tion in the blue channel and Kaede synthesis in the green The experiments described above correlate, but do not directly channel. We measured time constants and amplitudes for link an activity-related increase in local translation to degradative MOV10 degradation and the synthesis of the Lypla1 30UTR- control of MOV10. Therefore, we repeated measurements of driven translation reporter in individual puncta. activity-induced Lypla1 30UTR-mediated synthesis of Kaede This analysis led us to sort individual puncta into four after RNAi knockdown of MOV10 in hippocampal neurons. We categories depending on their degradation/synthesis kinetics first monitored the basal level of Kaede in these neurons for (Table 1): (1) puncta in which both MOV10 degradation and 20 min, then stimulated with glutamate (20 mM, 5 min), and Lypla1 30UTR-mediated synthesis of translation occurred (17%); we imaged these neurons for a further 20 min. We observed (2) puncta in which MOV10 was degraded without any reporter that RNAi-mediated loss of MOV10 abrogated the glutamate- synthesis (31.6%); (3) puncta in which synthesis of the translation induced increase of Kaede fluorescence (Figure 5F). This obser- reporter occurred without MOV10 degradation (6.9%); (4) puncta vation confirmed our hypothesis that localized synthesis of with essentially neither MOV10 degradation nor synthesis of Lypla1 mRNA is directly linked to the integrity of MOV10. the translation reporter, i.e., time constants >60 min (44.5%). Consistent with our hypothesis that proteasome-mediated Interestingly, of the synapses where MOV10 degradation took MOV10 degradation is required for release from translational place, 35% synthesized new proteins. Consistent with this silencing, we observed that pretreatment of neurons with lacta- observation, data from the double in situ hybridization (Figure 4E) cystin (10 mM, 10 min) blocked Lypla1 30UTR-driven new protein showed that a similar fraction of synapses (32%) contained both synthesis from the Kaede reporter upon stimulation with miR-138 and Lypla1 mRNA. Synthesis occurred in a significantly glutamate (20 mM, 5 min, Figures 5E and S5E). In similar experi- lower fraction of the puncta without MOV10-CFP degradation, ments using the Kaede reporter fused to the a-CaMKII 30UTR, and therefore, either other mechanisms contribute to regulate the total relative amount of synthesis was reduced by 30% local protein synthesis (Costa-Mattioli et al., 2009) or degrada- (p < 104, Wilcoxon test) upon NMDA stimulation (20 mM, tion of unlabeled endogenous MOV10 has occurred in this 5 min) as compared to stimulation without lactacystin pretreat- population. Thus, MOV10 degradation dramatically increased ment (Figures S5A and S5B). The less profound effect of the the fraction of puncta where protein synthesis took place. proteasome inhibitor on the a-CaMKII 30UTR-driven reporter Consistent with that observation was that puncta synthesizing translation could be due to additional translation control mecha- Kaede degraded more MOV10 (Figure 6B). Indeed, the ampli- nism, such as cytoplasmic polyadenylation (Wu et al., 1998). tudes of MOV10 degradation and Kaede synthesis in those puncta where both processes occurred were correlated (r = 0.5, Coordinated Degradation and Synthesis Figure 6C). Interestingly, in those puncta where both MOV10 in Single Puncta degradation and protein synthesis occurred, the rate of protein To pinpoint more precisely the correlation between MOV10 synthesis exceeded the rate of MOV10 degradation in 56% of degradation and new protein synthesis, we coexpressed MOV10 the puncta (Figure 6D). This observation is compatible with the

Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. 879 Neuron MOV10 Degradation and Translational Silencing

differential kinetics of MOV10 degradation as reported by loss of Table 1. The Simultaneous Monitoring of MOV10 Degradation the GFP signal, which occurs on the minute timescale, and the and Lypla1 30UTR-Driven Kaede Synthesis Allowed Us to Define rapid onset of Kaede synthesis. Four Classes of Synapses Puncta Characteristics Fraction All Puncta DISCUSSION Puncta degrading MOV10 48.6%

