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.

Rescue of Normal Excitability in LGI1-Deficient Epileptic Neurons.

Leucine-rich glioma inactivated 1 (LGI1) is a glycoprotein secreted by neurons, the deletion of which leads to autosomal dominant lateral temporal lobe epilepsy. We previously showed that LGI1 deficiency in a mouse model (i.e., knock-out for LGI1 or KO-Lgi1) decreased Kv1.1 channel density at the axon initial segment (AIS) and at presynaptic terminals, thus enhancing both intrinsic excitability and glutamate release. However, it is not known whether normal excitability can be restored in epileptic neurons. Here, we show that the selective expression of LGI1 in KO-Lgi1 neurons from mice of both sexes, using single-cell electroporation, reduces intrinsic excitability and restores both the Kv1.1-mediated D-type current and Kv1.1 channels at the AIS. In addition, we show that the homeostatic-like shortening of the AIS length observed in KO-Lgi1 neurons is prevented in neurons electroporated with the Lgi1 gene. Furthermore, we reveal a spatial gradient of intrinsic excitability that is centered on the electroporated neuron. We conclude that expression of LGI1 restores normal excitability through functional Kv1 channels at the AIS.SIGNIFICANCE STATEMENT The lack of leucine-rich glioma inactivated 1 (LGI1) protein induces severe epileptic seizures that leads to death. Enhanced intrinsic and synaptic excitation in KO-Lgi1 mice is because of the decrease in Kv1.1 channels in CA3 neurons. However, the conditions to restore normal excitability profile in epileptic neurons remain to be defined. We show here that the expression of LGI1 in KO-Lgi1 neurons in single neurons reduces intrinsic excitability, and restores both the Kv1.1-mediated D-type current and Kv1.1 channels at the axon initial segment (AIS). Furthermore, the homeostatic shortening of the AIS length observed in KO-Lgi1 neurons is prevented in neurons in which the Lgi1 gene has been rescued. We conclude that LGI1 constitutes a critical factor to restore normal excitability in epileptic neurons.

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.

An Epitope-Specific LGI1-Autoantibody Enhances Neuronal Excitability by Modulating Kv1.1 Channel.

Leucine-rich Glioma-Inactivated protein 1 (LGI1) is expressed in the central nervous system and its genetic loss of function is associated with epileptic disorders. Additionally, patients with LGI1-directed autoantibodies have frequent focal seizures as a key feature of their disease. LGI1 is composed of a Leucine-Rich Repeat (LRR) and an Epitempin (EPTP) domain. These domains are reported to interact with different members of the transsynaptic complex formed by LGI1 at excitatory synapses, including presynaptic Kv1 potassium channels. Patient-derived recombinant monoclonal antibodies (mAbs) are ideal reagents to study whether domain-specific LGI1-autoantibodies induce epileptiform activities in neurons and their downstream mechanisms. We measured the intrinsic excitability of CA3 pyramidal neurons in organotypic cultures from rat hippocampus treated with either an LRR- or an EPTP-reactive patient-derived mAb, or with IgG from control patients. We found an increase in intrinsic excitability correlated with a reduction of the sensitivity to a selective Kv1.1-channel blocker in neurons treated with the LRR mAb, but not in neurons treated with the EPTP mAb. Our findings suggest LRR mAbs are able to modulate neuronal excitability that could account for epileptiform activity observed in patients.

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.

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.

Distinctive binding properties of human monoclonal LGI1 autoantibodies determine pathogenic mechanisms.

Autoantibodies against leucine-rich glioma inactivated 1 (LGI1) are found in patients with limbic encephalitis and focal seizures. Here, we generate patient-derived monoclonal antibodies (mAbs) against LGI1. We explore their sequences and binding characteristics, plus their pathogenic potential using transfected HEK293T cells, rodent neuronal preparations, and behavioural and electrophysiological assessments in vivo after mAb injections into the rodent hippocampus. In live cell-based assays, LGI1 epitope recognition was examined with patient sera (n = 31), CSFs (n = 11), longitudinal serum samples (n = 15), and using mAbs (n = 14) generated from peripheral B cells of two patients. All sera and 9/11 CSFs bound both the leucine-rich repeat (LRR) and the epitempin repeat (EPTP) domains of LGI1, with stable ratios of LRR:EPTP antibody levels over time. By contrast, the mAbs derived from both patients recognized either the LRR or EPTP domain. mAbs against both domain specificities showed varied binding strengths, and marked genetic heterogeneity, with high mutation frequencies. LRR-specific mAbs recognized LGI1 docked to its interaction partners, ADAM22 and ADAM23, bound to rodent brain sections, and induced internalization of the LGI1-ADAM22/23 complex in both HEK293T cells and live hippocampal neurons. By contrast, few EPTP-specific mAbs bound to rodent brain sections or ADAM22/23-docked LGI1, but all inhibited the docking of LGI1 to ADAM22/23. After intrahippocampal injection, and by contrast to the LRR-directed mAbs, the EPTP-directed mAbs showed far less avid binding to brain tissue and were consistently detected in the serum. Post-injection, both domain-specific mAbs abrogated long-term potentiation induction, and LRR-directed antibodies with higher binding strengths induced memory impairment. Taken together, two largely dichotomous populations of LGI1 mAbs with distinct domain binding characteristics exist in the affinity matured peripheral autoantigen-specific memory pools of individuals, both of which have pathogenic potential. In human autoantibody-mediated diseases, the detailed characterization of patient mAbs provides a valuable method to dissect the molecular mechanisms within polyclonal populations.

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.

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.

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.

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.

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.

Optimization of neuronal cultures from rat superior cervical ganglia for dual patch recording.

Superior cervical ganglion neurons (SCGN) are often used to investigate neurotransmitter release mechanisms. In this study, we optimized the dissociation and culture conditions of rat SCGN cultures for dual patch clamp recordings. Two weeks in vitro are sufficient to achieve a significant CNTF-induced cholinergic switch and to develop mature and healthy neuronal profiles suited for detailed patch clamp analysis. One single pup provides sufficient material to prepare what was formerly obtained from 12 to 15 animals. The suitability of these cultures to study neurotransmitter release mechanisms was validated by presynaptically perturbing the interaction of the v-SNARE VAMP2 with the vesicular V-ATPase V0c subunit.

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.

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.

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.

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.

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.