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1.
Cells ; 12(5)2023 02 26.
Article in English | MEDLINE | ID: mdl-36899886

ABSTRACT

V-ATPase is an important factor in synaptic vesicle acidification and is implicated in synaptic transmission. Rotation in the extra-membranous V1 sector drives proton transfer through the membrane-embedded multi-subunit V0 sector of the V-ATPase. Intra-vesicular protons are then used to drive neurotransmitter uptake by synaptic vesicles. V0a and V0c, two membrane subunits of the V0 sector, have been shown to interact with SNARE proteins, and their photo-inactivation rapidly impairs synaptic transmission. V0d, a soluble subunit of the V0 sector strongly interacts with its membrane-embedded subunits and is crucial for the canonic proton transfer activity of the V-ATPase. Our investigations show that the loop 1.2 of V0c interacts with complexin, a major partner of the SNARE machinery and that V0d1 binding to V0c inhibits this interaction, as well as V0c association with SNARE complex. The injection of recombinant V0d1 in rat superior cervical ganglion neurons rapidly reduced neurotransmission. In chromaffin cells, V0d1 overexpression and V0c silencing modified in a comparable manner several parameters of unitary exocytotic events. Our data suggest that V0c subunit promotes exocytosis via interactions with complexin and SNAREs and that this activity can be antagonized by exogenous V0d.


Subject(s)
SNARE Proteins , Vacuolar Proton-Translocating ATPases , Rats , Animals , SNARE Proteins/metabolism , Protons , Synaptic Vesicles/metabolism , Membrane Fusion , Vacuolar Proton-Translocating ATPases/metabolism
2.
Brain ; 145(11): 3843-3858, 2022 11 21.
Article in English | MEDLINE | ID: mdl-35727946

ABSTRACT

Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localization of these features, and hence gain broader insights into LGI1's neurobiology, we analysed the detailed localization of LGI1 and the diversity of its protein interactome, in mouse brains using patient-derived recombinant monoclonal LGI1 antibodies. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus. LGI1 is secreted in both neuronal somatodendritic and axonal compartments, and occurs in oligodendrocytic, neuro-oligodendrocytic and astro-microglial protein complexes. Proteomic data support the presence of LGI1-Kv1-MAGUK complexes, but did not reveal LGI1 complexes with postsynaptic glutamate receptors. Our results extend our understanding of regional, cellular and subcellular LGI1 expression profiles and reveal novel LGI1-associated complexes, thus providing insights into the complex biology of LGI1 and its relationship to seizures and memory loss.


Subject(s)
Glioma , Intracellular Signaling Peptides and Proteins , Animals , Mice , Leucine , Proteomics , Autoantibodies , Seizures
3.
Proc Natl Acad Sci U S A ; 116(36): 18098-18108, 2019 09 03.
Article in English | MEDLINE | ID: mdl-31431523

ABSTRACT

Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals by binding to 2 receptor components: a polysialoganglioside, predominantly GT1b, and synaptotagmin 1/2. It is widely thought that BoNT/B initially binds to GT1b then diffuses in the plane of the membrane to interact with synaptotagmin. We have addressed the hypothesis that a GT1b-synaptotagmin cis complex forms the BoNT/B receptor. We identified a consensus glycosphingolipid-binding motif in the extracellular juxtamembrane domain of synaptotagmins 1/2 and confirmed by Langmuir monolayer, surface plasmon resonance, and circular dichroism that GT1b interacts with synaptotagmin peptides containing this sequence, inducing α-helical structure. Molecular modeling and tryptophan fluorescence spectroscopy were consistent with the intertwining of GT1b and synaptotagmin, involving cis interactions between the oligosaccharide and ceramide moieties of GT1b and the juxtamembrane and transmembrane domains of synaptotagmin, respectively. Furthermore, a point mutation on synaptotagmin, located outside of the BoNT/B-binding segment, inhibited GT1b binding and blocked GT1b-induced potentiation of BoNT/B binding to synaptotagmin-expressing cells. Our findings are consistent with a model in which a preassembled GT1b-synaptotagmin complex constitutes the high-affinity BoNT/B receptor.


