The downregulation of Kv1 channels in Lgi1-/-mice is accompanied by a profound modification of its interactome and a parallel decrease in Kv2 channels.

In animal models of LGI1-dependent autosomal dominant lateral temporal lobe epilepsy, Kv1 channels are downregulated, suggesting their crucial involvement in epileptogenesis. The molecular basis of Kv1 channel-downregulation in LGI1 knock-out mice has not been elucidated and how the absence of this extracellular protein induces an important modification in the expression of Kv1 remains unknown. In this study we analyse by immunofluorescence the modifications in neuronal Kv1.1 and Kv1.2 distribution throughout the hippocampal formation of LGI1 knock-out mice. We show that Kv1 downregulation is not restricted to the axonal compartment, but also takes place in the somatodendritic region and is accompanied by a drastic decrease in Kv2 expression levels. Moreover, we find that the downregulation of these Kv channels is associated with a marked increase in bursting patterns. Finally, mass spectrometry uncovered key modifications in the Kv1 interactome that highlight the epileptogenic implication of Kv1 downregulation in LGI1 knock-out animals.

Patient-derived antibodies reveal the subcellular distribution and heterogeneous interactome of LGI1.

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.

Molecular landscape of BoNT/B bound to a membrane-inserted synaptotagmin/ganglioside complex.

Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of BoNT/B bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagmin-ganglioside GT1b complex and a free ganglioside allowing a lipid-binding loop of BoNT/B to interact with the glycone part of the synaptotagmin-associated GT1b. Furthermore, our data provide molecular support for the decrease in BoNT/B sensitivity in Felidae that harbor the natural variant synaptotagmin2-N59Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex.

Gangliosides interact with synaptotagmin to form the high-affinity receptor complex for botulinum neurotoxin B.

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.

LGI1 tunes intrinsic excitability by regulating the density of axonal Kv1 channels.

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.

A chip-based assay for botulinum neurotoxin A activity in pharmaceutical preparations.

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.

A fast, single-vesicle fusion assay mimics physiological SNARE requirements.

Almost all known intracellular fusion reactions are driven by formation of trans-SNARE complexes through pairing of vesicle-associated v-SNAREs with complementary t-SNAREs on target membranes. However, the number of SNARE complexes required for fusion is unknown, and there is controversy about whether additional proteins are required to explain the fast fusion which can occur in cells. Here we show that single vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNAREs syntaxin-SNAP25. Fusion rates decreased dramatically when the number of externally oriented v-SNAREs per vesicle was reduced below 5-10, directly establishing this as the minimum number required for rapid fusion. Docking-to-fusion delay time distributions were consistent with a requirement that 5-11 t-SNAREs be recruited to achieve fusion, closely matching the v-SNARE requirement.

A Role for the V0 Sector of the V-ATPase in Neuroexocytosis: Exogenous V0d Blocks Complexin and SNARE Interactions with V0c.

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.

Calcium-dependent regulation of SNARE-mediated membrane fusion by calmodulin.

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.

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

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors)-mediated exocytotic release of neurotransmitters is a key process in neuronal communication, controlled by a number of molecular interactions. A synaptic vesicle v-SNARE protein (VAMP2 or synaptobrevin), in association with two plasma membrane t-SNAREs (syntaxin 1 and SNAP25), assemble to form a protein complex that is largely accepted as the minimal membrane fusion machine. Acidification of the synaptic vesicle lumen by the large multi-subunit vacuolar proton pump (V-ATPase) is required for loading with neurotransmitters. Recent data demonstrate a direct interaction between the c-subunit of the V-ATPase and VAMP2 that appears to play a role at a late step in transmitter release. In this review, we examine evidence suggesting that the V0 membrane sector of the V-ATPase not only participates in proton pumping, but plays a second distinct role in neurosecretion, downstream of filling and close to vesicle fusion.

A label-free biosensor assay for botulinum neurotoxin B in food and human serum.

Botulinum neurotoxins (BoNTs) are among the most toxic substances known. Surveillance and diagnostics require methods for rapid detection of BoNTs in complex media such as foodstuffs and human serum. We have developed in vitro assays to specifically detect the protease activity of botulinum neurotoxin B (BoNT/B) on a time scale of minutes. Cleavage of the BoNT/B substrate VAMP2, a membrane SNARE protein associated with synaptic vesicles, was monitored using real-time surface plasmon resonance to measure vesicle capture by specific antibodies coupled to microchips. The assay is functional in low-ionic-strength buffers and stable over a wide range of pH values (5.5-9.0). Endoproteolytic cleavage of VAMP2 was detected in 10 min with 2 pM native BoNT/B holotoxin. Contamination of liquid food products such as carrot juice, apple juice, and milk with low picomolar amounts of BoNT/B was revealed within 3h. BoNT/B activity was detected in sera from patients with type B botulism but not in healthy controls or patients with other neurological diseases. This robust, sensitive, and rapid protein chip assay is appropriate for monitoring BoNT/B in food products and diagnostic tests for type B botulism and could replace the current in vivo mouse bioassay.

Toxicity and endocytosis of spinocerebellar ataxia type 6 polyglutamine domains: role of myosin IIb.

Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited neurodegenerative disease caused by a small expansion of CAG repeats in the sequence coding for the cytoplasmic C-terminal region of the Ca(v)2.1 subunit of P/Q-type calcium channels. We have tested the toxicity of mutated Ca(v)2.1 C-terminal domains expressed in the plasma membrane. In COS-7 cells, CD4-green fluorescent protein fused to Ca(v)2.1 C-terminal domains containing expanded 24 polyglutamine (Q) tracts displayed increased toxicity and stronger expression at the cell surface relative to 'normal' 12 Q tracts, partially because of reduced endocytosis. Glutathione S-transferase pull-down and proteomic analysis indicated that Ca(v)2.1 C-termini interact with the heavy and light chains of cerebellar myosin IIB, a molecular motor protein. This interaction was confirmed by coimmunoprecipitation from rat cerebellum and COS-7 cells and shown to be direct by binding of in vitro-translated (35)S-myosin IIB heavy chain. In COS-7 cells, incremented polyglutamine tract length increased the interaction with myosin IIB. Furthermore, the myosin II inhibitor blebbistatin reversed the effects of polyglutamine expansion on plasma membrane expression. Our findings suggest a key role of myosin IIB in promoting accumulation of mutant Ca(v)2.1Ct at the plasma membrane and suggest that this gain of function might contribute to the pathogenesis of SCA6.

Chromophore-Assisted Light Inactivation of the V-ATPase V0c Subunit Inhibits Neurotransmitter Release Downstream of Synaptic Vesicle Acidification.

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.

Nuclear localization of a novel human syntaxin 1B isoform.

The syntaxins are proteins associated with various intracellular membrane compartments. They are major participants in a large variety of physiological processes where membrane fusion occurs, including exocytosis. We have identified a novel syntaxin isoform generated by alternative splicing of the human STX1B gene. In contrast with the canonical syntaxins, this isoform (STX1B-DeltaTMD) lacked the classical C-terminal transmembrane domain and localized to the nucleus of various tumoral and non-tumoral cell types including human brain cortical neurons in vivo. The reversible blockade of STX1B-DeltaTMD nuclear import demonstrated that nuclear import occurred via a Ran-dependent pathway. A specific and glycine-rich C-terminus of 15 amino acids served as an unconventional nuclear localization signal. STX1B-DeltaTMD colocalized with Lamin A/C and NuMA (NUclear Mitotic Apparatus protein) in interphasic nuclei, and with NuMA and gamma-tubulin in the pericentrosomal region of the mitotic spindle in dividing cells. In a series of 37 human primary brain tumors, the ratio of STX1B-DeltaTMD to Lamin A/C transcripts was a significant prognostic marker of survival, independent of tumor staging. The characterization of STX1B-DeltaTMD as the first nucleoplasmic syntaxin with no transmembrane domain, illustrates the importance of alternative splicing in the emergence of unsuspected properties of the syntaxins in human cells, in both physiological and pathological conditions.

Distinct evolution of calcium channel antibody types in Lambert-Eaton myasthenic syndrome.

To determine whether titers of anti-P/Q type and anti-N type calcium channel antibodies provide distinct information, both types of assay were performed during follow-up of 7 patients with Lambert-Eaton myasthenic syndrome (LEMS). In 4 patients with both antibody responses, titers evolved independently and often in an inverse relationship. Two patients with squamous cell lung carcinoma (SqCLC) produced anti-N type channel antibodies, but no detectable anti-P/Q channel responses. These results suggest that anti-N channel autoantibodies constitute an immune response distinct from the anti-P/Q type channel specificity and can also correlate with clinical evolution. Consequently combined assays may provide more comprehensive information during follow-up of LEMS.

A protein chip membrane-capture assay for botulinum neurotoxin activity.

Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC(50)s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC(50) of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.

Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor.

The development of simple molecular assays with membrane protein receptors in a native conformation still represents a challenging task. Exosomes are extracellular vesicles which, due to their stability and small size, are suited for analysis in various assay formats. Here, we describe a novel approach to sort recombinant fully native and functional membrane proteins to exosomes using a targeting peptide. Specific binding of high affinity ligands to the potassium channel Kv1.2, the G-protein coupled receptor CXCR4, and the botulinum neurotoxin type B (BoNT/B) receptor, indicated their correct assembly and outside out orientation in exosomes. We then developed, using a label-free optical biosensor, a new method to determine the kinetic constants of BoNT/B holotoxin binding to its receptor synaptotagmin2/GT1b ganglioside (kon = 2.3 ×105 M-1.s-1, koff = 1.3 10-4 s-1), yielding an affinity constant (KD = 0.6 nM) similar to values determined from native tissue. In addition, the recombinant binding domain of BoNT/B, a potential vector for neuronal delivery, bound quasi-irreversibly to synaptotagmin 2/GT1b exosomes. Engineered exosomes provide thus a novel means to study membrane proteins for biotechnology and clinical applications.

Synaptic vesicle chips to assay botulinum neurotoxins.

BoNTs (botulinum neurotoxins), considered to be the most toxic of all biological substances, inhibit neurotransmission through proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins [VAMP (vesicle-associated membrane protein, or synaptobrevin), SNAP-25 (25 kDa synaptosome-associated protein) or syntaxin]. Expansion in the use of BoNTs as therapeutic and cosmetic agents, and the potential threat they constitute as biological weapons, underlines the need for rapid and sensitive in vitro assays. Here, we present new automatized bioassays to detect VAMP cleavage by BoNT/B and F. Western blotting and SPR (surface plasmon resonance) methods revealed that BoNT/B and F totally cleave their substrate on immunoisolated SVs (synaptic vesicles). Real-time monitoring of the immunocapture of native SVs from crude lysates on SPR sensor chips enabled the detection of picogram amounts of different SV proteins. Pre-incubation of a membrane fraction containing SVs with BoNT specifically inhibited capture by anti-VAMP antibodies, and amounts as low as 0.1 pg of BoNT/B were detected. This automated SPR assay is approx. 200 times more sensitive, and 25 times more rapid, than the in vivo BoNT/B test currently used. Moreover, the method can be performed using a few thousand cultured neurons and constitutes a new screening assay for inhibitors. Our data indicate that native VAMP is an optimal substrate for in vitro BoNT assays that can be monitored by SPR.

An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E.

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.