Xiao-Min Min Zhang

Urielle François

Katlin Silm

Maria Florencia Angelo

Maria Victoria Fernandez-Busch

Mona Maged

Christelle Martin

Veronique Bernard

Fabrice P Cordelières

Melissa Deshors

Maria Victoria Fernández Busch

Véronique Bernard

Stéphanie Pons

Uwe Maskos

Alexis-Pierre Bemelmans

Sonja M Wojcik

Salah El Mestikawy

Yann Humeau

Etienne Herzog
email: etienne.herzog@u-bordeaux.fr

A proline-rich motif on VGLUT1 reduces synaptic vesicle super-pool and spontaneous release frequency

Keywords: proline-rich domain, synaptic vesicle, synaptic vesicle super-pool, vesicular glutamate transporter, liquid phase separation, EndophilinA1, quantal neurotransmitter release, fluorescence live cell microscopy, synaptic vesicle cycle

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Synaptic vesicles (SVs) engage in multiple protein interactions at the presynaptic active zone [START_REF] Südhof | Synaptic vesicle exocytosis[END_REF] and fuse with the presynaptic plasma membrane upon calcium influx, to release their neurotransmitter content [START_REF] Lisman | The sequence of events that underlie quantal transmission at central glutamatergic synapses[END_REF]. Within axon terminals, SVs are segregated from other organelles and grouped in a cluster behind the active zone [START_REF] Gray | Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study[END_REF]. In adult neurons, SV supply at synapses depends not only on de novo vesicle biogenesis, but also on the exchange of mobile SVs between en passant boutons along the axon. This exchange pool has been named "SV superpool" [START_REF] Kraszewski | Mobility of synaptic vesicles in nerve endings monitored by recovery from photobleaching of synaptic vesicleassociated fluorescence[END_REF][START_REF] Darcy | Constitutive sharing of recycling synaptic vesicles between presynaptic boutons[END_REF][START_REF] Westphal | Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement[END_REF][START_REF] Staras | A Vesicle Superpool Spans Multiple Presynaptic Terminals in Hippocampal Neurons[END_REF][START_REF] Herzog | In vivo imaging of intersynaptic vesicle exchange using VGLUT1 Venus knock-in mice[END_REF] and is probably a feature of both glutamatergic and GABAergic axons [START_REF] Wierenga | GABAergic synapses are formed without the involvement of dendritic protrusions[END_REF]. While the last steps in the regulation of SV release have been studied intensively in different models, the relationship between super-pool SVs, clustered SVs, and the fine-tuning of release at terminals is much less well understood. However, synapsins, a family of SV associated phospho-proteins, play a central role in the regulation of SV clustering and mobility [START_REF] Pieribone | Distinct pools of synaptic vesicles in neurotransmitter release[END_REF][START_REF] Song | Synapsin Isoforms and Synaptic Vesicle Trafficking[END_REF].

A growing body of evidence furthermore suggests that SV cluster formation may result from a liquid phase separation from other cytoplasmic elements [START_REF] Milovanovic | Synaptic Vesicle Clusters at Synapses: A Distinct Liquid Phase?[END_REF][START_REF] Milovanovic | A liquid phase of synapsin and lipid vesicles[END_REF]. Phase separation may be induced by the loose interaction of multiple proline-rich (or Poly-Proline; PRD) domains with multiple SH3 (Src Homology 3) domain proteins [START_REF] Li | Phase transitions in the assembly of multivalent signalling proteins[END_REF]. Indeed, PRD/SH3 interactions are numerous among the actors of SV trafficking such as synapsins and dephosphins [START_REF] Slepnev | Accessory factors in clathrin-dependent synaptic vesicle endocytosis[END_REF][START_REF] Pechstein | Taking a back seat: synaptic vesicle clustering in presynaptic terminals[END_REF]. In addition to these interactions, the actin cytoskeleton may contribute to the scaffolding of SV clusters and SV super-pool motility [START_REF] Darcy | Constitutive sharing of recycling synaptic vesicles between presynaptic boutons[END_REF][START_REF] Morales | Actin-dependent regulation of neurotransmitter release at central synapses[END_REF][START_REF] Sankaranarayanan | Actin has a molecular scaffolding, not propulsive, role in presynaptic function[END_REF][START_REF] Shupliakov | Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton[END_REF][START_REF] Gramlich | Actin/Myosin-V-and Activity-Dependent Inter-synaptic Vesicle Exchange in Central Neurons[END_REF].

Besides SV dynamics and release competence, SV loading with neurotransmitter is another important parameter for the fine-tuning of neurotransmission. To fulfill this function, each excitatory SV may contain between 4 and 14 molecules of Vesicular Glutamate Transporters [START_REF] Takamori | Molecular anatomy of a trafficking organelle[END_REF][START_REF] Mutch | Protein Quantification at the Single Vesicle Level Reveals That a Subset of Synaptic Vesicle Proteins Are Trafficked with High Precision[END_REF]. Three isoforms of VGLUTs have been identified, and named VGLUT1-3 [START_REF] Takamori | Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons[END_REF][START_REF] Bellocchio | Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter[END_REF][START_REF] Herzog | The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons[END_REF][START_REF] Fremeau | The expression of vesicular glutamate transporters defines two classes of excitatory synapse[END_REF][START_REF] Gras | A third vesicular glutamate transporter expressed by cholinergic and serotoninergic neurons[END_REF][START_REF] Schäfer | Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons[END_REF].

They share a nearly identical glutamate transport mechanism [START_REF] Schenck | A chloride conductance in VGLUT1 underlies maximal glutamate loading into synaptic vesicles[END_REF][START_REF] Preobraschenski | Vesicular glutamate transporters use flexible anion and cation binding sites for efficient accumulation of neurotransmitter[END_REF][START_REF] Eriksen | Protons Regulate Vesicular Glutamate Transporters through an Allosteric Mechanism[END_REF] but have distinct expression patterns [START_REF] Fremeau | VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate[END_REF]. VGLUT1 is predominantly expressed in pathways of the olfactory bulb, neo-cortex, hippocampus and cerebellum and is associated with low release probability, while VGLUT2 is strongly expressed in sub-cortical pathways of the thalamus and brainstem, and is preferentially associated with high release probability projections [START_REF] Varoqui | Identification of the differentiation-associated Na+/PI transporter as a novel vesicular glutamate transporter expressed in a distinct set of glutamatergic synapses[END_REF][START_REF] Fremeau | Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites[END_REF]. This observation raised questions regarding a potential role of the VGLUT transporters in tuning SV release probability.

Hence, soon after their initial characterization, VGLUT1 and -2 were suspected to bear additional functional features that influence neurotransmitter release beyond quantal size [START_REF] Fremeau | Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites[END_REF][START_REF] Wojcik | An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size[END_REF][START_REF] Moechars | Vesicular glutamate transporter VGLUT2 expression levels control quantal size and neuropathic pain[END_REF][START_REF] Wallén-Mackenzie | Vesicular glutamate transporter 2 is required for central respiratory rhythm generation but not for locomotor central pattern generation[END_REF]. We discovered that mammalian VGLUT1, but not -2 or -3, interacts with the SH3 domain of endophilinA1 via a proline-rich sequence [START_REF] Vinatier | Interaction between the vesicular glutamate transporter type 1 and endophilin A1, a protein essential for endocytosis[END_REF][START_REF] Voglmaier | Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling[END_REF][START_REF] De Gois | Identification of endophilins 1 and 3 as selective binding partners for VGLUT1 and their co-localization in neocortical glutamatergic synapses: implications for vesicular glutamate transporter trafficking and excitatory vesicle formation[END_REF]. The VGLUT1/EndophilinA1 interaction reduces SV release probability [START_REF] Weston | Interplay between VGLUT isoforms and endophilin A1 regulates neurotransmitter release and short-term plasticity[END_REF] and increases the speed of endocytosis of several SV proteins upon long trains of stimulation [START_REF] Voglmaier | Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling[END_REF][START_REF] Pan | Vesicular Glutamate Transporter 1 orchestrates recruitment of other synaptic vesicle cargo proteins during synaptic vesicle recycling[END_REF]. Furthermore, VGLUTs bear several di-leucine motifs on their N-and C-terminal sequences, which are responsible for efficient internalization after exocytosis [START_REF] Voglmaier | Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling[END_REF][START_REF] Pan | Vesicular Glutamate Transporter 1 orchestrates recruitment of other synaptic vesicle cargo proteins during synaptic vesicle recycling[END_REF][START_REF] Foss | Multiple Dileucine-like Motifs Direct VGLUT1 Trafficking[END_REF][START_REF] Li | VGLUT2 Trafficking Is Differentially Regulated by Adaptor Proteins AP-1 and AP-3[END_REF]. The functional relevance of these additional properties of VGLUTs is underscored by the 40% reduction in the number of SVs at Slc17a7 (VGLUT1) knock-out (VGLUT1 KO) hippocampal Schaffer collateral and cerebellar parallel fiber terminals [START_REF] Fremeau | Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites[END_REF][START_REF] Siksou | A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility[END_REF]. Despite this reduction in clustered SV numbers, SV protein expression is not diminished nor displaced to other subcellular compartments [START_REF] Siksou | A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility[END_REF].

