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.

The small fiber neuropathy NaV1.7 I228M mutation: impaired neurite integrity via bioenergetic and mitotoxic mechanisms, and protection by dexpramipexole.

Gain-of-function variants in voltage-gated sodium channel NaV1.7 that increase firing frequency and spontaneous firing of dorsal root ganglion (DRG) neurons have recently been identified in 5-10% of patients with idiopathic small fiber neuropathy (I-SFN). Our previous in vitro observations suggest that enhanced sodium channel activity can contribute to a decrease in length of peripheral sensory axons. We have hypothesized that sustained sodium influx due to the expression of SFN-associated sodium channel variants may trigger an energetic deficit in neurons that contributes to degeneration and loss of nerve fibers in SFN. Using an ATP FRET biosensor, we now demonstrate reduced steady-state levels of ATP and markedly faster ATP decay in response to membrane depolarization in cultured DRG neurons expressing an SFN-associated variant NaV1.7, I228M, compared with wild-type neurons. We also observed that I228M neurons show a significant reduction in mitochondrial density and size, indicating dysfunctional mitochondria and a reduced bioenergetic capacity. Finally, we report that exposure to dexpramipexole, a drug that improves mitochondrial energy metabolism, increases the neurite length of I228M-expressing neurons. Our data suggest that expression of gain-of-function variants of NaV1.7 can damage mitochondria and compromise cellular capacity for ATP production. The resulting bioenergetic crisis can consequently contribute to loss of axons in SFN. We suggest that, in addition to interventions that reduce ionic disturbance caused by mutant NaV1.7 channels, an alternative therapeutic strategy might target the bioenergetic burden and mitochondrial damage that occur in SFN associated with NaV1.7 gain-of-function mutations.NEW & NOTEWORTHY Sodium channel NaV1.7 mutations that increase dorsal root ganglion (DRG) neuron excitability have been identified in small fiber neuropathy (SFN). We demonstrate reduced steady-state ATP levels, faster depolarization-evoked ATP decay, and reduced mitochondrial density and size in cultured DRG neurons expressing SFN-associated variant NaV1.7 I228M. Dexpramipexole, which improves mitochondrial energy metabolism, has a protective effect. Because gain-of-function NaV1.7 variants can compromise bioenergetics, therapeutic strategies that target bioenergetic burden and mitochondrial damage merit study in SFN.

Sodium Channels in Human Pain Disorders: Genetics and Pharmacogenomics.

Acute pain is adaptive, but chronic pain is a global challenge. Many chronic pain syndromes are peripheral in origin and reflect hyperactivity of peripheral pain-signaling neurons. Current treatments are ineffective or only partially effective and in some cases can be addictive, underscoring the need for better therapies. Molecular genetic studies have now linked multiple human pain disorders to voltage-gated sodium channels, including disorders characterized by insensitivity or reduced sensitivity to pain and others characterized by exaggerated pain in response to normally innocuous stimuli. Here, we review recent developments that have enhanced our understanding of pathophysiological mechanisms in human pain and advances in targeting sodium channels in peripheral neurons for the treatment of pain using novel and existing sodium channel blockers.

The AMPK Activator A769662 Blocks Voltage-Gated Sodium Channels: Discovery of a Novel Pharmacophore with Potential Utility for Analgesic Development.

Voltage-gated sodium channels (VGSC) regulate neuronal excitability by governing action potential (AP) generation and propagation. Recent studies have revealed that AMP-activated protein kinase (AMPK) activators decrease sensory neuron excitability, potentially by preventing sodium (Na+) channel phosphorylation by kinases such as ERK or via modulation of translation regulation pathways. The direct positive allosteric modulator A769662 displays substantially greater efficacy than other AMPK activators in decreasing sensory neuron excitability suggesting additional mechanisms of action. Here, we show that A769662 acutely inhibits AP firing stimulated by ramp current injection in rat trigeminal ganglion (TG) neurons. PT1, a structurally dissimilar AMPK activator that reduces nerve growth factor (NGF) -induced hyperexcitability, has no influence on AP firing in TG neurons upon acute application. In voltage-clamp recordings, application of A769662 reduces VGSC current amplitudes. These findings, based on acute A769662 application, suggest a direct channel blocking effect. Indeed, A769662 dose-dependently blocks VGSC in rat TG neurons and in Nav1.7-transfected cells with an IC50 of ~ 10 µM. A769662 neither displayed use-dependent inhibition nor interacted with the local anesthetic (LA) binding site. Popliteal fossa administration of A769662 decreased noxious thermal responses with a peak effect at 5 mins demonstrating an analgesic effect. These data indicate that in addition to AMPK activation, A769662 acts as a direct blocker/modulator of VGSCs, a potential mechanism enhancing the analgesic property of this compound.

