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Received 3 Apr 2014 | Accepted 1 Sep 2014 | Published 14 Oct 2014

Cline Loriol1,2, Frdric Cass1,2, Anouar Khayachi1,2, Gwnola Poupon1,2, Magda Chafai2, Emmanuel Deval2, Carole Gwizdek1,2 & Stphane Martin1,2

Sumoylation plays important roles in the modulation of protein function, neurotransmission and plasticity, but the mechanisms regulating this post-translational system in neurons remain largely unknown. Here we demonstrate that the synaptic diffusion of Ubc9, the sole conjugating enzyme of the sumoylation pathway, is regulated by synaptic activity. We use restricted photobleaching/photoconversion of individual hippocampal spines to measure the diffusion properties of Ubc9 and show that it is regulated through an mGlu5R-dependent signalling pathway. Increasing synaptic activity with a GABAA receptor antagonist or directly activating mGlu5R increases the synaptic residency time of Ubc9 via a Gaq/PLC/Ca2 /PKC cascade. This activation promotes a transient synaptic trapping of Ubc9 through a PKC phosphorylation-dependent increase of Ubc9 recognition to phosphorylated substrates and consequently leads to the modulation of synaptic sumoylation. Our data demonstrate that Ubc9 diffusion is subject to activity-dependent regulatory processes and provide a mechanism for the dynamic changes in sumoylation occurring during synaptic transmission.

DOI: 10.1038/ncomms6113

mGlu5 receptors regulate synaptic sumoylation via a transient PKC-dependent diffusional trapping of Ubc9 into spines

1 University of Nice Sophia Antipolis, Institut de Pharmacologie Molculaire et Cellulaire, UMR7275 Laboratory of Excellence Network for Innovation on Signal Transduction Pathways in Life Sciences, UMR7275, 660 route des lucioles, 06560 Valbonne, France. 2 Centre National de la Recherche Scientique, Institut de Pharmacologie Molculaire et Cellulaire, UMR7275, 06560 Valbonne, France. Correspondence and requests for materials should be addressed to S.M. (email: mailto:[email protected]

Web End [email protected] ).

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Neurons are specialized cells, which connect through synapses for rapid information transfer in the brain. The efciency of synaptic transmission largely depends

on the correct targeting and arrangement of complex protein networks on both sides of the synapse. These processes are almost always regulated by post-translational modications such as phosphorylation or ubiquitination1. Sumoylation is emerging as a potent post-translational mechanism important for the regulation of synaptic transmission and plasticity24. The proper functioning of such modications requires a rigorous spatial and temporal control of the associated enzymatic machinery. However, the regulatory mechanisms of the sumoylation machinery are still largely unknown, especially at synapses.

Sumoylation consists in the covalent binding of the small ubiquitin-like modier peptide SUMO to specic lysine residues of target proteins5,6. Three SUMO paralogues have been identied so far. Protein sumoylation is mediated through a specic enzymatic cascade. Matured SUMO moieties are activated by the E1-activating complex. Then, SUMO is transferred onto the E2 Ubc9, the sole conjugating enzyme of the system. The covalent SUMO attachment to lysine residues requires the binding of Ubc9 to target proteins through its C93-containing catalytic domain7,8 and, either directly or in conjunction with one of the E3 enzymes Ubc9, catalyses SUMO conjugation to substrate proteins7,8. Many target proteins bear a phospho-dependent sumoylation motif (PDSM)9. The phosphorylation of this motif leads to an increase in the binding of Ubc9 to phosphorylated target proteins compared with their unmodied counterparts9,10. Importantly, sumoylation is a reversible process through the action of specic isopeptidases called sentrin-specic proteases11.

Molecular consequences of sumoylation are multiple. Sumoylation may mask proteinprotein interaction sites, create new binding interfaces or lead to conformational changes. Another emerging role for sumoylation in the central nervous system is the propensity to regulate protein aggregation3,1214. Sumoylation can also directly impact on other post-translational modications. For instance, sumoylation and ubiquitination processes can compete to modify the same lysine residue to dynamically control protein stability15,16.

In neurons, sumoylation inuences various aspects of neuronal activity2,3 and modies the stability and activity of several transcription factors known to regulate neuronal morphogenesis and postsynaptic differentiation10,1719. We reported the presence of multiple unidentied sumoylation substrates at synapses20. Since then, several studies including ours, reported that cytosolic and integral plasma membrane proteins important for synaptic transmission and plasticity are sumoylated, thereby modulating their stability, subcellular targeting, trafcking or interacting properties12,13,2025.

Although sumoylation has impacts on key neuronal processes, the regulatory mechanisms of this enzymatic system at synapses are still unknown. Therefore, investigating the spatiotemporal regulation of the SUMO system in the spines is of particular interest to better understand the synaptic function of sumoylation. We reported the developmental regulation of the SUMO system in the rat brain and showed an enrichment of sumoylation enzymes at synapses during neuronal maturation26. We further demonstrated that neuronal depolarization evokes a redistribution of the endogenous sumoylation machinery in spines, indicating that sumoylation is dynamically regulated by neuronal activity27.

Here we characterize the activity-dependent synaptic diffusion of Ubc9, the sole conjugating enzyme of the SUMO system, using a live-imaging approach on rat hippocampal neurons. We demonstrate that Ubc9 is highly mobile in neurons and that its synapto-dendritic mobility is not regulated by N-methyl-D-

aspartate receptors (NMDARs) but through a signalling cascade downstream of the activation of metabotropic glutamate 5 receptors (mGlu5R). Activation of mGlu5R leads to an increased synaptic residency time for the active form of Ubc9. This mGlu5R-induced diffusional trapping is prevented when the signalling pathway downstream of Gaq proteins, but not downstream of Gai, is blocked. We further show that the pharmacological blocking of phospholipase C (PLC) activity, calcium inux from L-type voltage-dependent calcium channels (L-VDCCs) or from internal stores or protein kinase C (PKC) activity abolishes the mGlu5R-dependent synaptic regulation of Ubc9. Interestingly, the transient diffusional trapping observed in mGlu5R- or phorbol 12-myristate 13-acetate (PMA)-activated spines is lost with the catalytically inactive and substrate-binding-decient Ubc9-C93S mutant or with the Ubc9-K65A enzyme, which, contrary to the WT Ubc9, loses its ability to preferentially recognize phosphorylated substrates. Altogether, our data demonstrate that the transient diffusional trapping of Ubc9 measured in mGlu5R-activated spines results from an increased Ubc9 recognition to PKC-phosphorylated synaptic substrate proteins, ultimately promoting synaptic sumoylation.

ResultsThe diffusion of Ubc9 is regulated by synaptic activity. To examine whether neuronal activity regulates the synaptic diffusion of the sumoylation machinery, we expressed the wild-type (WT) form of the SUMO-conjugating enzyme Ubc9 fused in frame to the green uorescent protein carboxy terminus (GFPUbc9) in cultured hippocampal neurons (Fig. 1). As previously demonstrated for the endogenous enzyme, we found high levels of GFP-Ubc9 uorescence in the nucleus, indicating that the GFP tag does not impair the nucleocytoplasmic transport of Ubc9. GFP-Ubc9 uorescence was also distributed throughout the dendrites where it displayed a clear synaptic distribution in addition to lower levels of diffuse staining in the shaft (Fig. 1a). We next evaluated whether the GFP tag would prevent Ubc9 activity. We co-expressed GFP-Ubc9 or its inactive C93S mutant with mCherry-SUMO1 or mCherry-SUMO2 in COS7 cells (Supplementary Fig. 1). We showed that contrary to the Ubc9-C93S inactive mutant, GFP-Ubc9 was active and able to increase protein sumoylation levels when co-expressed with mCherry-SUMOs. Together, these ndings indicated that GFP-Ubc9 is active with a subcellular localization comparable to the neuronal distribution reported for the endogenous enzyme20,26.

