Targeting the tamoxifen receptor within sodium channels to block osteoarthritic pain.

Voltage-gated sodium channels (NaV) in nociceptive neurons initiate action potentials required for transmission of aberrant painful stimuli observed in osteoarthritis (OA). Targeting NaV subtypes with drugs to produce analgesic effects for OA pain management is a developing therapeutic area. Previously, we determined the receptor site for the tamoxifen analog N-desmethyltamoxifen (ND-Tam) within a prokaryotic NaV. Here, we report the pharmacology of ND-Tam against eukaryotic NaVs natively expressed in nociceptive neurons. ND-Tam and analogs occupy two conserved intracellular receptor sites in domains II and IV of NaV1.7 to block ion entry using a "bind and plug" mechanism. We find that ND-Tam inhibition of the sodium current is state dependent, conferring a potent frequency- and voltage-dependent block of hyperexcitable nociceptive neuron action potentials implicated in OA pain. When evaluated using a mouse OA pain model, ND-Tam has long-lasting efficacy, which supports the potential of repurposing ND-Tam analogs as NaV antagonists for OA pain management.

Evaluation of the upper airway microbiome and immune response with nasal epithelial lining fluid absorption and nasal washes.

Despite being commonly used to collect upper airway epithelial lining fluid, nasal washes are poorly reproducible, not suitable for serial sampling, and limited by a dilution effect. In contrast, nasal filters lack these limitations and are an attractive alternative. To examine whether nasal filters are superior to nasal washes as a sampling method for the characterization of the upper airway microbiome and immune response, we collected paired nasal filters and washes from a group of 40 healthy children and adults. To characterize the upper airway microbiome, we used 16S ribosomal RNA and shotgun metagenomic sequencing. To characterize the immune response, we measured total protein using a BCA assay and 53 immune mediators using multiplex magnetic bead-based assays. We conducted statistical analyses to compare common microbial ecology indices and immune-mediator median fluorescence intensities (MFIs) between sample types. In general, nasal filters were more likely to pass quality control in both children and adults. There were no significant differences in microbiome community richness, α-diversity, or structure between pediatric samples types; however, these were all highly dissimilar between adult sample types. In addition, there were significant differences in the abundance of amplicon sequence variants between sample types in children and adults. In adults, total proteins were significantly higher in nasal filters than nasal washes; consequently, the immune-mediator MFIs were not well detected in nasal washes. Based on better quality control sequencing metrics and higher immunoassay sensitivity, our results suggest that nasal filters are a superior sampling method to characterize the upper airway microbiome and immune response in both children and adults.

Length-dependent MRI of hereditary neuropathy with liability to pressure palsies.

OBJECTIVE: Hereditary neuropathy with liability to pressure palsies (HNPP) is caused by heterozygous deletion of the peripheral myelin protein 22 (PMP22) gene. Patients with HNPP present multifocal, reversible sensory/motor deficits due to increased susceptibility to mechanical pressure. Additionally, age-dependent axonal degeneration is reported. We hypothesize that length-dependent axonal loss can be revealed by MRI, irrespective of the multifocal phenotype in HNPP. METHODS: Nerve and muscle MRI data were acquired in the proximal and distal leg of patients with HNPP (n = 10) and matched controls (n = 7). More specifically, nerve magnetization transfer ratios (MTR) were evaluated to assay proximal-to-distal gradients in nerve degeneration, while intramuscular fat percentages (Fper ) were evaluated to assay muscle fat replacement following denervation. Neurological disabilities were assessed via the Charcot-Marie-Tooth neuropathy score (CMTNS) for correlation with MRI. RESULTS: Fper values were elevated in HNPP proximal muscle (9.8 ± 2.2%, P = 0.01) compared to controls (6.9 ± 1.0%). We observed this same elevation of HNPP distal muscles (10.5 ± 2.5%, P < 0.01) relative to controls (6.3 ± 1.1%). Additionally, the amplitude of the proximal-to-distal gradient in Fper was more significant in HNPP patients than controls (P < 0.01), suggesting length-dependent axonal loss. In contrast, nerve MTR values were similar between HNPP subjects (sciatic/tibial nerves = 39.4 ± 2.0/34.2 ± 2.5%) and controls (sciatic/tibial nerves = 37.6 ± 3.8/35.5 ± 1.2%). Proximal muscle Fper values were related to CMTNS (r = 0.69, P = 0.03), while distal muscle Fper and sciatic/tibial nerve MTR values were not related to disability. INTERPRETATION: Despite the multifocal nature of the HNPP phenotype, muscle Fper measurements relate to disability and exhibit a proximal-to-distal gradient consistent with length-dependent axonal loss, suggesting that Fper may be a viable biomarker of disease progression in HNPP.

Myelin abnormality in Charcot-Marie-Tooth type 4J recapitulates features of acquired demyelination.

