Synaptic Vesicle Glycoprotein 2C Research Articles

Synaptic vesicle glycoprotein 2C enhances vesicular storage of dopamine and counters dopaminergic toxicity.

Dopaminergic neurons of the substantia nigra exist in a persistent state of vulnerability resulting from high baseline oxidative stress, high-energy demand, and broad unmyelinated axonal arborisations. Impairments in the storage of dopamine compound this stress because of cytosolic reactions that transform the vital neurotransmitter into an endogenous neurotoxicant, and this toxicity is thought to contribute to the dopamine neuron degeneration that occurs Parkinson's disease. We have previously identified synaptic vesicle glycoprotein 2C (SV2C) as a modifier of vesicular dopamine function, demonstrating that genetic ablation of SV2C in mice results in decreased dopamine content and evoked dopamine release in the striatum. Here, we adapted a previously published in vitro assay utilising false fluorescent neurotransmitter 206 (FFN206) to visualise how SV2C regulates vesicular dopamine dynamics and determined that SV2C promotes the uptake and retention of FFN206 within vesicles. In addition, we present data indicating that SV2C enhances the retention of dopamine in the vesicular compartment with radiolabelled dopamine in vesicles isolated from immortalised cells and from mouse brain. Further, we demonstrate that SV2C enhances the ability of vesicles to store the neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) and that genetic ablation of SV2C results in enhanced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced vulnerability in mice. Together, these findings suggest that SV2C functions to enhance vesicular storage of dopamine and neurotoxicants and helps maintain the integrity of dopaminergic neurons.

Biology activity and characterization of the functional L-HN fragment derivative of botulinum neurotoxin serotype E

ObjectivesThe mature botulinum neurotoxin (BoNT) is a long peptide chain consisting of a light chain (L) and a heavy chain (H) linked by a disulfide bond, where the heavy chain is divided into a translocation domain and an acceptor binding domain (Hc). In this study, we further explored the biology activity and characteristics of recombinant L-HN fragment (EL-HN) composed of the L and HN domains of BoNT/E in vivo and in vitro. MethodsNeurotoxicity of L-HN fragments from botulinum neurotoxins was assessed in mice. Cleavage of dichain EL-HN in vitro and in neuro-2a cells was assessed and compared with that of single chain EL-HN. Interaction of HN domain and the receptor synaptic vesicle glycoprotein 2C (SV2C) was explored in vitro and in neuro-2a cells only expressing SV2C. ResultsWe found that the 50% mouse lethal dose of the nicked dichain EL-HN fragment (EL–HN–DC) was 0.5 μg and its neurotoxicity was the highest among the L-HN's of the four serotypes of BoNT (A/B/E/F). The cleavage efficiency of EL–HN–DC toward synaptosome associated protein 25 (SNAP25) in vitro was 3-fold higher than that of the single chain at the cellular level, and showed 200-fold higher animal toxicity. The EL–HN–DC fragment might enter neuro-2a cells via binding to SV2C to efficiently cleave SNAP25. ConclusionsThe EL-HN fragment showed good biological activities in vivo and in vitro, and could be used as a drug screening model and to further explore the molecular mechanism of its transmembrane transport.

Behavioral and Transcriptomic Changes Following Brain-Specific Loss of Noradrenergic Transmission.