To assess the synaptic transport of Ubc9 in real time, we performed restricted photobleaching experiments to measure the uorescence recovery after photobleaching (FRAP) of individual spines from GFP-Ubc9-expressing neurons (Fig. 1bf). To quantitatively measure Ubc9 diffusion in spines, FRAP data were individually tted with a single-phase exponential function to obtain values for the mobile fraction and the rate of uorescence recovery (half-time of recovery; see the Methods section for details). The mobile fraction is the proportion of Ubc9 in the bleached spine that exchanged with unbleached Ubc9 from the dendritic shaft over the time course of the experiment. In absence of stimulation, there was only a small fraction of immobile Ubc9 in spines with B85% recovery of the initial uorescence, indicating that bleached GFP-Ubc9 were dynamically exchanged with uorescent GFP-Ubc9 from the shaft in resting conditions. In resting neurons, the rate of GFP-Ubc9 uorescence recovery in spines was 0.63 s with a diffusion coefcient of 0.59 mm2 s 1 (Fig. 1bf). Blocking synaptic activity with the Na channel blocker tetrodotoxin (TTX) did not have an impact on the diffusion of Ubc9 in spines with values obtained for TTX-treated neurons, similar to those measured in control conditions.

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Figure 1 | Activity-dependent synaptic diffusion of GFP-Ubc9 in hippocampal neurons. (a) Representative image of a 20 days in vitro (DIV) rat hippocampal neuron expressing GFP-Ubc9. Note that GFP-Ubc9 is mainly localized within the nucleus (inset). GFP-Ubc9 uorescence is also distributed in dendrites and spines. Scale bar, 20 mm. (b) Images from representative GFP-Ubc9 FRAP experimental time course in basal (Ctrl), bicuculline (BICU, 10 mM)

or tetrodotoxin (TTX, 2 mM)-treated conditions. (c) Normalized FRAP curves from spines in b showing recovery of GFP-Ubc9 uorescence after photobleaching over the course of 20 s in basal control, enhanced (BICU) or decreased (TTX) synaptic activity. Summary histograms of FRAP experiments showing the means (s.e.m.) of half-time of recovery (d), diffusion coefcients (e) and mobile fractions (f) from the number of bleached spines indicated on the bars (d). (d,e) *P 0.001; one-way analysis of variance.

Interestingly, increasing synaptic activity by blocking inhibitory GABAA receptors with bicuculline did not affect the ratio of mobile GFP-Ubc9 molecules (Fig. 1f) but signicantly increased the half-time of GFP-Ubc9 uorescence recovery (Fig. 1d; Control, 0.630.05 s; Bicu, 1.10.12 s, meanss.e.m.). The direct consequence of this effect is the concomitant decrease in GFP-Ubc9 diffusion coefcient in spines from bicuculline-treated neurons (Fig. 1e; Control, 0.590.05 mm2 s 1; Bicu,0.330.02 mm2 s 1, meanss.e.m.). The fact that the mobile fraction of GFP-Ubc9 remains unaffected in stimulated conditions concurrently to a delayed exchange of the enzyme between the shaft and the spine imply that there is an increase in the synaptic residency time of Ubc9 in activated neurons. We also showed that in contrast to GFP-Ubc9, the synaptic diffusion of free diffusing GFP molecules in spines was not regulated by activity (Supplementary Fig. 2).

Ubc9 diffusion is not regulated via NMDAR activation. Cytosolic proteins such as the Ca2 /calmodulin-dependent protein kinase II or the proteasome subunit Rpt1 are targeted to spines in activated cells predominantly via activation of NMDARs28,29. We therefore examined whether the increased synaptic residency time

of Ubc9 measured in bicuculline-treated neurons was sensitive to NMDAR inhibition (Fig. 2). We performed synaptic FRAP experiments on GFP-Ubc9 expressing neurons and the recovery curves were recorded under bicuculline treatment in the presence of the NMDAR antagonist D-AP5. Inhibition of NMDAR with D-AP5 did not modify the decrease in the rate of GFP-Ubc9 uorescence recovery when co-incubated with bicuculline (Fig. 2), indicating that the regulation of Ubc9 diffusion in spines is independent of NMDAR activation.

Ubc9 diffusion is regulated via an mGlu5R-dependent pathway. Activation of metabotropic glutamate receptors (mGluRs) results in diverse actions on neuronal excitability, synaptic transmission and plasticity by modulating the activity of a variety of ion channels and regulatory proteins30,31. We therefore assessed whether mGluRs are involved in the regulation of Ubc9 diffusion in activated spines (Fig. 2). We showed that preincubation of bicuculline-activated neurons with the specic mGlu5R antagonist 2-methyl-6-(phenylethynyl) pyridine (MPEP) occluded the increase in the half-time of uorescence recovery (Fig. 2b,c; Bicu, 1.10.12 s; Bicu MPEP,

0.700.1 s, meanss.e.m.) and the subsequent decrease in Ubc9

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Figure 2 | mGlu5R-dependent, NMDAR-independent synaptic regulation of Ubc9 diffusion. (a) Sequential images from GFP-Ubc9 FRAP experiments in individual spine (arrowheads). The NMDA receptor antagonist D-AP5 (50 mM) or the specic mGlu5R antagonist MPEP (30 mM) were tested in combination with the GABAA receptor antagonist bicuculline (10 mM) to determine the involvement of NMDA and mGlu receptors on Ubc9 diffusion.

(b) Normalized FRAP curves from spines in a showing recovery of GFP-Ubc9 uorescence after photobleaching over the time course of the experiment in bicuculline-treated cells. Histograms of FRAP experiments showing the means (s.e.m.) of half-time of uorescence recovery (c), diffusion coefcients (d) and mobile fractions (e) from the number of bleached spines indicated on the bars (c). One-way analysis of variance was performed with a Newman Keuls post test for multiple comparison data sets. (c) **P 0.001 compared with Ctrl, D-AP5, MPEP; *Po0.01 compared with BICU, D-AP5 BICU;

(d) **P 0.001 compared with Ctrl, D-AP5, MPEP; *Po0.01 compared with BICU, D-AP5 BICU.

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diffusion coefcient in activated neurons (Fig. 2d; Bicu, 0.330.02 mm2 s 1; Bicu MPEP, 0.500.03 mm2 s 1, means

s.e.m.), while it showed no effect on its own on control non-stimulated cells. Mobile fractions were not modied by the treatments (Fig. 2e). Our data therefore suggest that the activation of mGlu5R is involved in the synaptic regulation of Ubc9 diffusion.

To test whether a direct activation of mGlu5R is required to regulate the synaptic diffusion of Ubc9, GFP-Ubc9-expressing neurons were stimulated with the specic group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) and GFP-Ubc9-labelled spines were photobleached during the treatment (Fig. 3ae). Interestingly, although the mobile fraction was unaffected by the stimulation, there was an increase in the half time of uorescence recovery (Fig. 3c; Control, 0.690.04 s; DHPG, 1.020.05 s, meanss.e.m.) with the concurrent decrease in the diffusion coefcient values (Fig. 3d; Control, 0.520.02 mm2 s 1; DHPG,0.340.03 mm2 s 1, meanss.e.m.). This decreased synaptic diffusion of Ubc9 in DHPG condition was prevented when neurons were co-incubated with the specic mGlu5R antagonist MPEP (Fig. 3ad). As the mobile fraction is unmodied by the mGlu5R activation (Fig. 3e), it implies that there is no signicant accumulation of Ubc9 into spines. Consistently, although there was only a small increase in synaptic GFP-Ubc9 uorescence during DHPG treatment, we did not detect any modications in endogenous synaptic Ubc9 immunoreactivity in mGlu5R-activated neurons (Supplementary Fig. 3). Altogether, our data indicate that mGlu5R activation dynamically regulates the synapto-dendritic diffusion of Ubc9.

