Stem cell-derived sensory neurons modelling inherited erythromelalgia: normalization of excitability.

Effective treatment of pain remains an unmet healthcare need that requires new and effective therapeutic approaches. NaV1.7 has been genetically and functionally validated as a mediator of pain. Preclinical studies of NaV1.7-selective blockers have shown limited success and translation to clinical studies has been limited. The degree of NaV1.7 channel blockade necessary to attenuate neuronal excitability and ameliorate pain is an unanswered question important for drug discovery. Here, we utilize dynamic clamp electrophysiology and induced pluripotent stem cell-derived sensory neurons (iPSC-SNs) to answer this question for inherited erythromelalgia, a pain disorder caused by gain-of-function mutations in Nav1.7. We show that dynamic clamp can produce hyperexcitability in iPSC-SNs associated with two different inherited erythromelalgia mutations, NaV1.7-S241T and NaV1.7-I848T. We further show that blockade of approximately 50% of NaV1.7 currents can reverse neuronal hyperexcitability to baseline levels.

Depolarizing NaV and hyperpolarizing KV channels are co-trafficked in sensory neurons.

Neuronal excitability relies on coordinated action of functionally distinct ion channels. Voltage-gated sodium (NaV) and potassium (KV) channels have distinct but complementary roles in firing action potentials: NaV channels provide depolarizing current while KV channels provide hyperpolarizing current. Mutations and dysfunction of multiple NaV and KV channels underlie disorders of excitability, including pain and epilepsy. Modulating ion channel trafficking may offer a potential therapeutic strategy for these diseases. A fundamental question, however, is whether these channels with distinct functional roles are transported independently or packaged together in the same vesicles in sensory axons. We have used Optical Pulse-Chase Axonal Long-distance (OPAL) imaging to investigate trafficking of NaV and KV channels and other axonal proteins from distinct functional classes in live rodent sensory neurons (from male and female rats). We show that, similar to NaV1.7 channels, NaV1.8 and KV7.2 channels are transported in Rab6a-positive vesicles, and that each of the NaV channel isoforms expressed in healthy, mature sensory neurons - NaV1.6, NaV1.7, NaV1.8, and NaV1.9 - are co-transported in the same vesicles. Further, we show that multiple axonal membrane proteins with different physiological functions - NaV1.7, KV7.2, and TNFR1 - are co-transported in the same vesicles. However, vesicular packaging of axonal membrane proteins is not indiscriminate, since another axonal membrane protein - NCX2 - is transported in separate vesicles. These results shed new light on the development and organization of sensory neuron membranes, revealing complex sorting of axonal proteins with diverse physiological functions into specific transport vesicles.Significance StatementNormal neuronal excitability is dependent on precise regulation of membrane proteins including NaV and KV channels, and imbalance in the level of these channels at the plasma membrane could lead to excitability disorders. Ion channel trafficking could potentially be targeted therapeutically, which would require better understanding of the mechanisms underlying trafficking of functionally diverse channels. Optical Pulse-chase Axonal Long-distance (OPAL) imaging in live neurons permitted examination of the specificity of ion channel trafficking, revealing co-packaging of axonal proteins with opposing physiological functions into the same transport vesicles. This suggests that additional trafficking mechanisms are necessary to regulate levels of surface channels and reveals an important consideration for therapeutic strategies that target ion channel trafficking for the treatment of excitability disorders.

Paclitaxel increases axonal localization and vesicular trafficking of Nav1.7.

The microtubule-stabilizing chemotherapy drug paclitaxel (PTX) causes dose-limiting chemotherapy-induced peripheral neuropathy (CIPN), which is often accompanied by pain. Among the multifaceted effects of PTX is an increased expression of sodium channel Nav1.7 in rat and human sensory neurons, enhancing their excitability. However, the mechanisms underlying this increased Nav1.7 expression have not been explored, and the effects of PTX treatment on the dynamics of trafficking and localization of Nav1.7 channels in sensory axons have not been possible to investigate to date. In this study we used a recently developed live imaging approach that allows visualization of Nav1.7 surface channels and long-distance axonal vesicular transport in sensory neurons to fill this basic knowledge gap. We demonstrate concentration and time-dependent effects of PTX on vesicular trafficking and membrane localization of Nav1.7 in real-time in sensory axons. Low concentrations of PTX increase surface channel expression and vesicular flux (number of vesicles per axon). By contrast, treatment with a higher concentration of PTX decreases vesicular flux. Interestingly, vesicular velocity is increased for both concentrations of PTX. Treatment with PTX increased levels of endogenous Nav1.7 mRNA and current density in dorsal root ganglion neurons. However, the current produced by transfection of dorsal root ganglion neurons with Halo-tag Nav1.7 was not increased after exposure to PTX. Taken together, this suggests that the increased trafficking and surface localization of Halo-Nav1.7 that we observed by live imaging in transfected dorsal root ganglion neurons after treatment with PTX might be independent of an increased pool of Nav1.7 channels. After exposure to inflammatory mediators to mimic the inflammatory condition seen during chemotherapy, both Nav1.7 surface levels and vesicular transport are increased for both low and high concentrations of PTX. Overall, our results show that PTX treatment increases levels of functional endogenous Nav1.7 channels in dorsal root ganglion neurons and enhances trafficking and surface distribution of Nav1.7 in sensory axons, with outcomes that depend on the presence of an inflammatory milieu, providing a mechanistic explanation for increased excitability of primary afferents and pain in CIPN.