Noradrenaline (NE) plays an integral role in shaping behavioral outcomes including anxiety/depression, fear, learning and memory, attention and shifting behavior, sleep-wake state, pain, and addiction. However, it is unclear whether dysregulation of NE release is a cause or a consequence of maladaptive orientations of these behaviors, many of which associated with psychiatric disorders. To address this question, we used a unique genetic model in which the brain-specific vesicular monoamine transporter-2 (VMAT2) gene expression was removed in NE-positive neurons disabling NE release in the entire brain. We engineered VMAT2 gene splicing and NE depletion by crossing floxed VMAT2 mice with mice expressing the Cre-recombinase under the dopamine β-hydroxylase (DBH) gene promotor. In this study, we performed a comprehensive behavioral and transcriptomic characterization of the VMAT2DBHcre KO mice to evaluate the role of central NE in behavioral modulations. We demonstrated that NE depletion induces anxiolytic and antidepressant-like effects, improves contextual fear memory, alters shifting behavior, decreases the locomotor response to amphetamine, and induces deeper sleep during the non-rapid eye movement (NREM) phase. In contrast, NE depletion did not affect spatial learning and memory, working memory, response to cocaine, and the architecture of the sleep-wake cycle. Finally, we used this model to identify genes that could be up- or down-regulated in the absence of NE release. We found an up-regulation of the synaptic vesicle glycoprotein 2c (SV2c) gene expression in several brain regions, including the locus coeruleus (LC), and were able to validate this up-regulation as a marker of vulnerability to chronic social defeat. The NE system is a complex and challenging system involved in many behavioral orientations given it brain wide distribution. In our study, we unraveled specific role of NE neurotransmission in multiple behavior and link it to molecular underpinning, opening future direction to understand NE role in health and disease.

Nicotine-Mediated Rescue of α-Synuclein Toxicity Requires Synaptic Vesicle Glycoprotein 2 in Drosophila.

Parkinson's disease (PD) is characterized by α-synuclein aggregation and loss of dopamine neurons. Risk of PD arises due to a combination of genetic and environmental factors, which may interact, termed gene-environment (G×E) interactions. An inverse association between smoking and the risk of PD is well established, and a previous genome-wide G×E interaction study identified genetic variation in the synaptic-vesicle glycoprotein 2C (SV2C) locus as an important mediator of the degree to which smoking is inversely associated with PD. We sought to determine the mechanism of the smoking-SV2C interaction in a Drosophila model of PD. Flies expressing human α-synuclein in all neurons develop the hallmarks of PD, including motor dysfunction, loss of dopaminergic (DA) neurons, and formation of α-synuclein inclusions. We assessed the effects of increasing doses of nicotine on these parameters of neurodegeneration, in the presence or absence of knockdown of two Drosophila orthologues of SV2, hereafter referred to as SV2L1 and SV2L2. The α-synuclein-expressing flies treated with nicotine had improved locomotion, DA neuron counts, and α-synuclein aggregation. However, in α-synuclein-expressing flies in which SV2L1 and SV2L2 were knocked down, nicotine failed to rescue neurodegeneration. This work confirms a G×E interaction between nicotine and SV2, defines a role for this interaction in α-synuclein proteostasis, and suggests that future clinical trials on nicotine should consider genetic variation in SV2C. Furthermore, this provides proof of concept that our model can be used for the mechanistic study of G×E, paving the way for the investigation of additional G×E interactions or the identification of novel G×E. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Neuron-recognizable characteristics of peptides recombined using a neuronal binding domain of botulinum neurotoxin

Recombinant peptides were designed using the C-terminal domain (receptor binding domain, RBD) and its subdomain (peptide A2) of a heavy chain of botulinum neurotoxin A-type 1 (BoNT/A1), which can bind to the luminal domain of synaptic vesicle glycoprotein 2C (SV2C-LD). Peptide A2- or RBD-containing recombinant peptides linked to an enhanced green fluorescence protein (EGFP) were prepared by expression in Escherichia coli. A pull-down assay using SV2C-LD-covered resins showed that the recombinant peptides for CDC297 BoNT/A1, referred to EGFP-A2ʹ and EGFP-RBDʹ, exhibited ≥ 2.0-times stronger binding affinity to SV2C-LD than those for the wild-type BoNT/A1. Using bio-layer interferometry, an equilibrium dissociation rate constant (KD) of EGFP-RBDʹ to SV2C-LD was determined to be 5.45 μM, which is 33.87- and 15.67-times smaller than the KD values for EGFP and EGFP-A2ʹ, respectively. Based on confocal laser fluorescence micrometric analysis, the adsorption/absorption of EGFP-RBDʹ to/in differentiated PC-12 cells was 2.49- and 1.29-times faster than those of EGFP and EGFP-A2ʹ, respectively. Consequently, the recombinant peptides acquired reasonable neuron-specific binding/internalizing ability through the recruitment of RBDʹ. In conclusion, RBDs of BoNTs are versatile protein domains that can be used to mark neural systems and treat a range of disorders in neural systems.

