Bipartite Activation of Sensory Neurons by a TRPA1 Agonist Allyl Isothiocyanate Is Reflected by Complex Ca2+ Influx and CGRP Release Patterns: Enhancement by NGF and Inhibition with VAMP and SNAP-25 Cleaving Botulinum Neurotoxins.

The trafficking of transient receptor potential (TRP) channels to the plasma membrane and the release of calcitonin gene-related peptide (CGRP) from trigeminal ganglion neurons (TGNs) are implicated in some aspects of chronic migraines. These exocytotic processes are inhibited by cleavage of SNAREs with botulinum neurotoxins (BoNTs); moreover, type A toxin (/A) clinically reduces the frequency and severity of migraine attacks but not in all patients for unknown reasons. Herein, neonatal rat TGNs were stimulated with allyl isothiocyanate (AITC), a TRPA1 agonist, and dose relationships were established to link the resultant exocytosis of CGRP with Ca2+ influx. The CGRP release, quantified by ELISA, was best fit by a two-site model (EC50 of 6 and 93 µM) that correlates with elevations in intracellular Ca2+ [Ca2+]i revealed by time-lapse confocal microscopy of fluo-4-acetoxymethyl ester (Fluo-4 AM) loaded cells. These signals were all blocked by two TRPA1 antagonists, HC-030031 and A967079. At low [AITC], [Ca2+]i was limited because of desensitisation to the agonist but rose for concentrations > 0.1 mM due to a deduced non-desensitising second phase of Ca2+ influx. A recombinant BoNT chimera (/DA), which cleaves VAMP1/2/3, inhibited AITC-elicited CGRP release to a greater extent than SNAP-25-cleaving BoNT/A. /DA also proved more efficacious against CGRP efflux evoked by a TRPV1 agonist, capsaicin. Nerve growth factor (NGF), a pain-inducing sensitiser of TGNs, enhanced the CGRP exocytosis induced by low [AITC] only. Both toxins blocked NGF-induced neuropeptide secretion and its enhancement of the response to AITC. In conclusion, NGF sensitisation of sensory neurons involves TRPA1, elevated Ca2+ influx, and CGRP exocytosis, mediated by VAMP1/2/3 and SNAP-25 which can be attenuated by the BoNTs.

NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases.

Nerve growth factor (NGF) is known to intensify pain in various ways, so perturbing pertinent effects without negating its essential influences on neuronal functions could help the search for much-needed analgesics. Towards this goal, cultured neurons from neonatal rat trigeminal ganglia-a locus for craniofacial sensory nerves-were used to examine how NGF affects the Ca2+-dependent release of a pain mediator, calcitonin gene-related peptide (CGRP), that is triggered by activating a key signal transducer, transient receptor potential vanilloid 1 (TRPV1) with capsaicin (CAP). Measurements utilised neurons fed with or deprived of NGF for 2 days. Acute re-introduction of NGF induced Ca2+-dependent CGRP exocytosis that was inhibited by botulinum neurotoxin type A (BoNT/A) or a chimera of/E and/A (/EA), which truncated SNAP-25 (synaptosomal-associated protein with Mr = 25 k) at distinct sites. NGF additionally caused a Ca2+-independent enhancement of the neuropeptide release evoked by low concentrations (<100 nM) of CAP, but only marginally increased the peak response to ≥100 nM. Notably, BoNT/A inhibited CGRP exocytosis evoked by low but not high CAP concentrations, whereas/EA effectively reduced responses up to 1 µM CAP and inhibited to a greater extent its enhancement by NGF. In addition to establishing that sensitisation of sensory neurons to CAP by NGF is dependent on SNARE-mediated membrane fusion, insights were gleaned into the differential ability of two regions in the C-terminus of SNAP-25 (181-197 and 198-206) to support CAP-evoked Ca2+-dependent exocytosis at different intensities of stimulation.