The mGlu5R-dependent Ubc9 diffusion is restricted to spines. We then asked whether this mGlu5R-dependent regulation of Ubc9 diffusion could be specically restricted to the synaptic area. We measured the diffusion properties of GFP-Ubc9 in dendrites (Fig. 3fj). In marked contrast to the data obtained on spines, DHPG stimulation led to Ubc9 diffusion parameters in dendrites similar to those measured in non-stimulated neurons with no modication in the rate of GFP-Ubc9 uorescence recovery (Fig. 3fj), indicating that the mGlu5R-dependent regulation of Ubc9 diffusion is restricted to spines.

mGlu5R-dependent diffusional trapping of Ubc9 into spine. To conrm that the increased synaptic residency time of Ubc9 in activated neurons results from a brief retention of Ubc9 into spines, we designed experiments in which we could directly visualize the synaptic exit of Ubc9. To perform these experiments, we swapped the GFP from GFP-Ubc9 for the green-to-red photoactivatable Dendra2 (refs 32,33) protein (Dendra2-Ubc9). Using time-lapse microscopy, we rst visualized the loss of the red photoconverted Dendra2-Ubc9 uorescence from the spine head in basal condition (Fig. 4). We compared the half-time of the red uorescence decrease from two successive photoconversion events on the same spine (paired recordings) and showed that consecutive photoactivations do not affect the exit rate of Dendra2-Ubc9 in control conditions (Fig. 4bd). In marked contrast, a 10-min mGlu5R stimulation before the second photoconversion led to a signicant delay in the exit rate of the red photoconverted uorescence from the spines (Pre-DHPG, 1.630.16 s; post-DHPG,2.780.26 s, meanss.e.m.; Fig. 4ce), further conrming our initial observation using FRAP that mGlu5R activation regulates the synapto-dendritic diffusion of Ubc9.

Ca2 and PKC-dependent synaptic diffusional trapping of Ubc9. Our data indicate that mGlu5R activation regulates the diffusion of the SUMO-conjugating enzyme at hippocampal

synapses. We therefore investigated the signalling pathways downstream of mGlu5R that could participate in this regulatory process (Fig. 5). mGlu5R are coupled to Gai and Gaq proteins30,31.

Activation of Gai downstream of mGlu5R stimulates cAMP-dependent protein kinase A (PKA) activity. To evaluate the involvement of this pathway on Ubc9 diffusion, we performed synaptic FRAP experiments in the presence of Pertussis toxin (PTX), a potent blocker of Gai proteins34. We observed that the rate of GFP-Ubc9 uorescence recovery as well as the diffusion coefcient values were unaffected by PTX, indicating that activation of Gai proteins and the downstream activation of PKA is not required to regulate the synaptic diffusion of Ubc9 (Fig. 5ac). mGlu5R are also coupled to the activation of Gaq proteins, which stimulate PLC, resulting in the formation of intracellular inositol-1,4,5-trisphosphate (IP3) and diacylglycerol. IP3 binding to its intracellular receptor leads to the release of Ca2 from internal stores, and together with diacylglycerol they activate PKC30,31. We rst assessed the contribution of internal Ca2 stores to the regulation of Ubc9 diffusion in mGlu5R-activated spines by preincubating neurons with the potent inhibitor of the endoplasmic reticulum (ER) Ca2-ATPase pump, the cyclopiazonic acid (CPA)35 (Fig. 5df). We veried the effect of

CPA using calcium imaging (Supplementary Fig. 4) and then incubated GFP-Ubc9-expressing neurons in extracellular Ca2 -free medium to deplete internal Ca2 stores before DHPG activation in a CPA and Ca2 -containing buffer. Under these conditions, the mobile fractions remained unchanged by the pharmacological inhibition (Fig. 5f). Preincubation of neurons with CPA totally prevented the synaptic trapping of GFP-Ubc9 induced by DHPG (Fig. 5d,e; DHPG, 1.350.15 s; DHPG CPA, 0.810.08 s, meanss.e.m.).

In addition to the Ca2 rise from internal store that occurs in mGlu5R-stimulated cells, mGlu5R activation induces Ca2 inux through L-VDCC30,31. We therefore asked whether

Ca2 inux through L-VDCC could also contribute to the regulation of Ubc9 diffusion in spines. We used the L-type Ca2 channel blocker Nifedipine36; Supplementary Fig. 4) to inhibit

Ca2 entry through L-VDCC following mGlu5R activation (Fig. 5df). DHPG application resulted in a signicant increase in the half-time of GFP-Ubc9 uorescence recovery (Fig. 5d; Control, 0.730.07 s; DHPG, 1.350.15 s, meanss.e.m.) and this effect was totally abolished when GFP-Ubc9-transfected neurons were co-incubated with Nifedipine (DHPG, 1.350.15 s; DHPG Nife, 0.810.08 s, meanss.e.m.). Similar results

were obtained for the diffusion coefcient values (Fig. 5e; DHPG, 0.440.04 mm2 s 1; DHPG Nife, 0.280.03 mm2

s 1, meanss.e.m.), indicating that the Ca2 rise through L-VDCC in mGlu5R-stimulated neurons is required to regulate the synaptic mobility of Ubc9. These ndings reveal a central role for Ca2 ions from external and internal sources in the regulation of Ubc9 diffusion in activated spines.

We next assessed the involvement of the PLC/PKC signalling module downstream of mGlu5R in the regulation of Ubc9 diffusion in spines (Fig. 5gi). We rst incubated GFP-Ubc9-expressing neurons with the potent PLC antagonist U73122 (ref. 37) and performed FRAP to measure the synaptic properties of Ubc9 diffusion. Inhibiting PLC with U73122 occluded the effect of DHPG on the rate of GFP-Ubc9 uorescence recovery (Fig. 5g; Control, 0.750.07 s; DHPG, 1.140.02 s, means s.e.m.) without any signicant modulation of the mobile fraction (Fig. 5i). Accordingly, the diffusion coefcient in DHPG-stimulated neurons in the presence of U73122 were not different from control conditions, indicating that PLC activation is necessary to regulate the mobility of Ubc9 in spines (Fig. 5h).