Subject(s)
Botulinum Toxins, Type A , Gangliosides , Synaptotagmin I , Animals , Binding Sites , Botulinum Toxins, Type A/chemistry , Botulinum Toxins, Type A/metabolism , Gangliosides/chemistry , Gangliosides/pharmacology , Protein Conformation, alpha-Helical , Protein Domains , Rats , Synaptotagmin I/chemistry , Synaptotagmin I/genetics , Synaptotagmin I/metabolism , Synaptotagmin II/chemistry , Synaptotagmin II/genetics , Synaptotagmin II/metabolism
4.
Mol Neurobiol ; 56(5): 3591-3602, 2019 May.
Article in English | MEDLINE | ID: mdl-30155790

ABSTRACT

Synaptic vesicle proton V-ATPase is an essential component in synaptic vesicle function. Active acidification of synaptic vesicles, triggered by the V-ATPase, is necessary for neurotransmitter storage. Independently from its proton transport activity, an additional important function of the membrane-embedded sector of the V-ATPase has been uncovered over recent years. Subunits a and c of the membrane sector of this multi-molecular complex have been shown to interact with SNARE proteins and to be involved in modulating neurotransmitter release. The c-subunit interacts with the v-SNARE VAMP2 and facilitates neurotransmission. In this study, we used chromophore-assisted light inactivation and monitored the consequences on neurotransmission on line in CA3 pyramidal neurons. We show that V-ATPase c-subunit V0c is a key element in modulating neurotransmission and that its specific inactivation rapidly inhibited neurotransmission.


Subject(s)
Acids/metabolism , Chromophore-Assisted Light Inactivation , Neurotransmitter Agents/metabolism , Protein Subunits/metabolism , Synaptic Vesicles/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Down-Regulation , Fluorescence , Neurons/metabolism , RNA, Small Interfering/metabolism , Rats, Wistar , Synaptic Transmission , Vacuolar Proton-Translocating ATPases/chemistry , Vesicle-Associated Membrane Protein 2/metabolism
5.
Proc Natl Acad Sci U S A ; 114(29): 7719-7724, 2017 07 18.
Article in English | MEDLINE | ID: mdl-28673977

ABSTRACT

Autosomal dominant epilepsy with auditory features results from mutations in leucine-rich glioma-inactivated 1 (LGI1), a soluble glycoprotein secreted by neurons. Animal models of LGI1 depletion display spontaneous seizures, however, the function of LGI1 and the mechanisms by which deficiency leads to epilepsy are unknown. We investigated the effects of pure recombinant LGI1 and genetic depletion on intrinsic excitability, in the absence of synaptic input, in hippocampal CA3 neurons, a classical focus for epileptogenesis. Our data indicate that LGI1 is expressed at the axonal initial segment and regulates action potential firing by setting the density of the axonal Kv1.1 channels that underlie dendrotoxin-sensitive D-type potassium current. LGI1 deficiency incurs a >50% down-regulation of the expression of Kv1.1 and Kv1.2 via a posttranscriptional mechanism, resulting in a reduction in the capacity of axonal D-type current to limit glutamate release, thus contributing to epileptogenesis.


Subject(s)
Axons/metabolism , Proteins/metabolism , Shaker Superfamily of Potassium Channels/metabolism , Action Potentials , Animals , Elapid Venoms/pharmacology , Hippocampus/drug effects , Hippocampus/metabolism , Intracellular Signaling Peptides and Proteins , Kv1.2 Potassium Channel/metabolism , Mice, Mutant Strains , Neurons/drug effects , Neurons/metabolism , Organ Culture Techniques , Patch-Clamp Techniques , Proteins/genetics , Proteins/pharmacology , Rats, Wistar , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology
6.
Sci Rep ; 5: 17953, 2015 Dec 09.
Article in English | MEDLINE | ID: mdl-26648139

ABSTRACT

The enzymatic activity of the pathogenic botulinum neurotoxins type A and E (BoNT/A and E) leads to potentially lethal paralytic symptoms in humans and their prompt detection is of crucial importance. A chip assay based on Surface Plasmon Resonance monitoring of the cleavage products is a simple method that we have previously established to detect BoNT/A activity. We have now developed a similar format assay to measure BoNT/E activity. A monoclonal antibody specifically recognizing SNAP25 cleaved by BoNT/E was generated and used to measure the appearance of the neo-epitope following injection of BoNT/E over SNAP-25 immobilized on a chip. This assay detects BoNT/E activity at 1 LD50/ml within minutes and linear dose-responses curves were obtained using a multiplexed biosensor. A threshold of 0.01 LD50/ml was achieved after 5 h of cleavage. This assay is 10-fold more sensitive than the in vivo assay for direct detection of BoNT/E in serum samples. The SNAP25 chip assay is able to discriminate in an automated manner the presence of BoNT/E, BoNT/A or a combination of both toxins.