Population Coding of Capsaicin Concentration by Sensory Neurons Revealed Using Ca2+ Imaging of Dorsal Root Ganglia Explants from Adult pirt-GCaMP3 Mouse.

BACKGROUND/AIMS: Nociceptors detect noxious capsaicin (CAPS) via the transient receptor potential vanilloid 1 (TRPV1) ion channel, but coding mechanisms for relaying CAPS concentration [CAPS] remain obscure. Prolonged (up to 1h.) exposure to CAPS is used clinically to desensitise sensory fibres for treatment of neuropathic pain, but its signalling has typically been studied in cultures of dissociated sensory neurons employing low cell numbers and very short exposure times. Thus, it was pertinent to examine responses to longer CAPS exposures in large populations of adult neurons. METHODS: Confocal fluorescence microscopy was used to monitor the simultaneous excitation by CAPS of neuronal populations in intact L3/4 dorsal root ganglia (DRG) explants from adult pirt-GCaMP3 mice that express a cytoplasmic, genetically-encoded Ca2+ sensor in almost all primary sensory neurons. Peak analysis was performed using GraphPad Prism 9 to deconstruct the heterogenous and complex fluorescence signals observed into informative, readily-comparable measurements: number of signals, their lag time, maximum intensity relative to baseline (Max.) and duration. RESULTS: Exposure for 5 min. to CAPS activated plasmalemmal TRPV1 and led to increased fluorescence due to Ca2+ entry into DRG neurons (DRGNs), as it was prevented by capsazepine or removal of extracellular Ca2+. Increasing [CAPS] (0.3, 1 and 10 µM, respectively) evoked signals from more neurons (123, 275 and 390 from 5 DRG) with shorter average lag (6.4 ± 0.4, 3.3 ± 0.2 and 1.9 ± 0.1 min.) and longer duration (1.4 ± 0.2, 2.9 ± 0.2 and 4.8 ± 0.3 min.). Whilst raising [CAPS] produced a modest augmentation of Max. for individual neurons, those with large increases were selectively expedited; this contributed to a faster onset and higher peak of cumulative fluorescence for an enlarged responding neuronal population. CAPS caused many cells to fluctuate between high and low levels of fluorescence, with consecutive pulses increasing Max. and duration especially when exposure was extended from 5 to 20 min. Such signal facilitation counteracted tachyphylaxis, observed upon repeated exposure to 1 µM CAPS, preserving the cumulative fluorescence over time (signal density) in the population. CONCLUSION: Individual neurons within DRG differed extensively in the dynamics of response to CAPS, but systematic changes elicited by elevating [CAPS] increased signal density in a graded manner, unveiling a possible mechanism for population coding of responses to noxious chemicals. Signal density is sustained during prolonged and repeated exposure to CAPS, despite profound tachyphylaxis in some neurons, by signal facilitation in others. This may explain the burning sensation that persists for several hours when CAPS is used clinically.

Ca2+ Signalling Induced by NGF Identifies a Subset of Capsaicin-Excitable Neurons Displaying Enhanced Chemo-Nociception in Dorsal Root Ganglion Explants from Adult pirt-GCaMP3 Mouse.

