Cligosiban, A Novel Brain-Penetrant, Selective Oxytocin Receptor Antagonist, Inhibits Ejaculatory Physiology in Rodents.

INTRODUCTION: Few treatments are available for men with premature ejaculation (PE); oxytocin (OT) receptor antagonism in the central nervous system (CNS) is a potential new approach. AIM: To determine if cligosiban selectively inhibits human OT receptors, penetrates the CNS, shows pharmacology in the CNS, and effects ejaculatory physiology in pre-clinical systems. METHODS: Experiments complied with United Kingdom legislation and were subject to local ethical review. In vitro potency and selectivity of cligosiban was assessed using recombinant and native OT receptor systems including both neuronal and non-neuronal cell types. Selectivity was determined over neighboring V1A, V1B, and V2 vasopressin receptors using a combination of recombinant and native vasopressin receptor assay systems. To determine an effect on central OT receptors and on ejaculation, cligosiban was evaluated in 2 anesthetized rat models-the electromyography model of ejaculatory physiology and a model of OT-mediated CNS neuronal firing. The CNS penetration of cligosiban was also determined by measuring cerebrospinal fluid and plasma drug concentrations following an intravenous (IV) infusion in rats. MAIN OUTCOME MEASURE: These were functional measures of pharmacology in vitro, in cell lines and tissues, and in vivo in rats. RESULTS: Cligosiban is a potent OT receptor antagonist, with a base dissociation constant of 5.7 nmol/L against native human uterine smooth muscle cell OT receptors. Cligosiban displays similar antagonistic potency against human recombinant and rat native OT receptors, including neuronal OT receptors. Cligosiban demonstrates >100-fold selectivity over human V1A, V1B, and V2 vasopressin receptors. In the electromyography model, cligosiban (0.9 mg/kg, IV bolus) reduced the bulbospongiosum burst pattern and contraction amplitude associated with ejaculation. In the anesthetized CNS neuronal firing model, the same dosing regimen of cligosiban (0.9 mg/kg IV bolus) modulated the OT-mediated response in the nucleus tractus solitarius. After systemic dosing to rats, cligosiban showed good CNS penetration. CLINICAL IMPLICATIONS: As the first highly selective and centrally penetrant OT receptor antagonist, cligosiban represents a promising compound to test the clinical hypothesis that antagonism of central OT receptors may be of therapeutic benefit in the treatment of PE. STRENGTH & LIMITATIONS: The pharmacology and selectivity of cligosiban is determined using functional assays in recombinant cell lines, native cell lines, and tissue. Functional outcomes in in vivo systems are linked to CNS measures of pharmacology. The translation of the animal models of ejaculation to PE in man is unproven. CONCLUSION: Cligosiban, a potent, selective OT receptor antagonist, demonstrated CNS penetration and pharmacology and, using the same dosing regimen, inhibited apomorphine-induced ejaculation in rats. Cligosiban is a promising compound to test the clinical hypothesis that antagonism of central OT receptors may be of therapeutic benefit in the treatment of PE. Wayman C, Russell R, Tang K, et al. Cligosiban, A Novel Brain Penetrant Selective Oxytocin Receptor Antagonist, Inhibits Ejaculatory Physiology in Rodents. J Sex Med 2018;15:1698-1706.

Physical and functional convergence of the autism risk genes Scn2a and Ank2 in neocortical pyramidal cell dendrites.

Dysfunction in sodium channels and their ankyrin scaffolding partners have both been implicated in neurodevelopmental disorders, including autism spectrum disorder (ASD). In particular, the genes SCN2A, which encodes the sodium channel NaV1.2, and ANK2, which encodes ankyrin-B, have strong ASD association. Recent studies indicate that ASD-associated haploinsufficiency in Scn2a impairs dendritic excitability and synaptic function in neocortical pyramidal cells, but how NaV1.2 is anchored within dendritic regions is unknown. Here, we show that ankyrin-B is essential for scaffolding NaV1.2 to the dendritic membrane of mouse neocortical neurons and that haploinsufficiency of Ank2 phenocopies intrinsic dendritic excitability and synaptic deficits observed in Scn2a+/- conditions. These results establish a direct, convergent link between two major ASD risk genes and reinforce an emerging framework suggesting that neocortical pyramidal cell dendritic dysfunction can contribute to neurodevelopmental disorder pathophysiology.

