Brian Skriver Nielsen

STUDY QUESTION: Does paracetamol (N-acetyl-para-aminophenol (APAP) also known as acetaminophen) interfere with cell division and thereby disrupt pre-implantation embryonic development? SUMMARY ANSWER: Our findings suggest that APAP exposure inhibits cell cycling during pre-implantation development (PID) through the reduction of DNA synthesis, potentially resulting in early embryonic loss. WHAT IS KNOWN ALREADY: It is estimated that 10–40% of all human conceptions fail around the time of implantation. Genetic factors explain ∼50% of early embryonic loss, leaving a substantial portion of early losses without a known cause. Smoking and alcohol are established risk factors for spontaneous abortion, underscoring the importance of the chemical environment during embryonic development. STUDY DESIGN, SIZE, DURATION: To address the challenges in determining the mechanism of action and the effects of APAP during PID, we utilized a range of approaches, including in vitro, ex vivo, and in vivo methods across various models ranging from yeasts to human embryos and women of fertile age. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 90 human embryos were exposed in vitro (22 cleavage stage and 68 blastocyst-stage embryos). Endometrial tissue and uterine fluid were collected from seven women as part of an endometrial scratching procedure. Follicular fluid was collected from 26 women during transvaginal ultrasound guided aspiration of the pre-ovulatory follicles. All human material was sampled in accordance with relevant guidelines and regulations with consent from the regional scientific ethical committee of the Capital Region of Denmark and signed informed patient consent given prior to donation. All mouse experiments were approved by the Danish Animal Experiments Inspectorate and under EU directive 2010/63/EU on the protection of animals used for scientific purposes. The cultivation of the human embryonic stem cell lines H1 and HUES4 was conducted in compliance with relevant guidelines and regulations, following approval from the regional scientific ethical committee of the Capital Region of Denmark. MAIN RESULTS AND THE ROLE OF CHANCE: After exposure to APAP, we found an unequivocal repression of cell division across all used model systems. APAP exposure hindered cell cycle progression, likely by inhibiting ribonucleotide reductase, leading to reduced DNA synthesis and accumulation in the S-phase. At concentrations found in the reproductive system of women after standard dosing, APAP exposure decreased cell numbers in mouse and human cleavage-stage embryos or caused direct embryonic death. Similar exposure to mouse and human blastocyst-stage embryos resulted in a reduced inner cell mass and decreased DNA synthesis, respectively. LIMITATIONS, REASONS FOR CAUTION: A limitation of the study is the low number of available human cleavage-stage embryos. However, the high number of human blastocysts and our translational approach, which demonstrated reproducibility across various model systems, partly addressed this limitation. Further studies are needed to confirm the potential association between APAP use and pregnancy loss in prospective cohorts. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that the widely used mild analgesic APAP could contribute to early embryonic loss by impairing initial cell divisions. These results suggest that APAP should be used with caution by women attempting to conceive. Given that cell division is fundamental to all development, further investigation is now warranted to substantiate these findings and to elucidate possible implications for other developmental processes, such as gonadal and brain differentiation. STUDY FUNDING/COMPETING INTEREST(S): The research was funded by the Lundbeck Foundation (R324-2019-1881). Authors P.S. and H.K.M. were affiliated with the Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW; NNF21CC0073729). H.K.M. received a fellowship from the Novo Nordisk Foundation as part of the Copenhagen Bioscience PhD Program, supported by grant NNF19SA003544. M.H and A.D. are part of the National French Research Infrastructure France Exposome and have received funding from the European Regional Development Fund and Britanny region (Contrat Plan Etat Region, project Exposome, AIDEN 106201). TRIAL REGISTRATION NUMBER: N/A.

Background: Migraine is a common primary headache disorder with a significant genetic component influencing its pathophysiology, in which the trigeminal system plays a central role. The spontaneous trigeminal allodynia (STA) inbred rat strain is a validated migraine model that exhibits a chronic cephalic hypersensitive phenotype, responsive to specific migraine treatments. The heritable STA trait presents a unique opportunity to dissect the genetic component of migraine. Methods: STA rats were backcrossed twice with wild-type (WT) Sprague–Dawley (SD) rats and whole-genome sequencing was performed on 47 rats exhibiting either the STA or WT phenotype. mRNA and protein expression analyses were conducted in the trigeminovascular system of both rats and humans. Based on data from the STA rats, we performed an F-SKAT (i.e. sequence kernel association test for family data) analysis to investigate a potential link between families with clustering of migraine and our findings from the STA rats. Results: Sequencing of STA rats revealed a risk locus near the gene for adhesion G protein-coupled receptor L3 ( Adgrl3 ). In humans, the ADGRL3 gene showed an increased burden of rare variants segregating with migraine in families with a clustering of the condition ( p = 0.046). We found similar associations between migraine and the ADGRL3 when expanding the analyses to a genome-wide analysis including rare variants from more than one million individuals with migraine. Expression analyses of rat and human tissues confirmed that Adgrl3 is expressed in the migraine-associated trigeminovascular system. Conclusions: In this translational study, ADGRL3 was associated with both cephalic hypersensitivity in STA rats and an increased burden of rare variants in humans with migraine. The gene was expressed in the trigeminovascular system, a central pathophysiological component of cephalic pain. ADGRL3 provides novel insights into the pathophysiology of chronic cephalic pain in migraine.