Nociceptors sense hazards via plasmalemmal cation channels, including transient receptor potential vanilloid 1 (TRPV1). Nerve growth factor (NGF) sensitises TRPV1 to capsaicin (CAPS), modulates nociceptor excitability and induces thermal hyperalgesia, but cellular mechanisms remain unclear. Confocal microscopy was used to image changes in intracellular Ca2+ concentration ([Ca2+]i) across neuronal populations in dorsal root ganglia (DRG) explants from pirt-GCaMP3 adult mice, which express a fluorescent reporter in their sensory neurons. Raised [Ca2+]i was detected in 84 neurons of three DRG explants exposed to NGF (100 ng/mL) and most (96%) of these were also excited by 1 µM CAPS. NGF elevated [Ca2+]i in about one-third of the neurons stimulated by 1 µM CAPS, whether applied before or after the latter. In neurons excitable by NGF, CAPS-evoked [Ca2+]i signals appeared significantly sooner (e.g., respective lags of 1.0 ± 0.1 and 1.9 ± 0.1 min), were much (>30%) brighter and lasted longer (6.6 ± 0.4 vs. 3.9 ± 0.2 min) relative to those non-responsive to the neurotrophin. CAPS tachyphylaxis lowered signal intensity by ~60% but was largely prevented by NGF. Increasing CAPS from 1 to 10 µM nearly doubled the number of cells activated but only modestly increased the amount co-activated by NGF. In conclusion, a sub-population of the CAPS-sensitive neurons in adult mouse DRG that can be excited by NGF is more sensitive to CAPS, responds with stronger signals and is further sensitised by transient exposure to the neurotrophin.

Synaptic vesicle cycle and amyloid ß: Biting the hand that feeds.

The synaptic vesicle cycle (SVC) holds center stage in the biology of presynaptic terminals. Through recurrent exocytosis and endocytosis, it facilitates a sequence of events enabling chemical neurotransmission between functionally related neurons. As a fundamental process that links the interior of nerve cells with their environment, the SVC is also critical for signaling and provides an entry route for a range of pathogens and toxins, enabling detrimental effects. In Alzheimer's disease, the SVC is both the prime site of amyloid ß production and toxicity. In this study, we discuss the emerging evidence for physiological and pathological effects of Aß on various stages of the SVC, from postfusion membrane recovery to trafficking, docking, and priming of vesicles for fusion and transmitter release. Understanding of the mechanisms of Aß interaction with the SVC within the unifying calcium hypothesis of aging and Alzheimer's disease should further elucidate the fundamental biology of the presynaptic terminal and reveal novel therapeutic targets for Alzheimer's disease and other age-related dementias.

Conjugate of an IgG Binding Domain with Botulinum Neurotoxin A Lacking the Acceptor Moiety Targets Its SNARE Protease into TrkA-Expressing Cells When Coupled to Anti-TrkA IgG or Fc-ßNGF.

Numerous naturally occurring toxins can perturb biological systems when they invade susceptible cells. Coupling of pertinent targeting ligands to the active domains of such proteins provides a strategy for directing these to particular cellular populations implicated in disease. A novel approach described herein involved fusion of one mutated immunoglobulin G (IgG) binding moiety of staphylococcal protein A to the SNARE protease and translocation domain of botulinum neurotoxin A (BoNT/A). This chimera could be monovalently coupled to IgG or via its Fc region to recombinant targeting ligands. The utility of the resulting conjugates is demonstrated by the delivery of a SNARE protease into a cell line expressing tropomyosin receptor kinase A (TrkA) through coupling to anti-TrkA IgG or a fusion of Fc and nerve-growth factor. Thus, this is a versitile and innovative technology for conjugating toxins to diverse ligands for retargeted cell delivery of potential therapeutics.

Targeted delivery of a SNARE protease to sensory neurons using a single chain antibody (scFv) against the extracellular domain of P2X(3) inhibits the release of a pain mediator.

P2X3 (P2X purinoceptor 3) is predominantly expressed on nociceptive sensory neurons and plays a crucial role in signalling leading to chronic inflammatory pain and some features of neuropathic pain. Thus it represents a potential target for pain therapeutics. BoNT/A (botulinum neurooxin type A) effectively relieves certain types of pain through inhibiting the neuronal release of pain peptides. A recombinant single-chain variable fragment (scFv) antibody designated MH7C was generated against the extracellular domain of P2X3 using phage display. The genes encoding the scFv and activated di-chain form of BoNT/A without the C-terminal-binding subdomain (LC-HN-HCN/A) were ligated and expressed in Escherichia coli cells as a composite fusion protein. The purified protein bound and entered P2X3-containing sensory neurons, cleaved synaptosomal-associated protein of 25 kDa and inhibited the release of a pain peptide. This novel fusion protein designated 'LC-HN-HCN/A-MH7C' has potential clinical applications in the treatment of chronic inflammatory and sympathetically maintained neuropathic pain.