The experiments presented here demonstrate that the RISC Puncta not degrading MOV10 51.4% protein MOV10 is a pivotally positioned control element in the Puncta synthesizing Kaede 23.9% regulation of local protein synthesis in dendrites. By suppressing Puncta not synthesizing Kaede 76.1% MOV10 with RNAi, we set a translational trap to capture an mRNA Puncta degrading MOV10 and synthesizing Kaede 17.0% repertoire regulated by the miRNA system. Among these mRNAs Puncta degrading MOV10 and not synthesizing Kaede 31.6% was Lypla1, a depalmitoylating enzyme, and its miRNA silencer, Puncta not degrading MOV10 and synthesizing Kaede 6.9% miR-138. Palmitoylation is a widely used modification of Puncta not degrading MOV10 and not synthesizing Kaede 44.5% many synaptic proteins, including Cdc42, that can potentially MOV10 Degrading Puncta regulate postsynaptic structures (Kang et al., 2008). Interestingly, miR-138-mediated regulation of Lypla1 can also modulate spine Puncta synthesizing Kaede 35.0% morphology through Rho-dependent pathways (Siegel et al., Puncta not synthesizing Kaede 65.0% 2009). In addition to the depalmitoylating enzyme, Lypla1, two Puncta Not Degrading MOV10 palmitoylating enzymes, Zdhhc2 and Zdhhc17, appeared in our Puncta synthesizing Kaede 13.4% screen. Thus, activity-dependent regulation of the synthesis of Puncta not synthesizing Kaede 86.6% these palmitoylating and depalmitoylating enzymes represents Kaede Synthesizing Puncta an important facet of the mRNA repertoire under the control of Puncta degrading MOV10 71.1% the RISC in the context of synaptic stimulation. Among the Puncta not degrading MOV10 28.9% mRNAs found in the screen was the activity-dependent micro- RNA target—Limk1—which can modulate structural and func- tional plasticity by targeting cytoskeletal effectors (Schratt degraded by the proteasome presents a counterintuitive role for et al., 2006). Taken together, the set of mRNAs that are relieved degradation. In this case, degradation is linked to increased of their silence (Figure 3C) represents a coordinately regulated local mRNA translation and thus an increase in the levels of those response to synaptic stimulation. Whether changes in the levels proteins regulated in the RISC (Figure 7). Degradation of MOV10 of these enzymes are rate limiting will be important when weigh- and possibly other components of the RISC may underlie obser- ing the broader functional significance of these findings. vations that both degradation and synthesis are required for Just under half the MOV10 puncta underwent degradation synaptic plasticity (Ashraf et al., 2006; Fonseca et al., 2006). following neuronal activation (Table 1). Those puncta in which MOV10 is functionally required to mediate miRNA-guided MOV10 did not degrade following activation may reside in a func- mRNA cleavage (Meister et al., 2005). Tomari et al. (Tomari tionally incompetent pool due to saturation from overexpression. et al., 2004) proposed that Drosophila Armi facilitates the ATP- Those puncta that degraded MOV10 without concomitant dependent incorporation of siRNA into the RISC, and its associ- synthesis may contain endogenous mRNAs that are not tagged. ation/dissociation with the core RISC serves as a control element Protein synthesis in the absence of MOV10 degradation was over RISC maturation and competence. Interestingly, Cook et al. relatively uncommon. The known distal-proximal NMDA gradient (Cook et al., 2004) showed Armitage function in translational (Major et al., 2008) was not evident in the broad pattern of regulation of oskar and kinesin heavy chain mRNAs and also NMDA-induced MOV10 degradation. The likely reasons are (1) suggested miRNA regulation of these mRNAs. The high our bath application of NMDA may saturate its endogenous homology in the helicase domain between MOV10 and Armitage concentration, and (2) we used cultured hippocampal neurons suggests functional conservation. as opposed to neocortical pyramidal neurons on which such Many possible activity-related changes in the structure of gradients have been described. the RISC could lead to dissociation of MOV10 from the RISC, Many telltale signs of protein degradation at the synapse have and these changes could precede the measured experimental been noted. Among these signs are the association of ubiquitin end point of MOV10 degradation. MOV10 degradation requires ligases with synapses (Hegde et al., 1997) and activity-dependent that the protein is recognized by an as yet unknown ubiquitin movement of the proteasome into the spine (Bingol and Schu- ligase and is delivered to the proteasome. MOV10 degradation man, 2006), but few specific substrates have been described. kinetics appeared as first order, indicating that the pool of Degradation of spine-associated Rap guanosine triphosphatase MOV10 destined to be degraded interacted with the degradation activating protein (SPAR), PSD-95, and Bassoon were linked to machinery on the minute timescale, whereas Kaede synthesis the induction of serum-inducible kinase (SNK) (Pak and Sheng, rises rapidly after stimulation. The degradation time constant 2003), and in Drosophila, ubiquitin-dependent mechanisms we measured reflects the processing of MOV10 by the degrada- regulate synaptic development at the neuromuscular junction tion machinery, which we showed is likely to depend on local (DiAntonio et al., 2001; Fulga and Van Vactor, 2008). The proteasome availability. It is in accordance with the rapid rise discovery of MOV10 as ubiquitin-dependent client protein of Kaede synthesis after stimulation.

