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

1. Selective protein–protein interactions between neurotransmitter transporters and their synaptic targets play important roles in regulating chemical neurotransmission. We screened a yeast two-hybrid library with bait containing the C-terminal amino acids of VGLUT1 and obtained clones that encode endophilin 1 and endophilin 3, proteins considered to play an integral role in glutamatergic vesicle formation.2. Using a modified yeast plasmid vector to enable more cost-effective screens, we analyzed the selectivity and specificity of this interaction. Endophilins 1 and 3 selectively recognize only VGLUT1 as the C-terminus of VGLUT2 and VGLUT3 do not interact with either endophilin isoform. We mutagenized four conserved stretches of primary sequence in VGLUT1 that includes two polyproline motifs (Pro1, PPAPPP, and Pro2, PPRPPPP), found only in VGLUT1, and two conserved stretches (SEEK, SYGAT), found also in VGLUT2 and VGLUT3. The absence of the VGLUT conserved regions does not affect VGLUT1–endophilin association. Of the two polyproline stretches, only one (Pro2) is required for binding specificity to both endophilin 1 and endophilin 3.3. We also show that endophilin 1 and endophilin 3 co-localize with VGLUT1 in synaptic terminals of differentiated rat neocortical neurons in primary culture. These results indicate that VGLUT1 and both endophilins are enriched in a class of excitatory synaptic terminals in cortical neurons and there, may interact to play an important role affecting the vesicular sequestration and synaptic release of glutamate.     

Differential expression of endophilin 1 and 2 dimers at central nervous system synapses

Endophilin 1 is proposed to participate in synaptic vesicle biogenesis through SH3 domain-mediated interactions with the polyphosphoinositide phosphatase synaptojanin and the GTPase dynamin. Endophilin family members have also been identified as binding partners for a number of diverse cellular proteins. We define here the endophilin 1-binding site within synaptojanin 1 and show that this sequence independently and selectively purifies from brain extracts endophilin 1 and a closely related protein, endophilin 2. Endophilin 2, like endophilin 1, is highly expressed in brain, concentrated in nerve terminals, and found in complexes with synaptojanin and dynamin. Although a fraction of endophilins 1 and 2 coexist in the same complex, the distribution of these endophilin isoforms among central synapses only partially overlaps. Endophilins 1 and 2 are found predominantly as stable dimers through a predicted coiled-coil domain in their conserved NH2-terminal moiety. Dimerization may allow endophilins to link a number of different cellular targets to the endocytic machinery.     

Identification and characterization of a synaptojanin 2 splice isoform predominantly expressed in nerve terminals

We have previously identified synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of synaptojanin 2 that shares several properties with synaptojanin 1. This isoform, synaptojanin 2B, undergoes further alternative splicing to generate synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners synaptojanin 1, bind synaptojanin 2B2, whereas only amphiphysin binds synaptojanin 2B1. Sequence similar to the endophilin-binding site in synaptojanin 1 is present only in synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of synaptojanin 2, but not of synaptojanin 1. These results suggest that synaptojanin 2B has a partially overlapping function with synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.     

Developmental Up-Regulation of Vesicular Glutamate Transporter-1 Promotes Neocortical Presynaptic Terminal Development

Presynaptic terminal formation is a complex process that requires assembly of proteins responsible for synaptic transmission at sites of axo-dendritic contact. Accumulation of presynaptic proteins at developing terminals is facilitated by glutamate receptor activation. Glutamate is loaded into synaptic vesicles for release via the vesicular glutamate transporters VGLUT1 and VGLUT2. During postnatal development there is a switch from predominantly VGLUT2 expression to high VGLUT1 and low VGLUT2, raising the question of whether the developmental increase in VGLUT1 is important for presynaptic development. Here, we addressed this question using confocal microscopy and quantitative immunocytochemistry in primary cultures of rat neocortical neurons. First, in order to understand the extent to which the developmental switch from VGLUT2 to VGLUT1 occurs through an increase in VGLUT1 at individual presynaptic terminals or through addition of VGLUT1-positive presynaptic terminals, we examined the spatio-temporal dynamics of VGLUT1 and VGLUT2 expression. Between 5 and 12 days in culture, the percentage of presynaptic terminals that expressed VGLUT1 increased during synapse formation, as did expression of VGLUT1 at individual terminals. A subset of VGLUT1-positive terminals also expressed VGLUT2, which decreased at these terminals. At individual terminals, the increase in VGLUT1 correlated with greater accumulation of other synaptic vesicle proteins, such as synapsin and synaptophysin. When the developmental increase in VGLUT1 was prevented using VGLUT1-shRNA, the density of presynaptic terminals and accumulation of synapsin and synaptophysin at terminals were decreased. Since VGLUT1 knock-down was limited to a small number of neurons, the observed effects were cell-autonomous and independent of changes in overall network activity. These results demonstrate that up-regulation of VGLUT1 is important for development of presynaptic terminals in the cortex.     

