The CRTC1-SIK1 pathway regulates entrainment of the circadian clock.

Retinal photoreceptors entrain the circadian system to the solar day. This photic resetting involves cAMP response element binding protein (CREB)-mediated upregulation of Per genes within individual cells of the suprachiasmatic nuclei (SCN). Our detailed understanding of this pathway is poor, and it remains unclear why entrainment to a new time zone takes several days. By analyzing the light-regulated transcriptome of the SCN, we have identified a key role for salt inducible kinase 1 (SIK1) and CREB-regulated transcription coactivator 1 (CRTC1) in clock re-setting. An entrainment stimulus causes CRTC1 to coactivate CREB, inducing the expression of Per1 and Sik1. SIK1 then inhibits further shifts of the clock by phosphorylation and deactivation of CRTC1. Knockdown of Sik1 within the SCN results in increased behavioral phase shifts and rapid re-entrainment following experimental jet lag. Thus SIK1 provides negative feedback, acting to suppress the effects of light on the clock. This pathway provides a potential target for the regulation of circadian rhythms.

Patient fibroblast circadian rhythms predict lithium sensitivity in bipolar disorder.

Bipolar disorder is a chronic neuropsychiatric condition associated with mood instability, where patients present significant sleep and circadian rhythm abnormalities. Currently, the pathophysiology of bipolar disorder remains elusive, but treatment with lithium continues as the benchmark pharmacotherapy, functioning as a potent mood stabilizer in most, but not all patients. Lithium is well documented to induce period lengthening and amplitude enhancement of the circadian clock. Based on this, we sought to investigate whether lithium differentially impacts circadian rhythms in bipolar patient cell lines and crucially if lithium's effect on the clock is fundamental to its mood-stabilizing effects. We analyzed the circadian rhythms of bipolar patient-derived fibroblasts (n = 39) and their responses to lithium and three further chronomodulators. Here we show, relative to controls (n = 23), patients exhibited a wider distribution of circadian period (p < 0.05), and that patients with longer periods were medicated with a wider range of drugs, suggesting lower effectiveness of lithium. In agreement, patient fibroblasts with longer periods displayed muted circadian responses to lithium as well as to other chronomodulators that phenocopy lithium. These results show that lithium differentially impacts the circadian system in a patient-specific manner and its effect is dependent on the patient's circadian phenotype. We also found that lithium-induced behavioral changes in mice were phenocopied by modulation of the circadian system with drugs that target the clock, and that a dysfunctional clock ablates this response. Thus, chronomodulatory compounds offer a promising route to a novel treatment paradigm. These findings, upon larger-scale validation, could facilitate the implementation of a personalized approach for mood stabilization.

The genetics of circadian rhythms, sleep and health.

Circadian rhythms are 24-h rhythms in physiology and behaviour generated by molecular clocks, which serve to coordinate internal time with the external world. The circadian system is a master regulator of nearly all physiology and its disruption has major consequences on health. Sleep and circadian rhythm disruption (SCRD) is a ubiquitous feature in today's 24/7 society, and studies on shift-workers have shown that SCRD can lead not only to cognitive impairment, but also metabolic syndrome and psychiatric illness including depression (1,2). Mouse models of clock mutants recapitulate these deficits, implicating mechanistic and causal links between SCRD and disease pathophysiology (3-5). Importantly, treating clock disruption reverses and attenuates these adverse health states in animal models (6,7), thus establishing the circadian system as a novel therapeutic target. Significantly, circadian and clock-controlled gene mutations have recently been identified by Genome-Wide Association Studies (GWAS) in the aetiology of sleep, mental health and metabolic disorders. This review will focus upon the genetics of circadian rhythms in sleep and health.

Scaffold hopping with virtual screening from IP3 to a drug-like partial agonist of the inositol trisphosphate receptor.

