A next generation peripherally restricted Cavα2δ-1 ligand with inhibitory action on Cav2.2 channels and utility in neuropathic pain.

The Voltage-Gated Calcium Channel (VGCC) auxiliary subunit Cavα2δ-1 (CACNA2D1) is the target/receptor of gabapentinoids which are known therapeutics in epilepsy and neuropathic pain. Following damage to the peripheral sensory nervous system, Cavα2δ-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of chronic neuropathic pain. Gabapentinoids, such as gabapentin and pregabalin, engage with Cavα2δ-1 via binding an arginine residue (R241) within an RRR motif located at the N-terminus of human Cavα2δ-1. A novel, next generation gabapentinoid, engineered not to penetrate the brain, was able to generate a strong analgesic response in Chronic Constriction Injury animal model of chronic neuropathic pain and showed binding specificity for Cavα2δ-1 versus the Cavα2δ-2 subunit. This novel non-brain penetrant gabapentinoid, binds to R241 and a novel binding site on Cavα2δ-1, which is located within the VGCC_α2 domain, identified as a lysine residue within an IKAK amino acid motif (K634). The overall whole cell current amplitudes were diminished by the compound, with these inhibitory effects being diminished in R241A mutant Cavα2δ-1 subunits. The functional effects occurred at lower concentrations than those needed for inhibition by gabapentin or pregabalin, which apparently bound the Cavα2δ-1 subunit only on the R241 and not on the K634 residue. Our work sets the stage for the identification and characterisation of novel compounds with therapeutic properties in neuropathic pain and possibly in other disorders and conditions which require engagement of the Cavα2δ-1 target.

Differential regulation of Cav 3.2 and Cav 2.2 calcium channels by CB1 receptors and cannabidiol.

BACKGROUND AND PURPOSE: Cannabinoids are a promising therapeutic avenue for chronic pain. However, clinical trials often fail to report analgesic efficacy of cannabinoids. Inhibition of voltage gate calcium (Cav ) channels is one mechanism through which cannabinoids may produce analgesia. We hypothesized that cannabinoids and cannabinoid receptor agonists target different types of Cav channels through distinct mechanisms. EXPERIMENTAL APPROACH: Electrophysiological recordings from tsA-201 cells expressing either Cav 3.2 or Cav 2.2 were used to assess inhibition by HU-210 or cannabidiol (CBD) in the absence and presence of the CB1 receptor. Homology modelling assessed potential interaction sites for CBD in both Cav 2.2 and Cav 3.2. Analgesic effects of CBD were assessed in mouse models of inflammatory and neuropathic pain. KEY RESULTS: HU-210 (1 µM) inhibited Cav 2.2 function in the presence of CB1 receptor but had no effect on Cav 3.2 regardless of co-expression of CB1 receptor. By contrast, CBD (3 µM) produced no inhibition of Cav 2.2 and instead inhibited Cav 3.2 independently of CB1 receptors. Homology modelling supported these findings, indicating that CBD binds to and occludes the pore of Cav 3.2, but not Cav 2.2. Intrathecal CBD alleviated thermal and mechanical hypersensitivity in both male and female mice, and this effect was absent in Cav 3.2 null mice. CONCLUSION AND IMPLICATIONS: Our findings reveal differential modulation of Cav 2.2 and Cav 3.2 channels by CB1 receptors and CBD. This advances our understanding of how different cannabinoids produce analgesia through action at different voltage-gated calcium channels and could influence the development of novel cannabinoid-based therapeutics for treatment of chronic pain.

Doru luteipes (Dermaptera: Forficulidae) and Orius insidiosus (Hemiptera: Anthocoridae) as Nocturnal and Diurnal Predators of Thrips.