Neuronal activity in the central nervous system is associated with a [K⁺]_o transient that is swiftly cleared from the extracellular space, predominantly by the Na⁺/K⁺‐ATPase. The temporal contribution of the glial (α2β2) and the neuronal (α3β1) isoform complexes remains unresolved due to the lack of an isoform‐specific inhibitor. The role of the two main brain isoform complexes in spreading depression (SD) also remains unresolved, but an SD‐mediated increase in [K⁺]_o may suppress Na⁺/K⁺‐ATPase activity and thereby promote SD propagation. As demonstrated here, inhibitor assays of purified recombinant human and heterologously expressed rat Na⁺/K⁺‐ATPase isoforms demonstrated significant selectivity for inhibition of α2β2 compared to α3β1 isoform complexes by a cyclobutyl perhydro‐1,4‐oxazepine derivative of digoxin (DcB). This phenomenon was utilized to demonstrate the temporal role of α2β2 and α3β1 in [K⁺]_o clearance in electrically stimulated rat hippocampal slices, as monitored with ion‐sensitive microelectrodes. The observations demonstrate a role of α2β2 in regulating the [K⁺]_o during electrical stimulus of hippocampal slices, whereas α3β1 serves to restore [K⁺]_o to baseline post‐stimulus. SD can be triggered by elevated [K⁺]_o but elevated [K⁺]_o did not reduce the activity of the Na⁺/K⁺‐ATPase or the glutamate transporters in hippocampal brain slices or upon heterologous expression of individual isoforms in Xenopus oocytes. Our results demonstrate the temporal contribution of the glial and neuronal Na⁺/K⁺‐ATPase isoform complexes to clearance of [K⁺]_o but do not support the concept that direct effects of elevated [K⁺]_o on Na⁺/K⁺‐ATPase activity or glutamate transport underlie SD propagation.

Do paracetamol (N-acetyl-para-aminophenol (APAP) or acetaminophen) and/or its metabolites affect human sperm Ca²⁺-signalling and function? While APAP itself does not interact with Ca²⁺-signalling in human sperm, its metabolite N-arachidonoyl phenolamine (AM404), produced via fatty acid amide hydrolase (FAAH), interferes with human sperm Ca²⁺-signalling and function through a suggested CatSper channel-dependent action. Studies have shown that adult men with high urinary levels of over-the-counter mild analgesic APAP have impaired sperm motility and increased time-to-pregnancy. This study consists of (i) an in vivo human pharmaceutical APAP exposure experiment to understand to what degree APAP reaches the sperm cells in the seminal fluid; (ii) in vitro calcium imaging and functional experiments in freshly donated human sperm cells to investigate CatSper channel-dependent activation by APAP and its metabolites; and (iii) experiments to understand the in situ capabilities of human sperm cells to form APAP metabolite AM404. Three healthy young males participated in the in vivo human exposure experiment after prior consent. Human semen samples were provided by healthy young volunteer donors after prior consent on the day of the in vitro experiments. Pharmaceutical APAP exposure reaches the seminal plasma in high micromolar concentrations and accumulates in the seminal plasma between 3 and 5 days of exposure (P-value 0.023). APAP and its primary metabolite 4-aminophenol (4AP) do not interact with human sperm Ca²⁺-signalling. Instead, the APAP metabolite AM404 produced via FAAH interferes with human sperm Ca²⁺-signalling through a CatSper-dependent action. Also, AM404 significantly increases sperm cell penetration into viscous mucous (P-value of 0.003). FAAH is functionally expressed in human sperm cells in the neck/midpiece region, as evidenced by immunohistochemical staining and the ability of human sperm cells to hydrolyse the fluorogenic FAAH substrate arachidonyl 7-amino, 4-methyl coumarin amide in an FAAH-dependent manner. Importantly, human sperm cells have the capacity to form AM404 in situ after exposure to 4AP (P-value 0.0402 compared to vehicle-treated sperm cells). The experiments were conducted largely in vitro. Future studies are needed to test whether APAP can disrupt human sperm function in vivo through the action of AM404. We hypothesize that these observations could, at least in part, be responsible for the negative association between male urinary APAP concentrations, sperm motility and time-to-pregnancy. D.M.K. is funded by the Lundbeck Foundation, grant number R324-2019-1881, and the Svend Andersen Foundation. A.R. is funded by a BRIDGE—Translational Excellence Programme grant funded by the Novo Nordisk Foundation, grant agreement number: NNF18SA0034956. All authors declare no competing interests. N/A.