Subject(s)
Biosensing Techniques , Botulinum Toxins, Type A , Botulinum Toxins , Antibodies, Monoclonal/immunology , Botulinum Toxins/immunology , Botulinum Toxins, Type A/immunology , Enzyme Activation , Epitopes/immunology , Humans , Kinetics , Lab-On-A-Chip Devices , Microchip Analytical Procedures , Sensitivity and Specificity , Substrate Specificity
7.
Appl Microbiol Biotechnol ; 99(10): 4355-60, 2015 May.
Article in English | MEDLINE | ID: mdl-25672850

ABSTRACT

The production of botulinum neurotoxin A (BoNT/A) for therapeutic and cosmetic applications requires precise determination of batch potency, and the enzymatic activity of BoNT/A light chain is a crucial index that can be measured in vitro. We previously established a SNAP-25 chip-based assay using surface plasmon resonance (SPR) that is more sensitive than the standard mouse bioassay for the quantification of BoNT/A activity. We have now adapted this procedure for pharmaceutical preparations. The optimized SPR assay allowed multiple measurements on a single chip, including the kinetics of substrate cleavage. The activity of five different batches of a pharmaceutical BoNT/A preparation was determined in a blind study by SPR and found to be in agreement with data from the in vivo mouse lethality assay. Biosensor detection of specific proteolytic products has the potential to accurately monitor the activity of pharmaceutical BoNT/A preparations, and a single chip can be used to assay more than 100 samples.


Subject(s)
Biosensing Techniques/methods , Botulinum Toxins, Type A/analysis , Surface Plasmon Resonance/methods , Animals , Biosensing Techniques/instrumentation , Botulinum Toxins, Type A/toxicity , Mice , Surface Plasmon Resonance/instrumentation
8.
Biosens Bioelectron ; 57: 207-12, 2014 Jul 15.
Article in English | MEDLINE | ID: mdl-24583693

ABSTRACT

Botulinum neurotoxin A (BoNT/A) has intrinsic endoprotease activity specific for SNAP-25, a key protein for presynaptic neurotransmitter release. The inactivation of SNAP-25 by BoNT/A underlies botulism, a rare but potentially fatal disease. There is a crucial need for a rapid and sensitive in vitro serological test for BoNT/A to replace the current in vivo mouse bioassay. Cleavage of SNAP-25 by BoNT/A generates neo-epitopes which can be detected by binding of a monoclonal antibody (mAb10F12) and thus measured by surface plasmon resonance (SPR). We have explored two SPR assay formats, with either mAb10F12 or His6-SNAP-25 coupled to the biosensor chip. When BoNT/A was incubated with SNAP-25 in solution and the reaction products were captured on a mAb-coated chip, a sensitivity of 5 fM (0.1LD50/ml serum) was obtained. However, this configuration required prior immunoprecipitation of BoNT/A. A sensitivity of 0.5 fM in 10% serum (0.1 LD50/ml serum) was attained when SNAP-25 was coupled directly to the chip, followed by sequential injection of BoNT/A samples and mAb10F12 into the flow system to achieve on-chip cleavage and detection, respectively. This latter format detected BoNT/A endoprotease activity in 50-100 µl serum samples from all patients (11/11) with type A botulism within 5h. No false positives occurred in sera from healthy subjects or patients with other neurological diseases. The automated chip-based procedure has excellent specificity and sensitivity, with significant advantages over the mouse bioassay in terms of rapidity, required sample volume and animal ethics.


Subject(s)
Biosensing Techniques/methods , Botulinum Toxins, Type A/blood , Botulism/blood , Animals , Antibodies, Immobilized/chemistry , Antibodies, Monoclonal/chemistry , Botulinum Toxins, Type A/metabolism , Botulism/diagnosis , Botulism/metabolism , Humans , Limit of Detection , Mice , Peptide Hydrolases/blood , Peptide Hydrolases/metabolism , Protein Array Analysis/methods
9.
Biosens Bioelectron ; 49: 276-81, 2013 Nov 15.
Article in English | MEDLINE | ID: mdl-23787358

