Increased Expression of the<i>Drosophila</i>Vesicular Glutamate Transporter Leads to Excess Glutamate Release and a Compensatory Decrease in Quantal Content

Daniels, Richard W.; Collins, Catherine A.; Gelfand, Maria V.; Dant, Jaime; Brooks, Elizabeth S.; Krantz, David E.; DiAntonio, Aaron

doi:10.1523/jneurosci.3001-04.2004

Cited by 315 publications

(418 citation statements)

References 43 publications

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“…Thus, either dDAB does not participate in dSYT1 sorting or sufficient redundancy is provided by interactions of SYT1 with at least two other CLATHRIN-associated adaptor proteins, AP-2 and Stonins (4,36). Furthermore, the distributions of other synaptic vesicle proteins at dab mutant synapses, including neuronal SYN-APTOBREVIN (38) and the vesicular glutamate transporter (39), were similar to those of WT (Fig. S6 K-T and U-D′, respectively).…”

Section: Discussionmentioning

confidence: 89%

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

Kawasaki

Iyer

Posey

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Members of the DISABLED (DAB) family of proteins are known to play a conserved role in endocytic trafficking of cell surface receptors by functioning as monomeric CLATHRIN-associated sorting proteins that recruit cargo proteins into endocytic vesicles. Here, we report a Drosophila disabled mutant revealing a novel role for DAB proteins in chemical synaptic transmission. This mutant exhibits impaired synaptic function, including a rapid activitydependent reduction in neurotransmitter release and disruption of synaptic vesicle endocytosis. In presynaptic boutons, Drosophila DAB and CLATHRIN were highly colocalized within two distinct classes of puncta, including relatively dim puncta that were located at active zones and may reflect endocytic mechanisms operating at neurotransmitter release sites. Finally, broader analysis of endocytic proteins, including DYNAMIN, supported a general role for CLATHRIN-mediated endocytic mechanisms in rapid clearance of neurotransmitter release sites for subsequent vesicle priming and refilling of the release-ready vesicle pool.I ntensive study of chemical synaptic transmission has led to detailed molecular models of synaptic vesicle endocytosis (1-4). Ongoing efforts seek to define the underlying molecular components and interactions further as well as their spatial organization with respect to neurotransmitter release sites. The present study reveals novel molecular mechanisms and interactions in synaptic vesicle endocytosis involving a member of the DISABLED family of Phosphotyrosine Binding (PTB) domain proteins. In Drosophila, the disabled (dab) gene, the founding member of the dab gene family, was first identified in a screen for genetic modifiers of abelson (5). Although putative dab mutations were subsequently mapped to a different gene (6), recent studies have established functional interactions of Drosophila DISABLED (dDAB) and ABELSON (7). Proteins closely related to dDAB, including mouse DAB-2 (mDAB-2) and the single Caenorhabditis elegans family member ceDAB-1, function as CLATHRIN-associated adaptors in endocytic trafficking of cell surface receptors (8-10). These DABs have been shown to interact with CLATHRIN, the α-ADAPTIN (AP-2) adaptor complex, and other components of the endocytic machinery through conserved binding motifs (8-10) (Fig. 1A). Notably, mDAB-2, ceDAB-1, and dDAB share a PTB/DAB Homology (DH) domain near the N terminus (Fig. 1B), which interacts with specific vesicle cargo proteins and phosphatidylinositol-4,5-diphosphate (11, 12). Neither DAB proteins nor PTB domain interactions have been previously implicated in synaptic vesicle trafficking.The present study reveals a role for DAB proteins in synaptic vesicle endocytosis and extends previous work on interactions of endocytic and exocytic mechanisms in neurotransmitter release. Here, we use a glutamatergic neuromuscular synapse of the Drosophila adult as a model for genetic analysis of molecular mechanisms determining conserved properties of glutamatergic synapse function (13,14). Previous analysis i...

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Section: Discussionmentioning

confidence: 89%

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

Kawasaki

Iyer

Posey

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Indeed, CSP, n-Syb, Syt I, and the Drosophila vesicular glutamate transporter (VGluT, Daniels et al, 2004) were respectively found to colocalize within these varicosities ( Fig. 6C-F).…”