We nally evaluated the involvement of PKC activity on the synaptic diffusion of Ubc9. We rst blocked PKC activation using

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Figure 3 | mGlu5R-dependent regulation of Ubc9 diffusion at hippocampal synapses. (a) Images from representative GFP-Ubc9 FRAP experimental time course in basal (Ctrl), DHPG, MPEP or DHPG MPEP conditions. (b) Normalized curves from spines in a showing recovery of GFP-Ubc9 uorescence after

photobleaching over the course of the experiments. Summary histograms of FRAP experiments in panel a showing the means (s.e.m.) of half-time of uorescence recovery (c), diffusion coefcients (d) and mobile fractions (e) from the number of bleached spines indicated on the bars (c). (c) *P 0.001;

(d) ***P 0.001, **Po0.01, *Po0.05. Images (f) or normalized curves (g) from representative FRAP experiments performed on dendrites of GFP-Ubc9-

expressing neurons in basal and DHPG-stimulated conditions. Histograms showing the means (s.e.m.) of half-time of recovery (h), diffusion coefcients (i) and mobile fractions (j) from the number of bleached dendritic areas indicated on the bars (h). Photobleached GFP-Ubc9 molecules in the shaft were similarly exchanged for uorescent enzymes in basal and DHPG conditions.

the cell-permeable inhibitor chelerythrine (Fig. 5gl), which specically binds to the catalytic domain of the kinase38. Incubation of GFP-Ubc9-transfected neurons with chelerythrine before mGlu5R activation fully abolished the mGlu5R-dependent

increase in synaptic Ubc9 residency time evoked by DHPG (Fig. 5g,h; rates of recovery: DHPG, 1.140.02 s; DHPG

Cheler, 0.670.05 s; Diffusion coefcients: DHPG, 0.240.03 mm2 s 1; DHPG Cheler, 0.410.03 mm2 s 1, meanss.e.m.),

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Figure 4 | mGlu5R activation promotes a transient diffusional trapping of Ubc9 in spines. (a) Time-lapse series of confocal images of photoconverted Dendra2-Ubc9 red uorescence in spines in control and mGlu5R-stimulated conditions. Images of spine before Dendra2-Ubc9 photoconversion are shown in green (left). Following the rst synaptic green-to-red photoconversion, neurons were incubated with control solution (vehicle) or with DHPG (50 mM) for 10 min and spines were photoconverted a second time. The red photoconverted uorescence was then monitored as described in the Methods section.

Scale bar, 1 mm. (b) Representative sample paired recording traces of normalized uorescence from photoconverted Dendra2-Ubc9 in individual spines before (pre) and after mGlu5R activation (post) as shown in a. The thin traces (black) represent the corresponding ts. (c) Scatter plots of computed half-time of photoconverted Dendra2-Ubc9 uorescence diffusion in spines pre- and post-DHPG application. Paired t-test: vehicle, n 21; NS, not signicant;

DHPG, n 23, ***P 0.0001 compared with unstimulated conditions. (d) Histograms showing the means.e.m. of the half-time constant in seconds

computed for the exponential decay t from independent synaptic Dendra2-Ubc9 photoconversion experiments. Paired t-test: vehicle, n 21; DHPG,

n 23, ***P 0.0001 compared with unstimulated (pre) conditions. (e) Scatter plots showing the means.e.m. of half-time constant ratio of (post/pre) in

control vehicle (1.0010.025; n 21) and DHPG (1.8240.156; n 23) conditions. Unpaired t-test; ***P 0.0001.

therefore indicating that PKC activation is involved in the regulation of Ubc9 diffusion in mGlu5R-activated neurons.

To go further into the molecular mechanisms underlying the role of PKC in the regulation of Ubc9 diffusion, we incubated GFP-Ubc9-expressing neurons directly with the phorbol ester PMA39 to activate PKC (Fig. 5jl). PMA incubation led to synaptic FRAP parameters that were similar to those measured in

DHPG-treated neurons with a signicant increase in the half-time of GFP-Ubc9 uorescence recovery (Fig. 5j and Supplementary Fig. 5a,b) and the concomitant decrease of Ubc9 diffusion coefcient in PMA-treated cells (Fig. 5k). Importantly, the PMA-induced Ubc9 trapping in spine was totally abolished when neurons were co-incubated with either the cell-permeable PKC inhibitor chelerythrine or the potent PKC blocker

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Figure 5 | The transient Ubc9 synaptic trapping involves PKC activation and requires Ca2 inux. Hippocampal neurons were treated with DHPG or not (Control) in the absence or in the presence of the Gai blocker PTX (ac), the voltage-dependent calcium channel (VDCC) blocker nifedipine (Nife., df) or CPA, a potent inhibitor of the Ca2 -ATPase pumps (df), the PLC inhibitor U73122 (gi) and the PKC inhibitor chelerythrine (Cheler., gi) as described in the Methods section. Neurons were treated with the phorbol ester PMA in the presence of selective PKC inhibitors, chelerythrine (Cheler.) or dihydrosphingosine (Dihydro). Histograms show the means (s.e.m.) of half-time of recovery (a,d,g,j), diffusion coefcients (b,e,h,k) and mobile fractions (c,f,i,l) from the number of bleached spines indicated on the bars (a,d,g,j). One-way analysis of variance was performed with a NewmanKeuls post test for multiple comparison data sets. (a,b) *Po0.05 compared with Ctrl and PTX. (d) *Po0.001 compared with all treatments. (e) ***P 0.001 compared with

DHPG and **Po0.01 compared with all treatments except CPA. (g) *Po0.01. (h) **Po0.001 compared with all treatments. (j,k) *Po0.01 PMA compared with all conditions. Mobile fractions (c,f,i,l) were not signicantly different in all tested conditions.

dihydrosphingosine40 (Fig. 5j,k and Supplementary Fig. 5a,b). We then showed using synaptic photoconversion of Dendra2-Ubc9 in paired recordings that PKC activation with PMA led to a

signicant delay in the synaptic exit rate of the red photoconverted Dendra2-Ubc9 uorescence (Supplementary Fig. 5cf), further indicating that PKC activation is essential to

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promote the transient diffusional trapping of Ubc9 in mGlu5R-activated spines.

mGlu5R- and PKC-dependent regulation of synaptic sumoylation. Our data suggest that activation of mGlu5R triggers a PKC-dependent diffusional trapping of Ubc9 into spines that may lead to changes in synaptic sumoylation levels. To assess the direct impact of mGlu5R activation on synaptic sumoylation levels, we prepared synaptoneurosomes from P7 rat brains and pharmacologically stimulated in vitro these preparations before SUMO immunoblotting (Fig. 6). Activation of mGlu5R using DHPG evoked an overall increase in synaptic sumoylation. Interestingly, there was a marked increase in synaptic SUMO1 sumoylation (DHPG, 145% of control) that was fully blocked in the presence of the specic mGlu5R antagonist MPEP (Fig. 6a,b)

or using the PKC inhibitor chelerythrin. The direct activation of PKC with PMA also promoted a signicant increase in synaptic SUMO1 immunoreactivity (PMA, 144% of control) that was fully prevented when neurons were co-incubated with chelerythrine. We were unable to detect free SUMO1 in synaptoneurosomes (Fig. 6c). However, as previously demonstrated26,27, the unmodied Ubc9 enzyme was slightly detectable in P7 rat synaptoneurosomes, whereas its SUMO1-modied form represented the main form of the enzyme and may therefore constitute a pool of SUMO1 directly available for conjugation (Fig. 6a). Free SUMO2 and SUMO2-modied protein levels remained unchanged throughout the experiments, although a slight increase was measured in the levels of SUMO2-modied proteins following stimulation with either DHPG or PMA (Fig. 6a,b).

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Figure 6 | mGlu5R activation leads to a PKC-dependent increase in synaptic sumoylation. (a) Representative immunoblottings showing immunoreactivities of synaptic sumoylated proteins, mGlu5R, SUMO1-Ubc9 and Ubc9 in basal and activated synaptosomes (n 5). Control b-actin was

used as a loading control. (b) Summary histograms show meanss.e.m. of ve independent experiments. One-way analysis of variance was performed with a NewmanKeuls post test. **Po0.001; *Po0.01. (c) Representative immunoblottings showing immunoreactivities of free SUMO moieties in basal and stimulated synaptosomes. Post-nuclear fractions obtained from P7 rat brain were used as a control (rst lane) to show that free SUMO1 is detectable in our brain extracts but not in synaptosomes.