A novel gain-of-function sodium channel ß2 subunit mutation in idiopathic small fiber neuropathy.

Small fiber neuropathy (SFN) is a common condition affecting thinly myelinated Aδ and unmyelinated C fibers, often resulting in excruciating pain and dysautonomia. SFN has been associated with several conditions, but a significant number of cases have no discernible cause. Recent genetic studies have identified potentially pathogenic gain-of-function mutations in several pore-forming voltage-gated sodium channel α subunits (NaV) in a subset of patients with SFN, but the auxiliary sodium channel ß subunits have been less implicated in the development of the disease. ß subunits modulate NaV trafficking and gating, and several mutations have been linked to epilepsy and cardiac dysfunction. Recently, we provided the first evidence for the contribution of a mutation in the ß2 subunit to pain in human painful diabetic neuropathy. Here, we provide the first evidence for the involvement of a sodium channel ß subunit mutation in the pathogenesis of SFN with no other known causes. We show, through current-clamp analysis, that the newly identified Y69H variant of the ß2 subunit induces neuronal hyperexcitability in dorsal root ganglion neurons, lowering the threshold for action potential firing and allowing for increased repetitive action potential spiking. Underlying the hyperexcitability induced by the ß2-Y69H variant, we demonstrate an upregulation in tetrodotoxin-sensitive, but not tetrodotoxin-resistant sodium currents. This provides the first evidence for the involvement of ß2 subunits in SFN and strengthens the link between sodium channel ß subunits and the development of neuropathic pain in humans.NEW & NOTEWORTHY Small fiber neuropathy (SFN) often has no discernible cause, although mutations in the voltage-gated sodium channel α subunits have been implicated in some cases. We identify a patient suffering from SFN with a mutation in the auxiliary ß2 subunit and no other discernible causes for SFN. Functional assessment confirms this mutation renders dorsal root ganglion neurons hyperexcitable and upregulates tetrodotoxin-sensitive sodium currents. This study strengthens a newly emerging link between sodium channel ß2 subunit mutations and human pain disorders.

A gain-of-function sodium channel ß2-subunit mutation in painful diabetic neuropathy.

Diabetes mellitus is a global challenge with many diverse health sequelae, of which diabetic peripheral neuropathy is one of the most common. A substantial number of patients with diabetic peripheral neuropathy develop chronic pain, but the genetic and epigenetic factors that predispose diabetic peripheral neuropathy patients to develop neuropathic pain are poorly understood. Recent targeted genetic studies have identified mutations in α-subunits of voltage-gated sodium channels (Navs) in patients with painful diabetic peripheral neuropathy. Mutations in proteins that regulate trafficking or functional properties of Navs could expand the spectrum of patients with Nav-related peripheral neuropathies. The auxiliary sodium channel ß-subunits (ß1-4) have been reported to increase current density, alter inactivation kinetics, and modulate subcellular localization of Nav. Mutations in ß-subunits have been associated with several diseases, including epilepsy, cancer, and diseases of the cardiac conducting system. However, mutations in ß-subunits have never been shown previously to contribute to neuropathic pain. We report here a patient with painful diabetic peripheral neuropathy and negative genetic screening for mutations in SCN9A, SCN10A, and SCN11A-genes encoding sodium channel α-subunit that have been previously linked to the development of neuropathic pain. Genetic analysis revealed an aspartic acid to asparagine mutation, D109N, in the ß2-subunit. Functional analysis using current-clamp revealed that the ß2-D109N rendered dorsal root ganglion neurons hyperexcitable, especially in response to repetitive stimulation. Underlying the hyperexcitability induced by the ß2-subunit mutation, as evidenced by voltage-clamp analysis, we found a depolarizing shift in the voltage dependence of Nav1.7 fast inactivation and reduced use-dependent inhibition of the Nav1.7 channel.

A Molecular Determinant of Subtype-Specific Desensitization in Ionotropic Glutamate Receptors.