Changes in the Gene Expression Profiles of the Inferior Colliculus Following Unilateral Cochlear Ablation in Adult Rats.

This study aimed to explore gene expression changes in the inferior colliculus (IC) after single-sided deafness (SSD). Forty 8-week-old female Sprague-Dawley rats were used. Twenty rats underwent right-side cochlear ablation, and IC tissues were harvested after 2weeks (SSD 2-week group). Twenty rats underwent a sham operation and were sacrificed after 2weeks (control group). Both sides of the IC were analyzed using a gene expression array. Pathway analyses were performed on genes that were differentially expressed compared with their levels in the control group. The expression levels of genes involved in the candidate pathways were confirmed using reverse transcription polymerase chain reaction (RT-PCR). Among the genes with ≥ 1.5-fold changes in expression levels and P < 0.05, there were 7 and 9 genes with increased and decreased expression, respectively, in the ipsilateral IC and 10 and 12 genes with increased and decreased expression, respectively, in the contralateral IC. The pathway analysis did not identify significantly related pathway. In the bilateral analysis, a total of 14 genes were ≥ 1.3-fold downregulated in both the ipsilateral and contralateral IC in the SSD 2-week group compared with their expression in the control group. Pathway analyses of these 14 genes included 7 genes, namely, amine compound solute carrier (Slc)5a7; Slc18a3; Slc6a5; synaptic vesicle glycoprotein 2C (Sv2c); S100 calcium binding protein A10 (S100a10); a gene with sequence similarity to family 111, member A (Fam111a); and peripherin (Prph), that were related to the acetylcholine neurotransmitter release cycle, SLC transporters, and the neurotransmitter release cycle pathways. RT-PCR showed reduced expression of Slc5a7, Sv2c, and Prph in the contralateral IC and Slc18a3 and Slc6a5 in the ipsilateral IC of the SSD 2-week group compared with that in the control group.

Current and emerging therapeutic targets for Parkinson's disease.

Parkinson's disease (PD) is characterized by gradual neurodegeneration and forfeiture of dopamine neurons in substantia nigra pars compacta which ultimately leads to depletion of dopamine levels. PD patients not only display motor features such as rigidity, tremor, and bradykinesia but also non-motor features such as depression, anxiety, etc. Various treatments are available for PD patients such as dopamine replacement are well established but it is only partially or transiently effective. As these therapies not able to restore dopaminergic neurons and delay the development of Parkinson's disease, therefore, the need for an effective therapeutic approach is crucial. The present review discusses a comprehensive overview of current novel targets for PD which includes molecular chaperone, neuroinflammation, mitochondrial dysfunction, neuromelanin, Ubiquitin-proteasome system, protein Abelson, Synaptic vesicle glycoprotein 2C, and Cocaine-amphetamine-regulated transcript, etc. These approaches will help to identify new targets for the treatment of disease and may provide a ray of hope for PD patient treatment. Graphical abstract.

Commentary: Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson disease.

Commentary: Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson disease.