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Our data indicate that activation of mGlu5R promotes synaptic sumoylation through a PKC-dependent pathway. However, we showed that mGlu5R stimulation (Fig. 4) or direct activation of PKC (Supplementary Fig. 5c,f) promotes a decrease in the exit rate of Ubc9 from spines. As synaptoneurosomes is a closed system, it suggests that the synaptic PKC-dependent SUMO conjugation is not directly due to the modulation of Ubc9 synapto-dendritic mobility, but rather results from an increased recognition of Ubc9 to PKC-phosphorylated substrates. The direct consequence of this increased recognition process in neurons would therefore be a transient diffusional trapping of Ubc9 in activated spines.

Synaptic Ubc9 trapping implies a functional catalytic domain. To validate our hypothesis that the synaptic diffusional trapping of Ubc9 results from an increased recognition of target proteins in activated neurons, we investigated the synaptic diffusion properties of the catalytically inactive and substrate-binding-decient Ubc9-C93S mutant. Indeed, substrate recognition and enzymatic activity of Ubc9 are tightly linked, as its catalytic domain is essential for both processes4143. It was shown that the cysteine residue in position 93 in Ubc9 forms the catalytic domain holding the SUMO moiety via a thioester bond before its catalytic transfer onto a specic lysine residue of a target protein. Therefore, Ubc9 binds directly to consensus sumoylation sequences of target proteins via its catalytic domain in a way that aligns the active C93-containing site directly next to the lysine residue to modify42. Importantly, the C93S substitution totally prevents sumoylation4244. To assess whether the substrate recognition/catalytic activity of Ubc9 could be important for its synaptic regulation, we analysed the diffusion properties of the WT-active and the inactive GFP-Ubc9-C93S in mGlu5R-activated spines (Fig. 7ad). We rst ruled out potential side effects of the inactive form of GFP-Ubc9 on mGlu5R signalling pathway by investigating the levels of mitogen-activated protein kinase phosphorylation in response to DHPG31, in neurons expressing the active or inactive form of Ubc9. DHPG stimulation robustly induced Erk phosphorylation and this was not prevented on GFP-Ubc9-C93S mutant expression (Fig. 7e), indicating that the inactive Ubc9 does not occlude the downstream signalling of mGlu5R.

We then performed FRAP experiments on spines from WT and Ubc9-C93S-expressing neurons, and compared their diffusion properties (Fig. 7ad). Mobile fractions remained unaffected by the C93S mutation (Fig. 7d). As expected, DHPG induced an increase in the half-time of uorescence recovery for the WT Ubc9, while there was no difference when the inactive C93S mutant was analysed (Fig. 7a,b). These data were conrmed by comparing the diffusion coefcients where a signicant drop was measured only in DHPG-treated cells expressing GFP-Ubc9 but not with its inactive C93S mutant (Fig. 7c; Control WT, 0.440.04 mm2 s 1;

DHPG WT, 0.280.03 mm2 s 1; and DHPG WT versus DHPG C93S, 0.410.04 mm2 s 1, meanss.e.m.), indicating that the enzymatic activity and/or the substrate recognition is important for the synaptic regulation of Ubc9 diffusion. Interestingly, PKC activation with PMA had no effect on the diffusion of the C93S mutant (Fig. 7ad), invalidating a direct effect of PKC on Ubc9. Our data rather support our initial hypothesis that the transient diffusional trapping of Ubc9 into activated spine is due to the preferential anchoring of Ubc9 to synaptic PKC-phosphorylated protein substrates.

Ubc9-binding to phosphoproteins causes synaptic trapping. Many synaptic proteins are phosphorylated by PKC following mGlu5R stimulation31 and it is now clear that many SUMO

targets carry a PDSM9. This motif regulates the phosphorylation-dependent sumoylation of multiple substrates, including heat-shock factors and myocyte enhancer factor 2 (refs 9,10). To reinforce our hypothesis that mGlu5R activation leads to an increased synaptic anchoring of Ubc9 to PKC-phosphorylated substrates, we used the well-described PDSM discrimination-decient Ubc9-K65A mutant10. Importantly, Ubc9-K65A loses its ability to discriminate between PDSM phosphorylation statuses but presents the ability to equally sumoylate phosphorylated and non-phosphorylated proteins in vitro10. Ubc9-K65A presented a subcellular distribution similar to the WT enzyme (Fig. 8a). We conrmed that the Ubc9-K65A mutant is able to sumoylate proteins (Fig. 8b). We then compared the diffusion properties of GFP-Ubc9 with the PDSM discrimination-decient Ubc9-K65A mutant in mGlu5R-activated spines (Fig. 8cg). DHPG stimulation induced a signicant increase in the half-time of uorescence recovery for the WT Ubc9 (Fig. 8d,e; Control WT,0.630.04s; DHPG WT, 1.190.12s, meanss.e.m.), while there was no difference for the PDSM discrimination-decient mutant (Fig. 8e). Accordingly, a signicant drop in the diffusion coefcient was measured in DHPG-treated neurons expressing WT GFP-Ubc9 (Fig. 8f; Control WT, 0.360.02 mm2 s 1; DHPG

WT, 0.210.02 mm2 s 1, meanss.e.m.) but not when the PDSM discrimination-decient K65A mutant was analysed.

To further highlight the central role of PKC activity in the diffusional trapping of Ubc9 in activated spines, we compared the diffusion values measured from neurons expressing GFP-Ubc9 or its K65A mutant in the presence of the PKC activator PMA (Fig. 8cg). Treating GFP-Ubc9-expressing neurons with PMA resulted in a signicant increase in the half-time of uorescence recovery and the concurrent decrease in the diffusion coefcient, whereas PKC activation had no effect on the synaptic mobility of the PDSM discrimination-decient K65A mutant (Fig. 8df).

To conrm the functional impact of a tightly controlled Ubc9 regulation in spines, we analysed the excitability of neurons expressing the PDSM discrimination-decient K65A mutant (Supplementary Fig. 6). We showed that the neuronal excitability measured for the K65A mutant is signicantly decreased when compared with the WT enzyme. Indeed, although the resting potentials in WT and mutant conditions were almost similar (Supplementary Fig. 6c), the frequency of spontaneous action potentials in K65A-expressing neurons was signicantly lower than in cells expressing the WT Ubc9 (Supplementary Fig. 6a,b; K65A, 0.490.11 Hz; WT, 1.050.20 Hz, meanss.e.m.).