Yet, we unexpectedly discovered a significantly larger SV super-pool in the axons of VGLUT1 KO neurons [START_REF] Siksou | A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility[END_REF]. Also, VGLUT1 KO SVs appear pleomorphic under hyperosmotic chemical fixation [START_REF] Siksou | A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility[END_REF][START_REF] Herman | Vesicular glutamate transporter expression level affects synaptic vesicle release probability at hippocampal synapses in culture[END_REF], however this latter phenotype is most likely a direct consequence of a major change in the ionic composition of the SV lumen [START_REF] Schenck | A chloride conductance in VGLUT1 underlies maximal glutamate loading into synaptic vesicles[END_REF][START_REF] Preobraschenski | Vesicular glutamate transporters use flexible anion and cation binding sites for efficient accumulation of neurotransmitter[END_REF][START_REF] Eriksen | Protons Regulate Vesicular Glutamate Transporters through an Allosteric Mechanism[END_REF][START_REF] Martineau | VGLUT1 functions as a glutamate/proton exchanger with chloride channel activity in hippocampal glutamatergic synapses[END_REF].

In the present work, we determined the minimal domain responsible for the influence of VGLUT1 on the axonal super-pool in mammals. To this end, we generated mutants that disconnect the transport function of VGLUT1 from its trafficking function. We observed that VGLUT1 reduces SV super-pool size as well as the frequency of miniature Excitatory Post-Synaptic Currents (mEPSC) exclusively through the interaction of its PRD2 motif with EndophilinA1. These effects were further mediated by the non-canonical interaction of the VGLUT1/EndophilinA1 complex with the SH3B domain of the presynaptic scaffold protein intersectin1 [START_REF] Slepnev | Accessory factors in clathrin-dependent synaptic vesicle endocytosis[END_REF]. Taken together, our data support the idea that VGLUT1 fine-tunes SV release by strengthening the liquid phase-separation between clustered and super-pool SVs.

RESULTS

A specific and dose dependent reduction of super-pool size by VGLUT1

We first compared SV exchange between clusters and the axonal super-pool in wild type and VGLUT1 KO littermate primary neuron cultures. To this end, we expressed a tagged synaptobrevin protein (Syb2 EGFP ) as a reporter (Figure 1A). Through FRAP (fluorescence recovery after photo-bleaching) of boutons we measured higher exchange rates of Syb2 EGFP with the axonal compartment, for VGLUT1 KO neurons compared to wild-type neurons (Figure 1B, C, supplementary file 1). A higher exchange rate of VGLUT1 KO SVs with the axonal super-pool is in line with our previous findings [START_REF] Siksou | A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility[END_REF]. We then transduced VGLUT1 Venus cDNA [START_REF] Herzog | In vivo imaging of intersynaptic vesicle exchange using VGLUT1 Venus knock-in mice[END_REF] to rescue VGLUT1 KO neurons or over-express VGLUT1 in wild type neurons (Figure 1D). FRAP of VGLUT1 venus fluorescence revealed that VGLUT1 overexpression further reduces SV exchange with axonal pools compared to the rescue of the knock-out to endogenous levels (Figure 1E). Finally, VGLUT2 venus expression didn't reduce the VGLUT1 KO larger super-pool phenotype (Figure1-figure supplement 1, supplementary file 1). Therefore, VGLUT1 expression in neurons reduces the size of the SV super-pool in a dose dependent manner.

Structure analysis of VGLUT1

To uncover the molecular mechanism by which VGLUT1 regulates SV super-pool size, we generated a series of mutants spanning the sequence of the transporter (Figure 2A and Table 1). VGLUT1 contains 12 trans-membrane domains with both termini on the cytoplasmic side and N-glycosylation on the first luminal loop [START_REF] Almqvist | Docking and homology modeling explain inhibition of the human vesicular glutamate transporters[END_REF]. As vesicular glutamate transport strongly impacts SV tonicity, we first aimed at determining whether the SV loading state impacts SV mobility between SV clusters and the axonal super-pool. A triple point mutant R80Q, R176K, R314Q was generated to produce a glutamate transport deficient transporter as previously reported for VGLUT2 [START_REF] Herman | Vesicular glutamate transporter expression level affects synaptic vesicle release probability at hippocampal synapses in culture[END_REF][START_REF] Almqvist | Docking and homology modeling explain inhibition of the human vesicular glutamate transporters[END_REF]sVGLUT1 for silent VGLUT1; Figure 2A blue residues; 57).

We then focused our efforts on several conserved patterns at the VGLUT1 C-terminus. Indeed, mammalian VGLUT1 displays a unique double proline-rich (PRD1 530-540; PRD2 550-556)

pattern conserved in all mammals and absent in VGLUT2 and -3 or in invertebrate orthologs of VGLUT1 [START_REF] Vinatier | Interaction between the vesicular glutamate transporter type 1 and endophilin A1, a protein essential for endocytosis[END_REF]. We thus generated deletions and point mutations to test the function of the PRD motifs (Figure 2A and Table 1). A conserved 540SYGAT sequence between PRD1 and PRD2 is present in all VGLUT isoforms including invertebrates [START_REF] Vinatier | Interaction between the vesicular glutamate transporter type 1 and endophilin A1, a protein essential for endocytosis[END_REF]. In addition to the deletion mutants, we also generated S540 and T544 to alanine mutations (Figure 2A and Table 1). We furthermore tested the full deletion of the C-terminus and point mutations in a putative PDZ-type3 binding domain (DQL514; PDZ: Post-synaptic density protein/Drosophila disc large tumor suppressor/Zonula occludens-1; see Figure 2A and Table 1).

All mutants were tagged using our successful c-terminal strategy ( 8) and the expression level Vesicular glutamate uptake function does not influence SV super-pool size.

VGLUT1 KO neurons display hypotonic SVs due to the loss of glutamate transport function [START_REF] Li | VGLUT2 Trafficking Is Differentially Regulated by Adaptor Proteins AP-1 and AP-3[END_REF]. To monitor a potential effect of SV lumen tonicity on SV mobility, we transduced VGLUT1 mCherry or the triple mutation sVGLUT1 mCherry together with Syb2 EGFP in VGLUT1 KO neurons (Figure 3A) and probed SV turn over at synapses using Syb2 EGFP FRAP. While we had a very high percentage of co-transduced neurons, we could find fibers with no VGLUT1 mCherry signal and used these as a negative control. Both rescue conditions lowered the exchange of SVs compared to VGLUT1 KO synapses of the same cultures significantly and to the same degree (Figure 3B, supplementary file 1). In patch clamp experiments, a remaining spontaneous activity was found in the knock-out cultures that can be attributed to a minor but significant expression of VGLUT2 in hippocampal neurons [START_REF] Wojcik | An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size[END_REF][START_REF] Miyazaki | Subtype switching of vesicular glutamate transporters at parallel fibre-Purkinje cell synapses in developing mouse cerebellum[END_REF][START_REF] Herzog | Synaptic and vesicular colocalization of the glutamate transporters VGLUT1 and VGLUT2 in the mouse hippocampus[END_REF]. To minimize this contribution we monitored VGLUT2 levels in the culture at several ages and established that the lower plateau is reached between DIV17 and DIV22 when we performed our imaging and patch clamp experiments (see Figure 3-figure supplement 1). As anticipated, sVGLUT1 was unable to rescue the mEPSC amplitude to the same level as the wild type control (Figure 3C,D). The deficiency in the rescue of glutamate transport by sVGLUT1 is also apparent in the frequency of mEPSC events, as empty SVs also cycle [START_REF] Wojcik | An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size[END_REF][START_REF] Schuske | Neuroscience. Vesicular glutamate transporter--shooting blanks[END_REF]. No impact of sVGLUT1 was seen on mIPSC features (Figure 3-figure supplement 2, supplementary file 1). Hence, the triple mutation sVGLUT1 failed to rescue SV loading with glutamate, but reduced SV super-pool size to the same extent as the wild-type control.