Immunochemical analysis of the expression of SV2C in mouse, macaque and human brain

The synaptic vesicle glycoprotein 2C (SV2C) is an undercharacterized protein with enriched expression in phylogenetically old brain regions. Its precise role within the brain is unclear, though various lines of evidence suggest that SV2C is involved in the function of synaptic vesicles through the regulation of vesicular trafficking, calcium-induced exocytosis, or synaptotagmin function. SV2C has been linked to multiple neurological disorders, including Parkinson’s disease and psychiatric conditions. SV2C is expressed in various cell types—primarily dopaminergic, GABAergic, and cholinergic cells. In mice, it is most highly expressed in nuclei within the basal ganglia, though it is unknown if this pattern of expression is consistent across species. Here, we use a custom SV2C-specific antiserum to describe localization within the brain of mouse, nonhuman primate, and human, including cell-type localization. We found that the immunoreactivity with this antiserum is consistent with previously-published antibodies, and confirmed localization of SV2C in the basal ganglia of rodent, rhesus macaque, and human. We observed strongest expression of SV2C in the substantia nigra, ventral tegmental area, dorsal striatum, pallidum, and nucleus accumbens of each species. Further, we demonstrate colocalization between SV2C and markers of dopaminergic, GABAergic, and cholinergic neurons within these brain regions. SV2C has been increasingly linked to dopamine and basal ganglia function. These antisera will be an important resource moving forward in our understanding of the role of SV2C in vesicle dynamics and neurological disease.

Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson disease.

Members of the synaptic vesicle glycoprotein 2 (SV2) family of proteins are involved in synaptic function throughout the brain. The ubiquitously expressed SV2A has been widely implicated in epilepsy, although SV2C with its restricted basal ganglia distribution is poorly characterized. SV2C is emerging as a potentially relevant protein in Parkinson disease (PD), because it is a genetic modifier of sensitivity to l-DOPA and of nicotine neuroprotection in PD. Here we identify SV2C as a mediator of dopamine homeostasis and report that disrupted expression of SV2C within the basal ganglia is a pathological feature of PD. Genetic deletion of SV2C leads to reduced dopamine release in the dorsal striatum as measured by fast-scan cyclic voltammetry, reduced striatal dopamine content, disrupted α-synuclein expression, deficits in motor function, and alterations in neurochemical effects of nicotine. Furthermore, SV2C expression is dramatically altered in postmortem brain tissue from PD cases but not in Alzheimer disease, progressive supranuclear palsy, or multiple system atrophy. This disruption was paralleled in mice overexpressing mutated α-synuclein. These data establish SV2C as a mediator of dopamine neuron function and suggest that SV2C disruption is a unique feature of PD that likely contributes to dopaminergic dysfunction.

Only the complex N559-glycan in the synaptic vesicle glycoprotein 2C mediates high affinity binding to botulinum neurotoxin serotype A1.

The extraordinary potency of botulinum neurotoxins (BoNTs) is mediated by their high neurospecificity, targeting peripheral cholinergic motoneurons leading to flaccid paralysis and successive respiratory failure. Complex polysialo gangliosides accumulate BoNTs on the plasma membrane and facilitate subsequent binding to synaptic vesicle membrane proteins which results in toxin endocytosis. The luminal domain 4 (LD4) of the three synaptic vesicle glycoprotein 2 (SV2) isoforms A-C mediates uptake of the clinically most relevant serotype BoNT/A1. SV2C-LD4 exhibits the strongest protein-protein interaction and comprises five putative N-glycosylation sites (PNG sites). Here, we expressed human SV2C-LD4 fused to human IgG-Fc in prokaryotic and eukaryotic expression systems to analyse the effect of N-glycosylation of SV2C on the interaction with BoNT/A1. Mass spectrometric analysis of gSV2CLD-Fc demonstrates glycosylation of N534, N559 and N565, the latter two residing at the BoNT/A interface. Mutational analysis demonstrates that only the N559-glycan, but not N565-glycan increases affinity of BoNT/A for human gSV2C-LD4. The N559-glycan was characterised as a complex core-fucosylated typewith a heterogeneity ranging up to tetra-antennary structure with bisecting N-acetylglucosamine which can establish extensive interactions with BoNT/A. The mutant gSV2CLD-Fc N559A displayed a 50-fold increased dissociation rate kd resulting in an overall 12-fold decreased binding affinity in surface plasmon resonance (SPR) experiments. The delayed dissociation might provide BoNT/A more time for endocytosis into synaptic vesicles. In conclusion, we show the importance of the complex N559-glycan of SV2C-LD4, adding a third anchor point beside a ganglioside and the SV2C-LD4 peptide, for BoNT/A neuronal cell surface binding and uptake.