OBJECTIVE: Charcot-Marie-Tooth type 4J (CMT4J) is a rare autosomal recessive neuropathy caused by mutations in FIG4 that result in loss of FIG4 protein. This study investigates the natural history and mechanisms of segmental demyelination in CMT4J. METHODS: Over the past 9 years, we have enrolled and studied a cohort of 12 CMT4J patients, including 6 novel FIG4 mutations. We evaluated these patients and related mouse models using morphological, electrophysiological, and biochemical approaches. RESULTS: We found sensory motor demyelinating polyneuropathy consistently in all patients. This underlying myelin pathology was associated with nonuniform slowing of conduction velocities, conduction block, and temporal dispersion on nerve conduction studies, which resemble those features in acquired demyelinating peripheral nerve diseases. Segmental demyelination was also confirmed in mice without Fig4 (Fig4-/- ). The demyelination was associated with an increase of Schwann cell dedifferentiation and macrophages in spinal roots where nerve-blood barriers are weak. Schwann cell dedifferentiation was induced by the increasing intracellular Ca2+ . Suppression of Ca2+ level by a chelator reduced dedifferentiation and demyelination of Schwann cells in vitro and in vivo. Interestingly, cell-specific knockout of Fig4 in mouse Schwann cells or neurons failed to cause segmental demyelination. INTERPRETATION: Myelin change in CMT4J recapitulates the features of acquired demyelinating neuropathies. This pathology is not Schwann cell autonomous. Instead, it relates to systemic processes involving interactions of multiple cell types and abnormally elevated intracellular Ca2+ . Injection of a Ca2+ chelator into Fig4-/- mice improved segmental demyelination, thereby providing a therapeutic strategy against demyelination. Ann Neurol 2018;83:756-770.

The E Loop of the Transmitter Binding Site Is a Key Determinant of the Modulatory Effects of Physostigmine on Neuronal Nicotinic α4ß2 Receptors.

Physostigmine is a well known inhibitor of acetylcholinesterase, which can also activate, potentiate, and inhibit acetylcholine receptors, including neuronal nicotinic receptors comprising α4 and ß2 subunits. We have found that the two stoichiometric forms of this receptor differ in the effects of physostigmine. The form containing three copies of α4 and two of ß2 was potentiated at low concentrations of acetylcholine chloride (ACh) and physostigmine, whereas the form containing two copies of α4 and three of ß2 was inhibited. Chimeric constructs of subunits indicated that the presence of inhibition or potentiation depended on the source of the extracellular ligand binding domain of the subunit. Further sets of chimeric constructs demonstrated that a portion of the ACh binding domain, the E loop, is a key determinant. Transferring the E loop from the ß2 subunit to the α4 subunit resulted in strong inhibition, whereas the reciprocal transfer reduced inhibition. To control the number and position of the incorporated chimeric subunits, we expressed chimeric constructs with subunit dimers. Surprisingly, incorporation of a subunit with an altered E loop had similar effects whether it contributed either to an intersubunit interface containing a canonical ACh binding site or to an alternative interface. The observation that the α4 E loop is involved suggests that physostigmine interacts with regions of subunits that contribute to the ACh binding site, whereas the lack of interface specificity indicates that interaction with a particular ACh binding site is not the critical factor.

Functional characterization of the α5(Asn398) variant associated with risk for nicotine dependence in the α3ß4α5 nicotinic receptor.

Smoking is a major cause for premature death. Work aimed at identifying genetic factors that contribute to nicotine addiction has revealed several single nucleotide polymorphisms (SNPs) that are linked to smoking-related behaviors such as nicotine dependence and level of smoking. One of these SNPs leads to an aspartic acid-to-asparagine substitution in the nicotinic receptor α5 subunit at amino acid position 398 [rs16969968; α5(Asn398)]. The α5 subunit is expressed both in the brain and in the periphery. In the brain, it associates with the α4 and ß2 subunits to form α4ß2α5 receptors. In the periphery, the α5 subunit combines with the α3 and ß4 subunits to form the major ganglionic postsynaptic nicotinic receptor subtype. The α3ß4α5 receptor regulates a variety of autonomic responses such as control of cardiac rate, blood pressure, and perfusion. In this paradigm, the α5(Asn398) variant may act by regulating autonomic responses that may affect nicotine intake by humans. Here, we have investigated the effect of the α5(Asn398) variant on the function of the α3ß4α5 receptor. The wild-type or variant α5 subunits were coexpressed with the α3 and ß4 subunits in human embryonic kidney 293 cells. The properties of the receptors were studied using whole-cell and single-channel electrophysiology. The data indicate that the introduction of the α5(Asn398) mutation has little effect on the pharmacology of receptor activation, receptor desensitization, or single-channel properties. We propose that the effect of the α5(Asn398) variant on nicotine use is not mediated by an action on the physiological or pharmacological properties of the α3ß4α5 subtype.

Occupation of either site for the neurosteroid allopregnanolone potentiates the opening of the GABAA receptor induced from either transmitter binding site.

Potentiating neuroactive steroids are potent and efficacious modulators of the GABA(A) receptor that act by allosterically enhancing channel activation elicited by GABA. Steroids interact with the membrane-spanning domains of the α subunits of the receptor, whereas GABA binds to pockets in the interfaces between ß and α subunits. Steroid interaction with a single site is known to be sufficient to produce potentiation, but it is not clear whether effects within the same ß-α pair mediate potentiation. Here, we have investigated whether the sites for GABA and steroids are functionally linked (i.e., whether the occupancy of a steroid site selectively affects activation elicited by GABA binding to the transmitter binding site within the same ß-α pair). For that, we used receptors formed of mutated concatenated subunits to selectively eliminate one of the two GABA sites and one of the two steroid sites. The data demonstrate that receptors containing a single functional GABA site are potentiated by the neurosteroid allopregnanolone regardless of whether the steroid interacts with the α subunit from the same or the other ß-α pair. We conclude that steroids potentiate the opening of the GABA(A) receptor induced by either agonist binding site.