880 Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. Neuron MOV10 Degradation and Translational Silencing

NMDA stimulation Figure 7. Model for NMDA-Mediated Pro- teasomal Control of RISC-Regulated New Synthesis of Dendritic mRNA

NMDA receptor mRNA NMDA receptor activation respectively), packaging plasmid psPAX2 and RISC translational envelop plasmid pMD2.G as described in www. Ub repression lentiweb.com. Titers were determined by infecting MOV10 293T cells followed by FACS analysis. Titers were typically 1–3 3 107 TU/ml after virus concentration. Lentiviral infection procedures were described in Ub newly synthesized Supplemental Data. proteins proteasome-dependent Polyribosomal Fractionation MOV10 degradation and Quantitative PCR Analysis translating Polyribosomal fractionation was performed as mRNA described previously (Stefani et al., 2004) with minor modification (see Supplemental Data). Poly- ribosome-containing fractions were pooled, and RNA was isolated by precipitation as well as Abso- lutely RNA RT-PCR Miniprep kit (Stratagene). Double-stranded cDNA was synthesized using Interestingly, degrading MOV10 rather than recycling it places First Strand Superscript II kit (Invitrogen). Quantitative PCR (qPCR) was per- distinct kinetic boundaries on the window of translational formed on a 7500 Fast Real-Time PCR system using the SYBR-green-contain- competence at the synapse. While translation is relieved, the ing PCR kit (Applied Biosystems) with GAPDH and 18S rRNA as internal stan- polysomes will be competent to synthesize a potentially comput- dard. able number of copies of a protein until resilenced by reformation In Situ Hybridization of the holo-RISC that includes MOV10. Ultimately, synaptic In situ hybridization was performed using antisense-Lock Nucleic Acid (LNA) plasticity will be understood in the context of a full repertoire of oligonucleotide (Exiqon) using digoxigenin-labeled (DIG Oligonucleotide local responses to neuronal activation. Variation in the comple- Tailing Kit, 2nd generation, Roche) Lypla1 and Scramble-mRNA probes or ment of silenced mRNAs among different synapses and different biotin-labeled rno-miR-138 and Scramble-miR probes. After hybridization, neurons will be a critical future direction. neurons were incubated with cy3-labeled anti-DIG antibody (Jackson Immunoresearch). Synaptic localization of mRNA or miRNA was detected by anti-synapsin I antibody (Chemicon). To visualize endogenous MOV10 EXPERIMENTAL PROCEDURES colocalization with Lypla1 mRNA, in situ hybridization was performed followed by immunostaining with anti-MOV10 antibody (Bethyl Laboratories). Cell Culture For dual in situ hybridization, neurons were first hybridized with biotin- Rat hippocampal neuron cultures were prepared as described previously labeled rno-miR-138 probe followed by DIG-labeled Lypla1 probe. After (Goslin and Banker, 1991). Briefly, the hippocampus was removed from E18 hybridization, probes were detected by FITC-conjugated anti-biotin (Jackson 2 rat embryos, dissociated neurons were plated at 2800–8850 cells/cm in Immunoresearch) and cy3-conjugated anti-DIG (Jackson Immunoresearch) 60 mm dish containing five glass coverslips coated with poly-L-lysine (1 mg/ml) antibodies, respectively. Synaptic localization was detected as described 2 or 22 mm Willco glass bottom dishes (5000–6000 cells/cm ). Neurons were above. Detailed protocol for in situ hybridizations is described in Supplemental cocultured with glial cells in Neurobasal medium containing B27 supplement Experimental Procedures. up to DIV 24. Similarly, cortical neuronal cultures were prepared from E18 rat embryos, were plated onto poly-L-lysine-coated 150 mm dishes (9000– Stimulation of Hippocampal Neurons 2 10000 cells/cm ), and grown in Neurobasal medium containing B27 supple- Hippocampal neurons at DIV 21–24 were used for stimulation experiments as ment up to DIV 18–19. described previously (Bingol and Schuman, 2006) with minor modifications. Prior to stimulation, neurons were incubated in HBS (290 mOSm) (110 mM