Thrips (Thysanoptera, Thripidae) are pests of several crops and their chemical control is mainly hindered by their thigmotactic habits, which in turn allows the use of biological control agents with similar habits. Orius (Hemiptera: Anthocoridae) are effective control agents for thrips and are commercialized in many countries. Habitat overlap exists between Doru luteipes (Scudder) (Dermaptera: Forficulidae) and thrips, making D. luteipes a potential predator in the control of these insects. Our goals were to confirm the predatory ability of D. luteipes when exposed to thrips, Caliothrips phaseoli (Hood), and to evaluate the interaction between D. luteipes and Orius insidiosus Say for the control of thrips using behavioral and feeding preference tests. The ability of D. luteipes and O. insidiosus to prey on thrips at all stages was tested by predation bioassays; adults of D. luteipes consumed 210.9 ± 23.2 thrips per day, while adults of O. insidiosus consumed 32.4 ± 3.6 thrips per day. Intraguild predation was absent, and the predatory behavior feeding of the two predatory species was not altered in the presence of the other predator. In addition, these predators forage at different times-O. insidiosus during the day and D. luteipes at night, indicating that both predators do not interact negatively, allowing the use of both in a biological pest control program for thrips.

Regulation of N-type calcium channels by nociceptin receptors and its possible role in neurological disorders.

Activation of nociceptin opioid peptide receptors (NOP, a.k.a. opioid-like receptor-1, ORL-1) by the ligand nociceptin/orphanin FQ, leads to G protein-dependent regulation of Cav2.2 (N-type) voltage-gated calcium channels (VGCCs). This typically causes a reduction in calcium currents, triggering changes in presynaptic calcium levels and thus neurotransmission. Because of the widespread expression patterns of NOP and VGCCs across multiple brain regions, the dorsal horn of the spinal cord, and the dorsal root ganglia, this results in the alteration of numerous neurophysiological features. Here we review the regulation of N-type calcium channels by the NOP-nociceptin system in the context of neurological conditions such as anxiety, addiction, and pain.

Electrophysiological and computational analysis of Cav3.2 channel variants associated with familial trigeminal neuralgia.

Trigeminal neuralgia (TN) is a rare form of chronic neuropathic pain characterized by spontaneous or elicited paroxysms of electric shock-like or stabbing pain in a region of the face. While most cases occur in a sporadic manner and are accompanied by intracranial vascular compression of the trigeminal nerve root, alteration of ion channels has emerged as a potential exacerbating factor. Recently, whole exome sequencing analysis of familial TN patients identified 19 rare variants in the gene CACNA1H encoding for Cav3.2T-type calcium channels. An initial analysis of 4 of these variants pointed to a pathogenic role. In this study, we assessed the electrophysiological properties of 13 additional TN-associated Cav3.2 variants expressed in tsA-201 cells. Our data indicate that 6 out of the 13 variants analyzed display alteration of their gating properties as evidenced by a hyperpolarizing shift of their voltage dependence of activation and/or inactivation resulting in an enhanced window current supported by Cav3.2 channels. An additional variant enhanced the recovery from inactivation. Simulation of neuronal electrical membrane potential using a computational model of reticular thalamic neuron suggests that TN-associated Cav3.2 variants could enhance neuronal excitability. Altogether, the present study adds to the notion that ion channel polymorphisms could contribute to the etiology of some cases of TN and further support a role for Cav3.2 channels.

Ca V 3.2 calcium channels contribute to trigeminal neuralgia.

ABSTRACT: Trigeminal neuralgia (TN) is a rare but debilitating disorder characterized by excruciating facial pain, with a higher incidence in women. Recent studies demonstrated that TN patients present mutations in the gene encoding the Ca V 3.2 T-type calcium channel, an important player in peripheral pain pathways. We characterize the role of Ca V 3.2 channels in TN at 2 levels. First, we examined the biophysical properties of CACNA1H variants found in TN patients. Second, we investigated the role of Ca V 3.2 in an animal model of trigeminal neuropathic pain. Whole-cell patch-clamp recordings from 4 different mutants expressed in tsA-201 cells (E286K in the pore loop of domain I, H526Y, G563R, and P566T in the domain I-II linker) identified a loss of function in activation in the E286K mutation and gain of function in the G563R and P566T mutations. Moreover, a loss of function in inactivation was observed with the E286K and H526Y mutations. Cell surface biotinylation revealed no difference in channel trafficking among the variants. The G563R mutant also caused a gain of function in the firing properties of transfected trigeminal ganglion neurons. In female and male mice, constriction of the infraorbital nerve induced facial thermal heat hyperalgesia. Block of T-type channels with Z944 resulted in antihyperalgesia. The effect of Z944 was absent in Ca V 3.2 -/- mice, indicating that Ca V 3.2 is the molecular target of the antihyperalgesic Z944 effect. Finally, enzyme-linked immunosorbent assay analysis revealed increased Ca V 3.2 channel expression in the spinal trigeminal subnucleus caudalis. Altogether, the present study demonstrates an important role of Ca V 3.2 channels in trigeminal pain.