Protein Interactions of the Vesicular Glutamate Transporter VGLUT1

Exocytotic release of glutamate depends upon loading of the neurotransmitter into synaptic vesicles by vesicular glutamate transporters, VGLUTs. The major isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in synapses of the adult rodent brain that correlates with the probability of release and potential for plasticity. Indeed, expression of different VGLUT protein isoforms confers different properties of release probability. Expression of VGLUT1 or 2 protein also determines the kinetics of synaptic vesicle recycling. To identify molecular determinants that may be related to reported differences in VGLUT trafficking and glutamate release properties, we investigated some of the intrinsic differences between the two isoforms. VGLUT1 and 2 exhibit a high degree of sequence homology, but differ in their N- and C-termini. While the C-termini of VGLUT1 and 2 share a dileucine-like trafficking motif and a proline-, glutamate-, serine-, and threonine-rich PEST domain, only VGLUT1 contains two polyproline domains and a phosphorylation consensus sequence in a region of acidic amino acids. The interaction of a VGLUT1 polyproline domain with the endocytic protein endophilin recruits VGLUT1 to a fast recycling pathway. To identify trans-acting cellular proteins that interact with the distinct motifs found in the C-terminus of VGLUT1, we performed a series of in vitro biochemical screening assays using the region encompassing the polyproline motifs, phosphorylation consensus sites, and PEST domain. We identify interactors that belong to several classes of proteins that modulate cellular function, including actin cytoskeletal adaptors, ubiquitin ligases, and tyrosine kinases. The nature of these interactions suggests novel avenues to investigate the modulation of synaptic vesicle protein recycling.     

Interplay between VGLUT isoforms and endophilin A1 regulates neurotransmitter release and short-term plasticity

Vesicular glutamate transporters (VGLUTs) are essential for filling synaptic vesicles with glutamate and mammals express three VGLUT isoforms (VGLUT1-3) with distinct spatiotemporal expression patterns. Here, we find that neurons expressing VGLUT1 have lower release probability and less short-term depression than neurons expressing VGLUT2 or VGLUT3. Investigation of the underlying mechanism identified endophilin A1 as a positive regulator of exocytosis whose expression levels are positively correlated with release efficiency and showed that the differences in release efficiency between VGLUT1- and VGLUT2-expressing neurons are due to VGLUT1's ability to bind endophilin A1 and inhibit endophilin-induced enhancement of release probability.     

VGLUT1 and VGAT are sorted to the same population of synaptic vesicles in subsets of cortical axon terminals

Glutamate and GABA mediate most of the excitatory and inhibitory synaptic transmission; they are taken up and accumulated in synaptic vesicles by specific vesicular transporters named VGLUT1-3 and VGAT, respectively. Recent studies show that VGLUT2 and VGLUT3 are co-expressed with VGAT. Because of the relevance this information has for our understanding of synaptic physiology and plasticity, we investigated whether VGLUT1 and VGAT are co-expressed in rat cortical neurons. In cortical cultures and layer V cortical terminals we observed a population of terminals expressing VGLUT1 and VGAT. Post-embedding immunogold studies showed that VGLUT1+/VGAT+ terminals formed both symmetric and asymmetric synapses. Triple-labeling studies revealed GABAergic synapses expressing VGLUT1 and glutamatergic synapses expressing VGAT. Immunoisolation studies showed that anti-VGAT immunoisolated vesicles contained VGLUT1 and anti-VGLUT1 immunoisolated vesicles contained VGAT. Finally, vesicles containing VGAT resident in glutamatergic terminals undergo active recycling. In conclusion, we demonstrate that in neocortex VGLUT1 and VGAT are co-expressed in a subset of axon terminals forming both symmetric and asymmetric synapses, that VGLUT1 and VGAT are sorted to the same vesicles and that vesicles at synapses expressing the vesicular heterotransporter participate in the exo-endocytotic cycle.     

Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling

Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.     