NaV1.7: stress-induced changes in immunoreactivity within magnocellular neurosecretory neurons of the supraoptic nucleus.

BACKGROUND: NaV1.7 is preferentially expressed, at relatively high levels, in peripheral neurons, and is often referred to as a "peripheral" sodium channel, and NaV1.7-specific blockers are under study as potential pain therapeutics which might be expected to have minimal CNS side effects. However, occasional reports of patients with NaV1.7 gain-of-function mutations and apparent hypothalamic dysfunction have appeared. The two sodium channels previously studied within the rat hypothalamic supraoptic nucleus, NaV1.2 and NaV1.6, display up-regulated expression in response to osmotic stress. RESULTS: Here we show that NaV1.7 is present within vasopressin-producing neurons and oxytocin-producing neurons within the rat hypothalamus, and demonstrate that the level of Nav1.7 immunoreactivity is increased in these cells in response to osmotic stress. CONCLUSIONS: NaV1.7 is present within neurosecretory neurons of rat supraoptic nucleus, where the level of immunoreactivity is dynamic, increasing in response to osmotic stress. Whether NaV1.7 levels are up-regulated within the human hypothalamus in response to environmental factors or stress, and whether NaV1.7 plays a functional role in human hypothalamus, is not yet known. Until these questions are resolved, the present findings suggest the need for careful assessment of hypothalamic function in patients with NaV1.7 mutations, especially when subjected to stress, and for monitoring of hypothalamic function as NaV1.7 blocking agents are studied.

Sodium channels, the electrogenisome and the electrogenistat: lessons and questions from the clinic.

In the six decades that have followed the work of Hodgkin and Huxley, multiple generations of neuroscientists and biophysicists have built upon their pivotal contributions. It is now clear that, in mammals, nine genes encode nine distinct voltage-gated sodium channels with different amino acid sequences and different physiological and pharmacological properties. The different sodium channel isoforms produce a multiplicity of distinct sodium currents with different time-dependent characteristics and voltage dependencies, which interact with each other and with the currents produced by other channels (including calcium and potassium channels) to shape neuronal firing patterns. Expression of these sodium channel isoforms is highly dynamic, both in the normal nervous system, and in the injured nervous system. Recent research has shed light on the roles of sodium channels in human disease, a development that may open up new therapeutic strategies. This article examines the pain-signalling system as an example of a neuronal network where multiple sodium channel isoforms play complementary roles in electrogenesis and a strong link with human disease has been established. Recent research suggests that it may be possible to target specific sodium channel isoforms that drive hyperexcitability in pain-signalling neurons, thereby providing new therapeutic strategies for chronic pain, and providing an illustration of the impact of the Hodgkin-Huxley legacy in the clinical domain.

Neuropathic pain in diabetes--evidence for a central mechanism.

Hyperexcitability of and aberrant spontaneous impulse generation by damaged first-order sensory neurons and their peripheral axons are well-established processes that strongly contribute to pain associated with diabetic neuropathy. Studies in the past 5 years, however, suggest that, as in many neuropathic pain disorders, central neuropathic mechanisms can also contribute to pain experienced with diabetes. These studies have demonstrated that thalamic dysfunction occurs in patients with diabetes mellitus, and that in experimental models of this disease neurons in the ventral posterolateral thalamus can become hyperexcitable, firing at abnormally high frequencies and generating aberrant spontaneous activity. In this article, we discuss these findings, which suggest that thalamic neurons can act as central generators or amplifiers of pain in diabetes.