Lentiviral and Mammalian Constructs NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 10 mM D-glucose, 10 mM shRNA cassettes for MOV10 and control RNAi were cloned into lentiviral vector, HEPES-NaOH pH 7.4) for 60 min. Neurons were stimulated for 5 min with pLVTHM, and pLKO.1 (Addgene). Translation reporters were constructed by high KCl-HBS (same as HBS except for 55 mM NaCl and 60 mM KCl) subcloning Kaede-a-CamKII/Lypla1 30UTR into lentiviral vector, pWPXL and HBS containing 20 mM NMDA or 20 mM glutamate. After stimulation, (Addgene). MOV10-PA-GFP construct was obtained by inserting PA-GFP neurons were recovered in HBS for 30–60 min. Neurons were pretreated cassette into pWPXL followed by insertion of MOV10 coding sequence before with AP5 (50 mM, 50 min), lactacystin A (10 mM, 10 min), anisomycin (40 mM, KO PA-GFP cassette. Lentiviral MOV10-CFP, GFP-Ub, and GFP-Ub constructs 20 min), actinomycin D (5 mM, 10 min) to antagonize NMDA, proteasome were subcloned into pWPXL. A myc epitope-tagged MOV10 at its C terminus activity, protein synthesis, and transcription, respectively. Antagonists re- was constructed by subcloning MOV10 coding sequence into pcDNA4/myc- mained in the incubation media for the duration of the experiment. Western His A (Invitrogen). The luciferase reporter was constructed by inserting Lypla1 blot analysis of stimulated neurons is described in Supplemental Experimental 30UTR into pMIR-REPORT (Ambion). The mutant Lypla1 30UTR fused Kaede Procedures. was obtained by site directed mutagenesis. Slice Stimulation Lentiviral Transduction Hippocampal slices were stimulated as described previously (Bingol and Lentiviruses were produced by cotransfection of HEK293T cells with transfer Schuman, 2006; Murase et al., 2002) with minor modifications. Briefly, slices vector (pLVTHM or pWPXL containing shRNA or fusion protein cassette, were stimulated with ACSF buffer containing KCl (60 mM, 10 min). After

Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. 881 Neuron MOV10 Degradation and Translational Silencing

stimulation, slices were recovered in ACSF for 30 min. Slices were pretreated To visualize whether MOV10 was degraded at an activated synapse, with AP5 (50 mM, 60 min) and lactacystin (10 mM, 15 min) when needed. Stim- neurons were imaged for 10 min after stimulation and then stained with FM4- ulated slices were then processed for synaptoneurosome preparation as 64 dye (1 mM, 1 min). Neurons were further imaged for 15 min after FM4-64 described previously (Rao and Steward, 1991). Synaptoneurosome fractions staining. were then analyzed by western blot analysis using antibodies against PSD95 (NeuroMab, clone K28/43), synapsin I (Chemicon), GFAP (Chemicon), histone Image Analysis H1 (Chemicon), aCamKII (Chemicon), TUJ1 (Covance), and MOV10 (Bethyl Image analysis was done with custom-written scripts in Matlab (MathWorks). Laboratories and PTG lab). First, images were corrected for any drift in the focal plane by comparing to the first image of a series. We segmented the first image of a series. To do so, we Analysis of MOV10 Ubiquitination subtracted the average intensity of 18 3 18 pixel neighborhood considered as Hippocampal neurons were infected at DIV 12–14 with lentivirus construct background, the resulting image was filtered using a 2 3 2 pixel neighborhood expressing MOV10-PA-GFP. Neurons (DIV 21–24) were stimulated with KCl to reduce noise. Puncta were extracted from the resulting image by threshold- (60 mM, 5 min). Neurons were pretreated with lactacystin (10 mM, 10 min) ing. The thresholding value was chosen to avoid false positives. Puncta and remained in the incubation media for the duration of the experiment. After positions were determined, and their intensity was computed for all images recovery, immunoprecipitation was performed according to the protocol by integrating the signal from the raw image over a 10 3 10 pixel neighbor- described in the GFP antibody (Clone 1E4 from MBL International) data sheet. hood. We measured photobleaching by imaging a neuron without stimulation Briefly, neurons were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 250 mM at a higher imaging frequency. Puncta intensities were then corrected for NaCl, 0.1% NP40, 2 mM EDTA, and 10% glycerol) containing protease inhib- photobleaching, and their temporal variation was adjusted using a monoexpo- itor cocktail (complete mini EDTA free). The total protein quantity of the lysate nential law. For Kaede, the puncta fluorescence intensity was normalized was measured by BCA protein assay kit (Pierce). MOV10-PA-GFP was immu- to the green signal after photoconversion. For MOV10, the puncta fluores- noprecipitated from stimulated or unstimulated lysate (3 mg of total protein) cence intensity was normalized to the signal after PA-GFP photoactivation using anti-GFP antibody (30 mg). Immunoprecipitated MOV10-PA-GFP was and neuronal stimulation in most of the cases. Data analysis was done with then probed with a specific antibody for polyubiquitin conjugates (clone FK1 custom-written scripts in Python. Modeling of MOV10 degradation and Kaede from Biomol International) as well as GFP. synthesis is described in Supplemental Data.