Molecular components required for resting and stimulated endocytosis of botulinum neurotoxins by glutamatergic and peptidergic neurons.

Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synaptic vesicles (SCSVs) or large dense-core vesicles (LDCVs) are revealed herein, using probes that exploit surface-exposed vesicle proteins as acceptors for internalization. Basal uptake of botulinum neurotoxins (BoNTs) by both SCSV-releasing cerebellar granule neurons (CGNs) and LDCV-enriched trigeminal ganglionic neurons (TGNs) was found to require protein acceptors and acidic compartments. In addition, dynamin, clathrin, adaptor protein complex-2 (AP2), and amphiphysin contribute to the depolarization-evoked entry. For fast recycling of SCSVs, knockdown and knockout strategies demonstrated that CGNs use predominantly dynamin 1, whereas isoform 2 and, to a smaller extent, isoform 3 support a less rapid mode of stimulated endocytosis. Accordingly, proximity ligation assay confirmed that dynamin 1 and 2 colocalize with amphiphysin 1 in CGNs, and the latter copurified with both dynamins from cell extracts. In contrast, LDCV-releasing TGNs preferentially employ dynamins 2 and 3 and amphiphysin 1 for evoked endocytosis and lack the fast phase. Hence, stimulation recruits dynamin, clathrin, AP2, and amphiphysin to augment BoNT internalization, and neurons match endocytosis mediators to the different demands for locally recycling SCSVs or replenishing distally synthesized LDCVs.

Chapter 3: Molecular basis for the therapeutic effectiveness of botulinum neurotoxin type A.

The utility of botulinum neurotoxin type A (BoNT/A) for treating overactive muscles and endocrine glands is attributable to a unique conflation of properties honed to exploit and inactivate synaptic transmission. Specific, high-affinity coincident binding to gangliosides plus an intraluminal loop of synaptic vesicle protein 2 (SV2) by the heavy chain (HC) of BoNT/A confers selectivity for presynaptic nerve terminals and subsequent uptake by endocytosis. Upon vesicle acidification, the HC forms a channel for transmembrane transfer of the light chain to the cytosol, as observed by single channel recordings. The light chain is a Zn(2+) -dependent endoprotease that cleaves and inactivates SNAP-25, thereby blocking exocytotic release of transmitters, a discovery that revealed the pivotal role of the latter in synaptic vesicle fusion. A di-leucine motif in BoNT/A light chain stabilizes this protease, contributing to its longevity inside nerves. The ubiquity of SV2 and SNAP-25 has prompted re-evaluation of the nerve types susceptible to BoNT/A. In urology, there is emerging evidence that BoNT/A blocks neuropeptide release from afferent nerves, exocytosis of acetylcholine and purines from efferent nerves, and possibly ATP release from the urothelium. Suppression by BoNT/A of the surface expression of nociceptor channels on bladder afferents might also contribute to its improvement of urological sensory symptoms.

Pharmacological characteristics of Kv1.1- and Kv1.2-containing channels are influenced by the stoichiometry and positioning of their α subunits.