Inositol 1,4,5-trisphosphate (IP3 ) is a universal signalling molecule that releases calcium from stores within cells by activating the IP3 receptor. Although chemical tools that modulate the IP3 receptor exist, none is ideal due to trade offs between potency, selectivity and cell permeability, and their chemical properties make them challenging starting points for optimisation. Therefore, to find new leads, we used virtual screening to scaffold hop from IP3 by using the program ROCS to perform a 3D ligand-based screen of the ZINC database of purchasable compounds. We then used the program FRED to dock the top-ranking hits into the IP3 binding pocket of the receptor. We tested the 12 highest-scoring hits in a calcium-release bioassay and identified SI-9 as a partial agonist. SI-9 competed with [(3) H]IP3 binding, and reduced histamine-induced calcium signalling in HeLa cells. SI-9 has a novel 2D scaffold that represents a tractable lead for designing improved IP3 receptor modulators.

NAADP regulates human platelet function.

Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid-adenine dinucleotide phosphate) is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45Ca2+ from intracellular stores and that total platelet Ca2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.

Synthesis and use of cell-permeant cyclic ADP-ribose.

Cyclic ADP-ribose (cADPR) is a second messenger that acts on ryanodine receptors to mobilize Ca(2+). cADPR has a net negative charge at physiological pH making it not passively membrane permeant thereby requiring it to be injected, electroporated or loaded via liposomes. Such membrane impermeance of other charged intracellular messengers (including cyclic AMP, inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate) and fluorescent dyes (including fura-2 and fluorescein) has been overcome by synthesizing masked analogs (prodrugs), which are passively permeant and hydrolyzed to the parent compound inside cells. We now report the synthesis and biological activity of acetoxymethyl (AM) and butoxymethyl (BM) analogs of cADPR. Extracellular addition of cADPR-AM or cADPR-BM to neuronal cells in primary culture or PC12 neuroblastoma cells induced increases in cytosolic Ca(2+). Pre-incubation of PC12 cells with thapsigargin, ryanodine or caffeine eliminated the response to cADPR-AM, whereas the response still occurred in the absence of extracellular Ca(2+). Combined, these data demonstrate that masked cADPR analogs are cell-permeant and biologically active. We hope these cell-permeant tools will facilitate cADPR research and reveal its diverse physiological functions.

The calcium-mobilizing messenger nicotinic acid adenine dinucleotide phosphate participates in sperm activation by mediating the acrosome reaction.

Before a sperm can fertilize an egg it must undergo a final activation step induced by the egg termed the acrosome reaction. During the acrosome reaction a lysosome-related organelle, the acrosome, fuses with the plasma membrane to release hydrolytic enzymes and expose an egg-binding protein. Because NAADP (nicotinic acid adenine dinucleotide phosphate) releases Ca(2+) from acidic lysosome-related organelles in other cell types, we investigated a possible role for NAADP in mediating the acrosome reaction. We report that NAADP binds with high affinity to permeabilized sea urchin sperm. Moreover, we used Mn(2+) quenching of luminal fura-2 and (45)Ca(2+) to directly demonstrate NAADP regulation of a cation channel on the acrosome. Additionally, we show that NAADP synthesis occurs through base exchange and is driven by an increase in Ca(2+). We propose a new model for acrosome reaction signaling in which Ca(2+) influx initiated by egg jelly stimulates NAADP synthesis and that this NAADP acts on its receptor/channel on the acrosome to release Ca(2+) to drive acrosomal exocytosis.

Identification of a chemical probe for NAADP by virtual screening.

Research into the biological role of the Ca(2+)-releasing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) has been hampered by a lack of chemical probes. To find new chemical probes for exploring NAADP signaling, we turned to virtual screening, which can evaluate millions of molecules rapidly and inexpensively. We used NAADP as the query ligand to screen the chemical library ZINC for compounds with similar three-dimensional shape and electrostatic properties. We tested the top-ranking hits in a sea urchin egg bioassay and found that one hit, Ned-19, blocks NAADP signaling at nanomolar concentrations. In intact cells, Ned-19 blocked NAADP signaling and fluorescently labeled NAADP receptors. Moreover, we show the utility of Ned-19 as a chemical probe by using it to demonstrate that NAADP is a key causal link between glucose sensing and Ca(2+) increases in mouse pancreatic beta cells.

Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection.