AMPA and NMDA receptors are glutamate-gated ion channels that mediate fast excitatory synaptic transmission throughout the nervous system. In the continual presence of glutamate, AMPA and NMDA receptors containing the GluN2A or GluN2B subunit enter into a nonconducting, desensitized state that can impact synaptic responses and glutamate-mediated excitotoxicity. The process of desensitization is dramatically different between subtypes, but the basis for these differences is unknown. We generated an extensive sequence alignment of ionotropic glutamate receptors (iGluRs) from diverse animal phyla and identified a highly conserved motif, which we termed the "hydrophobic box," located at the extracellular interface of transmembrane helices. A single position in the hydrophobic box differed between mammalian AMPA and NMDA receptors. Surprisingly, we find that an NMDAR-to-AMPAR exchange mutation at this position in the rat GluN2A or GluN2B subunit had a dramatic and highly specific effect on NMDAR desensitization, making it AMPAR-like. In contrast, a reverse exchange mutation in AMPARs had minimal effects on desensitization. These experiments highlight differences in desensitization between iGluR subtypes and the highly specific contribution of the GluN2 subunit to this process. SIGNIFICANCE STATEMENT: Rapid communication between cells in the nervous system depends on ion channels that are directly activated by neurotransmitter molecules. Here, we studied ionotropic glutamate receptors (iGluRs), which are ion channels activated by the neurotransmitter glutamate. By comparing the sequences of a vast number of iGluR proteins from diverse animal species, assisted by available structural information, we identified a highly conserved motif. We showed that a single amino acid difference in this motif between mammalian iGluR subtypes has dramatic effects on receptor function. These results have implications in both the evolution of synaptic function, as well as the role of iGluRs in health and disease.

The impact of topical treatment for chalazia on the odds of procedural management.

Chalazia in pediatric patients are often treated with topical antibiotics or steroids, although no strong evidence supports their use. This retrospective review of pediatric patients with chalazia did not find a decreased odds of undergoing procedural treatment (incision and curettage and/or intralesional steroid injection) with initial topical antibiotics and/or steroids compared to conservative measures. Inflamed chalazia may benefit from topical treatment, but small sample size limits this subgroup analysis. Shorter pre-topical treatment chalazion duration correlated with a lower risk of procedural intervention. Regimens that included steroids were not found to be more effective than topical antibiotics alone.

iPSCs and DRGs: stepping stones to new pain therapies.

There is a pressing need for more effective nonaddictive treatment options for pain. Pain signals are transmitted from the periphery into the spinal cord via dorsal root ganglion (DRG) neurons, whose excitability is driven by voltage-gated sodium (NaV) channels. Three NaV channels (NaV1.7, NaV1.8, and NaV1.9), preferentially expressed in DRG neurons, play important roles in pain signaling in humans. Blockade of these channels may provide a novel approach to the treatment of pain, but clinical translation of preclinical results has been challenging, in part due to differences between rodent and human DRG neurons. Human DRG neurons and iPSC-derived sensory neurons (iPSC-SNs) provide new preclinical platforms that may facilitate the development of novel pain therapeutics.

Evaluation of the Content of Ophthalmology Fellowship Program Web sites.

Purpose To assess the availability and content of fellowship program Web sites (FPWs) among ophthalmology subspecialties. Design This is a cross-sectional study. Subjects Web sites of all Association of University Professors of Ophthalmology-accredited fellowship programs in five subspecialties (i.e., surgical retina and vitreous; cornea, external disease, and refractive surgery; glaucoma; neuro-ophthalmology; and pediatric ophthalmology). Methods FPWs were assessed for the presence of 26 key content criteria encompassing program demographics ( n = 13), features ( n = 10), and social life ( n = 3). The presence of each content criterion as well as the content criteria groups were compared across subspecialties. Main Outcome Measures The main outcome measured is the average percentage of key content criteria present among ophthalmology fellowship Web sites. Results Among 266 accredited fellowship programs, 240 (90.2%) had Web sites. On average, Web sites reported 14.9 of 26 key content criteria (57.2%), 8.29 of 13 demographic criteria (63.8%), 5.84 of the 10 program features criteria (58.4%), and 0.705 of the 3 social life criteria (23.5%). Significant differences were identified among subspecialties in the presence of program description ( p = 0.046), hospital affiliation ( p < 0.001), names of current fellows ( p = 0.004), case diversity ( p = 0.001), and surgical statistics ( p = 0.015). The average number of key criteria differed between subspecialties ( p < 0.001). Conclusion There is significant heterogeneity in program fellowship Web site content among ophthalmology subspecialties. Information regarding social life, such as wellness programs and community information, was largely absent across all disciplines. Addressing missing information on ophthalmology FPWs may help optimize program-applicant fit.

