VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission

Synaptic vesicles in the brain harbor several soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins. With the exception of synaptobrevin2, or VAMP2 (syb2), which is directly involved in vesicle fusion, the role of these SNAREs in neurotransmission is unclear. Here we show that in mice syb2 drives rapid Ca2+-dependent synchronous neurotransmission, whereas the structurally homologous SNARE protein VAMP4 selectively maintains bulk Ca2+-dependent asynchronous release. At inhibitory nerve terminals, up- or downregulation of VAMP4 causes a correlated change in asynchronous release. Biochemically, VAMP4 forms a stable complex with SNAREs syntaxin-1 and SNAP-25 that does not interact with complexins or synaptotagmin-1, proteins essential for synchronous neurotransmission. Optical imaging of individual synapses indicates that trafficking of VAMP4 and syb2 show minimal overlap. Taken together, these findings suggest that VAMP4 and syb2 diverge functionally, traffic independently and support distinct forms of neurotransmission. These results provide molecular insight into how synapses diversify their release properties by taking advantage of distinct synaptic vesicle–associated SNAREs

Fast subplasma membrane Ca2+ transients control exo-endocytosis of synaptic-like microvesicles in astrocytes

Astrocytes are the most abundant glial cell type in the brain. Although not apposite for long-range rapid electrical communication, astrocytes share with neurons the capacity of chemical signaling via Ca(2+)-dependent transmitter exocytosis. Despite this recent finding, little is known about the specific properties of regulated secretion and vesicle recycling in astrocytes. Important differences may exist with the neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca(2+) from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We here take advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses (Voglmaier et al., 2006; Balaji and Ryan, 2007); we combine epifluorescence and total internal reflection fluorescence imaging to investigate with unprecedented temporal and spatial resolution, the stimulus-secretion coupling underlying exo-endocytosis of glutamatergic synaptic-like microvesicles (SLMVs) in astrocytes. Our main findings indicate that (1) exo-endocytosis in astrocytes proceeds with a time course on the millisecond time scale (tau(exocytosis) = 0.24 +/- 0.017 s; tau(endocytosis) = 0.26 +/- 0.03 s) and (2) exocytosis is controlled by local Ca(2+) microdomains. We identified submicrometer cytosolic compartments delimited by endoplasmic reticulum tubuli reaching beneath the plasma membrane and containing SLMVs at which fast (time-to-peak, approximately 50 ms) Ca(2+) events occurred in precise spatial-temporal correlation with exocytic fusion events. Overall, the above characteristics of transmitter exocytosis from astrocytes support a role of this process in fast synaptic modulation

VGLUT2 Trafficking Is Differentially Regulated by Adaptor Proteins AP-1 and AP-3.

Release of the major excitatory neurotransmitter glutamate by synaptic vesicle exocytosis depends on glutamate loading into synaptic vesicles by vesicular glutamate transporters (VGLUTs). The two principal isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in adult brain that broadly distinguishes cortical (VGLUT1) and subcortical (VGLUT2) systems, and correlates with distinct physiological properties in synapses expressing these isoforms. Differential trafficking of VGLUT1 and 2 has been suggested to underlie their functional diversity. Increasing evidence suggests individual synaptic vesicle proteins use specific sorting signals to engage specialized biochemical mechanisms to regulate their recycling. We observed that VGLUT2 recycles differently in response to high frequency stimulation than VGLUT1. Here we further explore the trafficking of VGLUT2 using a pHluorin-based reporter, VGLUT2-pH. VGLUT2-pH exhibits slower rates of both exocytosis and endocytosis than VGLUT1-pH. VGLUT2-pH recycling is slower than VGLUT1-pH in both hippocampal neurons, which endogenously express mostly VGLUT1, and thalamic neurons, which endogenously express mostly VGLUT2, indicating that protein identity, not synaptic vesicle membrane or neuronal cell type, controls sorting. We characterize sorting signals in the C-terminal dileucine-like motif, which plays a crucial role in VGLUT2 trafficking. Disruption of this motif abolishes synaptic targeting of VGLUT2 and essentially eliminates endocytosis of the transporter. Mutational and biochemical analysis demonstrates that clathrin adaptor proteins (APs) interact with VGLUT2 at the dileucine-like motif. VGLUT2 interacts with AP-2, a well-studied adaptor protein for clathrin mediated endocytosis. In addition, VGLUT2 also interacts with the alternate adaptors, AP-1 and AP-3. VGLUT2 relies on distinct recycling mechanisms from VGLUT1. Abrogation of these differences by pharmacological and molecular inhibition reveals that these mechanisms are dependent on the adaptor proteins AP-1 and AP-3. Further, shRNA-mediated knockdown reveals differential roles for AP-1 and AP-3 in VGLUT2 recycling