Immunocytochemistry Statistical Analysis Neurons (DIV 21) were immunostained as described previously (Wu et al., We used two-tailed Student’s t test (Welch t test was used when appropriate) 1998) with minor modifications. Neurons were stained with antibodies against to compare protein levels from western blot experiments and quantitative PCR MOV10 (Bethyl Laboratories), PSD95 (clone 6G6-1C9; ABR Affinity Biore- experiment results. The Kolmogorov-Smirnov test was used to compare time agent), MAP2 (clone SMI-52; Covance), and GFP (clone 1E4; MBL Interna- constant distributions and degradation or synthesis amplitudes distributions. tional). Specificity of MOV10 antibody (Bethyl Laboratories) was analyzed by The Wilcoxon signed-rank test was used to compare Kaede synthesis extents. a blocking peptide as well as shRNA-mediated RNAi against MOV10 (Figures Significance level was set at 0.05. S1A and S1B and S2C and S2D). Imaging was performed on an Oympus Fluoview FV500 microscope using an Olympus UPLFLN 403 NA = 1.3 oil- immersion objective. Illumination conditions were kept constant throughout each experiment and between experiments. SUPPLEMENTAL DATA

Live Imaging Supplemental Data include six supplemental figures, supplemental Experi- Hippocampal neurons infected at DIV 14 with lentivirus constructs (MOV10– mental Procedures, details of modeling for MOV10 degradation and local PA-GFP, Kaede-a-CaMKII 30UTR, Kaede-Lypla1 30UTR, MOV10–CFP) were protein synthesis and can be found with this article online at http://www.cell. stimulated at DIV 21–24. Prior to imaging, neurons were incubated in HBS com/neuron/supplemental/S0896-6273(09)00939-8. for 60 min, PA-GFP or Kaede were globally photoactivated by illumination with a benchtop 365 nm UV lamp (8W 365 nm, Spectroline) for 10 min. Neurons were identified prior to stimulation and their morphology checked immediately ACKNOWLEDGMENTS after stimulation. Stimulation was carried out by applying KCl (60 mM), N-methyl-D-aspartic acid (NMDA, 20 mM) or glutamate (20 mM) in HBS for We thank Jennifer Lippincott-Schwartz for providing PA-GFP construct, 5 min. When needed, neurons were pretreated with (R)-2-amino-5-phospho- Addgene for lentiviral vectors, NeuroMab for PSD95 antibody, Zissimos Mour- nopentanoic acid (AP5, 50 mM, 50 min), lactacystin (10 mM, 10 min), anisomy- elatos for pan Argonaute antibody (2A8), Snigdha Chatterjee for preparing cin (40 mM, 20 min), or actinomycin D (5 mM, 10 min). Neurons were washed in hippocampal cultures, Boris Shraiman and the members of the Kosik lab for HBS (with 50 mM AP5, 10 mM lactacystin, 40 mM anisomycin, or 5 mM actino- their insightful discussion and generosity. This work was funded by W.M. mycin D when needed). Imaging was done at 30C on an Oympus Fluoview Keck Foundation Grant to K.S.K. (SB 060811). P.N. research is supported in FV500 confocal inverted microscope using an Olympus UPLFLN 403 NA = part by the National Science Foundation under Grant No. PHY05-51164. 1.3 oil-immersion objective. Illumination conditions were kept constant throughout each experiment and between experiments. Images were taken Accepted: November 20, 2009 every minute for 45–60 min starting 1 min after the end of stimulation. To Published: December 23, 2009 monitor Kaede synthesis, the green form of Kaede was imaged after deter- mining that stimulation did not induce any changes in the red channel (filters were from Chroma or Semrock; 500/20 excitation, 515LP dichroic, 535/20 emission for the green channel; 532/22 excitation, 560LP dichroic, and REFERENCES 617/73 emission for the red channel). To analyze activity-dependent translation after RNAi against MOV10, hippo- Aakalu, G., Smith, W.B., Nguyen, N., Jiang, C., and Schuman, E.M. (2001). campal neurons at DIV 7 were infected with lentivirus expressing shRNA Dynamic visualization of local protein synthesis in hippocampal neurons. against MOV10. Neurons (DIV 17) were further infected with lentivirus express- Neuron 30, 489–502. ing Kaede fused with Lypla1 30UTR and then stimulated at DIV 22. Prior to Ageta, H., Kato, A., Fukazawa, Y., Inokuchi, K., and Sugiyama, H. (2001). stimulation, images were captured for 20 min. After stimulation with glutamate Effects of proteasome inhibitors on the synaptic localization of Vesl-1S/ (20 mM, 5 min), neurons were further imaged for 20 min. Homer-1a proteins. Brain Res. Mol. Brain Res. 97, 186–189.

882 Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. Neuron MOV10 Degradation and Translational Silencing

Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., and Miyawaki, A. (2002). Lagos-Quintana, M., Rauhut, R., Yalcin, A., Meyer, J., Lendeckel, W., and An optical marker based on the UV-induced green-to-red photoconversion of Tuschl, T. (2002). Identification of tissue-specific microRNAs from mouse. a fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 12651–12656. Curr. Biol. 12, 735–739. Ashraf, S.I., McLoon, A.L., Sclarsic, S.M., and Kunes, S. (2006). Synaptic Leung, K.M., van Horck, F.P., Lin, A.C., Allison, R., Standart, N., and Holt, C.E. protein synthesis associated with memory is regulated by the RISC pathway (2006). Asymmetrical beta-actin mRNA translation in growth cones mediates in Drosophila. Cell 124, 191–205. attractive turning to netrin-1. Nat. Neurosci. 9, 1247–1256. Bergink, S., Salomons, F.A., Hoogstraten, D., Groothuis, T.A., de Waard, H., Lopez-Salon, M., Alonso, M., Vianna, M.R., Viola, H., Mello e Souza, T., Wu, J., Yuan, L., Citterio, E., Houtsmuller, A.B., Neefjes, J., et al. (2006). Izquierdo, I., Pasquini, J.M., and Medina, J.H. (2001). The ubiquitin-protea- DNA damage triggers nucleotide excision repair-dependent monoubiquityla- some cascade is required for mammalian long-term memory formation. Eur. tion of histone H2A. Genes Dev. 20, 1343–1352. J. Neurosci. 14, 1820–1826. Major, G., Polsky, A., Denk, W., Schiller, J., and Tank, D.W. (2008). Spatiotem- Bingol, B., and Schuman, E.M. (2005). Synaptic protein degradation by the porally graded NMDA spike/plateau potentials in basal dendrites of neocor- ubiquitin proteasome system. Curr. Opin. Neurobiol. 15, 536–541. tical pyramidal neurons. J. Neurophysiol. 99, 2584–2601. Bingol, B., and Schuman, E.M. (2006). Activity-dependent dynamics Meister, G., Landthaler, M., Peters, L., Chen, P.Y., Urlaub, H., Lu¨ hrmann, R., and sequestration of proteasomes in dendritic spines. Nature 441, 1144– and Tuschl, T. (2005). Identification of novel argonaute-associated proteins. 1148. Curr. Biol. 15, 2149–2155. Chendrimada, T.P., Finn, K.J., Ji, X., Baillat, D., Gregory, R.I., Liebhaber, S.A., Mukai, J., Dhilla, A., Drew, L.J., Stark, K.L., Cao, L., MacDermott, A.B., Pasquinelli, A.E., and Shiekhattar, R. (2007). MicroRNA silencing through RISC Karayiorgou, M., and Gogos, J.A. (2008). Palmitoylation-dependent neurode- recruitment of eIF6. Nature 447, 823–828. velopmental deficits in a mouse model of 22q11 microdeletion. Nat. Neurosci. Cook, H.A., Koppetsch, B.S., Wu, J., and Theurkauf, W.E. (2004). The 11, 1302–1310. Drosophila SDE3 homolog armitage is required for oskar mRNA silencing Murase, S., Mosser, E., and Schuman, E.M. (2002). Depolarization drives beta- and embryonic axis specification. Cell 116, 817–829. Catenin into neuronal spines promoting changes in synaptic structure and Costa-Mattioli, M., Sossin, W.S., Klann, E., and Sonenberg, N. (2009). function. Neuron 35, 91–105. Translational control of long-lasting synaptic plasticity and memory. Neuron Nelson, P.T., De