Development of a selective inhibitor for Kv1.1 channels prevalent in demyelinated nerves.

Members of the voltage-gated K+ channel subfamily (Kv1), involved in regulating transmission between neurons or to muscles, are associated with human diseases and, thus, putative targets for neurotherapeutics. This applies especially to those containing Kv1.1 α subunits which become prevalent in murine demyelinated axons and appear abnormally at inter-nodes, underlying the perturbed propagation of nerve signals. To overcome this dysfunction, akin to the consequential debilitation in multiple sclerosis (MS), small inhibitors were sought that are selective for the culpable hyper-polarising K+ currents. Herein, we report a new semi-podand - compound 3 - that was designed based on the modelling of its interactions with the extracellular pore region in a deduced Kv1.1 channel structure. After synthesis, purification, and structural characterisation, compound 3 was found to potently (IC50 = 8 µM) and selectively block Kv1.1 and 1.6 channels. The tested compound showed no apparent effect on native Nav and Cav channels expressed in F-11 cells. Compound 3 also extensively and selectively inhibited MS-related Kv1.1 homomer but not the brain native Kv1.1- or 1.6-containing channels. These collective findings highlight the therapeutic potential of compound 3 to block currents mediated by Kv1.1 channels enriched in demyelinated central neurons.

Alleviation of Trigeminal Nociception Using p75 Neurotrophin Receptor Targeted Lentiviral Interference Therapy.

Acute and chronic trigeminal (TG) neuropathies are the cause of considerable distress, with limited treatments available at present. Nociceptive neurons enriched with the vanilloid type 1 receptor (VR1) partake in pain sensation and sensitization in the TG system. While VR1 blockers with anti-nociceptive potential are of substantial medical interest, their use remains limited due to poor selectivity and lack of cell-targeting capabilities. This study describes a methodology for the alleviation of nociception via targeted depletion of VR1 in TG sensory neurons in rats. In cultured TG ganglion neurons, VR1 expression was virtually abolished by lentiviral short hairpin RNA (LV-VR1). By decorating GFP encoding LV (LV-GFP) and LV-VR1 with IgG192 for targeting TG sensory neurons enriched with the p75 neurotrophin receptor (p75NTR), transduction of a reporter GFP and VR1 depletion was achieved after injection of targeted vectors into the whisker pad. In IgG192/LV-VR1-injected rats, the behavioral response to capsaicin exposure as well as Erk 1/2 phosphorylation and VR1 current activation by capsaicin were significantly reduced. This pioneering investigation, thus, provides a proof of principle for a means of attenuating TG nociception, revealing therapeutic potential.

Distinctive role of KV1.1 subunit in the biology and functions of low threshold K(+) channels with implications for neurological disease.

The diversity of pore-forming subunits of KV1 channels (KV1.1-KV1.8) affords their physiological versatility and predicts a range of functional impairments resulting from genetic aberrations. Curiously, identified so far human neurological conditions associated with dysfunctions of KV1 channels have been linked exclusively to mutations in the KCNA1 gene encoding for the KV1.1 subunit. The absence of phenotypes related to irregularities in other subunits, including the prevalent KV1.2 subunit of neurons is highly perplexing given that deletion of the corresponding kcna2 gene in mouse models precipitates symptoms reminiscent to those of KV1.1 knockouts. Herein, we critically evaluate the molecular and biophysical characteristics of the KV1.1 protein in comparison with others and discuss their role in the greater penetrance of KCNA1 mutations in humans leading to the neurological signs of episodic ataxia type 1 (EA1). Future research and interpretation of emerging data should afford new insights towards a better understanding of the role of KV1.1 in integrative mechanisms of neurons and synaptic functions under normal and disease conditions.

Internalization and retrograde axonal trafficking of tetanus toxin in motor neurons and trans-synaptic propagation at central synapses exceed those of its C-terminal-binding fragments.

The prominent tropism of tetanus toxin (TeTx) towards peripheral nerves with retrograde transport and transfer to central neurons render it an invaluable probe for exploring fundamental neuronal processes such as endocytosis, retrograde trafficking and trans-synaptic transport to central neurons. While the specificity of TeTx to nerve cells has been attributed to its binding domains (HC and HCC), molecular determinants of the long-range trafficking that ensure its central delivery and induction of spastic paralysis remain elusive. Here, we report that a protease-inactive TeTx mutant (TeTIM) fused to core streptavidin (CS) proved superior to CS-HC and CS-HCC fragments in antagonizing the internalization of the active toxin in cultured spinal cord neurons. Also, in comparison to CS-HC and CS-HCC, CS-TeTIM undergoes faster clearance from motor nerve terminals after peripheral injection, and is detected in a greater number of neurons in the spinal cord and brain stem ipsi-lateral to the administration site. Consistent with trans-synaptic transfer from motor neurons to inter-neurons, CS-TeTIM infiltrated non-cholinergic cells in the spinal cord; in contrast, the retrograde spread of CS-HC was largely restricted to neurons stained for choline acetyltransferase. Peripheral injection of CS-TeTIM conjugated to a lentivirus encoding mutated SNAP-25, resistant to cleavage by botulinum neurotoxin A, E and C1, rendered spontaneous excitatory postsynaptic currents in motor neurons resilient to challenge by type A toxin in vitro, whereas the same virus conjugated to CS-HC proved ineffective. These findings indicate that full-length inactive TeTx greatly exceeds HC and HCC in targeting and invading motor nerve terminals at the periphery and exploits more efficiently the retrograde transport and trans-synaptic transfer mechanisms of motor neurons to arrive at central neurons. Such qualities render TeTIM a more suitable research probe and neuron-targeting vehicle for retro-axonal delivery of viral vectors to the CNS.

