The elusive Nav1.7: From pain to cancer.

Voltage-gated sodium channels (Nav) are protein complexes that play fundamental roles in the transmission of signals in the nervous system, at the neuromuscular junction and in the heart. They are mainly present in excitable cells where they are responsible for triggering action potentials. Dysfunctions in Nav ion conduction give rise to a wide range of conditions, including neurological disorders, hypertension, arrhythmia, pain and cancer. Nav family 1 is composed of nine members, named numerically from 1 to 9. A Nax family also exists and is involved in body-fluid homeostasis. Of particular interest is Nav1.7 which is highly expressed in the sensory neurons of the dorsal root ganglions, where it is involved in the propagation of pain sensation. Gain-of-function mutations in Nav1.7 cause pathologies associated with increased pain sensitivity, while loss-of-function mutations cause reduced sensitivity to pain. The last decade has seen considerable effort in developing highly specific Nav1.7 blockers as pain medications, nonetheless, sufficient efficacy has yet to be achieved. Evidence is now conclusively showing that Navs are also present in many types of cancer cells, where they are involved in cell migration and invasiveness. Nav1.7 is anomalously expressed in endometrial, ovarian and lung cancers. Nav1.7 is also involved in Chemotherapy Induced Peripheral Neuropathy (CIPN). We propose that the knowledge and tools developed to study the role of Nav1.7 in pain can be exploited to develop novel cancer therapies. In this chapter, we illustrate the various aspects of Nav1.7 function in pain, cancer and CIPN, and outline therapeutic approaches.

Stimulation of Neurite Outgrowth in Cerebrocortical Neurons by Sodium Channel Activator Brevetoxin-2 Requires Both N-Methyl-D-aspartate Receptor 2B (GluN2B) and p21 Protein (Cdc42/Rac)-Activated Kinase 1 (PAK1).

N-methyl-D-aspartate (NMDA) receptors play a critical role in activity-dependent dendritic arborization, spinogenesis, and synapse formation by stimulating calcium-dependent signaling pathways. Previously, we have shown that brevetoxin 2 (PbTx-2), a voltage-gated sodium channel (VGSC) activator, produces a concentration-dependent increase in intracellular sodium [Na+]I and increases NMDA receptor (NMDAR) open probabilities and NMDA-induced calcium (Ca2+) influxes. The objective of this study is to elucidate the downstream signaling mechanisms by which the sodium channel activator PbTx-2 influences neuronal morphology in murine cerebrocortical neurons. PbTx-2 and NMDA triggered distinct Ca2+-influx pathways, both of which involved the NMDA receptor 2B (GluN2B). PbTx-2-induced neurite outgrowth in day in vitro 1 (DIV-1) neurons required the small Rho GTPase Rac1 and was inhibited by both a PAK1 inhibitor and a PAK1 siRNA. PbTx-2 exposure increased the phosphorylation of PAK1 at Thr-212. At DIV-5, PbTx-2 induced increases in dendritic protrusion density, p-cofilin levels, and F-actin throughout the dendritic arbor and soma. Moreover, PbTx-2 increased miniature excitatory post-synaptic currents (mEPSCs). These data suggest that the stimulation of neurite outgrowth, spinogenesis, and synapse formation produced by PbTx-2 are mediated by GluN2B and PAK1 signaling.

Diversity and frequency of kdr mutations within Anopheles sinensis populations from Guangxi, China.