Voltage-sensitive neuronal Kv1 channels composed of four α subunits and four associated auxiliary ß subunits control neuronal excitability and neurotransmission. Limited information exists on the combinations of α subunit isoforms (i.e. Kv1.1-1.6) or their positions in the oligomers, and how these affect sensitivity to blockers. It is known that TEA (tetraethylammonium) inhibits Kv1.1 channels largely due to binding a critical tyrosine (Tyr379) in the pore, whereas Val381 at the equivalent location in Kv1.2 makes it insensitive. With the eventual aim of developing blockers for therapeutic purposes, Kv1.1 and 1.2 α subunit genes were concatenated to form combinations representing those in central neurons, followed by surface expression in HEK (human embryonic kidney)-293 cells as single-chain functional proteins. Patch-clamp recordings demonstrated the influences of the ratios and positioning of these α subunits on the biophysical and pharmacological properties of oligomeric K+ channels. Raising the ratio of Kv1.1 to Kv1.2 in Kv1.2-1.2-1.1-1.2 led to the resultant channels being more sensitive to TEA and also affected their biophysical parameters. Moreover, mutagenesis of one or more residues in the first Kv1.2 to resemble those in Kv1.1 increased TEA sensitivity only when it is adjacent to a Kv1.1 subunit, whereas placing a non-interactive subunit between these two diminished susceptibility. The findings of the present study support the possibility of α subunits being precisely arranged in Kv1 channels, rather than being randomly assembled. This is important in designing drugs with abilities to inhibit particular oligomeric Kv1 subtypes, with the goal of elevating neuronal excitability and improving neurotransmission in certain diseases.

Dendritic SNAREs add a new twist to the old neuron theory.

Dendritic exocytosis underpins a broad range of integrative and homeostatic synaptic functions. Emerging data highlight the essential role of SNAREs in trafficking and fusion of secretory organelles with release of peptides and neurotransmitters from dendrites. This Perspective analyzes recent evidence inferring axo-dendritic polarization of vesicular release machinery and pinpoints progress made with existing challenges in this rapidly progressing field of dendritic research. Interpreting the relation of new molecular data to physiological results on secretion from dendrites would greatly advance our understanding of this facet of neuronal mechanisms.

A defined heteromeric KV1 channel stabilizes the intrinsic pacemaking and regulates the output of deep cerebellar nuclear neurons to thalamic targets.

The output of the cerebellum to the motor axis of the central nervous system is orchestrated mainly by synaptic inputs and intrinsic pacemaker activity of deep cerebellar nuclear (DCN) projection neurons. Herein, we demonstrate that the soma of these cells is enriched with K(V)1 channels produced by mandatory multi-merization of K(V)1.1, 1.2 α and KV ß2 subunits. Being constitutively active, the K(+) current (IK(V)1) mediated by these channels stabilizes the rate and regulates the temporal precision of self-sustained firing of these neurons. Placed strategically, IK(V)1 provides a powerful counter-balance to prolonged depolarizing inputs, attenuates the rebound excitation, and dampens the membrane potential bi-stability. Somatic location with low activation threshold render IK(V)1 instrumental in voltage-dependent de-coupling of the axon initial segment from the cell body of projection neurons, impeding invasion of back-propagating action potentials into the somato-dendritic compartment. The latter is also demonstrated to secure the dominance of clock-like somatic pacemaking in driving the regenerative firing activity of these neurons, to encode time variant inputs with high fidelity. Through the use of multi-compartmental modelling and retro-axonal labelling, the physiological significance of the described functions for processing and communication of information from the lateral DCN to thalamic relay nuclei is established.

Novel chimeras of botulinum and tetanus neurotoxins yield insights into their distinct sites of neuroparalysis.

Botulinum neurotoxin (BoNT) A or E and tetanus toxin (TeTx) bind to motor-nerve endings and undergo distinct trafficking; their light-chain (LC) proteases cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) peripherally or centrally and cause flaccid or spastic paralysis, respectively. To seek protein domains responsible for local blockade of transmitter release (BoNTs) rather than retroaxonal transport to spinal neurons (TeTx), their acceptor-binding moieties (H(C))--or in one case, heavy chain (HC)--were exchanged by gene recombination. Each chimera, expressed and purified from Escherichia coli, entered rat cerebellar neurons to cleave their substrates, blocked in vitro nerve-induced muscle contractions, and produced only flaccid paralysis in mice. Thus, the local cytosolic delivery of BoNT/A or BoNT/E proteases and the contrasting retrograde transport of TeTx are not specified solely by their HC or H(C); BoNT/A LC translocated locally irrespective of being targeted by either of the latter TeTx domains. In contrast, BoNT/E protease fused to a TeTx enzymatically inactive mutant (TeTIM) caused spastic paralysis and cleaved SNAP-25 in spinal cord but not the injected muscle. Apparently, TeTIM precludes cytosolic release of BoNT/E protease at motor nerve endings. It is deduced that the LCs of the toxins, acting in conjunction with HC domains, dictate their local or distant destinations.