DiscussionHere we have investigated the regulatory mechanisms of Ubc9, the sole conjugating enzyme of the sumoylation system during the modulation of synaptic activity. Our work demonstrates that Ubc9 diffusion at hippocampal synapses is impacted by activity providing several important insights into the mechanisms that underlie this activity dependence (Fig. 9). This regulation involves an mGlu5R-dependent signalling pathway. Neuronal activation using either the GABAAR antagonist bicuculline or the direct exposure of neurons to group I mGluR agonist increases the synaptic residency time of Ubc9 and this effect is blocked using the specic mGlu5R antagonist MPEP. The downstream mGlu5R-signalling pathway regulating the synaptic diffusion of Ubc9 involves a PLC/PKC activation cascade. Importantly, the direct PKC activation mimicked the effect of mGlu5R stimulation on Ubc9 diffusion, whereas blocking PKC activity abolished the diffusional trapping of Ubc9 in activated spines. Activation of mGlu5R promotes synaptic sumoylation through a PKC-dependent pathway, suggesting that Ubc9-driven SUMO conjugation results from an increased anchoring of Ubc9 to PKC-

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26 + + +Figure 7 | The integrity of the catalytic domain of Ubc9 is crucial for the regulation of Ubc9 diffusion in spines. (a) Neurons expressing GFP-Ubc9 or its inactive C93S form were treated or not with DHPG or PMA. Normalized FRAP curves from individual spine showing recovery of WTand C93S GFP-Ubc9 uorescence after photobleaching over the time course of the experiment. Summary histograms of FRAP experiments showing the means (s.e.m.)of half-time of uorescence recovery (b, **Po0.001 compared with all treatments except PMA; *Po0.05 compared with controls), diffusion coefcients (c, *Po0.01 DHPG, PMA compared with all treatments) and mobile fractions (d) from the number of bleached spines indicated on the bars (b). One-way analysis of variance with a NewmanKeuls post test. (e) Immunoblottings using anti-phospho- and total ERK antibodies of total proteins extracted from hippocampal neurons expressing the WT, the inactive C93S form of GFP-Ubc9 or free GFP as a control. Cells were treated or not for 10 min with DHPG. Note that the C93S mutation does not affect the DHPG-induced mGluR5 downstream signalling. The lower panel shows immunoblotting with anti-GFP antibodies to control for WT, mutant GFP-Ubc9 and free GFP protein levels.

phosphorylated proteins. Consistently, the catalytic domain of Ubc9 comprising the C93 residue, known to be essential for the activity and substrate recognition of the enzyme4244, is also central to the synaptic regulation of Ubc9 diffusion through the PKC-dependent phosphorylation of synaptic proteins. We conrmed this transient PKC-dependent anchoring of Ubc9 in activated spines using the phosphorylation discrimination-decient Ubc9-K65A mutant and demonstrate its impact on neuronal excitability.

Our data also demonstrated that blocking the intracellular Ca2 rise through L-VDCC or the Ca2 release from internal

ER store in mGlu5R-activated neurons abolished the synaptic regulation of Ubc9 diffusion. These results suggest that the Ca2

released from the ER is downstream of the VDCC-mediated Ca2 inux. This is consistent with recent data showing that large synaptic complexes comprising mGlu5R, IP3 receptors and

L-VDCC play key roles in the calcium-current facilitation evoked by mGlu5R activation45. The mGlu5R-induced calcium-current facilitation requires the activation of calcium-induced calcium release that was triggered by the L-VDCC opening45. It was also reported using L-VDCC knockout mice that activation of mGlu5R sensitized both IP3 and ryanodin receptors onto the ER to Ca2 entry through the L-type voltage-gated Cav1.3 channels, and that this event promoted calcium-induced calcium release that was essential to promote synaptic plasticity46. It is therefore not surprising to see that the blockade of Ca2 inux

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Figure 8 | Ubc9 trapping occurs via its recognition to PKC-phosphorylated proteins. (a) Representative image of a 20 DIV hippocampal neuron expressing the GFP-tagged PDSM discrimination-decient Ubc9-K65A mutant. Insets show the nucleus and an enlarged dendrite. Scale bar, 20 mm.

(b) Immunoblottings using anti-mCherry (top panel), anti-GFP (middle panel) or anti-b-actin antibodies (loading control) of total proteins extracted from COS7 cells expressing GFP-Ubc9, GFP-Ubc9-K65A mutant or free GFP together with mCherry-SUMO1. Molecular weight in kDa is shown. Sequential images (c) or normalized curves (d) from representative FRAP experiments on individual spine (arrowheads) from neurons expressing GFP-Ubc9 or GFPUbc9-K65A treated or not (Ctrl) with DHPG or PMA. Summary histograms of FRAP experiments showing the means (s.e.m.) of half-time of uorescence recovery (e, **P 0.001 and *P 0.01), diffusion coefcients (f, *Po0.01 compared with all conditions) and mobile fractions (g) from the

number of bleached spines indicated on the bars (e). One-way analysis of variance with a NewmanKeuls post test.

from both L-VDCC and internal ER stores prevent the synaptic trapping of Ubc9 and reinforce our conclusion that a Ca2 rise is critical for the synaptic regulation of sumoylation.

Another potential mechanism for the regulation of Ubc9 function could be the direct phosphorylation of the enzyme. It was reported that Ubc9 could be phosphorylated in vitro on

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Agonist

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serine 71 by the CDK1/cyclin B kinase but not by other kinases including PKA, ERK, JNK2 or CDK2. The direct effect of Ubc9 phosphorylation by CDK1 is an increased SUMO conjugation47. However, the authors cannot rule out the possibility that this increased sumoylation may also result from the CDK1-dependent phosphorylation of other proteins, thereby increasing Ubc9 binding/recognition and subsequently sumoylation of CDK1-phosphorylated substrates.

Intriguingly, the Ubc9-K65A mutant showed diffusion parameters similar to those measured for the WT form of the enzyme in mGlu5R- or PMA-activated conditions. These results suggest that contrary to WT Ubc9, the K65A mutant is able to recognize and bind to additional non-phosphorylated synaptic substrates in resting conditions, leading to an apparent reduced diffusion in non-activated spines. As Ubc9-K65A is unable to discriminate the phosphorylation status of the substrates, it is not surprising that we cannot measure any additional diffusional trapping in mGlu5R or PMA-stimulated conditions. Moreover, the neuronal expression of the K65A mutant led to a decrease in neuronal excitability highlighting a functional role for the regulation of the sumoylation process in synaptic communication. The fact that neuronal excitability is decreased in PDSM discrimination-decient Ubc9-expressing neurons will lead to many exciting research avenues, as numerous unidentied synaptic proteins may be targeted for sumoylation in a non-regulated manner. It also implies that the balance between sumoylation and desumoylation is impacted when the Ubc9-K65A mutant is expressed. Further work will now be required to identify synaptically activated sumoylation substrates and to characterize the regulatory mechanisms of desumoylation enzymes in spines.

Interestingly, sumoylation of some synaptic proteins has already been shown to require phosphorylation before sumoylation. Konopacki et al. reported the agonist-induced PKC phosphorylation of the kainate receptor GluK2 C-terminus and showed that this event is required for GluK2 sumoylation23. Both events were shown to be essential for the endocytosis of GluK2 that occurs during kainate receptor-mediated long term depression at hippocampal mossy bre synapses25 and are consistent with our work showing that the mGlu5R-driven synaptic phosphorylation triggers the binding of Ubc9 to de novo PKC-phosphorylated proteins and the transient synaptic trapping of Ubc9.

Another interesting aspect of our work is that sumoylation can act as a ubiquitin antagonist by competing at specic lysine residues on target proteins to inhibit protein degradation15,16. For

instance, Bingol and Schuman29 demonstrated that the synaptic trafcking of the proteasome is activity regulated. Indeed, NMDAR activation induces the translocation of the proteasome into spines, leading to ubiquitination and degradation of synaptic proteins29. The direct consequence of this is the shaping of the synaptic protein content. Here we demonstrate that the synaptic regulation of Ubc9 does not involve NMDAR but mGlu5R. Our data therefore add an additional level of regulation in the control of the synaptic protein content, as these two post-translational modications can target the same residue to regulate protein degradation.