Planell-Saguer, M., Lamprinaki, S., Kiriakidou, M., Zhang, P., 61, 10–26. O’Doherty, U., and Mourelatos, Z. (2007). A novel monoclonal antibody against human Argonaute proteins reveals unexpected characteristics of miRNAs in DiAntonio, A., Haghighi, A.P., Portman, S.L., Lee, J.D., Amaranto, A.M., and human blood cells. RNA 13, 1787–1792. Goodman, C.S. (2001). Ubiquitination-dependent mechanisms regulate synaptic growth and function. Nature 412, 449–452. Ostroff, L.E., Fiala, J.C., Allwardt, B., and Harris, K.M. (2002). Polyribosomes redistribute from dendritic shafts into spines with enlarged synapses during Dittrich, P.S., Schafer, S.P., and Schwille, P. (2005). Characterization of the LTP in developing rat hippocampal slices. Neuron 35, 535–545. photoconversion on reaction of the fluorescent protein Kaede on the single- Pak, D.T., and Sheng, M. (2003). Targeted protein degradation and synapse molecule level. Biophys. J. 89, 3446–3455. remodeling by an inducible protein kinase. Science 302, 1368–1373. Duncan, J.A., and Gilman, A.G. (1998). A cytoplasmic acyl-protein thioester- Patrick, G.N. (2006). Synapse formation and plasticity: recent insights from ase that removes palmitate from G protein alpha subunits and p21(RAS). the perspective of the ubiquitin proteasome system. Curr. Opin. Neurobiol. J. Biol. Chem. 273, 15830–15837. 16, 90–94. Ehlers, M.D. (2003). Activity level controls postsynaptic composition and Patterson, G.H., and Lippincott-Schwartz, J. (2002). A photoactivatable GFP signaling via the ubiquitin-proteasome system. Nat. Neurosci. 6, 231–242. for selective photolabeling of proteins and cells. Science 297, 1873–1877. el-Husseini, A.-D., and Bredt, D.S. (2002). Protein palmitoylation: a regulator of Poon, M.M., Choi, S.H., Jamieson, C.A., Geschwind, D.H., and Martin, K.C. neuronal development and function. Nat. Rev. Neurosci. 3, 791–802. (2006). Identification of process-localized mRNAs from cultured rodent hippo- Fonseca, R., Vabulas, R.M., Hartl, F.U., Bonhoeffer, T., and Na¨ gerl, U.V. campal neurons. J. Neurosci. 26, 13390–13399. (2006). A balance of protein synthesis and proteasome-dependent degrada- Raab-Graham, K.F., Haddick, P.C., Jan, Y.N., and Jan, L.Y. (2006). Activity- tion determines the maintenance of LTP. Neuron 52, 239–245. and mTOR-dependent suppression of Kv1.1 channel mRNA translation in dendrites. Science 314, 144–148. Fujimuro, M., Sawada, H., and Yokosawa, H. (1994). Production and charac- terization of monoclonal antibodies specific to multi-ubiquitin chains of polyu- Rao, A., and Steward, O. (1991). Evidence that protein constituents of postsyn- biquitinated proteins. FEBS Lett. 349, 173–180. aptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes. J. Neurosci. 