VGLUT1 PRD2 domain reduces SV super-pool size and the frequency of miniature EPSCs.

We then tested a series of VGLUT1 venus mutations spanning the C-terminal cytoplasmic tail of the transporter to rescue the VGLUT1 KO large SV super-pool phenotype (see Figure 2 and Table 1). In the FRAP paradigm, only the mutations disrupting the PRD2 sequence were not able to reduce SV exchange down to WT levels (Figure 4A,B). A point mutation in PRD2 (P554A) that blocks the interaction with endophilin is sufficient to increase both the plateau of recovery and the rate of exchange of SVs (shorter slow half-life for P554A, Figure 4A, supplementary file 1). Similar shifts were observed upon deletion of PRD2 (not shown). To further measure SV super-pool size, we performed time-lapse imaging at high sampling rates

(5 images per seconds) and tracked VGLUT1 venus axonal transport between synaptic boutons (Figure 4C-E). This assay confirmed a significantly larger mobile axonal super-pool of SVs for VGLUT1 P554A-venus but not for VGLUT1 S540A-venus compared to WT rescue (Figure 4D).

Yet, no difference in axonal transport speed could be measured between the 3 constructs (Figure 4E). We also investigated whether PRD2 function could be regulated by phosphorylation of the conserved 540-SYGAT sequence. We thus implemented the PhosTag assay that specifically shifts the electrophoretic mobility of phospho-proteins [START_REF] Kinoshita | Phosphate-binding tag, a new tool to visualize phosphorylated proteins[END_REF].

VGLUT1 venus constructs displayed an additional slow band in PhosTag gel migration, except when samples were digested with alkaline phosphatase (Figure 4-figure supplement 1C). All mutants including ∆C-term displayed this second slower band. Thus, VGLUT1 appears to be phosphorylated, but not at the C-terminal tail, and the function of the extremely conserved 540-SYGAT sequence therefore remains unclear.

In patch-clamp experiments, we tested the effect of VGLUT1 P554A-venus rescue on miniature events, compared to WT and empty vector controls (sham; Figure 4F). VGLUT1 P554A rescued miniature EPSC amplitudes to a similar level as the wild type transporter (Figure 4G).

Interestingly, VGLUT1 P554A increased the frequency of miniature events significantly more than the WT rescue (Figure 4H). In contrast to this, mIPSC frequencies were similar in all three conditions (Figure 4I and Figure 4-figure supplement 2, supplementary file 1). As intended in our rescue strategy, VENUS fluorescence intensity at boutons was not significantly different between groups tested by electrophysiology (Figure 4J). Therefore, the PRD2 sequence of mammalian VGLUT1 is sufficient to reduce SV super-pool size and mEPSC frequency.

Intersectin1 interaction with endophilin A1 mediates the reduction of the SV super-pool promoted by VGLUT1 expression.

Finally, we performed competition experiments with 3 SH3 domains to investigate the VGLUT1 PRD2 dependent pathway mediating SV super pool reduction (see Table 1 and Figure 5A). SH3 domains fused to the cyan fluorescent protein mCERULEAN3 (50) were overexpressed under control of the human synapsin promoter in neuron cultures from VGLUT1 venus mice (Figure 5B). Single boutons from fibers coexpressing mCERULEAN3 and VENUS were selected for FRAP experiments. An empty mCerulean3 sham construct was used as negative control and had a VGLUT1 venus FRAP curve similar to our previous measurements (Figure 5B,C). The SH3 domain of EndophilinA1 was used to displace endogenous full-length EndophilinA1 to test the contribution of the membrane binding Bin1, Amphiphysin, RVs (BAR) domain. The FRAP curve in this experiment matched the sham control recovery (Figure 5B,C). The SH3 domain of EndophilinA1 mutated at E329K and S336K was over-expressed to displace endogenous EndophilinA1 and block the interaction with the SH3B domain of intersectin1 [START_REF] Pechstein | Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses[END_REF]. Endo-SH3 E329K,S336K shifted the FRAP recovery to a significantly higher plateau, creating a phenocopy of the VGLUT1 P554A and VGLUT1 KO mutants (Figure 5B, C, supplementary file 1). Similarly, the SH3B domain of intersectin1 was over-expressed to displace endogenous intersectin1 from EndophilinA1. ITSN1-SH3B also shifted the FRAP recovery to a higher plateau. Hence, full length intersectin1 may be required for the VGLUT1 mediated reduction of SV super pool (Figure 5B,C). Taken together, these competition experiments reveal the involvement of EndophilinA1 and interectin1 in a complex with VGLUT1 in the negative regulation of SV super pool size in mammals.

DISCUSSION

In the present study we could observe that VGLUT1 PRD2 motif is a negative regulator of SV mobility and mEPSC frequency. Our data support that SV super-pool reduction is mediated by an interaction of endophilinA1 with both VGLUT1 PRD2 and intersectin1 SH3B domains.

Molecular dissection of VGLUT1 functions

Previous works had shown that several dileucine-like motifs are involved in VGLUT1

endocytosis [START_REF] Voglmaier | Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling[END_REF][START_REF] Pan | Vesicular Glutamate Transporter 1 orchestrates recruitment of other synaptic vesicle cargo proteins during synaptic vesicle recycling[END_REF][START_REF] Foss | Multiple Dileucine-like Motifs Direct VGLUT1 Trafficking[END_REF] and that the PRD2 of VGLUT1 regulates endocytosis during long trains of stimulation [START_REF] Voglmaier | Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling[END_REF]. The present data provide the first molecular separation of glutamate transport and SV trafficking functions of VGLUT1. The sVGLUT1 mutant allowed us to test the function of the VGLUT1 backbone structure without potential interference brought by the complex flow of ions occurring through glutamate loading [START_REF] Schenck | A chloride conductance in VGLUT1 underlies maximal glutamate loading into synaptic vesicles[END_REF][START_REF] Preobraschenski | Vesicular glutamate transporters use flexible anion and cation binding sites for efficient accumulation of neurotransmitter[END_REF][START_REF] Eriksen | Protons Regulate Vesicular Glutamate Transporters through an Allosteric Mechanism[END_REF][START_REF] Juge | Vesicular glutamate transporter contains two independent transport machineries[END_REF][START_REF] Goh | Presynaptic regulation of quantal size: K(+)/H(+) exchange stimulates vesicular glutamate transport[END_REF]. sVGLUT1 revealed that glutamate transport is not related to the negative regulation of SV super-pool size. Instead, our detailed structure-function analysis of VGLUT1 C-terminus showed that the PRD2 motif is necessary and sufficient to inhibit SV exchange between boutons and the axonal super-pool.

We further tested a conserved putative type 3 PDZ binding motif (DQL514) and the PRD1 pattern but found no significant effect on SV super-pool size. Though we brought evidence for VGLUT1 phosphorylation, putative phosphorylation is not at the C-terminus. It thus remains to be seen whether additional features of the C-terminus such as 540SYGAT can be shown to regulate VGLUT1 tuning of SV mobility.

Hence, distinctively from the dileucine based endocytosis of VGLUTs, the C-terminal proline-rich domain PRD2 supports the negative regulation of SV mobility and mEPSC frequency in mammals. SV mobility inhibition is operated independently of glutamate loading processed by the core trans-membrane domains. The structure-function relationship of other conserved sequences remain to be established.