BACKGROUND: Anopheles sinensis is a major vector of malaria in China and its control is under great threat as the development of insecticide resistance. Voltage-gated sodium channel (VGSC) is the target of several classes of insecticides. Genetic mutations of VGSC have been documented to confer knockdown resistance (kdr) to dichlorodiphenyltrichloroethane (DDT) and pyrethroids in mosquitoes. To control this vector efficiently, it is important to know the resistance-associated genetic mutations, their distribution frequencies and genealogical relations. METHODS: Three hundreds and thirteen (313) adults of An. sinensis collected from nine locations across Guangxi Zhuang Autonomous Region were used. The partial sequence of the An. sinensis voltage gated sodium channel gene (AS-VGSC) containing codon 1014 was sequenced. PHASE2.1 was used to construct the haplotypes of each individual, and the accuracy of haplotypes was further confirmed by clone sequencing. The genealogical relations of kdr mutations in AS-VGSC was analysed using TCS 2.1 and Network 5.0. RESULTS: Sixteen AS-VGSC haplotypes including seven haplotypes carrying non-synonymous mutations at codon 1014, and fifty-five AS-VGSC genotypes were identified from 313 mosquitoes collected from nine geographical locations across Guangxi. The number of haplotypes in each of the nine populations ranged from 5 to 13. The frequency of haplotypes carrying kdr mutations ranged from 2.7 to 80.0 % within the nine populations, of which 1014C was unexpectedly high in the northeast of Guangxi. Genealogical analysis suggested multiple origins of kdr mutations in An. sinensis. CONCLUSION: Diverse haplotypes of AS-VGSC are distributed in Guangxi. The presence of haplotypes carrying mutations at codon 1014 indicates a risk of pyrethroid and DDT resistance. The kdr mutations show differential distribution geographically, with high frequencies occurred in the northeast of Guangxi. Genealogical analysis suggests multiple origins of kdr mutations in An. sinensis populations in Guangxi. These findings have important practical implications for the sustainability of An. sinensis control programmes.

Widespread Mutations in Voltage-Gated Sodium Channel Gene of Cimex lectularius (Hemiptera: Cimicidae) Populations in Paris.

Bed bugs, Cimex lectularius and C. hemipterus, are common blood-sucking ectoparasites of humans with a large geographical distribution, worldwide. In France, little is known about the status of bed bugs' infestation and their resistance to insecticides, particularly, pyrethroids. Here, we aimed to find mutations in the kdr gene, known to be involved in resistance to insecticides. We gathered bed bugs from various infested locations, including 17 private houses, 12 HLM building complex, 29 apartments, 2 EHPAD, and 2 immigrants' residences. A total of 1211 bed bugs were collected and morphologically identified as C. lectularius. Two fragments of the kdr gene, encompassing codons V419L and L925I, were successfully amplified for 156 specimens. We recorded sense mutation in the first amplified fragment (kdr1) in 89 out of 156 (57%) samples, in which in 61 out of 89 (68.5%) sequences, a change of valine (V) into leucine (L) V419L was observed. Within the second fragment (kdr2), a homozygous mutation was recorded in 73 out of 156 (46.7%) specimens at the codon 925. At this position, 43 out of 73 (58.9%) specimens had a sense mutation leading to the replacement of leucine (L) by isoleucine (I). Among 162 mutant sequences analyzed (89 for the kdr1 fragment and 73 for the kdr2 one), we detected single point mutation in 26.6%, while 73.4% presented the mutation in both kdr1 and kdr2 fragments. All modifications recorded in bed bug populations of Paris are described to be involved in the knockdown resistance (kdr) against pyrethroids.

A survey of insecticide resistance-conferring mutations in multiple targets in Anopheles sinensis populations across Sichuan, China.

