Screening an In-House Isoquinoline Alkaloids Library for New Blockers of Voltage-Gated Na+ Channels Using Voltage Sensor Fluorescent Probes: Hits and Biases.

Voltage-gated Na+ (NaV) channels are significant therapeutic targets for the treatment of cardiac and neurological disorders, thus promoting the search for novel NaV channel ligands. With the objective of discovering new blockers of NaV channel ligands, we screened an In-House vegetal alkaloid library using fluorescence cell-based assays. We screened 62 isoquinoline alkaloids (IA) for their ability to decrease the FRET signal of voltage sensor probes (VSP), which were induced by the activation of NaV channels with batrachotoxin (BTX) in GH3b6 cells. This led to the selection of five IA: liriodenine, oxostephanine, thalmiculine, protopine, and bebeerine, inhibiting the BTX-induced VSP signal with micromolar IC50. These five alkaloids were then assayed using the Na+ fluorescent probe ANG-2 and the patch-clamp technique. Only oxostephanine and liriodenine were able to inhibit the BTX-induced ANG-2 signal in HEK293-hNaV1.3 cells. Indeed, liriodenine and oxostephanine decreased the effects of BTX on Na+ currents elicited by the hNaV1.3 channel, suggesting that conformation change induced by BTX binding could induce a bias in fluorescent assays. However, among the five IA selected in the VSP assay, only bebeerine exhibited strong inhibitory effects against Na+ currents elicited by the hNav1.2 and hNav1.6 channels, with IC50 values below 10 µM. So far, bebeerine is the first BBIQ to have been reported to block NaV channels, with promising therapeutical applications.

Neuronal maturation-dependent nano-neuro interaction and modulation.

Nanotechnology-enabled neuromodulation is a promising minimally-invasive tool in neuroscience and engineering for both fundamental studies and clinical applications. However, the nano-neuro interaction at different stages of maturation of a neural network and its implications for the nano-neuromodulation remain unclear. Here, we report heterogeneous to homogeneous transformation of neuromodulation in a progressively maturing neural network. Utilizing plasmonic-fluors as ultrabright fluorescent nanolabels, we reveal that negative surface charge of nanoparticles renders selective nano-neuro interaction with a strong correlation between the maturation stage of the individual neurons in the neural network and the density of the nanoparticles bound on the neurons. In stark contrast to homogeneous neuromodulation in a mature neural network reported so far, the maturation-dependent density of the nanoparticles bound to neurons in a developing neural network resulted in a heterogeneous optical neuromodulation (i.e., simultaneous excitation and inhibition of neural network activity). This study advances our understanding of nano-neuro interactions and nano-neuromodulation with potential applications in minimally-invasive technologies for treating neuronal disorders in parts of the mammalian brain where neurogenesis persists throughout aging.

Toxicity and mode of action of the aporphine plant alkaloid liriodenine on the insect GABA receptor.

Liriodenine is a biologically active plant alkaloid with multiple effects on mammals, fungi, and bacteria, but has never been evaluated for insecticidal activity. Accordingly, liriodenine was applied topically in ethanolic solutions to adult female Anopheles gambiae, and found to be mildly toxic. Its lethality was synergized in mixtures with dimethyl sulfoxide and piperonyl butoxide. Recordings from the ventral nerve cord of larval Drosophila melanogaster showed that liriodenine was neuroexcitatory and reversed the inhibitory effect of 1 mM GABA at effective concentrations of 20-30 µM. GABA antagonism on the larval nervous system was equally expressed on both susceptible and cyclodiene-resistant rdl preparations. Acutely isolated neurons from Periplaneta americana were studied under patch clamp and inhibition of GABA-induced currents with an IC50 value of about 1 µM were observed. In contrast, bicuculline did not reverse the effects of GABA on cockroach neurons, as expected. In silico molecular models suggested reasonable structural concordance of liriodenine and bicuculline and isosteric hydrogen bond acceptor sites. This study is the first assessing of the toxicology of liriodenine on insects and implicates the GABA receptor as one likely neuronal target, where liriodenine might be considered an active chemical analog of bicuculline.

Screening for Enhancement of Permethrin Toxicity by Plant Essential Oils Against Adult Females of the Yellow Fever Mosquito (Diptera: Culicidae).