Mammalian VGLUT1 is a molecular player of SV clustering at synapses

Synaptic vesicles are segregated from other organelles in the nerve terminal, and grouped in a cluster [START_REF] Gray | Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study[END_REF]. It has been proposed that SVs are recruited to the cluster by synapsins to an actin based cytoskeleton [START_REF] Hilfiker | Synapsins as regulators of neurotransmitter release[END_REF]. Yet, several lines of evidence suggest a different mechanism involving the low affinity binding of many partners forming a liquid phase separation to the rest of the cytosol [START_REF] Milovanovic | Synaptic Vesicle Clusters at Synapses: A Distinct Liquid Phase?[END_REF][START_REF] Pechstein | Taking a back seat: synaptic vesicle clustering in presynaptic terminals[END_REF][START_REF] Sankaranarayanan | Actin has a molecular scaffolding, not propulsive, role in presynaptic function[END_REF]. Indeed, endophilinA1, intersectin, amphiphysin are abundant within the vesicle cluster (Figure 5-figure supplement 1 and, 16,64) while the binding of SH3 domains to proline-rich motifs has been shown to generate liquid phase separations in a synthetic assay [START_REF] Li | Phase transitions in the assembly of multivalent signalling proteins[END_REF]. Further, synapsins were recently shown to induce liquid phase separation with lipid vesicles [START_REF] Milovanovic | A liquid phase of synapsin and lipid vesicles[END_REF]. Hence, the best model to date infers the liquid phase separation of SVs in a dynamic array of labile interactions between synapsins, dephosphins (endophilins, amphiphysins, EPS15, synaptojanins…) and intersectins.

Previously, a slower re-acidification of PRD2 deleted VGLUT1-phluorin probes during long trains of stimulation suggested that the recruitment of endophilinA1 increases the endocytosis efficiency of VGLUT1 SVs [START_REF] Voglmaier | Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling[END_REF]. However, recent reports indicate that endophilinA1 interaction with intersectin1 favors clathrin uncoating [START_REF] Pechstein | Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses[END_REF][START_REF] Milosevic | Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission[END_REF], whereas the clathrin coat was shown to inhibit SV acidification [START_REF] Farsi | Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity[END_REF]. Therefore, the recruitment of endophilinA1 and intersectin1 at SVs may speed up clathrin uncoating and SV acidification. This advocates for new experiments designed to discriminate between an impact of VGLUT1 PRD2 on SV endocytosis versus SV uncoating, consecutive acidification kinetics and clustering. More recently, intersectin1 (ITSN1) SH3B was shown to interact with the SH3 domain of endophilinA1 while preserving the interaction of endophilinA1 with its PRD targets on Dynamin and VGLUT1 [START_REF] Pechstein | Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses[END_REF]. Furthermore, intersectin1 may as well interact directly with VGLUT1 through the SH3A domain [START_REF] Santos | Protein Interactions of the Vesicular Glutamate Transporter VGLUT1[END_REF][START_REF] Richter | VGLUT1 binding to endophilin or intersectin1 and dynamin phosphorylation in a diurnal context[END_REF]. Our present data provide evidence for a reduction of the SV super-pool size to the benefit of the SV clusters mediated by a tripartite complex between VGLUT1 PRD2, endophilinA1 and intersectin1 (VGLUT1/EndoA1/ITSN1, Figure 5). The increase in SV exchange rate when PRD2 is mutated strongly suggests a reduced strength in the interactions scavenging SVs in the cluster [START_REF] Staras | A Vesicle Superpool Spans Multiple Presynaptic Terminals in Hippocampal Neurons[END_REF][START_REF] Herzog | In vivo imaging of intersynaptic vesicle exchange using VGLUT1 Venus knock-in mice[END_REF]). Yet further investigations using single particle tracking methods will be necessary to address whether VGLUT1 PRD2 reduces SV mobility within synaptic clusters as well [START_REF] Gramlich | Actin/Myosin-V-and Activity-Dependent Inter-synaptic Vesicle Exchange in Central Neurons[END_REF]. Our data also fit well with recent reports of intersectin1 function in the re-clustering of newly endocytosed SVs, and on the nano-scale organization of synapsins at terminals [START_REF] Gerth | Intersectin associates with synapsin and regulates its nanoscale localization and function[END_REF][START_REF] Winther | An Endocytic Scaffolding Protein together with Synapsin Regulates Synaptic Vesicle Clustering in the Drosophila Neuromuscular Junction[END_REF]. Downstream of VGLUT1/EndoA1/ITSN1, the SV super-pool reduction may be driven by interactions of intersectin1 with synapsins (13,70) and/or through remodeling of the actin cytoskeleton surrounding the cluster [START_REF] Humphries | Cdc42 and the Rho GEF intersectin-1 collaborate with Nck to promote N-WASP-dependent actin polymerisation[END_REF]. Yet further analysis will be required to discriminate between these pathways and evaluate a possible regulation by phosphorylation/dephosphorylation cycles.

Mammalian VGLUT1 acts as a dual regulator of glutamate release

Vesicular glutamate transporters are necessary and sufficient to generate a glutamatergic phenotype in neurons by loading secretory organelles with glutamate [START_REF] Takamori | Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons[END_REF]. Quantal size modulation has been reported upon changes in the level of VGLUT expression [START_REF] Wojcik | An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size[END_REF][START_REF] Moechars | Vesicular glutamate transporter VGLUT2 expression levels control quantal size and neuropathic pain[END_REF][START_REF] Wilson | Presynaptic regulation of quantal size by the vesicular glutamate transporter VGLUT1[END_REF] and is reproduced in our present work (Figure 3 and4). Furthermore, EndophilinA1 binding to the VGLUT1 PRD2 sequence was shown to reduce SV release probability [START_REF] Weston | Interplay between VGLUT isoforms and endophilin A1 regulates neurotransmitter release and short-term plasticity[END_REF]. Our dataset now supports the role of VGLUT1/EndoA1/ITSN1 in the reduction of mEPSC frequency and SV exchange between clusters and super-pool (Figure 4 and5). A discrepancy may arise from the fact that Weston et al. did not see an effect of VGLUT1 ∆PRD2 on mEPSC frequency but only on SV release probability. However, this may be explained by two differences in our recording conditions. First, the autaptic culture system from Weston et al. prevents the formation of a network. Autapses are powerful tools to dissect evoked activity, but mEPSCs in autaptic conditions arise from a single cell (the recorded cell), while in continental cultures they are generated by multiple cells targeting the recorded neuron. Additionally, continental cultures build a network that generates activity, which most likely leads to different set-points of SV super-pool sizes and homeostatic plasticity compared to autaptic micro-islands [START_REF] De Gois | Homeostatic scaling of vesicular glutamate and GABA transporter expression in rat neocortical circuits[END_REF].

Second, Weston and colleagues worked before 14 days in culture, whereas we worked after Beyond setting the quantal size, VGLUT1 influences glutamate release parameters most likely by changing the strength of SVs phase separation in clusters. It remains to establish how neurons take advantage of this feature to locally modulate the parameters of quantal release at selected synapses [START_REF] Staras | A Vesicle Superpool Spans Multiple Presynaptic Terminals in Hippocampal Neurons[END_REF][START_REF] Herzog | In vivo imaging of intersynaptic vesicle exchange using VGLUT1 Venus knock-in mice[END_REF][START_REF] Rothman | Physical determinants of vesicle mobility and supply at a central synapse[END_REF]. In this line, neuromodulation has been very recently proposed to tune SV pools in the axon through GPCR signaling and synapsin phosphorylation [START_REF] Patzke | Neuromodulator Signaling Bidirectionally Controls Vesicle Numbers in Human Synapses[END_REF]. All Slc17A7 -/-(VGLUT1 KO) [START_REF] Wojcik | An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size[END_REF] and Slc17A7 v/v (VGLUT1 venus knock-in) (8) and mice were maintained in C57BL/6N background and housed in 12/12 LD with ad libitum feeding.

MATERIALS & METHODS

Key Resources

Every effort was made to minimize the number of animals used and their suffering. The experimental design and all procedures were in accordance with the European guide for the care and use of laboratory animals and approved by the ethics committee of Bordeaux Universities (CE50) under the APAFIS n°1692.