Sorting of the Vesicular GABA Transporter to Functional Vesicle Pools by an Atypical Dileucine-like Motif

Increasing evidence indicates that individual synaptic vesicle proteins may use different signals, endocytic adaptors, and trafficking pathways for sorting to distinct pools of synaptic vesicles. Here, we report the identification of a unique amino acid motif in the vesicular GABA transporter (VGAT) that controls its synaptic localization and activity-dependent recycling. Mutational analysis of this atypical dileucine-like motif in rat VGAT indicates that the transporter recycles by interacting with the clathrin adaptor protein AP-2. However, mutation of a single acidic residue upstream of the dileucine-like motif leads to a shift to an AP-3-dependent trafficking pathway that preferentially targets the transporter to the readily releasable and recycling pool of vesicles. Real-time imaging with a VGAT-pHluorin fusion provides a useful approach to explore how unique sorting sequences target individual proteins to synaptic vesicles with distinct functional properties

Multiple Dileucine-like Motifs Direct VGLUT1 Trafficking

The vesicular glutamate transporters (VGLUTs) package glutamate into synaptic vesicles, and the two principal isoforms VGLUT1 and VGLUT2 have been suggested to influence the properties of release. To understand how a VGLUT isoform might influence transmitter release, we have studied their trafficking and previously identified a dileucine-like endocytic motif in the C terminus of VGLUT1. Disruption of this motif impairs the activity-dependent recycling of VGLUT1, but does not eliminate its endocytosis. We now report the identification of two additional dileucine-like motifs in the N terminus of VGLUT1 that are not well conserved in the other isoforms. In the absence of all three motifs, rat VGLUT1 shows limited accumulation at synaptic sites and no longer responds to stimulation. In addition, shRNA-mediated knockdown of clathrin adaptor proteins AP-1 and AP-2 shows that the C-terminal motif acts largely via AP-2, whereas the N-terminal motifs use AP-1. Without the C-terminal motif, knockdown of AP-1 reduces the proportion of VGLUT1 that responds to stimulation. VGLUT1 thus contains multiple sorting signals that engage distinct trafficking mechanisms. In contrast to VGLUT1, the trafficking of VGLUT2 depends almost entirely on the conserved C-terminal dileucine-like motif: without this motif, a substantial fraction of VGLUT2 redistributes to the plasma membrane and the transporter's synaptic localization is disrupted. Consistent with these differences in trafficking signals, wild-type VGLUT1 and VGLUT2 differ in their response to stimulation

VGLUT2 Trafficking Is Differentially Regulated by Adaptor Proteins AP-1 and AP-3

Release of the major excitatory neurotransmitter glutamate by synaptic vesicle exocytosis depends on glutamate loading into synaptic vesicles by vesicular glutamate transporters (VGLUTs). The two principal isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in adult brain that broadly distinguishes cortical (VGLUT1) and subcortical (VGLUT2) systems, and correlates with distinct physiological properties in synapses expressing these isoforms. Differential trafficking of VGLUT1 and 2 has been suggested to underlie their functional diversity. Increasing evidence suggests individual synaptic vesicle proteins use specific sorting signals to engage specialized biochemical mechanisms to regulate their recycling. We observed that VGLUT2 recycles differently in response to high frequency stimulation than VGLUT1. Here we further explore the trafficking of VGLUT2 using a pHluorin-based reporter, VGLUT2-pH. VGLUT2-pH exhibits slower rates of both exocytosis and endocytosis than VGLUT1-pH. VGLUT2-pH recycling is slower than VGLUT1-pH in both hippocampal neurons, which endogenously express mostly VGLUT1, and thalamic neurons, which endogenously express mostly VGLUT2, indicating that protein identity, not synaptic vesicle membrane or neuronal cell type, controls sorting. We characterize sorting signals in the C-terminal dileucine-like motif, which plays a crucial role in VGLUT2 trafficking. Disruption of this motif abolishes synaptic targeting of VGLUT2 and essentially eliminates endocytosis of the transporter. Mutational and biochemical analysis demonstrates that clathrin adaptor proteins (APs) interact with VGLUT2 at the dileucine-like motif. VGLUT2 interacts with AP-2, a well-studied adaptor protein for clathrin mediated endocytosis. In addition, VGLUT2 also interacts with the alternate adaptors, AP-1 and AP-3. VGLUT2 relies on distinct recycling mechanisms from VGLUT1. Abrogation of these differences by pharmacological and molecular inhibition reveals that these mechanisms are dependent on the adaptor proteins AP-1 and AP-3. Further, shRNA-mediated knockdown reveals differential roles for AP-1 and AP-3 in VGLUT2 recycling

Modulation of protein interactions by phosphomimetic mutations in VGLUT1.