ABSTRACT

Botulinum neurotoxin A (BoNT/A) induces muscle paralysis by enzymatically cleaving the presynaptic SNARE protein SNAP-25, which results in lasting inhibition of acetylcholine release at the neuromuscular junction. A rapid and sensitive in vitro assay for BoNT/A is required to replace the mouse lethality assay (LD50) in current use. We have developed a fully automated sensor to assay the endoprotease activity of BoNT/A. We produced monoclonal antibodies (mAbs) that recognize SNAP-25 neo-epitopes specifically generated by BoNT/A action. Recombinant SNAP-25 was coupled to the sensor surface of a surface plasmon resonance (SPR) system and samples containing BoNT/A were injected over the substrate sensor. Online substrate cleavage was monitored by measuring binding of mAb10F12 to a SNAP-25 neo-epitope. The SNAP-25-chip assay was toxin serotype-specific and detected 55 fM BoNT/A (1 LD50/ml) in 5 min and 0.4 fM (0.01 LD50/ml) in 5h. Time-course and dose-response curves were linear, yielding a limit of quantification of 0.03 LD50/ml. This label-free method is 100 times more sensitive than the mouse assay, potentially providing rapid read-out of small amounts of toxin for environmental surveillance and the quality control of pharmaceutical preparations.


Subject(s)
Botulinum Toxins, Type A/analysis , Botulinum Toxins, Type A/metabolism , Clostridium botulinum/metabolism , Neurotoxins/analysis , Neurotoxins/metabolism , Peptide Hydrolases/metabolism , Surface Plasmon Resonance/methods , Animals , Antibodies, Monoclonal/metabolism , Botulism/microbiology , Humans , Immobilized Proteins/metabolism , Limit of Detection , Mice , Protein Array Analysis/methods , Recombinant Proteins/metabolism , Synaptosomal-Associated Protein 25/metabolism
10.
Neuron ; 67(2): 268-79, 2010 Jul 29.
Article in English | MEDLINE | ID: mdl-20670834

ABSTRACT

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.


Subject(s)
Neurons/metabolism , Neurotransmitter Agents/metabolism , Synapses/physiology , Synaptic Vesicles/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Animals , Animals, Newborn , Calcium/metabolism , Cell Membrane/metabolism , Cerebral Cortex/cytology , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay/methods , Excitatory Postsynaptic Potentials/drug effects , In Vitro Techniques , Liposomes/metabolism , Macrolides/pharmacology , Mutation/genetics , Neurons/drug effects , Neurons/ultrastructure , Neurotransmitter Agents/pharmacology , Peptides/metabolism , Peptides/pharmacology , Protein Binding/drug effects , Protein Binding/physiology , Protein Subunits/genetics , Protein Subunits/metabolism , Proteolipids/metabolism , Rats , Rats, Wistar , SNARE Proteins/metabolism , Sequence Alignment/methods , Two-Hybrid System Techniques , Vacuolar Proton-Translocating ATPases/chemistry , Vesicle-Associated Membrane Protein 2/genetics , Vesicle-Associated Membrane Protein 2/metabolism
11.
J Biol Chem ; 285(31): 23665-75, 2010 Jul 30.
Article in English | MEDLINE | ID: mdl-20519509

ABSTRACT

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca(2+)/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K(D) = 500 nm) and syntaxin 1 (K(D) = 2 microm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca(2+) sensors act antagonistically in SNARE-mediated fusion.


Subject(s)
Calcium/metabolism , Calmodulin/chemistry , Gene Expression Regulation , Membrane Fusion , SNARE Proteins/chemistry , Animals , Calcium/chemistry , Cattle , Cell Membrane/metabolism , Exocytosis , Fluorescence Resonance Energy Transfer , Humans , Kinetics , Liposomes/chemistry , Synaptotagmin I/chemistry , Tetanus Toxin/chemistry
12.
Langmuir ; 20(14): 5885-90, 2004 Jul 06.
Article in English | MEDLINE | ID: mdl-16459605

ABSTRACT

Oriented stable binding of functional proteins on surfaces is of fundamental interest for receptor/ligand studies in atomic force microscopy (AFM) and surface plasmon resonance (SPR) experiments. Here we have chosen the His6-tagged carboxyl-tail (C-tail) of the alpha1c-subunit of the L-type Ca2+ channel and calmodulin (CaM) as its cognitive partner as a model system to develop a new functional surface. Covalently attached self-assembled monolayers on ultraflat gold containing NTA-thiols to which the His6-tagged C-tail was bound and thiols with triethylene-glycol groups as matrix-thiols represented the system of choice. The topography of this surface was characterized using AFM; its ability to bind C-tail proteins oriented and stable was confirmed by SPR measurements and by complementary force spectroscopy experiments with a CaM4-construct covalently attached to the tip. The developed anchoring strategy can now be used to study receptor/ligand interactions in general applying force spectroscopy and SPR on His6-tagged proteins oriented immobilized onto this new NTA-functionalized self-assembled monolayer.

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