We have optimised a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from extracted RNA for clinical application. We improved the stability and reliability of the RT-LAMP assay by the addition of a temperature-dependent switch oligonucleotide to reduce self- or off-target amplification. We then developed freeze-dried master mix for single step RT-LAMP reaction, simplifying the operation for end users and improving long-term storage and transportation. The assay can detect as low as 13 copies of SARS-CoV2 RNA per reaction (25-µL). Cross reactivity with other human coronaviruses was not observed. We have applied the new RT-LAMP assay for testing clinical extracted RNA samples extracted from swabs of 72 patients in the UK and 126 samples from Greece and demonstrated the overall sensitivity of 90.2% (95% CI 83.8-94.7%) and specificity of 92.4% (95% CI 83.2-97.5%). Among 115 positive samples which Ct values were less than 34, the RT-LAMP assay was able to detect 110 of them with 95.6% sensitivity. The specificity was 100% when RNA elution used RNase-free water. The outcome of RT-LAMP can be reported by both colorimetric detection and quantifiable fluorescent reading. Objective measures with a digitized reading data flow would allow for the sharing of results for local or national surveillance.

Adenosine integrates light and sleep signalling for the regulation of circadian timing in mice.

The accumulation of adenosine is strongly correlated with the need for sleep and the detection of sleep pressure is antagonised by caffeine. Caffeine also affects the circadian timing system directly and independently of sleep physiology, but how caffeine mediates these effects upon the circadian clock is unclear. Here we identify an adenosine-based regulatory mechanism that allows sleep and circadian processes to interact for the optimisation of sleep/wake timing in mice. Adenosine encodes sleep history and this signal modulates circadian entrainment by light. Pharmacological and genetic approaches demonstrate that adenosine acts upon the circadian clockwork via adenosine A1/A2A receptor signalling through the activation of the Ca2+ -ERK-AP-1 and CREB/CRTC1-CRE pathways to regulate the clock genes Per1 and Per2. We show that these signalling pathways converge upon and inhibit the same pathways activated by light. Thus, circadian entrainment by light is systematically modulated on a daily basis by sleep history. These findings contribute to our understanding of how adenosine integrates signalling from both light and sleep to regulate circadian timing in mice.

Analogues of the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist Ned-19 indicate two binding sites on the NAADP receptor.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+)-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca(2+) release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca(2+) release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca(2+) release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca(2+) release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one low-affinity) on the NAADP receptor.

TRESK is a key regulator of nocturnal suprachiasmatic nucleus dynamics and light adaptive responses.

The suprachiasmatic nucleus (SCN) is a complex structure dependent upon multiple mechanisms to ensure rhythmic electrical activity that varies between day and night, to determine circadian adaptation and behaviours. SCN neurons are exposed to glutamate from multiple sources including from the retino-hypothalamic tract and from astrocytes. However, the mechanism preventing inappropriate post-synaptic glutamatergic effects is unexplored and unknown. Unexpectedly we discovered that TRESK, a calcium regulated two-pore potassium channel, plays a crucial role in this system. We propose that glutamate activates TRESK through NMDA and AMPA mediated calcium influx and calcineurin activation to then oppose further membrane depolarisation and rising intracellular calcium. Hence, in the absence of TRESK, glutamatergic activity is unregulated leading to membrane depolarisation, increased nocturnal SCN firing, inverted basal calcium levels and impaired sensitivity in light induced phase delays. Our data reveals TRESK plays an essential part in SCN regulatory mechanisms and light induced adaptive behaviours.

Effects of the putative lithium mimetic ebselen on pilocarpine-induced neural activity.