Plasmids and viral vectors

From the Lentivector F(syn)W-RBN::VGLUT1 venus previously published [START_REF] Siksou | A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility[END_REF], we engineered a series of point and deletion mutations of VGLUT1 venus using conventional site directed mutagenesis protocols (see Table 1). Some experiments were performed using enhanced green fluorescent protein tagged synaptobrevin2 (F(syn)W-RBN::Syb2EGFP; 45). Lentiviral particles were generated by co-transfection of HEK-293T cells with the vector plasmid, a packaging plasmid (CMVD8.9 or CMV-8.74) and an envelope plasmid (CMV-VSVg) using Lipofectamine Plus (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. 48h after transfection, viral particles were harvested in the supernatant, treated with DNaseI and MgCl 2 , passed through a 0.45 m filter and concentrated by ultracentrifugation (ω 2 t = 3,2 10 10 rad 2 /s) and suspended in a small volume of PBS. Viral stocks were stored in 10µl aliquots at -80° C before use. Viral titres were estimated by quantification of the p24 capsid protein using the HIV-1 p24 antigen immunoassay (ZeptoMetrix Corporation, Buffalo, NY, USA) according to the manufacturer's instructions.

All productions were between 100 and 300ng/µl of p24. Alternatively, recombinant adenoassociated virus vectors (AAV) were engineered with either WT or the transport deficient sVGLUT1 inserts (see Table 1). We tagged these constructs with mCherry-miniSOG [START_REF] Qi | Photo-inducible cell ablation in Caenorhabditis elegans using the genetically encoded singlet oxygen generating protein miniSOG[END_REF]. Serotype 9 AAV particles were generated by transient transfection of HEK293T cells and viral stocks were tittered by QPCR on the recombinant genome as previously described [START_REF] Berger | Repair of rhodopsin mRNA by spliceosome-mediated RNA trans-splicing: a new approach for autosomal dominant retinitis pigmentosa[END_REF].

Both viral vectors control the expression of the inserts through the human synapsin promoter.

For competition experiments with endophilinA1 and intersectin1 SH3 domains, we built fusions of the endophilinA1 SH3 domain (aa 290 to aa 352) or the SH3B domain of intersectin1 (aa 903 to 971) with the fluorescent protein mCerulean3 positioned at their N-terminus [START_REF] Markwardt | An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching[END_REF]. All three SH3 domains were synthesized and the sequences checked (Eurofins genomics company). These fusions were cloned into the AAV shuttle plasmid.

Hippocampal cell cultures and transgene expression

Hippocampal primary dissociated cultures were prepared from P0 mice. The hippocampi were dissected in ice-cold Leibovitz's L-15 medium (11415064; Gibco), and then incubated in 0.05% trypsin-EDTA (25300054, Gibco) for 15 min at 37°C. The tissues were washed with Dulbecco's Modified Eagle's Medium (DMEM, 61965026, Gibco) containing 10% FBS (CVFSVF0001, Eurobio), 1% Penicillin-streptomycin (15140122, Gibco). Cells were mechanically dissociated by pipetting up and down, and plated onto poly-L-lysine (P2636, Sigma) coated coverslips at a density of 20 000 cells/cm 2 . Cells were grown in Neurobasal A medium (12349105, Gibco) containing 2% B27 supplement (17504044, Gibco), 0.

FRAP imaging

Fluorescence Recovery After Photobleaching (FRAP) experiments were performed to determine the mobility of Syb2 labeled SVs (for silent VGLUT1 mutant rescue experiments) or VGLUT1 labeled SVs (for VGLUT1 C-terminal mutants rescue experiments) at synapses.

The mobile fraction of Syb2/VGLUT1 labeled SVs is the proportion of fluorescent material that can be replenished after photo bleaching. FRAP was performed using a spinning-disk confocal head Yokogawa CSU-X1 (Yokogawa Electric Corporation, Tokyo, Japan) mounted on an inverted Leica DMI 6000 microscope (Leica Microsystems, Wetzlar, Germany) and equipped with a sensitive EM-CCD QuantEM camera (Photometrics, Tucson, USA), and a FRAP scanner system (Roper Scientific, Evry, France). Surrounding the setup, a thermal incubator was set to 37°C (Life Imaging Services, Switzerland). Z-stacks of 4.8 µm thickness were obtained with a piezo P721.LLQ (Physik Instrumente, Karlsruhe, Germany) at randomly selected fields from hippocampal cell culture with a 63×/1.4 numerical aperture oilimmersion objective. For each stack, five fluorescent boutons, distant from each other, were selected for bleaching. Three passes of the 491 nm laser (40 mW) for Syb2 EGFP or two laser passes using the 491 nm laser (30 mW) and the 405 nm laser (10 mW) for VGLUT1 venus , were applied on the mid-plane of the stack and resulted in an average bleaching of 50% of the initial fluorescence intensity at boutons. The bleaching protocol for Venus/EYFP prevents the spontaneous recovery of fluorescence from a dark reversible photochemical state as previously reported [START_REF] Herzog | In vivo imaging of intersynaptic vesicle exchange using VGLUT1 Venus knock-in mice[END_REF][START_REF] Mcananey | Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10C): a unifying mechanism[END_REF]. Additional recordings were performed at membrane potentials of -20mV, -40mV, -60mV and -80mV. For drug treatment, CNQX was used at 50µM and PTX at 100µM and 2minutes after drug addition, the condition was considered as stable.

Electrophysiology Analysis

Analyses were performed using Clampfit (Molecular Device), in which we created one miniexcitatory (-70mV) and one mini-inhibitory (0mV) template from a representative recording.

Those templates were used for all recordings and analysis was done blind to the experimental group. We measured the number of mEPSCs at -70mV and mIPSCs at 0mV and their mean amplitude. Cell properties were monitored to get a homogenous set of cells, i.e. we analyzed the seal-test recordings of every cell (see supplementary file 2) and calculated the capacitance from the Tau measured by Clampfit (supplementary file 2). Cells with a leak current over -200pA, and/or a membrane resistance over -100MOhms were excluded from the analysis.

Statistical analyses were performed using One-way ANOVA or Kruskal-Wallis test (* for p < 0.05, ** for p < 0.01 and *** for p < 0.001).

Antisera

The detection of wild type VGLUT1 was performed with rabbit polyclonal antiserum [START_REF] Herzog | In vivo imaging of intersynaptic vesicle exchange using VGLUT1 Venus knock-in mice[END_REF][START_REF] Herzog | The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons[END_REF] whereas the detection of VGLUT1 venus was done with a mouse monoclonal anti-GFP antibody (11814460001, Roche). Anti-VIAAT (131004, SYSY) and Anti-VGLUT2 (AB2251, Millipore) guinea pig polyclonal antisera were used. For electron microscopy we used a guinea pig anti-VGLUT1 polyclonal antiserum (AB5905, Merck) and a rabbit polyclonal antiserum against endophilinA1 [START_REF] Vinatier | Interaction between the vesicular glutamate transporter type 1 and endophilin A1, a protein essential for endocytosis[END_REF]. Secondary HRP-coupled anti-rabbit, anti-mouse and anti-guinea pig antibodies were used for western blot detection (711-035-152, 715-035-150, 706-035-148, respectively, Jackson ImmunoResearch).

Immunocytochemistry

Neuron cultures were washed with cold 1X PBS and fixed with 4% paraformaldehyde in 1X

PBS for 5min at room temperature. Immunostainings were performed as previously described [START_REF] Herzog | The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons[END_REF]. Wide-field pictures were acquired using an epifluorescence Nikon Eclipse NIS-element microscope with the 40x objective. The ImageJ software was used to quantify the fluorescence intensity. A mean filter, background subtraction, and threshold were applied to cover the punctate signals and generate a selection mask. The mean density values were extracted. The averages of 5 frames per culture were probed and more than 3 cultures per age were measured.

Immunohistochemistry and electron microscopy

VGLUT1 and EndophilinA1 were simultaneously detected by combination of immunoperoxidase and immunogold methods, respectively, on brain sections at the ultrastructural level.

Animals and tissue preparation

The mice were deeply anesthetized with sodium chloral hydrate and then perfused transcardially with 0.9% NaCl, followed by fixative consisting of 2% paraformaldehyde (PFA) with 0.2% glutaraldehyde in 0.1M phosphate buffer (PB), pH 7.4, at 4°C. The brain was quickly removed and left overnight in 2% PFA at 4°C.

