Silencing neurotransmission with membrane-tethered toxins.

At synaptic terminals, high voltage-activated Ca(v)2.1 and Ca(v)2.2 calcium channels have an essential and joint role in coupling the presynaptic action potential to neurotransmitter release. Here we show that membrane-tethered toxins allowed cell-autonomous blockade of each channel individually or simultaneously in mouse neurons in vivo. We report optimized constitutive, inducible and Cre recombinase-dependent lentiviral vectors encoding fluorescent recombinant toxins, and we also validated the toxin-based strategy in a transgenic mouse model. Toxins delivered by lentiviral vectors selectively inhibited the dopaminergic nigrostriatal pathway, and transgenic mice with targeted expression in nociceptive peripheral neurons displayed long-lasting suppression of chronic pain. Optimized tethered toxins are tools for cell-specific and temporal manipulation of ion channel-mediated activities in vivo, including blockade of neurotransmitter release.

Cross-reactivity of acid-sensing ion channel and Na⁺-H⁺ exchanger antagonists with nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the mammalian central and peripheral nervous systems, where they contribute to neuronal excitability and synaptic communication. It has been reported that nAChRs are modulated by BK channels and that BK channels, in turn, are inhibited by acid-sensing ion channels (ASICs). Here we investigate the possible functional interaction between these channels in medial habenula (MHb) neurones. We report that selective antagonists of large-conductance calcium-activated potassium channels and ASIC1a channels, paxilline and psalmotoxin 1, respectively, did not induce detectable changes in nicotine-evoked currents. In contrast, the non-selective ASIC and Na(+)-H(+) exchanger (NHE1) antagonists, amiloride and its analogues, suppressed nicotine-evoked responses in MHb neurones of wild-type and ASIC2 null mice, excluding a possible involvement of ASIC2 in the nAChR inhibition by amiloride. Zoniporide, a more selective inhibitor of NHE1, reversibly inhibited α3ß4-, α7- and α4-containing (*) nAChRs in Xenopus oocytes and in brain slices, as well as in PS120 cells deficient in NHE1 and virally transduced with nAChRs, suggesting a generalized effect of zoniporide in most neuronal nAChR subtypes. Independently from nAChR antagonism, zoniporide profoundly blocked synaptic transmission onto MHb neurones without affecting glutamatergic and GABA receptors. Taken together, these results indicate that amiloride and zoniporide, which are clinically used to treat hypertension and cardiovascular disease, have an inhibitory effect on neuronal nAChRs when used experimentally at high doses. The possible cross-reactivity of these compounds with nAChRs in vivo will require further investigation.

An in vivo tethered toxin approach for the cell-autonomous inactivation of voltage-gated sodium channel currents in nociceptors.

Understanding information flow in sensory pathways requires cell-selective approaches to manipulate the activity of defined neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in specific voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 +/- 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inflammatory mechanical hypersensitivity, cold pain insensitivity and reduced firing of cutaneous C-fibres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in specific cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of specific ionic currents to neuronal function and to behaviour.

Progress toward a universal H5N1 vaccine: a recombinant modified vaccinia virus Ankara-expressing trivalent hemagglutinin vaccine.

BACKGROUND: The rapid evolution of new sublineages of H5N1 influenza poses the greatest challenge in control of H5N1 infection by currently existing vaccines. To overcome this, an MVAtor vector expressing three H5HA antigens A/Vietnam/1203/04, A/Indonesia/669/06 and A/Anhui/01/05 (MVAtor-tri-HA vector) was developed to elicit broad cross-protection against diverse clades by covering amino acid variations in the major neutralizing epitopes of HA among H5N1 subtypes. METHODS: BALB/c mice and guinea pigs were immunized i.m. with 8×107 TCID50/animal of MVAtor-tri-HA vector. The immunogenicity and cross-protective immunity of the MVAtor-tri-HA vector was evaluated against diverse clades of H5N1 strains. RESULTS: The results showed that mice immunized with MVAtor-tri-HA vector induced robust cross-neutralizing immunity to diverse H5N1 clades. In addition, the MVAtor-tri-HA vector completely protected against 10 MLD50 of a divergent clade of H5N1 infection (clade 7). Importantly, the serological surveillance of post-vaccinated guinea pig sera demonstrated that MVAtor-tri-HA vector was able to elicit strong cross-clade neutralizing immunity against twenty different H5N1 strains from six clades that emerged between 1997 and 2012. CONCLUSIONS: The present findings revealed that incorporation of carefully selected HA genes from divergent H5N1 strains within a single vector could be an effective approach in developing a vaccine with broad coverage to prevent infection during a pandemic situation.

Aversion to nicotine is regulated by the balanced activity of ß4 and α5 nicotinic receptor subunits in the medial habenula.

Nicotine dependence is linked to single nucleotide polymorphisms in the CHRNB4-CHRNA3-CHRNA5 gene cluster encoding the α3ß4α5 nicotinic acetylcholine receptor (nAChR). Here we show that the ß4 subunit is rate limiting for receptor activity, and that current increase by ß4 is maximally competed by one of the most frequent variants associated with tobacco usage (D398N in α5). We identify a ß4-specific residue (S435), mapping to the intracellular vestibule of the α3ß4α5 receptor in close proximity to α5 D398N, that is essential for its ability to increase currents. Transgenic mice with targeted overexpression of Chrnb4 to endogenous sites display a strong aversion to nicotine that can be reversed by viral-mediated expression of the α5 D398N variant in the medial habenula (MHb). Thus, this study both provides insights into α3ß4α5 receptor-mediated mechanisms contributing to nicotine consumption, and identifies the MHb as a critical element in the circuitry controlling nicotine-dependent phenotypes.

"The King is dead": Checkmating ion channels with tethered toxins.

The quickest possible checkmate in the game of chess requires two moves using a pawn and the queen. Metaphorically speaking, the pawn (a membrane tether) and the queen (a toxin) work together to checkmate an ion channel within a neuronal circuit. This strategy termed "tethered toxin" (t-toxin) is based on the use of genetically encoded peptide toxins that are anchored to the cell-membrane via a glycolipid or transmembrane tether. Because of their mode of action at the cell surface, t-toxins act only on ion channels and receptors of the cell that is expressing the t-toxin, and not on identical receptors present in neighboring cells that do not express the t-toxin. In this mini-review we discuss the design of these genetic tools and their application for cell-specific and temporal manipulation of ion channel-mediated activities in vivo.

Manipulating neuronal circuits with endogenous and recombinant cell-surface tethered modulators.

Neuronal circuits depend on the precise regulation of cell-surface receptors and ion channels. An ongoing challenge in neuroscience research is deciphering the functional contribution of specific receptors and ion channels using engineered modulators. A novel strategy, termed "tethered toxins", was recently developed to characterize neuronal circuits using the evolutionary derived selectivity of venom peptide toxins and endogenous peptide ligands, such as lynx1 prototoxins. Herein, the discovery and engineering of cell-surface tethered peptides is reviewed, with particular attention given to their cell-autonomy, modular composition, and genetic targeting in different model organisms. The relative ease with which tethered peptides can be engineered, coupled with the increasing number of neuroactive venom toxins and ligand peptides being discovered, imply a multitude of potentially innovative applications for manipulating neuronal circuits and tissue-specific cell networks, including treatment of disorders caused by malfunction of receptors and ion channels.