Lithium, commonly used to treat bipolar disorder, potentiates the ability of the muscarinic agonist pilocarpine to induce seizures in rodents. As this potentiation by lithium is reversed by the administration of myo-inositol, the potentiation may be mediated by inhibition of inositol monophosphatase (IMPase), a known target of lithium. Recently, we demonstrated that ebselen is a 'lithium mimetic' in regard to behaviours in both mice and man. Ebselen inhibits IMPase in vitro and lowers myo-inositol in vivo in the brains of mice and men, making ebselen the only known inhibitor of IMPase, other than lithium, that penetrates the blood-brain barrier. Our objective was to determine the effects of ebselen on sensitization to pilocarpine-induced seizures and neural activity. We administered ebselen at different doses and time intervals to mice, followed by injection of a sub-seizure dose of pilocarpine. We assessed seizure and neural activity by a subjective seizure rating scale, by monitoring tremors, and by induction of the immediate early gene c-fos. In contrast to lithium, ebselen did not potentiate the ability of pilocarpine to induce seizures. Unexpectedly, ebselen inhibited pilocarpine-induced tremor as well as pilocarpine-induced increases in c-fos mRNA levels. Both lithium and ebselen inhibit a common target, IMPase, but only lithium potentiates pilocarpine-induced seizures, consistent with their polypharmacology at diverse molecular targets. We conclude that ebselen does not potentiate pilocarpine-induced seizures and instead, reduces pilocarpine-mediated neural activation. This lack of potentiation of muscarinic sensitization may be one reason for the lack of side-effects observed with ebselen treatment clinically.

Sperm express a Ca2+-regulated NAADP synthase.

NAADP (nicotinic acid-adenine dinucleotide phosphate), the most potent Ca2+-mobilizing second messenger, is active in a wide range of organisms and cell types. Until now, all NAADP-producing enzymes have been thought to be members of the ADP-ribosyl cyclase family. ADP-ribosyl cyclases exhibit promiscuous substrate selectivity, synthesize a variety of products and are regulated in a limited manner, which may be non-physiological. In the present paper, we report the presence of an enzyme on the surface of sea urchin sperm that exhibits bell-shaped regulation by Ca2+ over a range (EC(50) of 10 nM and IC(50) of 50 microM) that is physiologically relevant. Uniquely, this surface enzyme possesses complete selectivity for nucleotides with a 2'-phosphate group and exhibits only base-exchange activity without any detectable cyclase activity. Taken together, these findings indicate that this novel enzyme should be considered as the first true NAADP synthase.

Cell-permeant NAADP: a novel chemical tool enabling the study of Ca2+ signalling in intact cells.

NAADP (nicotinic acid adenine dinucleotide phosphate) is a recently discovered second messenger, and as such, we have much yet to learn about its functions in health and disease. A bottleneck in this basic research is due to NAADP, like all second messengers, being charged to prevent it from leaking out of cells. This makes for effective biology, but imposes difficulties in experiments, as it must be injected, loaded via liposomes, or electroporated, techniques that are highly technically demanding and are possible only in certain single cell preparations. For the better understood second messenger inositol 1,4,5-trisphosphate, great success has been obtained with cell-permeant derivatives where the charged groups are masked through esterification. We now report NAADP-AM as a cell-permeant analogue of NAADP that is taken up into cells and induces NAADP-mediated Ca(2+) signalling. NAADP-AM is a powerful chemical tool that will be of enormous biological utility in a wide range of systems and will greatly facilitate research into the role of NAADP in health and disease.

Effects of the potential lithium-mimetic, ebselen, on brain neurochemistry: a magnetic resonance spectroscopy study at 7 tesla.

RATIONALE: Lithium is an effective treatment for bipolar disorder, but safety issues complicate its clinical use. The antioxidant drug, ebselen, may be a possible lithium-mimetic based on its ability to inhibit inositol monophosphatase (IMPase), an action which it shares with lithium. OBJECTIVES: Our primary aim was to determine whether ebselen lowered levels of inositol in the human brain. We also assessed the effect of ebselen on other brain neurometabolites, including glutathione, glutamate, glutamine, and glutamate + glutamine (Glx) METHODS: Twenty healthy volunteers were tested on two occasions receiving either ebselen (3600 mg over 24 h) or identical placebo in a double-blind, random-order, crossover design. Two hours after the final dose of ebselen/placebo, participants underwent proton magnetic resonance spectroscopy ((1)H MRS) at 7 tesla (T) with voxels placed in the anterior cingulate and occipital cortex. Neurometabolite levels were calculated using an unsuppressed water signal as a reference and corrected for individual cerebrospinal fluid content in the voxel. RESULTS: Ebselen produced no effect on neurometabolite levels in the occipital cortex. In the anterior cingulate cortex, ebselen lowered concentrations of inositol (p = 0.028, Cohen's d = 0.60) as well as those of glutathione (p = 0.033, d = 0.58), glutamine (p = 0.024, d = 0.62), glutamate (p = 0.01, d = 0.73), and Glx (p = 0.001, d = 1.0). CONCLUSIONS: The study suggests that ebselen produces a functional inhibition of IMPase in the human brain. The effect of ebselen to lower glutamate is consistent with its reported ability to inhibit the enzyme, glutaminase. Ebselen may have potential as a repurposed treatment for bipolar disorder.