Sections from cerebellum and caudate putamen were cut on a vibrating microtome at 70mm and collected in PBS (0.01M phosphate, pH 7.4). To enhance the penetration of the immunoreagents in the preembedding procedures, the sections were equilibrated in a cryoprotectant solution (0.05MPB, pH 7.4, containing 25% sucrose and 10% glycerol) and freeze-thawed by freezing in isopentane cooled in liquid nitrogen andthawed in PBS.

The sections were then preincubated in 4% normal goat serum (NGS) in PBS. After washing (PBS; sodium acetate buffer, 0.1M, pH 7.0), the diameter of the gold immunoparticles was increased using a silver enhancement kit (HQ silver; Nanoprobes) for 5min at RT in the dark. After washing (2xPBS, 1xTB 0.05M, pH 7.6), the immunoreactive sites VGLUT1 were revealed using DAB. The sections were then stored in PB and processed for electronmicroscopy.

Double detection of VGLUT1 and

Preparation for electron microscopy The sections were post-fixed in osmium tetroxide (1% in PB, 0.1M,pH7.4) for 10 min at RT. After washing, they were dehydrated in an ascending series of dilutions of ethanol that included 1% uranyl acetate in 70% ethanol.

They were then treated with propylene oxide and equilibrated in resin overnight (Durcupan AC M; Fluka, Buchs, Switzerland), mounted on glass slides, and cured at 60°C for 48 hr. The immunoreactive areas identified on thick sections were cut in semithin sections (1-mm-thick), then in ultrathin sections on a Reichert Ultracut S.

Ultrathinsections were collected on pioloform-coated single slot copper grids. The sections were stained with lead citrate and examined in a Philips C M10 electron microscope.

Biochemistry

All steps were performed at 4°C or on ice. Brains of wild type adult mice were dissected for the collection of brain regions. The samples were treated with homogenization buffer (0.

SOURCE DATA FILES

Source data used to build figures are available in the following separate supplementary files: A: Schematic model of the competition experiment designed to test for the effective recruitment of intersectin1 at the VGLUT1/endophilinA1 complex [START_REF] Pechstein | Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses[END_REF]. The three SH3 domains over-expressed in this competition assay disrupt distinct parts of the tripartite complex. The SH3 domain of endophilinA1 displaces the endogenous endophilinA1 BAR domain (Endo SH3 WT ). The SH3 domain of endophilinA1 mutated at E329K and S336K disrupts the interaction of intersectin1 with the VGLUT1/endophilinA1 complex [START_REF] Pechstein | Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses[END_REF]

  after transduction was monitored to match the endogenous levels of VGLUT1 using immunoblot (not shown). During FRAP and time-lapse experiments, the first frame of each sequence was used to further check the expression level of each mutant compared to the wildtype control (see examples in Figure2Band quantification in Figure2C, D). None of the mutants displayed a significant qualitative or quantitative difference in expression compared to wild-type controls.

  17 days in culture when VGLUT2 expression reached a lower plateau and may generate less background mEPSC activity (Figure 3-figure supplement 1; 35,59). The molecular mechanism of SV super-pool reduction that we uncovered requires the EndoA1 SH3 domain, but not the BAR domain (Figure 5). Previously, Weston et al. showed that endophilinA1 promotes SV release probability through dimerization and BAR domain mediated membrane binding (41). Our current results complement this model by adding a pathway through which VGLUT1/EndoA1 recruit intersectin1 to promote SV clustering and mEPSC frequency reduction in a BAR domain independent fashion. Hence, free EndophilinA1 may actively promote SV exocytosis through membrane binding mechanisms while VGLUT1-bound EndophilinA1 may actively reduce SV mobility and exocytosis through an intersectin1 dependent pathway.

  5 mM Glutamax (35050038, Gibco), and 0.2% MycoZap plus-PR (VZA2021, Lonza) for 5 days invitro (DIV). From DIV 5-6, complete Neurobasal A medium was partially replaced to ½ by BrainPhys medium (51), every two or three days. Imaging of live dissociated neuron cultures was performed at DIV 17-21 in culture medium with Hepes buffer (40 mM). Neurons were transduced at DIV1 or -2 with viral vectors diluted by a factor 1/1000. The viral expression levels were controlled by both western-blot (not shown), and fluorescence intensity to rescue VGLUT1 expression to endogenous VGLUT1 level. The bouton size and fluorescent intensity from the different VGLUT1 mutants were analyzed from the first frames the FRAP sequences. Images were processed by "Find Edges", binary images were created through a threshold setting application to define the boundary of boutons. Area and background subtracted fluorescence intensity were measured. Integrated intenstity values were normalized to the value in WT boutons of the same group. The statistical significance of the bouton size and fluorescence intensity between the WT and VGLUT1 mutant rescues were evaluated with unpaired t-tests. For competition experiments with endophilinA1 and intersectin1 SH3 domains, plasmids were delivered by electroporation at DIV0 before plating (Nucleofector, Lonza).

Fluorescence

  recovery was monitored every 30 s during the first 3 min and then every 5 min during the next 70 min. The entire FRAP procedure was controlled by MetaMorph (Molecular Devices, Sunnyvale, USA). Image processing was automated using ImageJ macro commands(53) (available at https://github.com/fabricecordelieres/IJ-Macro_FRAP-MM).Sum projections of the individual stacks, assembly and x-y realignment were applied, resulting in 32 bits/pixel sequences. Integrated fluorescence intensities of the five bleached boutons, and the cells in the field, as well as one background area were extracted. The background signal was subtracted, and data were normalized to the average baseline before bleaching (100%) and corrected for photobleaching against the cells. Experiments were discarded if photobleaching exceeded 60% (risk of phototoxicity). Fluorescence intensity of boutons was normalized to 1 before bleaching, and 0 right after bleaching. A double exponential function was used to fit the average of all normalized FRAP traces and the extra sum-of-squares F test was applied to compare the different best fits. Unpaired t-test was applied to analysis of bouton fluorescence intensity between different mutants and the corresponding WT data sets.Live Cell imagingTime-lapse experiments using the spinning disk confocal microscope were performed to quantify the SVs moving along the inter-synaptic axonal segments. Images were sampled at 5 frames/s for 30 seconds with 200 ms exposure time (151 frames in total). Synaptic boutons were saturated in order to allow better visualization of the dimmer fluorescent material moving along the axons. Quantification of the speed of moving clusters was performed with /github.com/fabricecordelieres/IJ-Plugin_KymoToolBox)[START_REF] Zala | Vesicular Glycolysis Provides On-Board Energy for Fast Axonal Transport[END_REF]. In each sequence, 8 axons segments of 10-15 µm were selected for particle tracking. Furthermore, the traffic at inter-synaptic segments was quantified by drawing line ROIs perpendicularly to the axon, and cumulating the integrated density values in each of the 151 images. Background was subtracted, and all values were divided by the average of 10 lowest values of the sequence to normalize for the differences of fluorescence intensity between different sets of experiments.All normalized values from each line selection were summed to evaluate the total amount of material going through the given cross-section. The statistical significance of the differences in cumulative traffic between the WT and VGLUT1 mutants (S540A and P554A) were evaluated with unpaired t-test.ElectrophysiologyDual whole-cell patch-clamp recordings were performed from DIV17 to DIV21. Patch pipettes (2-4MΩ) were filled with the following intracellular solution (in mM): 125 CsMeSO 3 , 2 MgCl 2 , 1 CaCl 2 , 4 Na2ATP, 10 EGTA, 10 HEPES, 0.4 NaGTP and 5 QX-314-Cl, pH was adjusted to 7.3 with CsOH. Extracellular solution was a standard ACSF containing the following components (in mM): 124 NaCl, 1.25 NaH 2 PO 4 , 1.3 MgCl 2 , 2.7 KCL, 26 NaHCO 3 , 2 CaCl 2 , 18.6 Glucose and 2.25 Ascorbic acid. To record excitatory and inhibitory miniature currents (mEPSC and mIPSC), Tetrodotoxin (TTX) was added at 1µM into an aliquot of the standard ACSF (Alomone labs). Cultures were perfused at 35°C with an ACSF perfusion speed of 0.02mL/min and equilibrated with 95% O2/5% CO2. Signals were recorded at different membrane potentials under voltage clamp conditions for about 2 min (0mV for inhibitory events and -70mV for excitatory events) using a MultiClamp 700B amplifier (Molecular Devices, Foster City, CA) and Clampfit software. Recording at 0mV in voltage clamp allowed us to confirm the effect of TTX on network activity. The recording of miniature events began 2min after adding TTX and the extinction of synchronized IPSCs.