Aedes aegypti L. (Diptera: Culicidae) is one of the most medically important mosquito species, due to its ability to spread viruses of yellow fever, dengue fever, and Zika in humans. In this study, the insecticidal activity of 17 plant essential oils was evaluated via topical application against two strains of Ae. aegypti mosquito, Orlando (insecticide-susceptible) and Puerto Rico (pyrethroid-resistant). Initial screens with the Orlando strain showed that cucumber seed oil (2017 sample) was the most toxic, followed by sandalwood and thyme oil. When the essential oils were mixed with permethrin, they failed to show any significant synergism of insecticidal activity. Sandalwood and thyme oils displayed consistently high mortality against the resistant Puerto Rico strain, with low resistance ratios of 2.1 and 1.4, respectively. In contrast, cucumber seed oil showed significantly less activity against Puerto Rico mosquitoes, with a resistance ratio of 45. Bioactivity-guided fractionation of the 2017 sample of cucumber seed oil sample via flash column chromatography produced 11 fractions, and gas-chromatography/mass spectrometry analysis revealed that the three active fractions were contaminated with 0.33, 0.36, and 0.33% of chlorpyrifos-methyl, an organophosphorus insecticide, whereas inactive fractions did not show any trace of it. These results suggested that the insecticidal activity of cucumber seed oil was probably due to the presence of the insecticide, later confirmed with a clean batch of cucumber seed oil obtained in 2018, which showed negligible insecticidal activity. These findings demonstrate clearly the need for essential oil analysis to confirm purity before any claims are made about pesticidal potency.

Pyrethroid-Derived Acids and Alcohols: Bioactivity and Synergistic Effects on Mosquito Repellency and Toxicity.

Pyrethroids are one of the most commonly used classes of insecticides, and their acid and alcohol components are esterase degradation products, usually considered to be biologically inactive. In this study, it was found that several pyrethroid acids had a spatial repellent activity that was greater than DEET, often more active than the parent pyrethroids, and showed little cross resistance in a pyrethroid-resistant Puerto Rico strain of Aedes aegypti mosquitoes. Further investigation revealed that the acids can synergize not only contact repellent standards but also other pyrethroid components as well as the parent pyrethroids themselves. Synergism by the pyrethroid acids is expressed as both increased spatial repellency and vapor toxicity as well as human bite protection. Electrophysiological studies confirmed that pyrethroid acids (100 µM) had no effect on neuronal discharge in larval Drosophila melanogaster CNS and were detected by electroantennography, and there was little resistance to olfactory sensing of these acids in antennae from Puerto Rico strain mosquitoes carrying kdr mutations. Thus, the data suggest that the pyrethroid acids have a different mode of action than the parent pyrethroids, unrelated to the voltage-sensitive sodium channel. The results highlight the potential of pyrethroid acids to be useful in future repellent formulations.

Emerging role of autoantibodies against appetite-regulating neuropeptides in eating disorders.

OBJECTIVE: Recent findings of autoantibodies directed against melanocortin peptides suggest that these autoantibodies may represent a source of variability in peptidergic signaling that can be responsible for altered appetite and emotion in eating disorders. However, it is still unknown if autoantibodies directed against some other appetite-regulating neuropeptides and peptide hormones exist in healthy human subjects and if these autoantibodies can regulate appetite and emotion. METHODS: We determined the presence of autoantibodies against some key appetite-regulating neuropeptides and peptide hormones in sera of human subjects and in rats, and used animal models to study the role of alpha-melanocyte-stimulating hormone autoantibodies in food intake and anxiety. RESULTS: Immunoglobulin G and A autoantibodies against alpha-melanocyte-stimulating hormone, neuropeptide Y, agouti-related protein, ghrelin, leptin, and some other neuropeptides or peptide hormones involved in appetite control were present in healthy humans and rats. Animal models including active and passive transfer showed that alpha-melanocyte-stimulating hormone autoantibodies are involved in the regulation of feeding and anxiety. Sequence homology was found between neuropeptides and proteins from some members of intestinal microflora, whereas germ-free rats showed altered levels of autoantibodies directed against several neuropeptides. CONCLUSION: Autoantibodies directed against appetite-regulating neuropeptides and peptide hormones are emerging as important participants in the peptidergic mechanisms controlling motivated behavior. Furthermore, these autoantibodies could provide a link in the gut-brain axis and may represent new biological targets for the diagnosis and treatment of eating disorders.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster.