Thalamic neuron hyperexcitability and enlarged receptive fields in the STZ model of diabetic pain.

Distal limb pain in diabetes mellitus is frequently attributed to hyperexcitability of primary afferents associated with peripheral neuropathy. However, prior studies have demonstrated that, after traumatic nerve injury, hyperexcitability develops not only within primary afferents but also within pain-signalling neurons of the spinal cord dorsal horn and thalamic ventral posterolateral (VPL) nucleus, establishing a basis for tiered central pain generators or amplifiers. In this study we asked whether hyperexcitability develops within thalamic neurons in experimental painful diabetes. Diabetes was induced in adult male Sprague-Dawley rats with streptozotocin (STZ). Behavioral testing for tactile allodynia, performed one week prior to STZ injection and weekly thereafter, indicated that, by six weeks after STZ injection, mechanical allodynia had developed (mechanical withdrawal threshold <4 g, STZ; 21.75 g, control). Thalamic unit recordings were obtained from the VPL nucleus at seven weeks after STZ injection, in rats that met a criterion withdrawal threshold of <4 g, at a time when mean glucose level for control rats was 104.8+/-2.9, and for diabetic rats was 420.1+/-42.0. Our analysis shows that, in this model of diabetic neuropathic pain, thalamic VPL neurons develop hyperexcitability, with increased responses to phasic brush, press, and pinch stimuli applied to identified peripheral receptive fields. VPL neurons from diabetic rats also display enhanced spontaneous activity, independent of ascending afferent barrage, and enlarged receptive fields. These results suggest that aberrant levels of spontaneous activity and hyper-responsiveness of VPL thalamic neurons may contribute to diabetic neuropathic pain.

Sodium channel expression in the ventral posterolateral nucleus of the thalamus after peripheral nerve injury.

Peripheral nerve injury is known to up-regulate the expression of rapidly-repriming Nav1.3 sodium channel within first-order dorsal root ganglion neurons and second-order dorsal horn nociceptive neurons, but it is not known if pain-processing neurons higher along the neuraxis also undergo changes in sodium channel expression. In this study, we hypothesized that after peripheral nerve injury, third-order neurons in the ventral posterolateral (VPL) nucleus of the thalamus undergo changes in expression of sodium channels. To test this hypothesis, adult male Sprague-Dawley rats underwent chronic constriction injury (CCI) of the sciatic nerve. Ten days after CCI, when allodynia and hyperalgesia were evident, in situ hybridization and immunocytochemical analysis revealed up-regulation of Nav1.3 mRNA, but no changes in expression of Nav1.1, Nav1.2, or Nav1.6 in VPL neurons, and unit recordings demonstrated increased background firing, which persisted after spinal cord transection, and evoked hyperresponsiveness to peripheral stimuli. These results demonstrate that injury to the peripheral nervous system induces alterations in sodium channel expression within higher-order VPL neurons, and suggest that misexpression of the Nav1.3 sodium channel increases the excitability of VPL neurons injury, contributing to neuropathic pain.

Fire and phantoms after spinal cord injury: Na+ channels and central pain.

Neuropathic pain and phantom phenomena occur commonly after spinal cord injury (SCI) but their molecular basis is not yet fully understood. Recent findings demonstrate abnormal expression of the Nav1.3 Na(+) channel within second-order spinal cord dorsal horn neurons and third-order thalamic neurons along the pain pathway after SCI, and suggest that this change makes these neurons hyperexcitable so that they act as pain amplifiers and generators. Delineation of molecular changes that contribute to hyperexcitability of pain-signaling neurons might lead to identification of molecular targets that will be useful in the treatment of neuropathic pain after SCI and related nervous system injuries.

Changes in electrophysiological properties and sodium channel Nav1.3 expression in thalamic neurons after spinal cord injury.

