Concurrent Imaging of Synaptic Vesicle Recycling and Calcium Dynamics

Synaptic transmission involves the calcium dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-shifted reporter of vesicle recycling based on a vesicular glutamate transporter, VGLUT1-mOrange2 (VGLUT1-mOr2), and a presynaptically localized green calcium indicator, synaptophysin-GCaMP3 (SyGCaMP3) with a large dynamic range. The fluorescence of VGLUT1-mOr2 is quenched by the low pH of synaptic vesicles. Exocytosis upon electrical stimulation exposes the luminal mOr2 to the neutral extracellular pH and relieves fluorescence quenching. Reacidification of the vesicle upon endocytosis again reduces fluorescence intensity. Changes in fluorescence intensity thus monitor synaptic vesicle exo- and endocytosis, as demonstrated previously for the green VGLUT1-pHluorin. To monitor changes in calcium, we fused the synaptic vesicle protein synaptophysin to the recently improved calcium indicator GCaMP3. SyGCaMP3 is targeted to presynaptic varicosities, and exhibits changes in fluorescence in response to electrical stimulation consistent with changes in calcium concentration. Using real time imaging of both reporters expressed in the same synapses, we determine the time course of changes in VGLUT1 recycling in relation to changes in presynaptic calcium concentration. Inhibition of P/Q- and N-type calcium channels reduces calcium levels, as well as the rate of synaptic vesicle exocytosis and the fraction of vesicles released

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

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

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

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

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 phosphorylation.

<p>(<b>A</b>) Cultured rat cortical neurons transfected with HA-VGLUT1 were incubated with ³²P_i for 4 h prior to immunoprecipitation with rat anti-HA antibody or rat IgG, and detected by autoradiography. A radiolabeled band of approximately 52 kD (arrowhead) was immunoprecipitated specifically with rat HA antibody, but not IgG. One lane with 1/4 amount of input, HA(1/4), was also loaded on the gel for clarity. (<b>B</b>) Rat cortical neurons transfected with HA-VGLUT1 or a mutant substituting serines 519 and 522 with alanine (SS/AA) were radiolabeled and immunoprecipitated with antibody to HA. Decreased radiolabeling of a 52 kD band (arrowhead) is noted in the SS/AA mutant (0.4028±0.0131 a.u.). Top panel shows a representative immunoblot, lower panels shows the averaged quantification of band intensities from three independent experiments. ***p<0.0001, two-tailed t-test.</p

Interaction of the VGLUT1 C-terminus with SH3 and WW protein domains.

<p>GST fusions of (<b>A</b>) SH3 and (<b>B</b>) WW domains from proteins spotted in duplicate on membranes were incubated with soluble extracts from bacteria expressing a His-tagged VGLUT1 C-terminal peptide (amino acids 513-549). After washing, the bound His-VGLUT1 C-terminus was recognized by anti-His antibody coupled to horseradish peroxidase and detected by enhanced chemiluminescence. Proteins expressed at low levels in brain and those with an established function unrelated to trafficking or neurotransmitter transport are marked with an asterisk, and were excluded from further analysis.</p