James Oliver Dolly

TNFα induces co-trafficking of TRPV1/TRPA1 in VAMP1-containing vesicles to the plasmalemma via Munc18–1/syntaxin1/SNAP-25 mediated fusion

Transient receptor potential (TRP) A1 and V1 channels relay sensory signals, yet little is known about their transport to the plasmalemma during inflammation. Herein, TRPA1 and TRPV1 were found on vesicles containing calcitonin gene-related peptide (CGRP), accumulated at sites of exo- and endo-cytosis, and co-localised on fibres and cell bodies of cultured sensory neurons expressing both. A proinflammatory cytokine, TNFα, elevated their surface content, and both resided in close proximity, indicating co-trafficking. Syntaxin 1–interacting protein, Munc18–1, proved necessary for the response to TNFα, and for TRPV1-triggered CGRP release. TNFα-induced surface trafficking of TRPV1 and TRPA1 required a synaptic vesicle membrane protein VAMP1 (but not 2/3), which is essential for CGRP exocytosis from large dense-core vesicles. Inactivation of two proteins on the presynaptic plasma membrane, syntaxin-1 or SNAP-25, by botulinum neurotoxin (BoNT)/C1 or /A inhibited the TNFα-elevated delivery. Accordingly, enhancement by TNFα of Ca2+ influx through the upregulated surface-expressed TRPV1 and TRPA1 channels was abolished by BoNT/A. Thus, in addition, the neurotoxins’ known inhibition of the release of pain transmitters, their therapeutic potential is augmented by lowering the exocytotic delivery of transducing channels and the resultant hyper-sensitisation in inflammation.

SNAP-23 and VAMP-3 contribute to the release of IL-6 and TNFα from a human synovial sarcoma cell line

Fibroblast‐like synoviocytes are important mediators of inflammatory joint damage in arthritis through the release of cytokines, but it is unknown whether their exocytosis from these particular cells is SNARE‐dependent. Here, the complement of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) in human synovial sarcoma cells (SW982) was examined with respect to the secretion of interleukin‐6 (IL‐6) and tumour necrosis factor α (TNFα), before and after knockdown of a synaptosome‐associated protein of molecular mass 23 kDa (SNAP‐23) or the vesicle‐associated membrane protein 3 (VAMP‐3). Wild‐type SW982 cells expressed SNAP‐23, VAMP‐3, syntaxin isoforms 2–4 and synaptic vesicle protein 2C (SV2C). These cells showed Ca2+‐dependent secretion of IL‐6 and TNFα when stimulated by interleukin‐1β (IL‐1β) or in combination with K+ depolarization. Specific knockdown of SNAP‐23 or VAMP‐3 decreased the exocytosis of IL‐6 and TNFα; the reduced expression of SNAP‐23 caused accumulation of SV2 in the peri‐nuclear area. A monoclonal antibody specific for VAMP‐3 precipitated SNAP‐23 and syntaxin‐2 (and syntaxin‐3 to a lesser extent). The formation of SDS‐resistant complexes by SNAP‐23 and VAMP‐3 was reduced upon knockdown of SNAP‐23. Although the syntaxin isoforms 2, 3 and 4 are expressed in SW982 cells, knockdown of each did not affect the release of cytokines. Collectively, these results show that SNAP‐23 and VAMP‐3 participate in IL‐1β‐induced Ca2+‐dependent release of IL‐6 and TNFα from SW982 cells.

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.

Low-Affinity Neurotrophin Receptor p75 Promotes the Transduction of Targeted Lentiviral Vectors to Cholinergic Neurons of Rat Basal Forebrain

Basal forebrain cholinergic neurons (BFCNs) are one of the most affected neuronal types in Alzheimer’s disease (AD), with their extensive loss documented at late stages of the pathology. While discriminatory provision of neuroprotective agents and trophic factors to these cells is thought to be of substantial therapeutic potential, the intricate topography and structure of the forebrain cholinergic system imposes a major challenge. To overcome this, we took advantage of the physiological enrichment of BFCNs with a low-affinity p75 neurotrophin receptor (p75NTR) for their targeting by lentiviral vectors within the intact brain of adult rat. Herein, a method is described that affords selective and effective transduction of BFCNs with a green fluorescence protein (GFP) reporter, which combines streptavidin–biotin technology with anti-p75NTR antibody-coated lentiviral vectors. Specific GFP expression in cholinergic neurons was attained in the medial septum and nuclei of the diagonal band Broca after a single intraventricular administration of such targeted vectors. Bioelectrical activity of GFP-labeled neurons was proven to be unchanged. Thus, proof of principle is obtained for the utility of the low-affinity p75NTR for targeted transduction of vectors to BFCNs in vivo.

Neurobiology and therapeutic applications of neurotoxins targeting transmitter release

Synaptic transmission is a fundamental neurobiological process enabling exchange of signals between neurons as well as neurons and their non-neuronal effectors. The complex molecular machinery of the synaptic vesicle cycle and transmitter release has emerged and developed in the course of the evolutionary race, to ensure adaptive gain and survival of the fittest. In parallel, a generous arsenal of biomolecules and neuroactive peptides have co-evolved, which selectively target the transmitter release machinery, with the aim of subduing natural rivals or neutralizing prey. With advances in neuropharmacology and quantitative biology, neurotoxins targeting presynaptic mechanisms have attracted major interest, revealing considerable potential as carriers of molecular cargo and probes for meddling synaptic transmission mechanisms for research and medical benefit. In this review, we investigate and discuss key facets employed by the most prominent bacterial and animal toxins targeting the presynaptic secretory machinery. We explore the cellular basis and molecular grounds for their tremendous potency and selectivity, with effects on a wide range of neural functions. Finally, we consider the emerging preclinical and clinical data advocating the use of active ingredients of neurotoxins for the advancement of molecular medicine and development of restorative therapies.

Pharmacology of Botulinum Neurotoxins: Exploitation of Their Multifunctional Activities as Transmitter Release Inhibitors and Neuron-Targeted Delivery Vehicles

Quantal transmitter release from nerves is inhibited by all seven serotypes (A–G) of botulinum neurotoxin (BoNT), with some subtle but functional differences. Commonalities and dissimilarities in these proteins, and new recombinant forms, are highlighted in terms of their multiple activities and domains responsible for binding to the neuronal acceptors, subsequent endocytosis, translocation and proteolytic inactivation of intracellular soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) culminating in the blockade of neuro-exocytosis lasting for weeks or months. The neurotoxins bind to dual acceptors, gangliosides and intra-lumenal regions of vesicular proteins, and co-traffic into neurons. Subsequently, their proteases pass to the cytosol via a channel created in the endosomal limiting membrane and cleave distinct bonds in the substrate SNARE(s). Modification of these targets is responsible for their characteristic pharmacological activities. The prolonged duration of type A seems attributable to an identified stabilisation motif that extends the longevity of its protease. BoNTs have proved instrumental in deciphering a molecular basis for regulated exocytosis; now, emerging knowledge is helping to explain why synchronisation of released quanta of transmitter is perturbed by certain serotypes (/B, /D and /F) and not others (/A, /C1 and /E). Novel chimeras created by protein engineering are endowed with advantageous features of two serotypes for targeting sensory neurons and alleviating inflammatory pain (LC/E-BoTIM/A). Likewise, an innocuous mutant of /B (BoTIM/B) fused to core streptavidin (CS-BoTIM/B) has been exploited for guiding molecular cargo and viral vectors into nerve cells. These novel discoveries exemplify the versatility of BoNT in targeting and delivering therapeutics into neurons.