Anja Holm

Despite urgent need for better analgesics, drug development has been complicated by incomplete bench-to-bedside translation. A likely contributing factor is the poor connection between the experimenter-evoked pain-related measures often used in preclinical models, and the clinical symptoms, like broader functional impairment, reported by patients. Here, we asked whether vertical climbing, a species-specific elective behaviour in mice, could serve as a measure of pain-related impairment. We tested male and female mice in a wide range of pain-related models including models of post-operative pain, inflammatory injury, osteoarthritis, rheumatoid arthritis, cancer-induced bone pain, fibrous dysplasia, neuropathic pain, migraine-related pain and facial neuropathy. Vertical climbing was reduced in all models, across five different laboratories, demonstrating the robustness and consistency of this type of behaviour as a novel pain-related outcome measure. Reduced climbing correlated significantly with established pain-related measures, but its time course diverged from that of evoked mechanical allodynia, while aligned more closely with other non-evoked behaviours, like burrowing, home cage activity, wheel running or weight bearing. Pharmacological validation was bidirectional: gabapentin restored climbing in mice with neuropathic injury, and naltrexone reinstated the climbing deficit in mice that had recovered from intraplantar CFA, consistent with persistent pain held in remission by endogenous opioid signalling. Overall, our results indicate that voluntary vertical climbing is a stable, elective behaviour that provides a robust and translationally relevant measure of pain-related functional impairment in mice.

Adenosine triphosphate-sensitive potassium (K _ATP ) channel opener levcromakalim is a potent inducer of vasodilation, headache, and migraine attacks in humans and tactile hypersensitivity in mice. Other migraine-inducing agents, such as nitric oxide (NO) donors, calcitonin gene-related peptide, and pituitary adenylate cyclase-activating polypeptide, are thought to activate second messengers leading to K _ATP opening. However, the mechanism by which K _ATP channel opening leads to migraine remains unclear. In this study, we investigated the contribution of nitric oxide synthase (NOS) isoforms and downstream signaling cascades in a mouse model of migraine-relevant tactile hypersensitivity induced by repeated administration of levcromakalim. The nonselective NOS inhibitor N ^G -nitro-L-arginine methyl ester (L-NAME) effectively prevented levcromakalim-induced hypersensitivity. Gene expression analysis in the dura mater suggested contributions from endothelial NOS (eNOS) and inducible NOS (iNOS). Semi-selective neuronal NOS (nNOS) inhibition with S-methyl-L-thiocitrulline or genetic deletion of neuronal NOS had minimal effects on hypersensitivity and no effect on vasodilation. By contrast, eNOS ^−/− mice were partially protected from levcromakalim-induced hypersensitivity and exhibited impaired vascular response, highlighting eNOS as a key mediator. Inhibition of iNOS with S- methylisothiourea revealed a possible contribution from iNOS as well. Surprisingly, inhibition of soluble guanylate cyclase had no effect, while the peroxynitrite decomposition catalyst FeTPPS partially attenuated hypersensitivity, implicating nitrosative stress - rather than classical NO-soluble guanylate cyclase-cGMP signaling - as the critical downstream pathway. We propose that levcromakalim induces both coupled and uncoupled eNOS activity, enhanced NO production, and generation of reactive nitrogen species, including peroxynitrite. Our findings reveal a pivotal role for eNOS and peroxynitrite in K _ATP channel-induced migraine-relevant hypersensitivity and support the targeting of nitrosative stress as a potential therapeutic strategy.

MicroRNA-22 (miR-22) is a negative regulator of mitochondrial biogenesis, as well as lipid and glucose metabolism, in metabolically active tissues. Silencing miR-22 holds promise as a potential treatment of obesity and metabolic syndrome, as it restores metabolic capacity—enhancing oxidative metabolism—and reduces ectopic fat accumulation in chronic obesity, a driver of impaired metabolic flexibility and muscle mass loss. Intramuscular adipose accumulation and defective mitochondrial function are features associated with obese-mediated muscle atrophy and hallmarks of neuromuscular disorders such as Duchenne muscular dystrophy. Therefore, miR-22 could represent a compelling molecular target to improve muscle health across various muscle-wasting conditions. This study describes a pharmacological strategy for the inhibition of miR-22 in skeletal muscle by employing a mixmer antisense oligonucleotide (ASO, anti-miR-22). Administration of the ASO in a mouse model of obesity positively modulated myogenesis while protecting dystrophic mice from muscle function decline, enhancing fatigue resistance, and limiting pathological fibrotic remodeling. Mechanistically, we show that anti-miR-22 treatment promotes derepression of genes involved in mitochondrial homeostasis, favoring oxidative fiber content regardless of the disease model, thus promoting a more resilient phenotype. Furthermore, we suggest that miR-22 inhibition increases autophagy by transcriptional activation of multiple negative regulators of mammalian target of rapamycin (mTOR) signaling to decrease immune infiltration and fibrosis. These findings position miR-22 as a promising therapeutic target for muscle atrophy and support its potential to restore muscle health.

