Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport.

The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.

A slow excitatory postsynaptic current mediated by a novel metabotropic glutamate receptor in CA1 pyramidal neurons.

Slow excitatory postsynaptic currents (EPSCs) mediated by metabotropic glutamate receptors (mGlu receptors) have been reported in several neuronal subtypes, but their presence in hippocampal pyramidal neurons remains elusive. Here we find that in CA1 pyramidal neurons a slow EPSC is induced by repetitive stimulation while ionotropic glutamate receptors and glutamate-uptake are blocked whereas it is absent in the VGLUT1 knockout mouse in which presynaptic glutamate is lost, suggesting the slow EPSC is mediated by glutamate activating mGlu receptors. However, it is not inhibited by known mGlu receptor antagonists. These findings suggest that this slow EPSC is mediated by a novel mGlu receptor, and that it may be involved in neurological diseases associated with abnormal high-concentration of extracellular glutamate. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

Lentiviral delivery of a vesicular glutamate transporter 1 (VGLUT1)-targeting short hairpin RNA vector into the mouse hippocampus impairs cognition.

Glutamate is the principle excitatory neurotransmitter in the mammalian brain, and dysregulation of glutamatergic neurotransmission is implicated in the pathophysiology of several psychiatric and neurological diseases. This study utilized novel lentiviral short hairpin RNA (shRNA) vectors to target expression of the vesicular glutamate transporter 1 (VGLUT1) following injection into the dorsal hippocampus of adult mice, as partial reductions in VGLUT1 expression should attenuate glutamatergic signaling and similar reductions have been reported in schizophrenia. The VGLUT1-targeting vector attenuated tonic glutamate release in the dorsal hippocampus without affecting GABA, and selectively impaired novel object discrimination (NOD) and retention (but not acquisition) in the Morris water maze, without influencing contextual fear-motivated learning or causing any adverse locomotor or central immune effects. This pattern of cognitive impairment is consistent with the accumulating evidence for functional differentiation along the dorsoventral axis of the hippocampus, and supports the involvement of dorsal hippocampal glutamatergic neurotransmission in both spatial and nonspatial memory. Future use of this nonpharmacological VGLUT1 knockdown mouse model could improve our understanding of glutamatergic neurobiology and aid assessment of novel therapies for cognitive deficits such as those seen in schizophrenia.

Regulation of markers of synaptic function in mouse models of depression: chronic mild stress and decreased expression of VGLUT1.

Depression has been linked to failure in synaptic plasticity originating from environmental and/or genetic risk factors. The chronic mild stress model regulates the expression of synaptic markers of neurotransmitter function and associated depressive-like behaviour. Moreover, mice heterozygous for the synaptic vesicle protein vesicular glutamate transporter 1 (VGLUT1), have been proposed as a genetic model of deficient glutamate function linked to depressive-like behaviour. Here, we aimed to identify, in these two experimental models, mechanisms of failure in synaptic plasticity, common to stress and impaired glutamate function. First, we show that chronic mild stress induced a transient decrease of different plasticity markers (VGLUT1, synapsin 1, sinaptophysin, rab3A and activity regulated cytoskeletal protein - Arc) but a long-lasting decrease of the brain derived neurotrophic factor as well as depressive-like behaviour. The immediate early gene Arc was also down-regulated in VGLUT1+/- heterozygous mice. In contrast, an opposite regulation of synapsin 1 was observed. Finally, both models showed a marked increase of cortical Arc response to novelty. Increased Arc response to novelty could be suggested as a molecular mechanism underlying failure to adapt to environmental changes, common to chronic stress and altered glutamate function. Further studies should investigate whether these changes are associated to depressive-like behaviour both in animal models and in depressed patients.

Ca2+-dependent mobility of vesicles capturing anti-VGLUT1 antibodies.

Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca(2+)-dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca(2+) level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca(2+)-triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles.

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.