Regulation of CaV3.2 channels by the receptor for activated C kinase 1 (Rack-1).

This study describes the interaction between CaV3.2 calcium channels and the receptor for activated C kinase 1 (Rack-1), a scaffold protein which has recently been implicated in neuropathic pain. The coexpression of CaV3.2 and Rack-1 in tsA-201 cells led to a reduction in the magnitude of whole-cell CaV3.2 currents and CaV3.2 channel expression at the plasma membrane. Co-immunoprecipitations from transfected cells show the formation of a molecular protein complex between Cav3.2 channels and Rack-1. We determined that the interaction of Rack-1 occurs at the intracellular II-III loop and the C-terminus of the channel. Finally, the coexpression of PKCßII abolished the effect of Rack-1 on current densities. Altogether, our findings show that Rack-1 regulates CaV3.2-mediated calcium entry in a PKC-dependent manner.

A parasitoid's dilemma between food and host resources: the role of volatiles from nectar-providing marigolds and host-infested plants attracting Aphidius platensis.

The use of nectar-producing companion plants in crops is a well-known strategy of conserving natural enemies in biological control. However, the role of floral volatiles in attracting parasitoids and effects on host location via herbivore-induced plant volatiles is poorly known. Here, we examined the role of floral volatiles from marigold (Tagetes erecta), alone or in combination with volatiles from sweet pepper plant (Capsicum annuum), in recruiting Aphidius platensis, an important parasitoid of the green peach aphid Myzus persicae. We also investigated whether marigold floral volatiles are more attractive to the parasitoid than those emitted by sweet pepper plants infested by M. persicae. Olfactometry assays indicated that floral volatiles attracted A. platensis to the marigold plant and are more attractive than sweet pepper plant volatiles. However, volatiles emitted by aphid-infested sweet pepper were as attractive to the parasitoid as those of uninfested or aphid-infested blooming marigold. The composition of volatile blends released by uninfested and aphid-infested plants differed between both blooming marigold and sweet pepper, but the parasitoid did not discriminate aphid-infested from uninfested blooming marigold. Volatile released from blooming marigold and sweet pepper shared several compounds, but that of blooming marigold contained larger amounts of fatty-acid derivatives and a different composition of terpenes. We discuss the potential implications of the aphid parasitoid attraction in a diversified crop management strategy.

Splice-variant specific effects of a CACNA1H mutation associated with writer's cramp.

The CACNA1H gene encodes the α1 subunit of the low voltage-activated Cav3.2 T-type calcium channel, an important regulator of neuronal excitability. Alternative mRNA splicing can generate multiple channel variants with distinct biophysical properties and expression patterns. Two major splice variants, containing or lacking exon 26 (± 26) have been found in different human tissues. In this study, we report splice variant specific effects of a Cav3.2 mutation found in patients with autosomal dominant writer's cramp, a specific type of focal dystonia. We had previously reported that the R481C missense mutation caused a gain of function effect when expressed in Cav3.2 (+ 26) by accelerating its recovery from inactivation. Here, we show that when the mutation is expressed in the short variant of the channel (- 26), we observe a significant increase in current density when compared to wild-type Cav3.2 (- 26) but the effect on the recovery from inactivation is lost. Our data add to growing evidence that the functional expression of calcium channel mutations depends on which splice variant is being examined.