Synaptic vesicle glycoprotein 2C modulates vesicular dopamine release and contributes to behaviors associated with reward

Synaptic vesicle glycoprotein 2C modulates vesicular dopamine release and contributes to behaviors associated with reward

Effects of methylphenidate: the cellular point of view

The psychostimulant methylphenidate (MPH) is the first choice of treatment in attention-deficit hyperactivity disorder and is based mainly on inhibition of dopamine transporter (DAT). Nonetheless, the complete cellular effects of MPH are still unknown. We attempted to determine whether MPH influences neurotransmitter levels, synaptic gene expression, and cell proliferation in a dose-dependent manner in rat pheochromocytoma cells (PC12) lacking DAT. PC12 were treated in a dose-dependent manner with MPH. Gene expression level of synaptotagmin (Syt) 1 and 4, syntaxin 1a (Stx1a), and synaptic vesicle glycoprotein 2C (SV2C) was measured using quantitative real-time RT-PCR. Different Neurotransmitter release was measured using high-performance liquid chromatography (HPLC). Differences in cell proliferation were evaluated via BrdU incorporation. Treatment with low-dose MPH (1-100 nM) altered intra-/extracellular neurotransmitter levels, down-regulated all investigated genes as well as enhanced cell proliferation significantly. These data point to diverse effects of MPH on cell metabolism independent of inhibiting DAT.

Capture assay for Botulinum Neurotoxin Type A utilizing the receptor protein SV2c

Recent biochemical and molecular genetic studies have established synaptic vesicle glycoprotein 2C (SV2c) as the protein receptor for Botulinum Neurotoxin Type A (BoNT/A). The luminal domain loop of SV2c, between transmembrane domains 7 and 8, has been shown to be the location of BoNT/A binding. Utilizing this information, we have shown through GST pull-down experiments that we can detect binding of BoNT/A to SV2c using an immunoassay. This can be done with either the rat or the human forms of the SV2c protein. Upon specific binding of BoNT/A to its receptor protein, we can introduce our FRET peptide SNAPtide ® and detect enzymatic cleavage. We describe the reaction conditions and the detection limits using both rat and human forms of the SV2c binding region as well as multiple FRET substrates to determine the optimal conditions for this detection assay. There are three steps in the interruption of synaptic transmission by botulinum toxin. The first is binding to neuronal cells; the second is translocation of the enzymatic light chain out of the endosome; and finally cleavage of synaptosomal proteins to inhibit neurotransmitter release. With this assay we can monitor two of the three steps of toxin activity, binding and cleavage. In the future, we hope to establish this as a new functional assay for the detection and activity of BoNT/A and demonstrate a direct correlation to the gold standard, the mouse bioassay.

The synaptic vesicle protein 2C mediates the uptake of botulinum neurotoxin A into phrenic nerves

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by selectively cleaving core components of the vesicular fusion machinery. The synaptic vesicle proteins Synaptotagmin-I and -II act as receptors for BoNT/B and BoNT/G. Here we show that BoNT/A also interacts with a synaptic vesicle protein, the synaptic vesicle glycoprotein 2C (SV2C), but not with the homologous proteins SV2A and SV2B. Binding of BoNT/A occurs at the membrane juxtaposed region preceding transmembrane domain 8. A peptide comprising the intravesicular domain between transmembrane domains 7 and 8 specifically reduces the neurotoxicity of BoNT/A at phrenic nerve preparations demonstrating the physiological relevance of this interaction.