  EndophilinA1 by combined preembedding immunoperoxidase and immunogold methods. The brain sections were incubated in 4% NGS for 30 min and then in a mixture of VGLUT1 (1:5000) and EndophilinA1 (1:500) antibodies, supplemented with 1% NGS overnight at RT. The sections were then incubated with goat anti-guinea-pig IgGs conjugated to biotin (1:200), washed in PBS and incubated in a mixture of avidin-biotin-peroxidase complex (ABC), (1:100; Vector Laboratories, Burlingame, CA) and goat anti-rabbit IgGs conjugated to gold particles (1.4 nm diameter; Nanoprobes, Stony Brook, N Y; 1:100 in PBS/ BSA-C) for 2 hr in PBS/ BSA-C. The sections were then washed in PBS and post-fixed in 1% glutaraldehyde in PBS.

Figure 1 -

 1 Figure 1-source data 1.xlsx ; Figure 1-source data 2.xlsx ; Figure 1-figure supplement1 source

A:

  Expression of Synaptobrevin2 fused to Enhanced Green Fluorescent Protein (Syb2 EGFP ) in hippocampal neurons at 18 days in culture. B: examples of FRAP sequences from both Slc17a7 (VGLUT1) +/+ and -/-genotypes. Boutons are imaged for 3 min, bleached, and recovery is recorded for 73 min. C: average FRAP kinetics. 27 synapses from +/+ and 31 synapses from -/-were measured by FRAP and the average traces are displayed here (for +/+ N = 8 cultures; for -/-N = 11 cultures). The two traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test. The F test indicates that the traces are best fitted by 2 divergent models (F ratio = 19.32; P<0.0001). Fast FRAP recovery was monitored every 5 s in an independent set of experiments (inset). D: Expression of transduced VGLUT1 venus in hippocampal neurons. E: Average FRAP recovery of VGLUT1 venus at 68 minutes post-bleach in rescue or over-expression. Over-expression reduces the mobility of SVs (Unpaired t test, P = 0.0385, t = 2.176. For overexpression: N = 4 cultures, n = 14 synapses; for rescue: N = 3 cultures, n = 15 synapses). scale bar: 2µm in A and D, 1 µm in B.

Figure 2 :

 2 Figure 2: Structure of VGLUT1 and expression of mutants in neurons.

Figure 3 :

 3 Figure 3: Glutamate transport and SV tonicity are not involved in the reduction of SV superpool size. A: Expression of VGLUT1 mCherry , sVGLUT1 mCherry and Syb2 EGFP in hippocampal neurons. FRAP was performed on Syb2 EGFP at axons, both on those with, and those without mCherry signal. Scale bar 5µm. B: Average FRAP kinetics from knock-out cells rescued by VGLUT1 mCherry , sVGLUT1 mCherry , and VGLUT1 KO synapses not rescued. Synapses from each genotype were measured by FRAP and the average traces are displayed (N = 7 independent cultures, 16 synapses for -/-; N = 5 cultures, 23 synapses for WT rescue, and N = 10 cultures, 36 synapses for sVGLUT1 rescue). The 3 traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test. The F test indicates that the traces are best fitted by 2 divergent models (one for -/-synapses and the other one for both rescues, F ratio = 30.25; P<0.0001). FRAP kinetics for the 2 types of rescued synapses are best fitted by one convergent model (F ratio = 1.235; P = 0.294). C: Spontaneous excitatory activity in VGLUT1 KO rescued neurons. Example traces of mEPSC activity in wild type and sVGLUT1 rescue conditions. D: Comparison of the amplitude of mEPSC events in wild type and sVGLUT1 rescue conditions (N=3 independent cultures, n=31 cells, mean amplitude=15.3 pA ± 1.15 SEM for wild type rescue; N = 3 independent cultures, n=56 cells, mean amplitude = 13.12 pA ± 0.71SEM for sVGLUT1 rescue; unpaired t-test P= 0.011). E: Comparison of the frequency of mEPSC events in wild type and sVGLUT1 rescue conditions.(N = 3 independent cultures, n=31 cells, mean frequency= 3.51 Hz ± 0.6 SEM for wild type rescue; N = 3 independent cultures, n=56 cells, mean frequency = 2.51 Hz ± 0.58 SEM for sVGLUT1 rescue; unpaired t-test P= 0.008). Note that sVGLUT1 conditions are both significantly smaller than wild type rescue conditions.

Figure 4 :

 4 Figure 4: The VGLUT1 PRD2 domain mediates SV super-pool size and mEPSC frequency reductions A: Comparison of VGLUT1 venus and VGLUT1 P554A-venus rescues of VGLUT1 KO on SV exchange rates at synapses. 21 (WT) and 22 (P554A) synapses from each rescue were measured by FRAP and the average traces are displayed (N = 5 cultures for WT and N = 5 cultures for P554A). The two traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test. The F test indicates that the traces are best fitted by 2 divergent models (F ratio = 19.64; P < 0.0001). B: Similar experiments where performed for ∆PRD1+2, ∆PRD2,

Figure 5 :

 5 Figure 5: A tripartite complex between VGLUT1, endophilinA1 and intersectin1 mediates SV super-pool reduction.

  Finally, the SH3B domain of intersectin1 should displace the endogenous intersectin1 and allows us to assess whether the full-length intersectin1 is required. B: Over-expression of SH3 domains fused with mCerulean3 in VGLUT1 venus neurons. Axons filled with mCerulean3 were selected for FRAP experiments. Scale bar 2µm. C: FRAP measurement of SV super-pool in SH3 domains competition experiments. Synapses from each over-expression condition were measured by FRAP and the average recovery after 68 minutes are displayed (0.5346 ± 0.04128, N=4 and n=20 for sham; 0.6071 ± 0.03705, N=5 and n=25 for Endo-SH3 WT ; 0.7323 ± 0.04909, N=4 and n=18 for Endo-SH3 E329K,S336K ; 0.6894 ± 0.04036, N=5 and n=25 for ITSN1-SH3B). Sham and Endo-SH3 WT converged to a lower SV exchange recovery (Mann-Whitney test, P=0.141) while Endo-SH3 E329K,S336K (Mann-Whitney test, P=0.005 **) and ITSN1-SH3B (Mann-Whitney test, P=0.014 *) generated higher SV exchange recovery curves.

Figure 4 -figure supplement 1 :

 41 Figure 4-figure supplement 1: VGLUT1 is phosphorylated but not at the conserved 540-SYGAT motif A: Effect of alkaline phosphatase on the electrophoretic mobility of VGLUT1 from forebrain homogenates (and positive control VIAAT ; Vesicular Inhibitory Amino-Acid Transporter). B: regional distribution of VGLUT1 phosphorylation state. Homogenates of several brain regions where probed for VGLUT1 signal as in panel A. OB: olfactory bulb, St: Striatum, Hipp: Hippocampus, BS: Brain Stem, Thal: Thalamus, Cb: Cerebellum, Cx: Cortex. C:PhosTag assay on VGLUT1 KO primary neuron cultures rescued with VGLUT1 venus WT and C-terminal mutant constructs. Note that PhosTag assays prevent from using a size ladder. All constructs display a phospho-shifted band that disappeared upon desphosphorylation by alkaline phosphatase. PI : phosphatase inhibitors; AP: alkaline phosphatase. Selected lanes blotted on the same membranes were reorganized for display purpose.