Endophilin-1: a multifunctional protein

Endophilin-1, a cytoplasmic Src homology 3 (SH3) domain-containing protein, localises in brain presynaptic nerve termini. Endophilin dimerises through its N-terminus, and participates at multiple stages in clathrin-coated endocytosis, from early membrane invagination to synaptic vesicle uncoating. Both its C-terminal SH3 domain and N-terminus are required for endocytosis. Through its SH3 domain, endophilin bound to proline-rich domains (PRDs) in other endocytic proteins, including synaptojanin and dynamin. The N-terminal region possesses unique functions affecting lipid membrane curvature, through lysophosphatidic acid acyl transferase (LPAAT) activity and direct binding and tubulating activity. In addition to synaptic vesicle formation, endophilin-1 complexes with signalling molecules, including cell surface receptors, metalloprotease disintegrins and germinal centre kinase-like kinase (GLK). Therefore, endophilin-1 may serve to couple vesicle biogenesis with intracellular signalling cascades.     

Vesicular glutamate transporter 2 is expressed in different nerve fibre populations that selectively contact pulmonary neuroepithelial bodies

Pulmonary neuroepithelial body (NEB) receptors in rats receive at least four different nerve fibre populations. In addition to a spinal sensory innervation that contacts NEBs at their basal side, extensive vagal nodose sensory terminals and separate nitrergic and cholinergic nerve endings protrude between NEB cells. In the present study, antibodies against the vesicular glutamate transporter 2 (VGLUT2), a transmembrane protein responsible for loading glutamate into synaptic vesicles, were used to investigate whether some of the nerve terminals contacting NEBs in rat lungs might use glutamate as a neurotransmitter. VGLUT2 immunoreactivity (IR) was detected in extensive intraepithelial arborising nerve terminals that appeared to contact most of the NEBs. Multiple immunostaining showed VGLUT2 IR in the vagal nodose and spinal sensory nerve terminals contacting NEBs, and in another, most likely sensory, intraepithelial nerve fibre population, the origin and further characteristics of which remain to be elucidated. At least part of the VGLUT2-immunoreactive nerve fibres that contact NEBs were shown to be myelinated. The expression of VGLUT2 indicates that glutamate is stored and released as a neurotransmitter in terminals of several pulmonary (sensory) nerve fibre populations that selectively relate to the complex NEB receptors. The present study strongly suggests an involvement of glutamatergic mechanisms in the peripheral transduction of sensory stimuli from the lungs, via the release of glutamate from nerve terminals, thereby modulating the activity of NEB receptor cells or the excitability of afferent nerves.     

Docking and homology modeling explain inhibition of the human vesicular glutamate transporters

As membrane transporter proteins, VGLUT1-3 mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. This function is crucial for exocytosis and the role of glutamate as the major excitatory neurotransmitter in the central nervous system. The three transporters, sharing 76% amino acid sequence identity in humans, are highly homologous but differ in regional expression in the brain. Although little is known regarding their three-dimensional structures, hydropathy analysis on these proteins predicts 12 transmembrane segments connected by loops, a topology similar to other members in the major facilitator superfamily, where VGLUT1-3 have been phylogenetically classified. In this work, we present a three-dimensional model for the human VGLUT1 protein based on its distant bacterial homolog in the same superfamily, the glycerol-3-phosphate transporter from Escherichia coli. This structural model, stable during molecular dynamics simulations in phospholipid bilayers solvated by water, reveals amino acid residues that face its pore and are likely to affect substrate translocation. Docking of VGLUT1 substrates to this pore localizes two different binding sites, to which inhibitors also bind with an overall trend in binding affinity that is in agreement with previously published experimental data.     

Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis

Endophilin/SH3p4 is a protein highly enriched in nerve terminals that binds the GTPase dynamin and the polyphosphoinositide phosphatase synaptojanin, two proteins implicated in synaptic vesicle endocytosis. We show here that antibody-mediated disruption of endophilin function in a tonically stimulated synapse leads to a block in the invagination of clathrin-coated pits adjacent to the active zone and therefore to a block of synaptic vesicle recycling. We also show that in a cell-free system, endophilin is not associated with clathrin coats and is a functional partner of dynamin. Our findings suggest that endophilin is part of a biochemical machinery that acts in trans to the clathrin coat from early stages to vesicle fission.     