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Fatty Acid and Related Potassium Kv2 Channel Blockers: Toxicity and Physiological Actions on Mosquitoes.

Potassium channels constitute a very diverse group involved in neural signaling, neuronal activity, membrane potential maintenance, and action potential generation. Here, we tested the mammalian potassium channel blockers TRAM-34 and 5-hydroxydecanoate (5-HDC), as well as certain fatty acids (FA) that might fit in the lumen of the pore and block channel activity by obstructing K⁺ ion passage. Kv channel blockers could be leads for a novel pesticide type. Insecticidal activity was assessed by topical application to Anopheles gambiae adult mosquitoes, paralysis in a headless larval assay, at the cellular level with patch-clamp recordings of engineered HEK cells expressing AgKv2.1 channels, as well as central nervous system recordings from larval Drosophila melanogaster. With only one hydroxyl group difference, decanoic acid had a consistently greater effect than 5-HDC in blocking Kv channels, paralyzing larvae, and killing mosquitoes. The 11-dansylamino undecanoic acid (DAUDA) blockage of eukaryotic Kv channels is demonstrated for the first time, but it failed to kill adult mosquitoes. We synthesized alkyl esters from DAUDA and decanoic acid in an effort to improve cuticular penetration, but it had little impact upon adult toxicity. TRAM-34 and rolipram did not show activity on Kv channels nor potent insecticidal effect on adult mosquitoes. Furthermore, co-application of test compounds with permethrin did not increase mortality in adults. In conclusion, the compounds tested had modest insecticidal and synergistic activity.

Intestinal inflammation influences α-MSH reactive autoantibodies: relevance to food intake and body weight.

Autoantibodies reacting with alpha-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide, are involved in regulation of feeding. In this work we studied if intestinal inflammation (mucositis) may influence α-MSH autoantibodies production relevant to food intake and body weight. Mucositis and anorexia were produced in Sprague-Dawley rats by methotrexate (MTX, 2.5mg/kg/day, for three days, subcutaneously). Plasma levels of total IgG and of α-MSH autoantibodies were measured during and after MTX-induced mucositis and were compared with pair-fed and ad libitum-fed controls. Effects of intraperitoneal injections of rabbit anti-α-MSH IgG (3 or 10 µg/day/rat) on MTX-induced anorexia and on plasma α-MSH peptide concentration were separately studied. Here we show that in MTX rats, intestinal mucositis and anorexia were accompanied by decreased plasma levels of both total IgG and of α-MSH autoantibodies while refeeding was characterized by their elevated levels. In spite of similar food intake in MTX and pair-fed rats, recovery of body weight was delayed by at least 1 week in the MTX group. During refeeding and body weight deficit in MTX rats, α-MSH IgG autoantibody levels correlated negatively with food to water intake ratios. Injections of anti-α-MSH IgG induced a dose-dependent attenuation of food intake and body weight regain in MTX-treated rats accompanied by increased concentrations of α-MSH peptide which correlated positively with plasma levels of α-MSH autoantibodies. These data show that intestinal inflammation, independently from food restriction, affects general humoral immune response which may influence food intake and body weight control via modulation of α-MSH plasma concentration by α-MSH reactive autoantibodies.

Anti-neuropeptide Y plasma immunoglobulins in relation to mood and appetite in depressive disorder.