Contributions of NaV1.8 and NaV1.9 to excitability in human induced pluripotent stem-cell derived somatosensory neurons.

The inhibition of voltage-gated sodium (NaV) channels in somatosensory neurons presents a promising novel modality for the treatment of pain. However, the precise contribution of these channels to neuronal excitability, the cellular correlate of pain, is unknown; previous studies using genetic knockout models or pharmacologic block of NaV channels have identified general roles for distinct sodium channel isoforms, but have never quantified their exact contributions to these processes. To address this deficit, we have utilized dynamic clamp electrophysiology to precisely tune in varying levels of NaV1.8 and NaV1.9 currents into induced pluripotent stem cell-derived sensory neurons (iPSC-SNs), allowing us to quantify how graded changes in these currents affect different parameters of neuronal excitability and electrogenesis. We quantify and report direct relationships between NaV1.8 current density and action potential half-width, overshoot, and repetitive firing. We additionally quantify the effect varying NaV1.9 current densities have on neuronal membrane potential and rheobase. Furthermore, we examined the simultaneous interplay between NaV1.8 and NaV1.9 on neuronal excitability. Finally, we show that minor biophysical changes in the gating of NaV1.8 can render human iPSC-SNs hyperexcitable, in a first-of-its-kind investigation of a gain-of-function NaV1.8 mutation in a human neuronal background.

Lacosamide Inhibition of NaV1.7 Channels Depends on its Interaction With the Voltage Sensor Domain and the Channel Pore.

Lacosamide, developed as an anti-epileptic drug, has been used for the treatment of pain. Unlike typical anticonvulsants and local anesthetics which enhance fast-inactivation and bind within the pore of sodium channels, lacosamide enhances slow-inactivation of these channels, suggesting different binding mechanisms and mode of action. It has been reported that lacosamide's effect on NaV1.5 is sensitive to a mutation in the local anesthetic binding site, and that it binds with slow kinetics to the fast-inactivated state of NaV1.7. We recently showed that the NaV1.7-W1538R mutation in the voltage-sensing domain 4 completely abolishes NaV1.7 inhibition by clinically-achievable concentration of lacosamide. Our molecular docking analysis suggests a role for W1538 and pore residues as high affinity binding sites for lacosamide. Aryl sulfonamide sodium channel blockers are also sensitive to substitutions of the W1538 residue but not of pore residues. To elucidate the mechanism by which lacosamide exerts its effects, we used voltage-clamp recordings and show that lacosamide requires an intact local anesthetic binding site to inhibit NaV1.7 channels. Additionally, the W1538R mutation does not abrogate local anesthetic lidocaine-induced blockade. We also show that the naturally occurring arginine in NaV1.3 (NaV1.3-R1560), which corresponds to NaV1.7-W1538R, is not sufficient to explain the resistance of NaV1.3 to clinically-relevant concentrations of lacosamide. However, the NaV1.7-W1538R mutation conferred sensitivity to the NaV1.3-selective aryl-sulfonamide blocker ICA-121431. Together, the W1538 residue and an intact local anesthetic site are required for lacosamide's block of NaV1.7 at a clinically-achievable concentration. Moreover, the contribution of W1538 to lacosamide inhibitory effects appears to be isoform-specific.

Status of peripheral sodium channel blockers for non-addictive pain treatment.

The effective and safe treatment of pain is an unmet health-care need. Current medications used for pain management are often only partially effective, carry dose-limiting adverse effects and are potentially addictive, highlighting the need for improved therapeutic agents. Most common pain conditions originate in the periphery, where dorsal root ganglion and trigeminal ganglion neurons feed pain information into the CNS. Voltage-gated sodium (NaV) channels drive neuronal excitability and three subtypes - NaV1.7, NaV1.8 and NaV1.9 - are preferentially expressed in the peripheral nervous system, suggesting that their inhibition might treat pain while avoiding central and cardiac adverse effects. Genetic and functional studies of human pain disorders have identified NaV1.7, NaV1.8 and NaV1.9 as mediators of pain and validated them as targets for pain treatment. Consequently, multiple NaV1.7-specific and NaV1.8-specific blockers have undergone clinical trials, with others in preclinical development, and the targeting of NaV1.9, although hampered by technical constraints, might also be moving ahead. In this Review, we summarize the clinical and preclinical literature describing compounds that target peripheral NaV channels and discuss the challenges and future prospects for the field. Although the potential of peripheral NaV channel inhibition for the treatment of pain has yet to be realized, this remains a promising strategy to achieve non-addictive analgesia for multiple pain conditions.