Differential sorting of the vesicular glutamate transporter 1 into a defined vesicular pool is regulated by light signaling involving the clock gene Period2

Synaptic strength depends on the amount of neurotransmitter stored in synaptic vesicles. The vesicular transmitter content has recently been shown to be directly dependent on the expression levels of vesicular neurotransmitter transporters indicating that the transport capacity of synaptic vesicles is a critical determinant for synaptic efficacy. Using synaptic vesicles prepared from whole brain at different times of the day we now show that the amount of vesicular glutamate transporter (VGLUT) 1 undergoes strong diurnal cycling. VGLUT1 protein levels are high before the start of the light period, decline at noon, increase again before start of the dark period, and decline again at midnight. Mice kept in complete darkness showed within a 24-h period only a single peak of VGLUT1 expression in the middle of the rest phase. In contrast, mice lacking the period gene Period 2, a core component of the circadian clock, did not show any light-cycle-dependent changes of VGLUT1 levels. No other of several synaptic vesicle proteins examined underwent circadian cycling. Circadian cycling of VGLUT1 was not seen when analyzing homogenate or synaptosomes, the starting fraction for vesicle preparation. Circadian cycling of VGLUT1 was also not reflected at the mRNA level. We conclude that nerve terminals are endowed with mechanisms that regulate quantal size by changing the copy number of transporters in synaptic vesicles. A reduced amount of VGLUT1 per vesicle is probably achieved by means of selective sorting controlled by clock genes.     

中枢神经系统谷氨酸转运体的研究进展

在中枢神经系统,谷氨酸转运体在谷氨酸一谷氨酰胺循环中发挥着重要作用。谷氨酸转运体有高亲和力转运体,即兴奋性氨基酸转运体（excitatory amino acid transporters,EAATs）和低亲和力转运体,即囊泡谷氨酸转运体（vesicular glutamate transporters,VGLUTs）两种类型。其中,VGLUTs的功能是特异地将突触囊泡外的谷氨酸转运进入突触囊泡内,它包括三个成员,分别是VGLUT1、VGLUT2和VGLUT3。一方面,VGLUT1和VGLUT2标记了所有的谷氨酸能神经元,是谷氦酸能神经元和它们轴突末端高度特异的标志;另一方面,VGLUT1标志着皮质一皮质投射,而VGLUT2则标志着丘脑一皮层投射,VGLUT3则位于抑制性突触末端。     

Ataxin-2 associates with the endocytosis complex and affects EGF receptor trafficking

Ataxin-2 is a novel protein, where the unstable expansion of an internal polyglutamine domain can cause the neurodegenerative disease Spinocerebellar Ataxia type 2 (SCA2). To elucidate its cellular function, we have used full-length ataxin-2 as bait in a yeast two-hybrid screen of human adult brain cDNA. As binding partners we found endophilin A1 and A3, two brain-expressed members of the endophilin A family involved in synaptic vesicle endocytosis. Co-immunoprecipitation studies confirmed the binding of these proteins as an endogenous complex in mouse brain. In vitro binding experiments narrowed the binding interfaces down to two proline-rich domains on ataxin-2, which interacted with the SH3 domain of endophilin A1/A3. Ataxin-2 and endophilin associated at the endoplasmic reticulum as well as at the plasma membrane as determined by immunofluorescence microscopy of transfected cell lines, and by centrifugation fractionation studies of mouse brain. Importantly, the pattern observed in transfected cells was conserved in rat hippocampal neurons. In the mouse brain, an association of ataxin-2 with endocytic proteins such as the adaptor CIN85 and the ubiquitin ligase c-Cbl was also demonstrated. GST pull-down assays showed ataxin-2 to directly interact with the SH3 domains A and C of CIN85 and with the SH3 domain of Src, a kinase activated after receptor stimulation. Functional studies demonstrated that ataxin-2 affects endocytic trafficking of the epidermal growth factor receptor (EGFR). Taken together, these data implicate ataxin-2 to play a role in endocytic receptor cycling.     