De novo SCN8A and inherited rare CACNA1H variants associated with severe developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are a group of severe epilepsies that are characterized by seizures and developmental delay. DEEs are primarily attributed to genetic causes and an increasing number of cases have been correlated with variants in ion channel genes. In this study, we report a child with an early severe DEE. Whole exome sequencing showed a de novo heterozygous variant (c.4873-4881 duplication) in the SCN8A gene and an inherited heterozygous variant (c.952G > A) in the CACNA1H gene encoding for Nav1.6 voltage-gated sodium and Cav3.2 voltage-gated calcium channels, respectively. In vitro functional analysis of human Nav1.6 and Cav3.2 channel variants revealed mild but significant alterations of their gating properties that were in general consistent with a gain- and loss-of-channel function, respectively. Although additional studies will be required to confirm the actual pathogenic involvement of SCN8A and CACNA1H, these findings add to the notion that rare ion channel variants may contribute to the etiology of DEEs.

Myricetin improves metabolic outcomes but not cognitive deficit associated to metabolic syndrome in male mice.

Myricetin is a flavonol highly prevalent in edible vegetables and fruits, with recognized hypoglycemic and anti-obesity effects, besides great antioxidant capacity. Thus, this study sought to investigate whether myricetin is able to improve metabolic and behavioral outcomes found in monosodium l-glutamate (MSG) obese mice, a model of metabolic syndrome characterized by early hyperinsulinemia associated to obesity, dyslipidemia, hepatic steatosis, anxiety and cognitive deficit. Newborn male mice received MSG (4 mg kg-1 day-1, s.c.) on alternate days during the first 10 days of life for obesity induction, while control pups received equimolar saline solution. From postnatal day 90 to 135, MSG mice were orally treated with myricetin (50 mg kg-1 day-1) or distilled water, while control animals received vehicle. During the last week of treatment, all groups were submitted to behavioral tests: open field maze, elevated plus maze and Morris water maze. At the end of treatment, animals were euthanized for collection of liver, serum and adipose tissue fat pads. Myricetin treatment reduced the elevated serum levels of glucose and triglycerides, typically found in MSG mice, as well as restored peripheral insulin sensitivity and liver steatosis. Moreover, myricetin ameliorated the lack of thigmotaxis and exploratory behavior, but did not improve the cognitive deficit presented by MSG mice. Therefore, this study contributes to the pharmacological validation of myricetin as an affordable and healthy therapeutic adjuvant for the treatment of metabolic syndrome and most of its comorbidities.

The de novo CACNA1A pathogenic variant Y1384C associated with hemiplegic migraine, early onset cerebellar atrophy and developmental delay leads to a loss of Cav2.1 channel function.

CACNA1A pathogenic variants have been linked to several neurological disorders including familial hemiplegic migraine and cerebellar conditions. More recently, de novo variants have been associated with severe early onset developmental encephalopathies. CACNA1A is highly expressed in the central nervous system and encodes the pore-forming CaVα1 subunit of P/Q-type (Cav2.1) calcium channels. We have previously identified a patient with a de novo missense mutation in CACNA1A (p.Y1384C), characterized by hemiplegic migraine, cerebellar atrophy and developmental delay. The mutation is located at the transmembrane S5 segment of the third domain. Functional analysis in two predominant splice variants of the neuronal Cav2.1 channel showed a significant loss of function in current density and changes in gating properties. Moreover, Y1384 variants exhibit differential splice variant-specific effects on recovery from inactivation. Finally, structural analysis revealed structural damage caused by the tyrosine substitution and changes in electrostatic potentials.

Rare functional missense variants in CACNA1H: What can we learn from Writer's cramp?