Figure 4 -figure supplement 2 :

 42 Figure 4-figure supplement 2: VGLUT1 PRD2 domain removal does not affect mIPSCs

Figure 5 -figure supplement 1 :

 51 Figure 5-figure supplement 1: EndophilinA1 accumulates at synaptic vesicle clusters of

Table

	transfected construct (Rattus norvegicus) transfected construct (Rattus norvegicus) antibody	F(syn)W-RBN::VGLUT2-venus F(syn)W-RBN::VGLUT1 P554A-venus VGLUT2, polyclonal Guinea pig,	This paper This paper Millipore	available upon request to Dr. Etienne Herzog available upon request to Dr. Etienne Herzog Cat. AB2251 626 RRID:AB_1587	Fig1-S1 and related results part. Lentiviral vector expressing VGLUT2venus. VGLUT1P554A-Fig4 and related results part. expressing Lentiviral vector 1:2000
	transfected construct (Rattus norvegicus) transfected construct (Rattus norvegicus) antibody antibody	F(syn)W-RBN::VGLUT1 ∆C-term-venus VGLUT1, AAV9::VGLUT 1mCherry-miniSOG guinea pig, polyclonal EndophilinA1, rabbit, polyclonal	This paper This paper Merck PMID: 16606361	available upon request to Dr. Etienne Herzog Cat. AB5905 RRID:AB_2301 751 available upon request to Dr. Etienne Herzog available upon Etienne Herzog request to Dr.	venus. Fig4 and related results part. Lentiviral vector expressing VGLUT1∆C-term-venus. Fig4 and related 1:5000 Fig3 and related results part. Lentiviral vector expressing VGLUT1mCherry-miniSOG. 1:500
	transfected construct (Rattus norvegicus) transfected construct (Rattus norvegicus) peptide, recombinant protein	F(syn)W-RBN::VGLUT1 DQL514AQA-venus AAV9::sVGLU T1mCherry-miniSOG FastAP Phosphatase Thermosensitiv e Alkaline	This paper This paper Thermo Scientific	available upon request to Dr. Etienne Herzog available upon request to Dr. Etienne Herzog Cat. EF0651	results part. Fig3 and related Lentiviral vector expressing VGLUT1DQL514A QA-venus. results part. Lentiviral vector expressing sVGLUT1mCherry-miniSOG.
	Reagent type (species) or resource transfected construct (Rattus norvegicus) transfected construct (Homo peptide, recombinant protein sapiens) strain, strain background (Mus musculus) strain, strain background (Mus musculus) transfected construct (Homo sapiens) construct (Rattus norvegicus) transfected construct (Rattus norvegicus) antibody transfected transfected construct (Rattus norvegicus) transfected construct (Homo sapiens) chemical compound, drug software, algorithm transfected construct (Rattus norvegicus) transfected construct (Homo sapiens) software, algorithm transfected construct (Rattus norvegicus) antibody Animals	Designation F(syn)W-RBN::VGLUT1 ∆PRD1+2-venus AAV9::mCerule an3-EndophilinA1S H3 Halt TM phosphatase inhibitor cocktail Slc17a7 -/-(VGLUT1 KO) mice Slc17a7 v/v (VGLUT1venus KI) mice F(syn)W-RBN::Synaptob ervin2-EGFP F(syn)W-RBN::VGLUT1-venus F(syn)W-RBN::VGLUT1 ∆PRD2-venus VIAAT, Guinea pig, polyclonal F(syn)W-RBN::VGLUT1 PP534AA-venus Phos-tag Acrylamid AAV9::mCerule an3-EndophilinA1S H3 E329K, S336K FRAP Analysis Plugin F(syn)W-RBN::VGLUT1 S540A-venus AAV9::mCerule an3-IntersectinSH3 B KymoToolbox Plugin F(syn)W-RBN::VGLUT1 T544A-venus GFP, Mouse, monoclonal	Source or reference This paper This paper Thermo Scientific PMID: 15103023 PMID: 22031900 PMID: 23581566 PMID: 23581566 This paper SYSY This paper Wako This paper This paper This paper This paper PMID: 23374344 This paper Roche	Identifiers available upon request to Dr. Etienne Herzog available upon request to Dr. Cat. 78420 Etienne Herzog available upon request to Dr Sonja M. Wojcik wojcik@em.mp g.de available upon request to corresponding author available upon request to Dr. Etienne Herzog available upon request to Dr. Etienne Herzog available upon request to Dr. Etienne Herzog RRID:AB_8878 73 Cat. 131004 available upon request to Dr. Etienne Herzog Cat. AAL-107 available upon request to Dr. Etienne Herzog available upon request to Dr. Etienne Herzog available upon request to Dr. Etienne Herzog available upon Cat. request to Dr. Etienne Herzog 11814460001 RRID: AB_390913	Additional Fig4 and related results part. Lentiviral vector expressing VGLUT1∆PRD1+2-venus. Fig5 and related results part. Lentiviral vector expressing mCerulean3-information Fig1, Fig3 and related results part. Lentiviral vector expressing Syb2EGPF. Lentiviral vector expressing VGLUT1venus. VGLUT1∆PRD2-Lentiviral vector expressing 1:1000 results part. Fig4 and related related results part. Fig1, Fig4 and Fig4 and related results part. Lentiviral vector expressing VGLUT1PP534AA-venus. Fig4 and related EndophilinA1SH3. Fig5 and related results part. Lentiviral vector expressing The plugin is mCerulean3-EndophilinA1SH3 E3 29K, S336K available at: https://github.com/f abricecordelieres/IJ . results part. -Macro_FRAP-MM Lentiviral vector Fig5 and related expressing VGLUT1S540A-venus. Fig4 and related results part. The plugin is Lentiviral vector available at: expressing mCerulean3-https://github.com/f abricecordelieres/IJ -IntersectinSH3B. results part. Plugin_KymoToolB Lentiviral vector ox expressing VGLUT1T544A-venus. 1:1000
					venus.

Table 1 :

 1 List of mutant constructs tested.

		Mutation	Domain/Motif	Name	putative function
		R80Q/R176K/R314Q	TM1;TM4;TM7	sVGLUT1	Glutamate transport
		∆504-560	Whole C-terminus	∆C-term	SV/VGLUT Trafficking
	VGLUT1	DQL514AQA ∆530-560 PP534AA S540A T544A	PDZ type 3 binding Proline Rich Domains 1+2 Proline Rich Domain1 SYGAT SYGAT	DQL514AQA ∆PRD1+2 PP534AA S540A T544A	Unknown SH3 domain binding SH3 domain binding Unknown Unknown
		∆550-560	Proline Rich Domain 2	∆PRD2	Endophilin binding
		P554A	Proline Rich Domain 2	P554A	Endophilin binding
		EndoA1 290-352 EndoA1 290-352 E329K,S336K	SH3 SH3	SH3 SH3 E329K,S336K	Binds PRD and ITSN1 SH3B Binds PRD and ITSN1 SH3B
		ITSN1 903-971	SH3B	SH3B	Endo SH3 binding through E329 and S336

ACKNOWLEDGEMENTS

Elisa Luquet, Sally Wenger, and Charlène Josephine for excellent technical support. Nils Brose for a strong support throughout the project. Serge Marty and Martin Oheim for fruitful discussions. Christian Rosenmund, Prof Roger Tsien, Prof Mark A Rizzo, for sharing reagents. Several experiments required the use of Bordeaux University/CNRS/INSERM core facilities: Bordeaux Imaging Center (member of France BioImaging supported by the French National Research Agency; ANR-10-INBS-04); Vect'UB viral vector facility; Biochemistry and Biophysics of Proteins core facility; Mouse breeding facility. X.M.Z. was supported by the Erasmus Mundus ENC program and the labex BRAIN extension grant (ANR-10-LABX-43 BRAIN). Funding from the Agence Nationale de la Recherche (ANR-12-JSV4-0005-01 VGLUT-IQ ; ANR-10-LABX-43 BRAIN; ANR-10-IDEX-03-02 PEPS SV-PIT to E.H. and PSYVGLUT 09-MNPS-033 to S.E.M.). K.S. and M.F.A. were supported by the Fondation pour la Recherche Médicale (FDT20120925288 and ING20150532192 respectively).

CONFLICT OF INTEREST

The authors declare no conflict of interest. Supplementary file 1: Supplementary tables collating statistical analysis. Supplementary file 2: Seal test recording of every cell in the electrophysiology analysis.

Supporting Information :