Characterization of two distinct modes of endophilin in clathrin-mediated endocytosis

Endophilin, one of the main accessory proteins involved in clathrin-mediated endocytosis, interacts with other endocytic proteins, such as dynamin, by its SH3 domain. We previously reported that voltage-gated Ca(2+) channels are an integral part of the synaptic vesicle (SV) endocytosis machinery through their interaction with endophilin. Formation of the endophilin-channel complex is Ca(2+) dependent. A glutamate residue, E264, in endophilin is part of the primary Ca(2+) sensor for Ca(2+)-dependent formation of the channel-endophilin complex. We proposed that endophilin exists in two distinct modes (conformations), an open mode in the absence of Ca(2+), and a closed mode in the presence of Ca(2+). Binding of Ca(2+) switches endophilin from its open mode to the closed mode, resulting in dissociation of endophilin from other proteins. The present study is aimed at understanding the functional roles of endophilin in its two different modes, by creating two endophilin mutants, E264A and E264R, to mimic endophilin in its permanent open mode and permanent closed mode respectively. Here, we show that these two modes of endophilin have different effects on how endophilin interacts with other proteins, such as dynamin or β1-adrenergic receptors. In living cells, endophilin in its permanent closed mode does not show obvious effects on agonist-induced internalization of β1-adrenergic receptors. Endophilin, when in its permanent open mode, enhances the short-term synaptic depression in cultured hippocampal neurons, due partly to its failure to dissociate from Ca(2+) channels in the presence of Ca(2+). Our results show that modal switching by Ca(2+) allows endophilin to regulate, more effectively, the clathrin-mediated endocytosis of SV at the nerve terminal.     

Septin 3 (G-septin) is a developmentally regulated phosphoprotein enriched in presynaptic nerve terminals

The septins are GTPase enzymes with multiple roles in cytokinesis, cell polarity or exocytosis. The proteins from the mammalian septin genes are called Sept1-10. Most are expressed in multiple tissues, but the mRNA for Sept5 (CDCrel-1) and Sept3 (G-septin) appear to be primarily expressed in brain. Sept3 is phosphorylated by cGMP-dependent protein kinase I (PKG-I) and the cGMP/PKG pathway is involved in presynaptic plasticity. Therefore to determine whether Sept3 specifically associates with neurones and nerve terminals we investigated its distribution in rat brain and neuronal cultures. Sept3 protein was detected only in brain by immunoblot, but not in 12 other tissues examined. Levels were high in all adult brain regions, and reduced in those enriched in white matter. Expression was developmentally regulated, being absent in the early embryo, low in late embryonic rat brain and increasing after birth. Like dynamin I, Sept3 was specifically enriched in synaptosomes compared with whole brain, and was only found in a peripheral membrane extract and not in the soluble or membrane extracts. Sept3 was particularly abundant in mossy fibre nerve terminals in the hippocampus. In primary cultured hippocampal neurones Sept3 immunoreactivity was punctate in neurites and predominantly localized to presynaptic terminals, strongly colocalizing with synaptophysin and dynamin I. The specific nerve terminal localization was confirmed by immunogold electron microscopy. Together this shows that Sept3 is a neurone-specific protein highly enriched in nerve terminals which supports a secretory role in synaptic vesicle recycling.     

Vesicular glutamate transporter 2-immunoreactive afferent nerve terminals in the carotid body of the rat

The carotid body is a peripheral chemoreceptor that detects decreases in arterial pO₂ and subsequently activates the carotid sinus nerve. The hypoxia-evoked activity of the carotid sinus nerve has been suggested to be modulated by glutamate. In the present study, we investigate the immunohistochemical localization of vesicular glutamate transporters in the carotid body of the rat. Vesicular glutamate transporter 2 (VGLUT2) labeling was closely associated with glomus cells immunoreactive to tyrosine hydroxylase but was not in the cytoplasm of these cells. The VGLUT2 immunoreactivity was observed within nerve endings that were immunoreactive to P2X3 and densely localized inside P2X3-immunoreactive axon terminals. These results suggest that VGLUT2 is localized in the afferent nerve terminals of the carotid body. Glutamate may be released from afferent nerve terminals to modulate the chemosensory activity of the carotid body.     

Endophilin regulates JNK activation through its interaction with the germinal center kinase-like kinase

The endophilin family of proteins function in clathrin-mediated endocytosis. Here, we have identified and cloned the rat germinal center kinase-like kinase (rGLK), a member of the GCK (germinal center kinase) family of c-Jun N-terminal kinase (JNK) activating enzymes, as a novel endophilin I-binding partner. The interaction occurs both in vitro and in cells and is mediated by the Src homology 3 domain of endophilin I and a region of rGLK containing the endophilin consensus-binding sequence PPRPPPPR. Overlay analysis of rat brain extracts demonstrates that endophilin I is a major Src homology 3 domain-binding partner for rGLK. Overexpression of full-length endophilin I activates rGLK-mediated JNK activation, whereas N- and C-terminal fragments of endophilin I block JNK activation. Thus, endophilin I appears to have a novel function in JNK activation.