Depression and eating disorders are frequently associated, but the molecular pathways responsible for co-occurrence of altered mood, appetite and body weight are not yet fully understood. Neuropeptide Y (NPY) has potent antidepressant and orexigenic properties and low central NPY levels have been reported in major depression. In the present study, we hypothesized that in patients with major depression alteration of mood, appetite and body weight may be related to NPY-reactive autoantibodies (autoAbs). To test this hypothesis, we compared plasma levels and affinities of NPY-reactive autoAbs between patients with major depression and healthy controls. Then, to evaluate if changes of NPY autoAb properties can be causally related to altered mood and appetite, we developed central and peripheral passive transfer models of human autoAbs in mice and studied depressive-like behavior in forced-swim test and food intake. We found that plasma levels of NPY IgG autoAbs were lower in patients with moderate but not with mild depression correlating negatively with the Montgomery-Åsberg Depression Rating Scale scores and with immobility time of the forced-swim test in mice after peripheral injection of autoAbs. No significant differences in NPY IgG autoAb affinities between patients with depression and controls were found, but higher affinity of IgG autoAbs for NPY was associated with lower body mass index and prevented NPY-induced orexigenic response in mice after their central injection. These data suggest that changes of plasma levels of anti-NPY autoAbs are relevant to altered mood, while changes of their affinity may participate in altered appetite and body weight in patients with depressive disorder.

Autoantibodies reacting with vasopressin and oxytocin in relation to cortisol secretion in mild and moderate depression.

BACKGROUND: Abnormal vasopressin (VP) and oxytocin (OT) signaling may contribute to the altered activity of the hypothalamo-pituitary-adrenal (HPA) axis in major depression; however, the underlying mechanisms remain uncertain. This study characterized plasma levels and affinities of OT- and VP-reactive autoantibodies (autoAbs) in relation to disease severity and plasma cortisol response to physical exercise in patients with mild and moderate depression and healthy controls. METHODS: Physical exercise was used to elicit plasma cortisol response in 23 male patients with depression and 20 healthy controls and plasma samples were obtained before and after the exercise. Just before the exercise, patients and controls were evaluated by the Montgomery and Åsberg Depression Rating Scale (MADRS) and divided according to depression severity (14 mild and 9 moderate). Plasma levels of total and free VP- and OT-reactive IgG, IgA and IgM autoAbs were measured by ELISA and affinity of IgG and IgM autoAbs were measured by plasmon resonance technique at baseline before the exercise and analyzed with relation to the MADRS and cortisol response. Immunohistochemistry was used to evaluate autoAbs binding to the rat hypothalamus. RESULTS: Plasma levels of OT- and VP-reactive total IgG autoAbs were lower in patients with moderate depression vs. controls and patients with mild depression. Plasma levels of both OT- and VP-free IgG autoAbs were negatively correlated with MADRS scores. Affinity values of IgG and IgM autoAbs for both OT and VP displayed 100 fold variability among patients or controls but no significant group differences were found. Patients with moderate depression displayed blunted response of cortisol secretion to physical exercise. Baseline levels of VP total IgG and IgM autoAbs correlated negatively and VP-free IgG autoAbs correlated positively with plasma cortisol after physical exercise. Immunostaining of magnocellular hypothalamic neurons of the supraoptic and paraventricular nuclei by plasma IgG was present in 35% of the depression and in 14% of the controls groups, but this staining was not abolished by plasma preabsorption with OT or VP peptides. CONCLUSION: These data show that changes of levels but not affinity of OT- and VP-reactive autoAbs can be associated with the altered mood in subjects with moderate depression and that levels of VP-reactive autoAbs are associated with cortisol secretion.

Ghrelin reactive autoantibodies in restrictive anorexia nervosa.

OBJECTIVE: Subjects with restrictive anorexia nervosa (AN) display increased basal plasma levels of ghrelin that normalize after refeeding. The mechanism responsible for increased ghrelin levels in AN is unknown. We studied if changes of ghrelin reactive autoantibodies (autoAbs) could explain elevated plasma ghrelin in AN. METHODS: Plasma levels of autoAbs reactive with ghrelin and des-acyl ghrelin were measured by enzyme-linked immunosorbent assay in subjects with AN before and 1 mo after hospitalization (refeeding) and compared with healthy controls and with plasma levels of ghrelin peptides. RESULTS: Decreased levels of immunoglobulin (Ig) G, IgM, and IgA classes of autoAbs reacting with acyl ghrelin were found in patients with AN. Addition of des-acyl ghrelin but not of acyl ghrelin peptides at 10(-8) M to plasma before enzyme-linked immunosorbent assay showed in patients with AN but not in controls high levels of IgG autoAbs reacting with des-acyl ghrelin as a result of dissociation of des-acyl ghrelin autoAbs in immune complexes. Plasma levels of acyl and des-acyl ghrelin peptides correlated negatively with des-acyl ghrelin IgG autoAbs. Body mass index, which improved after refeeding, correlated with an increase of acyl ghrelin IgM autoAbs. CONCLUSION: These results show that in patients with AN, ghrelin IgG autoAbs exist mainly as immune complexes with des-acyl ghrelin accompanied by a decrease of a free fraction of these autoAbs binding acylated and des-acyl ghrelin. This decrease of bioavailable ghrelin autoAbs may underlie a long-term elevation of plasma ghrelin levels and the resulting phenomenon of ghrelin resistance in malnourished patients with AN.