BACKGROUND: Sichuan province is located in the southwest of China, and was previously a malaria-endemic region. Although no indigenous malaria case has been reported since 2011, the number of imported cases is on the rise. Insecticide-based vector control has played a central role in the prevention of malaria epidemics. However, the efficacy of this strategy is gravely challenged by the development of insecticide resistance. Regular monitoring of insecticide resistance is essential to inform evidence-based vector control. Unfortunately, almost no information is currently available on the status of insecticide resistance and associated mechanisms in Anopheles sinensis, the dominant malaria vector in Sichuan. In this study, efforts were invested in detecting the presence and frequency of insecticide resistance-associated mutations in three genes that encode target proteins of several classes of commonly used insecticides. METHODS: A total of 446 adults of An. sinensis, collected from 12 locations across Sichuan province of China, were inspected for resistance-conferring mutations in three genes that respectively encode acetylcholinesterase (AChE), voltage-gated sodium channel (VGSC), and GABA receptor (RDL) by DNA Sanger sequencing. RESULTS: The G119S mutation in AChE was detected at high frequencies (0.40-0.73). The predominant ace-1 genotype was GGC/AGC (119GS) heterozygotes. Diverse variations at codon 1014 were found in VGSC, leading to three different amino acid substitutions (L1014F/C/S). The 1014F was the predominant resistance allele and was distributed in all 12 populations at varying frequencies from 0.03 to 0.86. The A296S mutation in RDL was frequently present in Sichuan, with 296SS accounting for more than 80% of individuals in six of the 12 populations. Notably, in samples collected from Chengdu (DJY) and Deyang (DYMZ), almost 30% of individuals were found to be resistant homozygotes for all three targets. CONCLUSIONS: Resistance-related mutations in three target proteins of the four main classes of insecticides were prevalent in most populations. This survey reveals a worrisome situation of multiple resistance genotypes in Sichuan malaria vector. The data strengthen the need for regular monitoring of insecticide resistance and establishing a region-customized vector intervention strategy.

Target-site mutations (AChE-G119S and kdr) in Guangxi Anopheles sinensis populations along the China-Vietnam border.

BACKGROUND: In South Asia, the epidemiology of malaria is complex, and transmission mainly occurs in remote areas near international borders. Vector control has been implemented as a key strategy in malaria prevention for decades. A rising threat to the efficacy of vector control efforts is the development of insecticide resistance, thus it is important to monitor the type and frequency of insecticide resistant alleles in the disease vectors such as An. sinensis along the China-Vietnam border. Such information is needed to synthesize effective malaria vector control strategies. METHODS: A total of 208 adults of An. sinensis, collected from seven sites in southwest Guangxi along the China-Vietnam border, were inspected for the resistance-conferring G119S mutation in acetylcholinesterase (AChE) by PCR-RFLP (polymerase chain reaction restriction fragment length polymorphism) and kdr mutations in the voltage-gated sodium channel (VGSC) by sequencing. In addition, the evolutionary origin of An. sinensis vgsc gene haplotypes was analyzed using Network 5.0. RESULTS: The frequencies of mutant 119S of AChE were between 0.61-0.85 in the seven An. sinensis populations. No susceptible homozygote (119GG) was detected in three of the seven sites (DXEC, LZSK and FCGDX). Very low frequencies of kdr (0.00-0.01) were detected in the seven populations, with most individuals being susceptible homozygote (1014LL). The 1014F mutation was detected only in the southeast part (FCGDX) at a low frequency of 0.03. The 1014S mutation was distributed in six of the seven populations with frequencies ranging from 0.04 to 0.08, but absent in JXXW. Diverse haplotypes of 1014L and 1014S were found in An. sinensis along the China-Vietnam border, while only one 1014F haplotype was detected in this study. Consistent with a previous report, resistant 1014S haplotypes did not have a single origin. CONCLUSIONS: The G119S mutation of AChE was present at high frequencies (0.61-0.85) in the An. sinensis populations along the China-Vietnam border, suggesting that the vector control authorities should be cautious when considering carbamates and organophosphates as chemicals for vector control. The low frequencies (0.00-0.11) of kdr in these populations suggest that pyrethroids remain suitable for use against An. sinensis in these regions.

Sodium Channel SCN3A (NaV1.3) Regulation of Human Cerebral Cortical Folding and Oral Motor Development.

Channelopathies are disorders caused by abnormal ion channel function in differentiated excitable tissues. We discovered a unique neurodevelopmental channelopathy resulting from pathogenic variants in SCN3A, a gene encoding the voltage-gated sodium channel NaV1.3. Pathogenic NaV1.3 channels showed altered biophysical properties including increased persistent current. Remarkably, affected individuals showed disrupted folding (polymicrogyria) of the perisylvian cortex of the brain but did not typically exhibit epilepsy; they presented with prominent speech and oral motor dysfunction, implicating SCN3A in prenatal development of human cortical language areas. The development of this disorder parallels SCN3A expression, which we observed to be highest early in fetal cortical development in progenitor cells of the outer subventricular zone and cortical plate neurons and decreased postnatally, when SCN1A (NaV1.1) expression increased. Disrupted cerebral cortical folding and neuronal migration were recapitulated in ferrets expressing the mutant channel, underscoring the unexpected role of SCN3A in progenitor cells and migrating neurons.