Our work also raises intriguing questions as to the role of sumoylation in brain disorders involving altered group I mGluR-signalling pathways. Indeed, these receptors have been involved in the modulation of synaptic transmission and plasticity30,31, and in the pathogenesis of various central nervous system disorders, including but not restricted to schizophrenia or chronic pain, as well as several neurological diseases31. We show that the synaptic diffusion of Ubc9 is regulated through an mGlu5R-dependent signalling pathway. Thus, under pathological conditions, the deregulated mGlu5R signalling could modify Ubc9 diffusion and consequently induce changes in protein sumoylation, leading to altered synaptic function. Therefore, an important focus of future studies will be to characterize sumoylation in brain disorders where mGlu5R signalling is deregulated.

Methods

Constructs. The Gateway destination vector for the fusion of open reading frames to the amino terminus of mouse Ubc9 sequence is a generous gift from Dr Niedenthal48. The GFP-Ubc9 construct was made by inserting the GFP coding sequence in frame with the N-terminus of Ubc9 with the Gateway recombination technology (Invitrogen). Ubc9-C93S and K65A mutant constructs were made by site-directed mutagenesis using Quick-change mutagenesis (Agilent).

Cell culture. Hippocampal neurons were prepared from E18 pregnant Wistar rats as previously described27,49. Briey, cells were plated in Neurobasal medium (Invitrogen, France) supplemented with 2% B27 (Invitrogen), 0.5 mM glutamine and penicillin/streptomycin on 24-mm glass coverslips pre-coated with poly-L-

lysine (0.1 mg ml 1). Neurons (110,000 cells per coverslip) were fed once a week for 3 weeks in Neurobasal medium supplemented with 2% B27 and penicillin/

streptomycin. All procedures were approved by our local Animal Ethics Committee (Comit Institutionnel dthique Pour lAnimal de Laboratoire N28, Nice, France; Project reference NCE/2012-63).

Neuronal transfection. Hippocampal neurons (1820 days in vitro (DIV)) were transfected using Lipofectamin 2000 (Invitrogen), according to the manufacturers instructions, with 3 mg of plasmid DNA and used 4872 h post transfection.

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Immunocytochemistry. Hippocampal neurons (1921 DIV) treated or not for 10 min with 50 mM DHPG at 37 C were xed with paraformaldehyde 4% in PBS for 10 min at room temperature (RT) and washed three times for 5 min in PBS. Cells were then permeabilized for 20 min in PBS containing 0.1% Triton X-100 and 10% Horse Serum at RT. Neurons were immunostained with a mouse anti-Ubc9 (1/50; BD Bioscience, France) and a rabbit anti-Homer1 (1/200; Synaptic System, Germany) overnight at 4 C in PBS containing 0.05% Triton X-100 and 5% HS. Cells were washed three times in PBS and incubated with the appropriate secondary antibodies conjugated to Alexa488 or Alexa594, and mounted with Mowiol (Sigma). Confocal images (1,024 1,024 pixels) were acquired with a 63 oil-

immersion lens (numerical aperture (NA) 1.4) on an inverted TCS-SP5 confocal microscope (Leica Microsystems, France). Z-series of seven to eight images of randomly selected dendrites were compressed into two dimensions using the maximum projection algorithm of the Leica software. Quantication was performed using ImageJ software and the synaptic enzymatic staining was measured with the use of an in-house ImageJ macro26,27. Briey, confocal image of the synaptic marker was used to produce a mask after an automated intensity threshold. Masks were applied to the corresponding images, and the uorescence intensity within the synaptic area was measured.

Calcium imaging. Hippocampal neurons were loaded in neurobasal containing5 mM of Fura-2AM (Invitrogen) for 30 min and then washed and incubated for an additional 30 min period in neurobasal medium. After a further three washes in Earles buffer (25 mM HEPES-Tris pH 7.4, 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 0.9 g l 1 glucose), Fura-2AM-loaded neurons were continuously perfused (1 ml min 1, 37 C) on an inverted microscope (AxioObserver,

Carl Zeiss) equipped with a 300W Xenon lamp (Suttler Instruments) and a Fluar

40 (NA 1.4) oil-immersion objective. Fura-2AM was sequentially excited at 340 and 380 nm and the emission monitored at 510 nm. Images were acquired with a cascade 512 EMCCD camera every 5 s and digitized using Metauor software (Roper Scientic). The intracellular calcium concentration [Ca2 ]i was estimated by measuring the F340/380 nm uorescence ratio. Neurons were treated with various pharmacological drugs: 50 mM DHPG, 50 mM MPEP, 10 mM Nifedipine or 20 mM CPA diluted in Earles buffer.

Live cell imaging. Protocols were performed as previously described49,50. Briey, dendrites from live GFP- or GFP-Ubc9-transfected neurons (1920 DIV) were kept on a heated stage (set at 37 C) on a Nikon Ti inverted microscope. GFP uorescence was excited through a 100 oil-immersion lens (NA, 1.4) using a

488-nm laser light (50 mW, 12%) and time series (15 Hz) were collected for 25 s as a single image slice using a Perkin Elmer Ultra-View spinning disk solution.

Neurons were treated or not with 10 mM bicuculline, 2 mM TTX or 50 mM DHPG in Earles buffer (25 mM HEPES-Tris pH 7.4, 140 mM NaCl, 5 mM KCl,1.8 mM CaCl2, 0.8 mM MgCl2, 0.9 g l 1 glucose) for 1020 min in Earles buffer. A 1020 min preincubation at 37 C was achieved when specic inhibitors were used. Pharmacological drugs (MPEP 30 mM, D-APV 50 mM, PTX 0.5 mg ml 1, U73122 2 mM, chelerythrine 5 mM, dihydrosphingosine 50 mM, PMA 2 mM, nifedipine10 mM or CPA 20 mM) were diluted in Earles buffer.

FRAP measurements. Fluorescence intensity variations in spines were analysed using the FRAP module of the Volocity 6.3 software. Fluorescence recovery curve in FRAP allows the direct determination of two parameters. First, the difference between the basal level of uorescence and the recovered plateau level after photobleaching reects the immobile fraction of protein and conversely the mobile diffusible fraction. Immobile fractions reect the direct or indirect binding of the protein of interest to cytoskeleton constituents. Second, the half-time of recovery species the mobility of the diffusible fraction of the protein of interest. Fluorescence data were collected from regions of interest drawn around the uorescence of GFP- or GFP-Ubc9-expressing spines or shafts. FRAP data were expressed as a percentage of initial uorescence (average uorescence value from the 5-s imaging period immediately before photobleaching) over time and tted with a single-phase exponential function (f(t) y Ae kt) using the Volocity 6.3 software from Perkin

Elmer. Mobile fraction (in %), half-time of recovery (in seconds) and diffusion coefcient (in mm2 s 1; D photobleached spine area/4t1/2) values were extracted

for each experiment using the FRAP module of the Volocity 6.3 software.

Dendra2-Ubc9 photoconversion measurements. For Dendra2-Ubc9 experiments, the GFP tag from the GFP-Ubc9 construct was exchanged for the green-tored photoswitchable Dendra2 protein32,33 (Evrogen JSC, Russia). Individual Dendra2-Ubc9-expressing spines were photoconverted for 30 ms using a 405-nm laser light (50 mW, 100%). The red photoconverted Dendra2-Ubc9 was excited using a 561-nm laser light (50 mW, 20%) and time series (10 Hz) were collected for 20 s as a single image slice using a Perkin Elmer Ultra-View spinning disk solution. For paired recordings of synaptic Dendra2-Ubc9 photoconversion, spines were rst photoconverted and imaged for 20 s followed by a 10-min period in either control (vehicle) or the indicated drug solution at 37 C and then photoconverted/imaged a second time. The decrease in red uorescence from the Dendra2-Ubc9 photoconverted spines was measured over time using the Volocity 6.3 software and data expressed as a percentage of the initial red photoconverted uorescence (F/F0).