Increased immune complexes of hypocretin autoantibodies in narcolepsy.

BACKGROUND: Hypocretin peptides participate in the regulation of sleep-wake cycle while deficiency in hypocretin signaling and loss of hypocretin neurons are causative for narcolepsy-cataplexy. However, the mechanism responsible for alteration of the hypocretin system in narcolepsy-cataplexy and its relevance to other central hypersomnias remain unknown. Here we studied whether central hypersomnias can be associated with autoantibodies reacting with hypocretin-1 peptide present as immune complexes. METHODOLOGY: Serum levels of free and dissociated (total) autoantibodies reacting with hypocretin-1 peptide were measured by enzyme-linked immunosorbent assay and analyzed with regard to clinical parameters in 82 subjects with narcolepsy-cataplexy, narcolepsy without cataplexy or idiopathic hypersomnia and were compared to 25 healthy controls. PRINCIPAL FINDINGS: Serum levels of total but not free IgG autoantibodies against hypocretin-1 were increased in narcolepsy-cataplexy. Increased levels of complexed IgG autoantibodies against hypocretin-1 were found in all patients groups with a further increase in narcolepsy-cataplexy. Levels of total IgM hypocretin-1 autoantibodies were also elevated in all groups of patients. Increased levels of anti-idiotypic IgM autoantibodies reacting with hypocretin-1 IgG autoantibodies affinity purified from sera of subjects with narcolepsy-cataplexy were found in all three groups of patients. Disease duration correlated negatively with serum levels of hypocretin-1 IgG and IgM autoantibodies and with anti-idiotypic IgM autoantibodies. CONCLUSION: Central hypersomnias and particularly narcolepsy-cataplexy are characterized by higher serum levels of autoantibodies directed against hypocretin-1 which are present as immune complexes most likely with anti-idiotypic autoantibodies suggesting their relevance to the mechanism of sleep-wake cycle regulation.

Chemotherapy-induced anorexia is accompanied by activation of brain pathways signaling dehydration.

BACKGROUND AND AIMS: Cancer chemotherapy is accompanied by anorexia and mucositis. To clarify the mechanisms of chemotherapy-induced anorexia, we studied the expression of c-fos and appetite-regulating neuropeptidergic and inflammatory mediators in the hypothalamus of rats treated with methotrexate (MTX). METHODS: Sprague-Dawley rats received MTX (2.5mg/kg, subcutaneously) on three consecutive days and were compared with ad libitum- and pair-fed control rats five days after the first injection. RESULTS: MTX administration inhibited food and water intake and induced lean and fat mass losses. MTX also induced mucositis and diarrhea without changes in plasma osmolality. Pair-fed rats lost a similar amount of body weight but had no mucositis or diarrhea. Increased number of c-fos positive hypothalamic vasopressin neurosecretory neurons as well as numerous c-fos positive cells in the subfornical organ and in the organum vasculosum of the lamina terminalis were found in MTX-treated as compared to control or pair-fed rats. In both MTX and pair-fed rats, a decrease of hypothalamic proopiomelanocortin mRNA expression and low plasma levels of interleukin-1ß (IL-1ß) were found reflecting probably the energy deficit. No significant changes of IL-1ß mRNA expression and intensity of microglial staining in the hypothalamus were found in MTX-treated rats. CONCLUSION: The pattern of c-fos expression in the hypothalamus during MTX treatment is similar to that seen with systemic dehydration, which is known to cause anorexia. No evidence of inflammatory origin of anorexia was found, suggesting that chemotherapy accompanied by mucositis and diarrhea may cause anorexia associated with systemic dehydration.