Key points: The large‐pore channel pannexin 1 (Panx1) is expressed in many cell types and can open upon different, yet not fully established, stimuli. Panx1 permeability is often inferred from channel permeability to fluorescent dyes, but it is currently unknown whether dye permeability translates to permeability to other molecules. Cell shrinkage and C‐terminal cleavage led to a Panx1 open‐state with increased permeability to atomic ions (current), but did not alter ethidium uptake. Panx1 inhibitors affected Panx1‐mediated ion conduction differently from ethidium permeability, and inhibitor efficiency towards a given molecule therefore cannot be extrapolated to its effects on the permeability of another. We conclude that ethidium permeability does not reflect equal permeation of other molecules and thus is no measure of general Panx1 activity. Pannexin 1 (Panx1) is a large‐pore membrane channel connecting the extracellular milieu with the cell interior. While several activation regimes activate Panx1 in a variety of cell types, the selective permeability of an open Panx1 channel remains unresolved: does a given activation paradigm increase Panx1's permeability towards all permeants equally and does fluorescent dye flux serve as a proxy for biological permeation through an open channel? To explore permeant‐selectivity of Panx1 activation and inhibition, we employed Panx1‐expressing Xenopus laevis oocytes and HEK293T cells. We report that different mechanisms of activation of Panx1 differentially affected ethidium and atomic ion permeation. Most notably, C‐terminal truncation or cell shrinkage elevated Panx1‐mediated ion conductance, but had no effect on ethidium permeability. In contrast, extracellular pH changes predominantly affected ethidium permeability but not ionic conductance. High [K⁺]_o did not increase the flux of either of the two permeants. Once open, Panx1 demonstrated preference for anionic permeants, such as Cl⁻, lactate and glutamate, while not supporting osmotic water flow. Panx1 inhibitors displayed enhanced potency towards Panx1‐mediated currents compared to that of ethidium uptake. We conclude that activation or inhibition of Panx1 display permeant‐selectivity and that permeation of ethidium does not necessarily reflect an equal permeation of smaller biological molecules and atomic ions.

Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a ‘docked’ counterpart on the neighboring cell. A variety of stimuli are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate. Published studies about astrocyte hemichannel behavior, however, have been highly variable and/or contradictory. The field of connexin hemichannel research has been complicated by great variability in the experimental preparations employed, a lack of highly specific pharmacological inhibitors and by confounding changes associated with genetically modified animal models. This review attempts to critically assess the gating, inhibition and permeability of astrocytic connexin hemichannels and proposes that connexins in their hemichannel configuration act as gated pores with isoform-specific permeant selectivity. We expect that some, or all, of the controversies discussed here will be resolved by future research and sincerely hope that this review serves to motivate such clarifying investigations.

Ciliary membrane composition is controlled by transition zone (TZ) proteins such as RPGRIP1, RPGRIPL and NPHP4, which are vital for balanced coordination of diverse signalling systems like the Sonic hedgehog (Shh) pathway. Activation of this pathway involves Shh-induced ciliary accumulation of Smoothened (SMO), which is disrupted by disease-causing mutations in TZ components. Here we identify kinesin-3 motor protein KIF13B as a novel member of the RPGRIP1N-C2 domain-containing protein family and show that KIF13B regulates TZ membrane composition and ciliary SMO accumulation. KIF13B is upregulated during ciliogenesis and is recruited to the ciliary base by NPHP4, which binds to two distinct sites in the KIF13B tail region, including an RPGRIP1N-C2 domain. KIF13B and NPHP4 are both essential for establishment of a CAV1 membrane microdomain at the TZ, which in turn is required for Shh-induced ciliary SMO accumulation. Thus KIF13B is a novel regulator of ciliary TZ configuration, membrane composition and Shh signalling.