Curves were tted using a mono-exponential decay equation and data statistically analysed (paired and unpaired t-test as indicated) with Prism 4 (GraphPad software, Inc).

Synaptosomal preparation and stimulation. Brains (12 Wistar P7 rats) were homogenized at 4 C in 7 volumes (w/v) of 10 mM Tris buffer (pH 7.4) containing0.32 M sucrose and protease inhibitors (Complete EDTA-free; Roche, USA)51,52. The resulting homogenate was centrifuged at 1,000g for 5 min at 4 C to remove nuclei and cellular debris. Synaptosomal fractions were puried by centrifugation for 10 min at 14,000 r.p.m. (SW41Ti rotor) at 4 C using Percoll-sucrose density gradients (2-6-10-20%; v/v). Each fraction from the 1020% interface was collected, washed in 10 ml of a 5 mM HEPES buffer pH 7.4 (NaOH) containing 140 mM NaCl, 3 mM KCl, 1.2 mM MgSO4, 1.2 mM CaCl2, 1 mM NaH2PO4, 5 mM

NaHCO3, 10 mM glucose by centrifugation. Synaptosomes were resuspended in0.6 ml of the same buffer at RT and gently agitated at 37 C. After 10 min, antagonists, inhibitors or vehicle were added to synaptosomes for 10 min before synaptosomal stimulation with the indicated drug for a further 10 min at 37 C. Ice-cold HEPES medium (0.6 ml) containing freshly prepared NEM (40 mM) was then added to the stimulated synaptosomes and the suspension was immediately centrifuged at 10,000 g at 4 C for 5 min. Pellets were resuspended in 2 Laemlli

buffer containing 5% bME, boiled for 10 min and analysed by immunoblotting. Intensities of bands on the immunoblottings were quantied using Bio1D software (Vilber-Lourmat, France). Densitometric values measured from each lane were compared with the corresponding untreated control fraction.

Immunoblotting. Transfected COS7 cells (ATCC, Molsheim, France) were homogenized in lysis buffer (10 mM TrisHCl pH7.5, 10 mM EDTA, 150 mM NaCl, 1% Triton X100, 0.1% SDS) in the presence of a mammalian protease inhibitor cocktail (Sigma, 1/100) and 20 mM freshly prepared NEM (Sigma) to protect proteins from desumoylation. Protein extracts (30 mg) were resolved by

SDSPAGE and immunoblotted with primary antibodies: rabbit polyclonal anti-SUMO1 (ref. 53), 1.7 mg ml 1; rabbit anti-SUMO2/3 (ref. 27), 1/240; Invitrogen,

USA; mouse anti-Ubc9 (refs 20,26), 1/200, BD Bioscience, France; mouse anti-GFP, 1/1,000, Roche, Germany, or rat anti-mCherry54, 1/500, ChromoTek GmbH, Germany; mouse anti-phosphorylated-ERK and rabbit anti-ERK55, 1/1,000, Tebu-Bio, France. Standard b-actin loading controls were included using a mouse anti-bactin antibody (1/5,000, Sigma). Full-size blottings for cropped gels can be found in Supplementary Fig. 7.

Electrophysiological recordings. Neuronal excitability of transfected neurons (1921 DIV) was assessed using whole-cell conguration of the patch clamp technique. Membrane potentials were recorded for 3 min at RT using an axopatch 200B amplier (Axon Instruments) with a 2-kHz low-pass lter. Data were sampled at 10 kHz, digitized by a Digidata 1440 A-D/D-A converter (Axon Instruments), recorded and analysed using pClamp software. Patch pipettes (36 MO)

contained (in mM): 135 KCl, 2.5 Na2-ATP, 2 MgCl2, 2.1 CaCl2, 5 EGTA, 10 HEPES (pH 7.25 with KOH). Control bath solution contained (in mM): 145 NaCl,5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose (pH 7.4 with NaOH).

Statistical analysis. Statistical analyses were calculated using GraphPad Prism 4 (GraphPad software, Inc.). Data were expressed as means.e.m. One-way analysis of variance were performed with a NewmanKeuls post test for multiple comparison data sets. All data were tested for normal distribution. Statistical analyses for electrophysiological data were computed using a MannWhitney test.

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Acknowledgements

We thank John Hallenbeck and Yang-Ja Lee (NIH, Bethesda, USA) for the generous gift

of anti-SUMO1 antibody; H. Leonhardt for the gift of mCherry antibody; and R. Nie

denthal for UFDS-Ubc9 vector. We also thank F. Aguila for excellent artwork. We

gratefully acknowledge the Fondation pour la Recherche Mdicale (Equipe labellise

DEQ20111223747), the Agence Nationale de la Recherche (ANR-2011-JSV4-003 1), the

Jrme Lejeune and Bettencourt-Schueller foundations for nancial support. S.M. is a

fellow from the Young Investigator CNRS ATIP programme. We also thank the

French Government for the Investments for the Future LABEX SIGNALIFE (ANR-11-

LABX-0028-01).

Author contributions

C.L., F.C. and S.M. designed the experiments. C.L. performed most of the FRAP imaging.

F.C. performed calcium imaging and some FRAP and photoconversion experiments.

C.L., C.G., F.C., A.K. and G.P. performed biochemical experiments. C.L., G.P. and F.C.

prepared neuronal cultures. M.C. and E.D. performed electrophysiological recordings.

C.L., C.G., F.C. and S.M. analysed the data and wrote the manuscript. S.M. supervised

the project.

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How to cite this article: Loriol, C. et al. mGlu5 receptors regulate synaptic sumoylation

via a transient PKC-dependent diffusional trapping of Ubc9 into spines. Nat. Commun.

5:5113 doi: 10.1038/ncomms6113 (2014).

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Sumoylation plays important roles in the modulation of protein function, neurotransmission and plasticity, but the mechanisms regulating this post-translational system in neurons remain largely unknown. Here we demonstrate that the synaptic diffusion of Ubc9, the sole conjugating enzyme of the sumoylation pathway, is regulated by synaptic activity. We use restricted photobleaching/photoconversion of individual hippocampal spines to measure the diffusion properties of Ubc9 and show that it is regulated through an mGlu5R-dependent signalling pathway. Increasing synaptic activity with a GABA_A receptor antagonist or directly activating mGlu5R increases the synaptic residency time of Ubc9 via a Gα_q /PLC/Ca²⁺ /PKC cascade. This activation promotes a transient synaptic trapping of Ubc9 through a PKC phosphorylation-dependent increase of Ubc9 recognition to phosphorylated substrates and consequently leads to the modulation of synaptic sumoylation. Our data demonstrate that Ubc9 diffusion is subject to activity-dependent regulatory processes and provide a mechanism for the dynamic changes in sumoylation occurring during synaptic transmission.

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Title

mGlu5 receptors regulate synaptic sumoylation via a transient PKC-dependent diffusional trapping of Ubc9 into spines

Author

Loriol, Céline; Cassé, Frédéric; Khayachi, Anouar; Poupon, Gwénola; Chafai, Magda; Deval, Emmanuel; Gwizdek, Carole; Martin, Stéphane

Pages

5113

Publication year

2014

Publication date

Oct 2014

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms6113

ProQuest document ID

1610986412

mGlu5 receptors regulate synaptic sumoylation via a transient PKC-dependent diffusional trapping of Ubc9 into spines

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