11, 2881–2895. Fulga, T.A., and Van Vactor, D. (2008). Synapses and growth cones on two Schratt, G.M., Tuebing, F., Nigh, E.A., Kane, C.G., Sabatini, M.E., Kiebler, M., sides of a highwire. Neuron 57, 339–344. and Greenberg, M.E. (2006). A brain-specific microRNA regulates dendritic Goslin, K., and Banker, G. (1991). Rat hippocampal neurons in low-density spine development. Nature 439, 283–289. culture. In Culturing Nerve Cells, G. Banker and K. Goslin, eds. (Cambridge, Siegel, G., Obernosterer, G., Fiore, R., Oehmen, M., Bicker, S., Christensen, MA: The MIT Press), pp. 251–282. M., Khudayberdiev, S., Leuschner, P.F., Busch, C.J., Kane, C., et al. (2009). Hegde, A.N., Inokuchi, K., Pei, W., Casadio, A., Ghirardi, M., Chain, D.G., A functional screen implicates microRNA-138-dependent regulation of the Martin, K.C., Kandel, E.R., and Schwartz, J.H. (1997). Ubiquitin C-terminal depalmitoylation enzyme APT1 in dendritic spine morphogenesis. Nat. Cell hydrolase is an immediate-early gene essential for long-term facilitation in Biol. 11, 705–716. Aplysia. Cell 89, 115–126. Stefani, G., Fraser, C.E., Darnell, J.C., and Darnell, R.B. (2004). Fragile X Jiang, Y.H., Armstrong, D., Albrecht, U., Atkins, C.M., Noebels, J.L., Eichele, mental retardation protein is associated with translating polyribosomes in G., Sweatt, J.D., and Beaudet, A.L. (1998). Mutation of the Angelman ubiquitin neuronal cells. J. Neurosci. 24, 7272–7276. ligase in mice causes increased cytoplasmic p53 and deficits of contextual Tai, H.C., and Schuman, E.M. (2008). Ubiquitin, the proteasome and protein learning and long-term potentiation. Neuron 21, 799–811. degradation in neuronal function and dysfunction. Nat. Rev. Neurosci. 9, Kang, R., Wan, J., Arstikaitis, P., Takahashi, H., Huang, K., Bailey, A.O., 826–838. Thompson, J.X., Roth, A.F., Drisdel, R.C., Mastro, R., et al. (2008). Neural Tomari, Y., Du, T., Haley, B., Schwarz, D.S., Bennett, R., Cook, H.A., palmitoyl-proteomics reveals dynamic synaptic palmitoylation. Nature 456, Koppetsch, B.S., Theurkauf, W.E., and Zamore, P.D. (2004). RISC assembly 904–909. defects in the Drosophila RNAi mutant armitage. Cell 116, 831–841.

Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc. 883 Neuron MOV10 Degradation and Translational Silencing

Wu, L., Wells, D., Tay, J., Mendis, D., Abbott, M.A., Barnitt, A., Quinlan, E., Yi, J.J., and Ehlers, M.D. (2005). Ubiquitin and protein turnover in synapse Heynen, A., Fallon, J.R., and Richter, J.D. (1998). CPEB-mediated cytoplasmic function. Neuron 47, 629–632. polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses. Neuron 21, 1129–1139. Yi, J.J., and Ehlers, M.D. (2007). Emerging roles for ubiquitin and protein degradation in neuronal function. Pharmacol. Rev. 59, 14–39.

Yeh, D.C., Duncan, J.A., Yamashita, S., and Michel, T. (1999). Depalmitoyla- Zhong, J., Zhang, T., and Bloch, L.M. (2006). Dendritic mRNAs en- tion of endothelial nitric-oxide synthase by acyl-protein thioesterase 1 is code diversified functionalities in hippocampal pyramidal neurons. BMC potentiated by Ca(2+)-calmodulin. J. Biol. Chem. 274, 33148–33154. Neurosci. 7, 17.

884 Neuron 64, 871–884, December 24, 2009 ª2009 Elsevier Inc.