Writer's cramp (WC) is a task-specific focal dystonia that occurs selectively in the hand and arm during writing. Previous studies have shown a role for genetics in the pathology of task-specific focal dystonia. However, to date, no causal gene has been reported for task-specific focal dystonia, including WC. In this study, we investigated the genetic background of a large Dutch family with autosomal dominant‒inherited WC that was negative for mutations in known dystonia genes. Whole exome sequencing identified 4 rare variants of unknown significance that segregated in the family. One candidate gene was selected for follow-up, Calcium Voltage-Gated Channel Subunit Alpha1 H, CACNA1H, due to its links with the known dystonia gene Potassium Channel Tetramerization Domain Containing 17, KCTD17, and with paroxysmal movement disorders. Targeted resequencing of CACNA1H in 82 WC cases identified another rare, putative damaging variant in a familial WC case that did not segregate. Using structural modelling and functional studies in vitro, we show that both the segregating p.Arg481Cys variant and the non-segregating p.Glu1881Lys variant very likely cause structural changes to the Cav3.2 protein and lead to similar gains of function, as seen in an accelerated recovery from inactivation. Both mutant channels are thus available for re-activation earlier, which may lead to an increase in intracellular calcium and increased neuronal excitability. Overall, we conclude that rare functional variants in CACNA1H need to be interpreted very carefully, and additional studies are needed to prove that the p.Arg481Cys variant is the cause of WC in the large Dutch family.

A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels.

A novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.

Bioecology of Anagyrus saccharicola (Hymenoptera: Encyrtidae), a parasitoid of the pink sugarcane mealybug Saccharicoccus sacchari (Hemiptera: Pseudococcidae).

Parasitoids can be used as biological agents of pest control. Anagyrus saccharicola Timberlake (Hymenoptera: Encyrtidae) is a parasitoid of the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell) (Hemiptera: Pseudococcidae). Although this mealybug is present in all sugarcane-producing countries, there is limited information regarding this pest and its parasitoid. Aiming to elucidate information on bioecological parameters of A. saccharicola, were evaluated the survival of parasitoid females and males at three temperatures, the host preference of the parasitoid, and the fecundity and longevity of the host. In addition, the parasitism rate of A. saccharicola was estimated based on three factors, feeding, mating, and time. Survival was evaluated at 20, 25, and 30°C. Host preference was conducted on 15-, 20-, and 30-day-old mealybugs. And the parasitism rate was evaluated in fed and unfed, mated and unmated parasitoids and with 24 h and newly emerged. The temperature of 20°C was the most favorable for parasitoid survival. Parasitism occurred at all evaluated ages of the mealybug; however, the preference was for those that were 30-days-old. The parasitized mealybugs longevity was approximately 8 additional days after parasitization, and non-parasitized mealybugs lived for an additional 20 days for mealybugs aged 30 and 20 days at the outset of the tests, and a further 13 days for the 15 days. Feeding and mating after 24 h of emergence resulted in a higher parasitism rate. These findings can contribute to more efficient rearing of A. saccharicola and in the planning of the biological control of S. sacchari in the integrated pest management programs.

Functional identification of potential non-canonical N-glycosylation sites within Cav3.2 T-type calcium channels.

Low-voltage-activated T-type calcium channels are important contributors to nervous system function. Post-translational modification of these channels has emerged as an important mechanism to control channel activity. Previous studies have documented the importance of asparagine (N)-linked glycosylation and identified several asparagine residues within the canonical consensus sequence N-X-S/T that is essential for the expression and function of Cav3.2 channels. Here, we explored the functional role of non-canonical N-glycosylation motifs in the conformation N-X-C based on site directed mutagenesis. Using a combination of electrophysiological recordings and surface biotinylation assays, we show that asparagines N345 and N1780 located in the motifs NVC and NPC, respectively, are essential for the expression of the human Cav3.2 channel in the plasma membrane. Therefore, these newly identified asparagine residues within non-canonical motifs add to those previously reported in canonical sites and suggest that N-glycosylation of Cav3.2 may also occur at non-canonical motifs to control expression of the channel in the plasma membrane. It is also the first study to report the functional importance of non-canonical N-glycosylation motifs in an ion channel.