Ankyrin-B is lipid-modified by S-palmitoylation to promote dendritic membrane scaffolding of voltage-gated sodium channel NaV1.2 in neurons.

Neuronal ankyrin-B is an intracellular scaffolding protein that plays multiple roles in the axon. By contrast, relatively little is known about the function of ankyrin-B in dendrites, where ankyrin-B is also localized in mature neurons. Recently, we showed that ankyrin-B acts as a scaffold for the voltage-gated sodium channel, NaV1.2, in dendrites of neocortical pyramidal neurons. How ankyrin-B is itself targeted to the dendritic membrane is not well understood. Here, we report that ankyrin-B is lipid-modified by S-palmitoylation to promote dendritic localization of NaV1.2. We identify the palmitoyl acyl transferase zDHHC17 as a key mediator of ankyrin-B palmitoylation in heterologous cells and in neurons. Additionally, we find that zDHHC17 regulates ankyrin-B protein levels independently of its S-acylation function through a conserved binding mechanism between the ANK repeat domain of zDHHC17 and the zDHHC ankyrin-repeat binding motif of ankyrin-B. We subsequently identify five cysteines in the N-terminal ankyrin repeat domain of ankyrin-B that are necessary for ankyrin-B palmitoylation. Mutation of these five cysteines to alanines not only abolishes ankyrin-B palmitoylation, but also prevents ankyrin-B from scaffolding NaV1.2 at dendritic membranes of neurons due to ankyrin-B's inability to localize properly at dendrites. Thus, we show palmitoylation is critical for localization and function of ankyrin-B at dendrites. Strikingly, loss of ankyrin-B palmitoylation does not affect ankyrin-B-mediated axonal cargo transport of synaptic vesicle synaptotagmin-1 in neurons. This is the first demonstration of S-palmitoylation of ankyrin-B as an underlying mechanism required for ankyrin-B localization and function in scaffolding NaV1.2 at dendrites.

Synaptotagmin-7 facilitates acetylcholine release in splanchnic nerve-chromaffin cell synapses during nerve activity.

Disturbances that threaten homeostasis elicit activation of the sympathetic nervous system (SNS) and the adrenal medulla. The effectors discharge as a unit to drive global and immediate changes in whole-body physiology. Descending sympathetic information is conveyed to the adrenal medulla via preganglionic splanchnic fibers. These fibers pass into the gland and synapse onto chromaffin cells, which synthesize, store, and secrete catecholamines and vasoactive peptides. While the importance of the sympatho-adrenal branch of the autonomic nervous system has been appreciated for many decades, the mechanisms underlying transmission between presynaptic splanchnic neurons and postsynaptic chromaffin cells have remained obscure. In contrast to chromaffin cells, which have enjoyed sustained attention as a model system for exocytosis, even the Ca2+ sensors that are expressed within splanchnic terminals have not yet been identified. This study shows that a ubiquitous Ca2+-binding protein, synaptotagmin-7 (Syt7), is expressed within the fibers that innervate the adrenal medulla, and that its absence can alter synaptic transmission in the preganglionic terminals of chromaffin cells. The prevailing impact in synapses that lack Syt7 is a decrease in synaptic strength and neuronal short-term plasticity. Evoked excitatory postsynaptic currents (EPSCs) in Syt7 KO preganglionic terminals are smaller in amplitude than in wild-type synapses stimulated in an identical manner. Splanchnic inputs also display robust short-term presynaptic facilitation, which is compromised in the absence of Syt7. These data reveal, for the first time, a role for any synaptotagmin at the splanchnic-chromaffin cell synapse. They also suggest that Syt7 has actions at synaptic terminals that are conserved across central and peripheral branches of the nervous system.