Oussama El Far

V-ATPase membrane sector associates with synaptobrevin to modulate neurotransmitter release.

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.

Interaction of a synaptobrevin (VAMP)-syntaxin complex with presynaptic calcium channels

Nerve terminal protein complexes implicated in exocytosis were examined by immuno‐isolation from rat brain synaptosomes. Immunoprecipitation with anti‐syntaxin or anti‐VAMP antibodies revealed a syntaxin‐SNAP25‐VAMP‐synaptotagmin complex. Anti‐VAMP antibodies also trapped a distinct VAMP‐synaptophysin complex. A similar fraction (about 70%) of N‐type calcium channels ([125I]ω conotoxin GVIA receptors), was immunoprecipitated by either anti‐syntaxin or anti‐VAMP antibodies, but not by anti‐synaptophysin antibodies (< 4%). The majority of N‐ but not L‐type calcium channels ([3H]PN200‐110 receptors), appear to be associated with a synaptic vesicle prefusion complex.

A role for V-ATPase subunits in synaptic vesicle fusion?

J. Neurochem. (2011) 117, 603–612.

Expression of synaptotagmin and syntaxin associated with N-type calcium channels in small cell lung cancer.

The presence of synaptic proteins involved in excitation/secretion coupling was examined in ten small cell lung cancer lines. N‐Type calcium channels (ω)‐conotoxin receptors), synaptotagmin (p65) and syntaxin (HPC‐1) were detected in eight. Co‐immunoprecipitation experiments indicated that syntaxin can form a complex with synaptotagmin and calcium channels. The expression of synaptotagmin in small cell lung cancer may elicit an autoimmune response that reduces transmitter release at the nerve terminal.

Antigens Associated with N‐ and L‐Type Calcium Channels in Lambert‐Eaton Myasthenic Syndrome

Abstract: In Lambert‐Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N‐type (125I‐ω conotoxin GVIA) and L‐type ([3H]PN200‐110) calcium channels. Sera with a high antibody titer (>3 nM) against rat brain N‐type channels contained autoantibodies that immunoprecipitated neuronal and muscle L‐type channels. These IgG fractions stained a 55‐kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the β subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65‐kDa protein that is unrelated to the β subunit and displays properties similar to those of synaptotagmin.

A substrate sensor chip to assay the enzymatic activity of Botulinum neurotoxin A.

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.

Direct biosensor detection of botulinum neurotoxin endopeptidase activity in sera from patients with type A botulism.

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.

Interaction of synaptotagmin with voltage gated calcium channels: A role in Lambert-Eaton myasthenic syndrome?

Plasma from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, contains antibodies that bind to the synaptic vesicle protein synaptotagmin. Synaptotagmin associates with calcium channels and appears to regulate synaptic vesicle docking at the plasma membrane prior to rapid neurotransmitter release. Autoantibodies directed against a synaptotagmin-calcium channel complex may be involved in the etiology of LEMS. In the majority of patients LEMS is associated with small cell lung cancer (SCLC). We have detected the expression of proteins of the secretory pathway, including synaptotagmin, syntaxin and N-type calcium channels, in a panel of SCLC tumor lines. These observations are compatible with the hypothesis that the initial autoimmune response in LEMS is triggered by the tumor.

SNARE, V-ATPase et neurotransmission

> La communication entre neurones repose principalement sur les synapses chimiques et par consequent sur la liberation de neurotransmetteurs. Au niveau des terminaisons nerveuses, des vesicules synaptiques remplies de neurotransmetteurs s’arriment a la membrane plasmique et subissent des etapes de maturation qui les rendent competentes pour la fusion avec la membrane plasmique. La liberation de neurotransmetteurs s’effectue par un processus d’exocytose dependant du calcium. Quand l’influx calcique declenche la machinerie de fusion membranaire, un pore de fusion connectant la lumiere des vesicules synaptiques a la fente synaptique s’ouvre et permet la liberation des neurotransmetteurs.

The Synaptic Vesicle V-ATPase: A Regulatory Link Between Loading and Fusion?

The vacuolar proton pump (V-ATPase) is a huge multi-subunit complex composed of two distinct non-covalently associated sectors. The cytosolic V1 sector hydrolyses ATP, providing the energy for the V0 membrane sector to translocate protons into the vesicle lumen. The proton gradient is then used by vesicular transporters to load synaptic vesicles with specific neurotransmitters. The primary role of the V-ATPase in vesicle loading is widely accepted. However, multiple studies in a variety of model organisms point to an additional general role of the V0 sector in downstream events, notably in regulating SNARE-mediated membrane fusion. This chapter outlines the molecular pharmacology of the V-ATPase and its role in the synaptic vesicle cycle. It then focuses specifically on molecular interactions between V0 subunits and synaptic vesicle trafficking proteins and reviews their relevance to late steps in neurotransmitter release. While this secondary role for the V-ATPase membrane sector is not yet fully established, we speculate that it could provide a regulatory link between vesicle filling and fusion, acting as a filter that allows loaded vesicles to engage the fusion machinery.