Obesidade na infância e adolescência / Obesity in childhood and adolescence

A obesidade é uma doença crônica e multifatorial com sérias repercussões na saúde. O excesso de peso na infância aumenta o risco de obesidade na adolescência e na vida adulta. A obesidade é uma das principais causas de hipertensão arterial em crianças e adolescentes. No sexo feminino, os problemas ginecológicos relacionados com a obesidade incluem as desordens menstruais e a diminuição da fertilidade na adolescência e na vida adulta. O controle dessa patologia evita a sua evolução para formas crônicas e graves, que acarretaria novos transtornos e consequências para essas jovens. A mudança de hábitos alimentares e a realização de atividade física são a principal linha de tratamento. O tratamento medicamentoso é reservado para portadoras de obesidade grave que apresentam comorbidades associadas e que não respondem às mudanças do estilo de vida. (AU)

Obesity is a chronic and multifactorial disease with serious repercussions on health. Overweight in childhood increases the risk of obesity in adolescence and adulthood. Obesity is one of the main causes of high blood pressure in children and adolescents, among others. In women, gynecological problems related to obesity include menstrual disorders and decreased fertility in adolescence and adulthood. The control of this pathology prevents its evolution to chronic and severe forms that would cause new disorders and consequences for these young women. The main line of treatment is to change eating habits and encourage physical activity. Drug treatment is reserved for patients with severe obesity, who have associated comorbidities and who do not respond to changes in lifestyle.(AU)

Mechanisms of cadmium-stress avoidance by selenium in tomato plants.

Cadmium (Cd) is probably the most damaging metal to plant species; with a long biological half-life, it can be taken up by plants, disrupting the cell homeostasis and triggering several metabolic pathways. Selenium (Se) improves plant defence systems against stressful conditions, but the biochemical antioxidant responses to Cd stress in tomato plants is poorly understood. To further address the relationship of Cd-stress responses with Se mineral uptake, Cd and Se concentration, proline content, MDA and H2O2 production, and the activity of SOD, APX, CAT and GR enzymes were analyzed in Micro-Tom (MT) plants submitted to 0.5 mM Cd. The results revealed different responses according to Se combination and Cd application. For instance, roots and leaves of MT plants treated with Se exhibited an increase in dry mass and nutritional status, exhibited lower proline content and higher APX and GR activities when compared with plants with no Se application. Plants submitted to 0.5 mM Cd, irrespective of Se exposure, exhibited lower proline, MDA and H2O2 content and higher SOD, CAT and GR activities. Selenium may improve tolerance against Cd, which allowed MT plants exhibited less oxidative damage to the cell, even under elevated Cd accumulation in their tissues. The results suggest that Se application is an efficient management technique to alleviate the deleterious effects of Cd-stress, enhancing the nutritional value and activity of ROS-scavenging enzymes in tomato plants.

A rare CACNA1H variant associated with amyotrophic lateral sclerosis causes complete loss of Cav3.2 T-type channel activity.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of cortical, brain stem and spinal motor neurons that leads to muscle weakness and death. A previous study implicated CACNA1H encoding for Cav3.2 calcium channels as a susceptibility gene in ALS. In the present study, two heterozygous CACNA1H variants were identified by whole genome sequencing in a small cohort of ALS patients. These variants were functionally characterized using patch clamp electrophysiology, biochemistry assays, and molecular modeling. A previously unreported c.454GTAC > G variant produced an inframe deletion of a highly conserved isoleucine residue in Cav3.2 (p.ΔI153) and caused a complete loss-of-function of the channel, with an additional dominant-negative effect on the wild-type channel when expressed in trans. In contrast, the c.3629C > T variant caused a missense substitution of a proline with a leucine (p.P1210L) and produced a comparatively mild alteration of Cav3.2 channel activity. The newly identified ΔI153 variant is the first to be reported to cause a complete loss of Cav3.2 channel function. These findings add to the notion that loss-of-function of Cav3.2 channels associated with rare CACNA1H variants may be risk factors in the complex etiology of ALS.