Evaluación de primers reportados para detección de KDR en especies de Anopheles colectadas en los departamentos de Caaguazú y Alto Paraná en Paraguay / Evaluation of primers reported for KDR detection in Anopheles species collected in the departments of Caaguazú and Alto Paraná in Paraguay

Las mutaciones KDR en el gen del canal del sodio (VGSC) han sido ya detectadas en al menos 13 especies de mosquitos Anopheles en su mayoría especies de África, pero aún resta por determinar los cebadores específicos para la detección en especies de Latinoamérica. En nuestro país la especie Anopheles darlingi es el vector principal de la malaria, y el A. albitarsis, el vector secundario. Se emplearon muestras de mosquitos Anoheles de las especies A. strodei, A. albitarsis, A. fluminensis, A. evansae, A. nuneztovari, A. nyssorhynchela lutzi y A. oswaldoi capturadas en los departamentos de Caaguazú y Alto Paraná en Paraguay. Para la amplificación y secuenciación se usaron cebadores reportados para el gen VGSC de A. albimanus en Guatemala, que resultaron ser específicos solo para la especie A. strodei. La secuencia revela el codón TTA que codifica para una Leucina como la secuencia TTG, reportada para la versión susceptible en la posición L1014. El fragmento amplificado es de aproximadamente 225 pares de bases. A nuestro entender, esta es la primera caracterización del gen VGSC en mosquitos Anopheles del Paraguay y para la especie A. strodei

KDR mutations in the sodium channel gene (VGSC) have already been detected in at least 13 species of Anopheles mosquitoes, mostly African species, but the molecular techniques for detection in Latin American species have yet to be determined. In our country, Anopheles darlingi species is the main vector of Malaria, and A. albitarsis, the secondary vector. We used samples of Anoheles from the species A. strodei, A. albitarsis, A. fluminensis, A. evansae, A. nuneztovari, A. nyssorhynchela lutzi and A. oswaldoi collected at the departments of Caaguazú and Alto Paraná in Paraguay. For the amplification and sequentiation, primers reported for the VGSC gen of A. strodei in Guatemala were used and were specific only for A. strode in this case. The sequence revealed the TTA codon that codifies for a leucine as the TTG sequence, reported for the susceptible version at position L1014. The amplified fragment is approximately 225 base pairs. To our knowledge, this is the first characterization of the VGSC gene in Anopheles mosquitoes in Paraguay and for the species A. strodei

QSAR analyses of DDT analogues and their in silico validation using molecular docking study against voltage-gated sodium channel of Anopheles funestus.

DDT has enjoyed the reputation of a successful pesticide in disease control programme and agricultural practices along with the serious opposition and ban later on due to its biomagnification and toxic action against non-target organisms. The present work was carried out to develop QSAR models for analysing DDT analogues for their pesticidal activity and in silico validation of these models. A 2D-QSAR model was generated using stepwise with multiple regression, and the model with a value of r(2) = 0.7324; q(2) = 0.6215; pred r(2) = 0.7038, containing five descriptors, was selected for further study. The 3D QSAR with CoMFA analysis showed that steric contribution of 21% and electrostatic contribution of about 79% were required for larvicidal activity of DDT analogues. A set of 3430 molecules was generated using the basic DDT skeleton as template, and these were evaluated for their mosquito larvicidal activity using the generated QSAR models and DDT as standard. Eleven molecules were selected for in silico validation of these models. For this, a docking study of the selected molecules against the homology-modelled voltage-gated sodium channel of Anopheles funestus was conducted. The study showed that the activities of these analogues as predicted by 2D-QSAR, 3D-QSAR with CoMFA and dock score were observed to be well correlated.