Effect of the Putative Lithium Mimetic Ebselen on Brain Myo-Inositol, Sleep, and Emotional Processing in Humans.

Lithium remains the gold standard in treating bipolar disorder but has unwanted toxicity and side effects. We previously reported that ebselen inhibits inositol monophosphatase (IMPase) and exhibits lithium-like effects in animal models through lowering of inositol. Ebselen has been tested in clinical trials for other disorders, enabling us to determine for the first time the effect of a blood-brain barrier-penetrant IMPase inhibitor on human central nervous system (CNS) function. We now report that in a double-blind, placebo-controlled trial with healthy participants, acute oral ebselen reduced brain myo-inositol in the anterior cingulate cortex, consistent with CNS target engagement. Ebselen decreased slow-wave sleep and affected emotional processing by increasing recognition of some emotions, decreasing latency time in the acoustic startle paradigm, and decreasing the reinforcement of rewarding stimuli. In summary, ebselen affects the phosphoinositide cycle and has CNS effects on surrogate markers that may be relevant to the treatment of bipolar disorder that can be tested in future clinical trials.

Oxford Lithium Trial (OxLith) of the early affective, cognitive, neural and biochemical effects of lithium carbonate in bipolar disorder: study protocol for a randomised controlled trial.

BACKGROUND: Despite lithium's being the most effective drug for bipolar disorder and in clinical use for decades, we still know very little about its early effects relevant to its mode of action. METHODS/DESIGN: The Oxford Lithium Trial is a double-blind, randomised, placebo-controlled study of 6-week lithium treatment in participants with bipolar disorder and mood instability. Its aim is to identify early clinical, neurocognitive and biological effects. Participants (n = 40) will undergo an intensive battery of multi-modal investigations, including remote monitoring of mood, activity and physiology, as well as cognitive testing, fMRI and magnetoencephalography, together with biochemical and gene expression measurements to assess renal, inflammatory and circadian effects. DISCUSSION: The findings derived from this trial may be of value in predicting subsequent therapeutic response or side effects, not only relevant to the use of lithium but also providing a potential signature to help in more rapid evaluation of novel mood stabilisers. In this respect, OxLith is a step towards the development of a valid experimental medicine model for bipolar disorder. TRIAL REGISTRATION: ISRCTN91624955 . Registered on 22 January 2015.

Effects of the potential lithium-mimetic, ebselen, on impulsivity and emotional processing.

RATIONALE: Lithium remains the most effective treatment for bipolar disorder and also has important effects to lower suicidal behaviour, a property that may be linked to its ability to diminish impulsive, aggressive behaviour. The antioxidant drug, ebselen, has been proposed as a possible lithium-mimetic based on its ability in animals to inhibit inositol monophosphatase (IMPase), an action which it shares with lithium. OBJECTIVES: The aim of the study was to determine whether treatment with ebselen altered emotional processing and diminished measures of risk-taking behaviour. METHODS: We studied 20 healthy participants who were tested on two occasions receiving either ebselen (3600 mg over 24 h) or identical placebo in a double-blind, randomized, cross-over design. Three hours after the final dose of ebselen/placebo, participants completed the Cambridge Gambling Task (CGT) and a task that required the detection of emotional facial expressions (facial emotion recognition task (FERT)). RESULTS: On the CGT, relative to placebo, ebselen reduced delay aversion while on the FERT, it increased the recognition of positive vs negative facial expressions. CONCLUSIONS: The study suggests that at the dosage used, ebselen can decrease impulsivity and produce a positive bias in emotional processing. These findings have implications for the possible use of ebselen in the disorders characterized by impulsive behaviour and dysphoric mood.