Spinal cord contusion injury (SCI) is known to induce pain-related behaviour, as well as hyperresponsiveness in lumbar dorsal horn nociceptive neurons associated with the aberrant expression of Na(v)1.3, a rapidly repriming voltage-gated sodium channel. Many of these second-order dorsal horn neurons project to third-order neurons in the ventrobasal complex of the thalamus. In this study we hypothesized that, following SCI, neurons in the thalamus undergo electrophysiological changes linked to aberrant expression of Na(v)1.3. Adult male Sprague-Dawley rats underwent contusion SCI at the T9 thoracic level. Four weeks post-SCI, Na(v)1.3 protein was upregulated within thalamic neurons in ventroposterior lateral (VPL) and ventroposterior medial nuclei, where extracellular unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, and expansion of peripheral receptive fields. Altered electrophysiological properties of VPL neurons persisted after interruption of ascending spinal barrage by spinal cord transection above the level of the injury. Lumbar intrathecal administration of specific antisense oligodeoxynucleotides generated against Na(v)1.3 caused a significant reduction in Na(v)1.3 expression in thalamic neurons and reversed electrophysiological alterations. These results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after SCI.

The brain in diabetes: molecular changes in neurons and their implications for end-organ damage.

Although secondary end-organ damage in diabetes has generally been thought to result from long-term passive shunting of excess glucose through alternative metabolic pathways, recent studies have elucidated a second mechanism of pathogenesis that involves active changes in gene expression in neurons of the CNS. These changes in gene expression result in molecular and functional changes that can become maladaptive over time. In this review, we examine two neuronal populations in the brain that have been studied in human beings and animal models of diabetes. First, we discuss overactivation of magnocellular neurosecretory cells within the hypothalamus and how it relates to the development of diabetic nephropathy. And second, we describe how changes in hippocampal synaptic plasticity can lead to cognitive and behavioural deficits in chronic diabetes. Changes in neuronal gene expression in diabetes represent a new pathway for diabetic pathogenesis. This pathway may hold clues for the development of therapies that, via the targeting of neurons, can slow or prevent the development of diabetic end-organ damage.

Primary cortical motor neurons undergo apoptosis after axotomizing spinal cord injury.

Spinal cord injury (SCI) results in loss of voluntary motor control followed by incomplete recovery, which is partly mediated by the descending corticospinal tract (CST). This system is an important target for therapeutic repair strategies after SCI; however, the question of whether apoptotic cell death occurs in these axotomized neurons remains unanswered. In this study, adult (150-175 g) male Sprague-Dawley rats underwent T9 transection of the dorsal funiculus, which axotomizes the dorsal CST, and introduction of the retrograde tracer Fluoro-Gold into the lesion site. Primary motor cortex (M1) was then examined for evidence of apoptosis weekly for 4 weeks after injury. Axotomized pyramidal cells, identified by retrograde transport of Fluoro-Gold, were found in M1 (57.5 +/- 9.6/median section, 6127 +/- 292 total), and a significant proportion were terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate (dUTP)-rhodamine nick end labeling (TUNEL) -positive at 1 week after injury (39.3 +/- 5.6%), compared with animals undergoing sham surgery (1.2 +/- 1.4%). At 2-4 weeks, fewer cells were Fluoro-Gold-positive (24.6 +/- 65.06 to 25.3 +/- 6.4/median section, 2338 +/- 233 to 2393 +/- 124 total), of which very few were TUNEL-positive. In TUNEL-positive cells, Hoechst 33342 staining revealed nuclear morphology consistent with apoptosis, chromatin condensation, and formation of apoptotic bodies. Fluoro-Gold-positive cells showed increased caspase-3 and Bax immunoreactivity. Hematoxylin and eosin staining revealed similar nuclear changes and dystrophic cells. Internucleosomal DNA fragmentation was detected by gel electrophoresis at the 1-week time point. Lesioned animals not receiving Fluoro-Gold exhibited the same markers of apoptosis. These results document, for the first time, features of apoptotic cell death in a proportion of axotomized cortical motor neurons after SCI, suggesting that protection from apoptosis may be a prerequisite for regenerative approaches to SCI.