<p>GST pull-down assays were performed by incubating rat brain extracts with GST, or GST fusions of the WT VGLUT1 C-terminus (VG1), or mutants deleting both polyproline domains (ΔPP1&2), or mimicking the unphosphorylated (SS/AA) or phosphorylated state (SS/DD). (<b>A</b>) Bound proteins were detected by immunoblotting with antibodies against endophilin 1, endophilin 3, Nedd4, AIP4/Itch, Nck, and ponsin. Phosphomimetic mutations did not affect binding compared to VG1. Deletion of the polyproline motifs (ΔPP1&2) prevents binding of the polyproline domain interacting proteins. (<b>B</b>) Bound proteins were detected by immunoblotting with antibody against AP-2. The phosphomimetic SS/DD mutation promotes increased binding of VGLUT1 to AP-2 (1.761±0.2422 a.u.), while SS/AA mutation decreases binding of VGLUT1 to AP-2 (0.6745±0.0668 a.u.). (<b>C</b>) Bound proteins were detected by immunoblotting with antibody against AP-3. Binding of VGLUT1 to AP-3 is unaffected by serine mutations. Top panels show representative immunoblots, bottom panels show the averaged quantification of band intensity from at least three independent experiments. *p<0.05, **p<0.01, one-way ANOVA with Bonferroni's post-test.</p

VGLUT1 interacts with SH3 domain-containing proteins <i>in vitro</i>.

<p>(<b>A, B</b>) GST or GST fusions of SH3 domains from proteins identified in the array screen were incubated with rat brain extracts. Bound VGLUT1 was detected with specific antibody, quantified and expressed in arbitrary units (a.u.). (<b>A</b>) The three SH3 domains of Nck1 and 2 (D1-3) were screened independently. Interaction with VGLUT1 is strongest for the second SH3 domain of Nck1 (Nck1 D1 not detected; Nck1 D2, 0.7635±0.1104 a.u.; Nck1 D3, 0.1833±0.0649 a.u.; Nck2 D1, 0.1031±0.0595; Nck2 D2, 0.00976±0.00564 a.u.; Nck2 D3 not detected). (<b>B</b>) The SH3 domain of Lyn pulls down VGLUT1 from rat brain lysate (0.7182±0.0987 a.u.). No binding of VGLUT1 to the SH3 domains of EPS8, spectrin, or ArgBP2 was detected. Weak binding to SNX9 was detected in only one experiment (0.1107±0.1917 a.u.). (<b>C, D</b>) Rat brain extracts were incubated with GST or GST fusions of the VGLUT1 C-terminus (VG1), or VGLUT1 lacking both PP domains (ΔPP1&2), the first PP (ΔPP1) or second (ΔPP2). Both Nck (C) and ponsin (D) bound specifically to VG1 and ΔPP1, but not to ΔPP1&2 or ΔPP2 (Nck binding to ΔPP1: 1.114±0.261 a.u. Ponsin binding to: ΔPP1&2, 0.2249±0.1682 a.u.; ΔPP1, 2.243±0.447 a.u.; ΔPP2, 0.06198±0.03914 a.u.). (<b>E</b>) Extracts from COS7 cells transfected with <i>myc</i>-Lyn were incubated with GST or GST fusions of VGLUT1 as in (C, D). Binding to Lyn was detected with antibody to <i>myc</i> (ΔPP1&2, 0.03682±0.02458 a.u., ΔPP1, 1.119±0.189 a.u.; ΔPP2, 0.02823±0.01619 a.u.). (<b>F</b>) Rat brain extracts were incubated with GST or GST fusions of the SH3 domains of the kinases Src, Fyn, or Lyn. Immunoblots probed with antibody to VGLUT1 indicate specific binding to Lyn (0.8767±0.0644 a.u.) and significantly less to Fyn (0.3622±0.1034). Band intensities were quantified using ImageJ and normalized to lysate (A, B, F) or VG1 band (C, D, E). nd: not detected. Top panels show representative immunoblots, lower panels show the averaged quantification of band intensity from at least three independent experiments. **p<0.01, ***p<0.001, one-way ANOVA with Bonferroni's post-test.</p