Section: Synaptic Bouton-like Varicosities Also Form Within Axons Alomentioning

confidence: 92%

“…Following one hour incubation with a blocker solution (2-5% bovine serum albumin, 0.2% Tween-20 in PBS, PBT), the preparation was stained overnight at 4 ÂC using primary antisera, followed by one hour washes with PBT and 1-2 hours incubation with secondary antibodies at room temperature, and visualized using a Nikon fluorescence microscope. The following primary antibodies were used: rat anti-Syb (1:300, Wu et al, 1999), mAb anti-CSP (ab49, 1:20, Zinsmaier et al, 1994), rabbit anti-Syt (1:300, Mackler et al, 2002), mAb anti-Futsch (22C10, 1:100, Developmental Studies Hybridoma Bank), mAb anti-Hiw (6H4, 1:3, Wan et al, 2000), rabbit anti-DAP 160 (1:300, Roos and Kelly, 1999), mAb nc82 (1:20; Wagh et al, 2005), a polyclonal antibody to clathrin heavy chain (1:50) and to LAP (1:200, Zhang et al, 1998), a polyclonal antibody to glutamate receptor III (Marrus et al, 2004), a polyclonal antibody to the Drosophila vesicular glutamate transporter (VGluT, 1:500, Daniels et al, 2004), and a polyclonal antibody to HRP (Jan and Jan, 1982). Fluorescence-conjugated secondary antibodies were used 1:100 (Jackson Immunology Labs).…”

Section: Immunocytochemistry and Microscopymentioning

confidence: 99%

The atypical cadherin flamingo regulates synaptogenesis and helps prevent axonal and synaptic degeneration in Drosophila

Bao

Berlanga

Xue

et al. 2007

Molecular and Cellular Neuroscience

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The formation of synaptic connections with target cells and maintenance of axons are highly regulated and crucial for neuronal function. The atypical cadherin and G-protein-coupled receptor Flamingo and its orthologs in amphibians and mammals have been shown to regulate cell polarity, dendritic and axonal growth, and neural tube closure. However, the role of Flamingo in synapse formation and function and in axonal health remains poorly understood. Here we show that fmi mutations cause a significant increase in the number of ectopic synapses on muscles and result in the formation of novel en passant synapses along axons, and unique presynaptic varicosities, including active zones, within axons. fmi mutations also cause defective synaptic responses in a small subset of muscles, an agedependent loss of muscle innervation and a drastic degeneration of axons in 3 rd instar larvae without an apparent loss of neurons. Neuronal expression of Flamingo rescues all of these synaptic and axonal defects and larval lethality. Based on these observations, we propose that Flamingo is required in neurons for synaptic target selection, synaptogenesis, the survival of axons and synapses, and adult viability. These findings shed new light on a possible role for Flamingo in progressive neurodegenerative diseases.

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“…Bouin's fixative was used for vGLUT immunostaining, as reported by Daniels et al (2004). After permeabilization with 0.1% Triton X-100/PBS, preparations were incubated with primary antibodies at 4°C overnight.…”

Section: Immunocytochemistrymentioning

confidence: 99%

Stoned B mediates sorting of integral synaptic vesicle proteins

Mohrmann¹,

Matthies²,

Woodruff³

et al. 2008

Neuroscience

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A continuous supply of fusion-competent synaptic vesicles is essential for sustainable neurotransmission. Drosophila mutations of the dicistronic stoned locus disrupt normal vesicle cycling and cause functional deficits in synaptic transmission. Although both Stoned A and B proteins putatively participate in reconstituting synaptic vesicles, their precise function is still unclear. Here we investigate the effects of progressive depletion of Stoned B (STNB) on the release properties of neuromuscular synapses using a novel set of synthetic STNB hypomorphic alleles. Decreasing neuronal STNB expression to ≤35% of wildtype level causes a strong reduction in EJC amplitude at low stimulation frequencies and a marked slowing in synaptic depression during high-frequency stimulation, suggesting vesicle depletion is attenuated by decreased release probability. Recovery from synaptic depression after prolonged stimulation is also decelerated in mutants, indicating a delayed recovery of fusion-ready vesicles. These phenotypes appear not to be due to a diminished vesicle population, since the docked vesicle pool is ultrastructurally unaffected, and the total number of vesicles is only slightly reduced in these hypomorphs, unlike lethal stoned mutants. Therefore, we conclude that STNB not only functions as an essential component of the endocytic complex for vesicle reconstitution, as previously proposed, but also regulates the competence of recycled vesicles to undergo fusion. In support of such role of STNB, synaptic levels of the vesicular glutamate transporter (vGLUT) and synaptotagmin-1 are strongly reduced with diminishing STNB function, while other synaptic proteins are largely unaffected. We conclude that STNB organizes the endocytic sorting of a subset of integral synaptic vesicle proteins thereby regulating the fusion-competence of the recycled vesicle.

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Increased Expression of theDrosophilaVesicular Glutamate Transporter Leads to Excess Glutamate Release and a Compensatory Decrease in Quantal Content

Cited by 315 publications

References 43 publications

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

The atypical cadherin flamingo regulates synaptogenesis and helps prevent axonal and synaptic degeneration in Drosophila

Stoned B mediates sorting of integral synaptic vesicle proteins

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