Improved lentiviral transduction of ALS motoneurons in vivo via dual targeting.

Treatment of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, is hampered by its complex etiology and lack of efficient means for targeted transfer of therapeutics into motoneurons. The objective of this research was engineering of a versatile motoneuron targeting adapter--a full-length atoxic tetanus toxin fused to core-streptavidin (CS-TeTIM)--for retro-axonal transduction of viral vectors; validation of the targeting efficiency of CS-TeTIM in vivo, by expression of green fluorescence protein (GFP) reporter in motoneurons of presymptomatic and symptomatic ALS-like SOD1(G93A) mice, and comparison with age-matched controls; and appraisal of lentiviral transduction with CS-TeTIM relative to (1) a HC binding fragment of tetanus toxin CS-TeTx(HC), (2) rabies glycoprotein (RG), and (3) a CS-TeTIM-RG dual targeting approach. CS-TeTIM and CS-TeTx(HC) were engineered using recombinant technology and site-directed mutagenesis. Biotinylated vectors, pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) or RG, were linked to these adaptors and injected intraperitoneally (ip) into presymptomatic (12 weeks old), symptomatic SOD1(G93A) (22 weeks old) or wild type control mice, followed by monitoring of GFP expression in the spinal cord and supraspinal motor structures with quantitative PCR and immuno-histochemistry. Transcripts were detected in the spinal cord and supraspinal motor structures of all mice 2 weeks after receiving a single ip injection, although in symptomatic SOD1(G93A) animals reporter RNA levels were lower compared to presymptomatic and wild-type controls irrespective of the targeting approach. GFP transduction with CS-TeTIM proved more efficient than CS-TeTx(HC) across all groups while CS-TeTIM-RG dual-targeted vectors yielded the highest transcript numbers. Importantly, in both wild-type and presymptomatic SOD1(G93A) mice strong colabeling of choline-acetyltransferase (ChAT) and GFP was visualized in neurons of the brain stem and spinal cord. CS-TeTIM, a versatile adaptor protein for targeted lentiviral transduction of motoneurons, has been engineered and its competence assessed relative to CS-TeTx(HC) and RG. Evidence has been provided that highlights the potential usefulness of this novel recombinant tool for basic research with implications for improved transfer of therapeutic candidates into motoneurons for the amelioration of ALS and related diseases.

Extravesicular intraneuronal migration of internalized botulinum neurotoxins without detectable inhibition of distal neurotransmission.

Intracellular protein transport routes can be studied using toxins that exploit these to enter cells. BoNTA (botulinum neurotoxin type A) is a protease that binds to peripheral nerve terminals, becomes endocytosed and causes prolonged blockade of transmitter release by cleaving SNAP-25 (synaptosome-associated protein of 25 kDa). Retrograde transport of the toxin has been suggested, but not of the transient muscle relaxant, BoNTE (botulinum neurotoxin type E). In the present study, dispersal of these proteases in compartmented cultures of rat sympathetic neurons was examined after focal application of BoNTA or BoNTE to neurites. A majority of cleaved SNAP-25 was seen locally, but some appeared along neurites and accumulated in the soma over several weeks. BoNTE yielded less cleaved SNAP-25 at distal sites due to shorter-lived enzymic activity. Neurite transection prevented movement of BoNTA. The BoNTA protease could be detected only in the supernatants of neurites or cell body lysates, hence these proteases must move along neuronal processes in the axoplasm or are reversibly associated with membranes. Substitution into BoNTE of the BoNTA acceptor-binding domain did not alter its potency or mobility. Spontaneous or evoked transmission to cell bodies were not inhibited by retrogradely migrated BoNTA except with high doses, concurring with the lack of evidence for a direct central action when used clinically.