Neurotrophin receptor p75 mediates the uptake of the amyloid beta (Aß) peptide, guiding it to lysosomes for degradation in basal forebrain cholinergic neurons.

A fascinating yet perhaps overlooked trait of the p75 neurotrophin receptor (p75(NTR)) is its ability to bind ligands with no obvious neurotrophic function. Using cultured basal forebrain (BF) neurons, this study demonstrates selective internalization of amyloid ß (Aß) 1-42 in conjunction with p75(NTR) (labelled with IgG192-Cy3) by cholinergic cells. Active under resting conditions, this process was enhanced by high K(+) stimulation and was insensitive to inhibitors of regulated synaptic activity-tetrodotoxin or botulinum neurotoxins (BoNT type/A and/B). Blockade of sarco-endoplasmic reticulum (SERCA) Ca(2+) ATPase with thapsigargin and CPA or chelation of Ca(2+) with EGTA-AM strongly suppressed the endocytosis of p75(NTR), implicating the role of ER released Ca(2+). The uptake of IgG192-Cy3 was also reduced by T-type Ca(2+) channel blocker mibefradil but not Cd(2+), an indiscriminate blocker of high voltage-activated Ca(2+) currents. A strong co-localization of IgG192-Cy3 with late endosome (Rab7) or lysosome (Lamp1) qualifier proteins suggest these compartments as the primary destination for internalized IgG192 and Aß. Selective uptake and labeling of BF cholinergic cells with IgG192-Cy3 injected into the prefrontal cortex was verified also in vivo. The significance of these findings in relation to Aß clearance in the cerebral cortex and pathophysiology of Alzheimer's disease is discussed.

Insights into a basis for incomplete inhibition by botulinum toxin A of Ca2+-evoked exocytosis from permeabilised chromaffin cells.

Inhibition of regulated exocytosis by botulinum toxins type A and B was studied in chromaffin cells. Both virtually abolished catecholamine release triggered from intact cells by depolarising stimuli, whereas the blockade by type A, but not B, was only partial after cell permeabilisation and direct stimulation of exocytosis by Ca(2+). Botulinum toxin A did not alter the [Ca(2+)]-dependency of exocytosis in permeabilised cells but, rather, proportionally reduced the amount of release at each concentration tested. Likewise, this toxin decreased the extents of Ca(2+)-induced structural changes in SNAP-25, synaptobrevin and syntaxin (known collectively as SNAREs), whilst leaving their [Ca(2+)]-sensitivity unaltered. Thus, botulinum toxin A does not reduce the Ca(2+)-sensitivity of the exocytosis sensor, but hinders transmission of the signal to the SNAREs which mediate fusion.

Rescue of exocytosis in botulinum toxin A-poisoned chromaffin cells by expression of cleavage-resistant SNAP-25. Identification of the minimal essential C-terminal residues.

Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by cleavage of SNAP-25 and synaptobrevin, respectively; in humans, many months are required for full recovery from the resultant neuromuscular paralysis. To decipher the molecular basis for such prolonged poisoning, intoxication in adreno-chromaffin cells was monitored over 2 months. Exocytosis from BoNT/B-treated cells resumed after 56 days because of the appearance of intact synaptobrevin. However, inhibition continued in BoNT/A-treated cells, throughout the same interval, with a continued predominance of cleaved SNAP-25-(1-197) over the intact protein. When recovery from poisoning was attempted by transfection of the latter cells with the gene encoding full-length SNAP-25-(1-206), no restoration of exocytosis ensued even after 3 weeks. To ascertain if this failure was because of the persistence of the toxin's protease activity, the cells were transfected with BoNT/A-resistant SNAP-25 constructs; importantly, exocytosis was rescued. C-terminal truncation of the toxin-insensitive SNAP-25 revealed that residues 1-201, 1-202, 1-203 afforded a significant return of exocytosis, unlike shorter forms 1-197, -198, -199, or -200; accordingly, mutants M202A or L203A of full-length SNAP-25 rescued secretion. These findings give insights into the C-terminal functional domain of SNAP-25, demonstrate the longevity of BoNT/A protease, and provide the prospect of a therapy for botulism.

Relationship of acceptors for botulinum neurotoxins (types A and B) in rat CNS with the cholinergic marker, chol-I.

Localization in rat CNS of the acceptors for botulinum neurotoxin (types A and B) was examined by lesioning of cholinergic input to the cortex and immuno-affinity purification of cholinergic nerve terminals. Ibotenic acid lesions of the cortical cholinergic tract caused a small reduction in the content of high affinity binding sites for type A neurotoxin and a concomitant decrease in the activities of acetylcholinesterase and choline acetyltransferase. No such change was observed in the level of acceptors for BoNT B or the extent of immuno-labelling of Chol-I, a cholinergic ganglioside. Purification of cholinergic nerve terminals, using anti-(Chol-I) antibodies gave an equivalent enrichment in the acceptors (high and low affinity) for both toxin types and choline acetyltransferase. Neurotoxin type B (but not type A) inhibited binding of anti-(Chol-I) antibodies to this cholinergic ganglioside on nerve terminals and to semi-purified Chol-I. It can be deduced from these collective findings that the high affinity binding sites for BoNT A and possibly B are localized on cholinergic nerve terminals in the CNS and that the Chol-I ganglioside may be associated with the acceptor for type B toxin.