Longer-acting and highly potent chimaeric inhibitors of excessive exocytosis created with domains from botulinum neurotoxin A and B.

Various human neurogenic hyper-excitability disorders are successfully treated with type A or B BoNT (botulinum neurotoxin). The BoNT/A complex is widely used because of its longer-lasting benefits; also, autonomic side-effects are more often reported for BoNT/B. To establish if this distinct effect of BoNT/B could be exploited therapeutically, BoNT/A was modified so that it would bind the more abundant BoNT/B acceptor in rodents while retaining its desirable persistent action. The advantageous protease and translocation domain of BoNT/A were recombinantly combined with the acceptor-binding moiety of type B [H(C)/B (C-terminal half of BoNT/B heavy chain)], creating the chimaera AB. This purified protein bound the BoNT/B acceptor, displayed enhanced capability relative to type A for intraneuronally delivering its protease, cleaved SNAP-25 (synaptosome-associated protein of 25 kDa) and induced a more prolonged neuromuscular paralysis than BoNT/A in mice. The BA chimaera, generated by substituting H(C)/A (C-terminal half of BoNT/A heavy chain) into BoNT/B, exhibited an extremely high specific activity, delivered the BoNT/B protease via the BoNT/A acceptor into neurons, or fibroblast-like synoviocytes that lack SNAP-25, cleaving the requisite isoforms of VAMP (vesicle-associated membrane protein). Both chimaeras inhibited neurotransmission in murine bladder smooth muscle. BA has the unique ability to reduce exocytosis from non-neuronal cells expressing the BoNT/A-acceptor and utilising VAMP, but not SNAP-25, in exocytosis.

Botulinum neurotoxins: Future innovations.

Botulinum neurotoxins (BoNTs) are multi-domain proteins whose potent and selective actions on nerve endings have led to innovations in both basic and clinical science. The various BoNT domains are responsible for binding to gangliosides and proteins associated with nerve cell membranes, internalization into the cell, and cleavage of one or more SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins necessary for vesicle docking and fusion. Novel modifications to BoNT molecules, such as the creation of chimeras, helped identify the protein domains responsible for various aspects of BoNT action, such as localized effects. Other molecular modifications have been introduced in attempts to increase the specificity of BoNTs for autonomic or sensory neurons, with the ultimate goal of optimizing therapeutic selectivity. This research, in turn, has led to the development of BoNT-based proteins that can target non-SNARE substrates such as phosphatase and tensin homolog (PTEN). Still others are developing different BoNT serotypes, subtypes, or variants that are longer- or shorter-acting or have faster onset for various clinical purposes. New formulations of BoNTs that provide convenience for both patients and physicians are under investigation. Novel clinical uses are being evaluated for onabotulinumtoxinA, including in the prevention of post-operative atrial fibrillation. All these innovations capitalize on the unique properties of BoNTs, which continue to intrigue scientists and clinicians across numerous fields of study.

A dileucine in the protease of botulinum toxin A underlies its long-lived neuroparalysis: transfer of longevity to a novel potential therapeutic.

Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNT(A)) underlies the severe neuroparalytic symptoms of human botulism, which can last a few years. The structural basis for this remarkable persistence remains unclear. Herein, recombinant BoNT(A) was found to match the neurotoxicity of that from Clostridium botulinum, producing persistent cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) and neuromuscular paralysis. When two leucines near the C terminus of the protease light chain of A (LC(A)) were mutated, its inhibition of exocytosis was followed by fast recovery of intact SNAP-25 in cerebellar neurons and neuromuscular transmission in vivo. Deletion of 6-7 N terminus residues diminished BoNT(A) activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LC(E) to a BoNT(A) enzymically inactive mutant (BoTIM(A)) yielded a novel LC(E)-BoTIM(A) protein that targets neurons, and the BoTIM(A) moiety also delivers and stabilizes the inhibitory LC(E), giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNT(E). LC(E-)BoTIM(A)(AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNT(E), reaffirming that these residues are critical for the persistent action of LC(E)-BoTIM(A) as well as BoNT(A). LC(E)-BoTIM(A) inhibited release of calcitonin gene-related peptide from sensory neurons mediated by transient receptor potential vanilloid type 1 and attenuated capsaicin-evoked nociceptive behavior in rats, following intraplantar injection. Thus, a long acting, versatile composite toxin has been developed with therapeutic potential for pain and conditions caused by overactive cholinergic nerves.

Intrinsic voltage dynamics govern the diversity of spontaneous firing profiles in basal forebrain noncholinergic neurons.

Spontaneous firing and behavior-related changes in discharge profiles of basal forebrain (BF) neurons are well documented, albeit the mechanisms underlying the variety of activity modes and intermodal transitions remain elusive. With the use of cell-attached recordings, this study identifies a range of spiking patterns in diagonal band Broca (DBB) noncholinergic cells of rats and tentatively categorizes them into low-rate random, tonic, and cluster firing activities. It demonstrates further that the multiplicity of discharge profiles is sustained intrinsically and persists after blockade of glutamate-, glycine/GABA-, and cholinergic synaptic inputs. Stimulation of muscarinic receptors, blockade of voltage-gated Ca(2+)-, and small conductance (SK) Ca(2+)-activated K(+) currents as well as chelating of intracellular Ca(2+) concentration accelerate low-rate random and tonic firing and favor transition of neurons into cluster firing mode. A similar trend towards higher discharge rates with switch of neurons into cluster firing has been revealed by activation of neuropeptide Y (NPY) receptors with the NPY or NPY(1) receptor agonist [Leu(31),Pro(34)]-NPY. Whole cell current-clamp analysis demonstrates that the variety of spiking modes and intermodal transitions could be induced within the same neuronal population by injection of bias depolarizing or hyperpolarizing currents. Taken together, these data demonstrate the intrinsic and highly variable character of regenerative firing in BF noncholinergic cells, subject to powerful modulation by classical neurotransmitters, NPY, and small membrane currents.

Position-dependent attenuation by Kv1.6 of N-type inactivation of Kv1.4-containing channels.

Assembly of distinct α subunits of Kv1 (voltage-gated K(+) channels) into tetramers underlies the diversity of their outward currents in neurons. Kv1.4-containing channels normally exhibit N-type rapid inactivation, mediated through an NIB (N-terminal inactivation ball); this can be over-ridden if associated with a Kv1.6 α subunit, via its NIP (N-type inactivation prevention) domain. Herein, NIP function was shown to require positioning of Kv1.6 adjacent to the Kv1.4 subunit. Using a recently devised gene concatenation, heterotetrameric Kv1 channels were expressed as single-chain proteins on the plasmalemma of HEK (human embryonic kidney)-293 cells, so their constituents could be arranged in different positions. Placing the Kv1.4 and 1.6 genes together, followed by two copies of Kv1.2, yielded a K(+) current devoid of fast inactivation. Mutation of critical glutamates within the NIP endowed rapid inactivation. Moreover, separating Kv1.4 and 1.6 with a copy of Kv1.2 gave a fast-inactivating K(+) current with steady-state inactivation shifted to more negative potentials and exhibiting slower recovery, correlating with similar inactivation kinetics seen for Kv1.4-(1.2)(3). Alternatively, separating Kv1.4 and 1.6 with two copies of Kv1.2 yielded slow-inactivating currents, because in this concatamer Kv1.4 and 1.6 should be together. These findings also confirm that the gene concatenation can generate K(+) channels with α subunits in pre-determined positions.