ATP-sensitive potassium (K _ATP ) channel opener levcromakalim is a potent inducer of vasodilation, headache, and migraine attacks in humans and tactile hypersensitivity in mice. Other migraine-inducing agents such as nitric oxide (NO) donors, CGRP, and PACAP are thought to activate second messengers leading to K _ATP opening. Yet, how K _ATP channel opening leads to migraine remains unclear. Here, we investigated the contribution of nitric oxide synthase (NOS) isoforms and downstream signaling cascades in a mouse model of migraine-relevant tactile hypersensitivity induced by repeated administration of levcromakalim. The non-selective NOS inhibitor N ^G -nitro-L-arginine methyl ester (L-NAME) effectively prevented levcromakalim-induced hypersensitivity. Gene expression analysis in the dura mater suggested contributions from endothelial NOS (eNOS) and inducible NOS (iNOS). Semi-selective nNOS inhibition with S-methyl-L-Thiocitrulline (SMTC) or genetic deletion of neuronal NOS (nNOS) had minimal effect on hypersensitivity and no effect on vasodilation. In contrast, eNOS ^-/- mice were partially protected from levcromakalim-induced hypersensitivity and exhibited impaired vascular response, highlighting eNOS as a key mediator. Inhibition of iNOS with S- methylisothiourea (SMT) revealed a possible contribution from iNOS as well. Surprisingly, inhibition of soluble guanylate cyclase (sGC) had no effect, while the peroxynitrite decomposition catalyst FeTPPS partially attenuated hypersensitivity, implicating nitrosative stress—rather than classical NO-sGC-cGMP signaling—as the critical downstream pathway. We propose that levcromakalim induces both coupled and uncoupled eNOS activity, enhanced NO production and generation of reactive nitrogen species, including peroxynitrite. Our findings reveal a pivotal role for eNOS and peroxynitrite in K _ATP channel-induced migraine-relevant hypersensitivity and support targeting nitrosative stress as a potential therapeutic strategy.

Menstrual migraine (MM) is a debilitating neurological disorder triggered by fluctuations in ovarian hormones, particularly estrogen. While the calcitonin gene-related peptide (CGRP) system is central to migraine pathophysiology, the molecular mechanisms linking hormonal changes to CGRP signaling remain unclear. This study investigates how sex hormones regulate CGRP-related gene expression in the trigeminal ganglion (TG), with a focus on the receptor activity-modifying protein 1 (RAMP1), using both wild-type (WT) and Ramp1 knockout (KO) mice. We analyzed gene expression in the TG across the estrous cycle and following systemic estrogen or progesterone administration. Expression of Ramp1, Calca (encoding CGRPα), Calcrl (encoding CRLR), estrogen receptors, and related genes was assessed by RT-qPCR. WT and Ramp1 KO mice of both sexes were used to explore hormone-dependent gene regulation and RAMP1’s functional role. Ramp1 expression varied across the estrous cycle, peaking in proestrus and declining in diestrus, inversely correlated with Calca. Calcrl levels remained unchanged. Ramp1 expression correlated significantly with Esr2 (encoding ERβ), suggesting estrogen receptor–mediated regulation. Estrogen treatment upregulated Ramp1 in both sexes; Calca was downregulated in females but upregulated in males. Progesterone had more modest effects, primarily altering Ramp3 expression. In Ramp1 KO mice, the cyclical variation of Calca, Ramp2, and Ramp3 seen in WT mice was absent, and basal Calca expression was elevated in males, indicating that RAMP1 is essential for hormonal regulation of the CGRP system. Additionally, our findings support a role for estrogen-driven epigenetic mechanisms, such as DNA methylation, in the long-term regulation of Ramp1. This study highlights RAMP1 as a key mediator linking hormonal fluctuations to CGRP signaling in the TG. Hormone-dependent gene expression changes were sex-specific and disrupted in Ramp1 KO mice, supporting its role in migraine susceptibility. These findings provide mechanistic insight into hormonal migraine and suggest that both acute hormone signaling and long-term epigenetic regulation shape individual sensitivity to CGRP-based therapies.

Drug discovery in epilepsy began with the finding of potassium bromide by Sir Charles Locock in 1857. The following century witnessed the introduction of phenotypic screening tests for discovering antiseizure medications (ASMs). Despite the high success rate of developing ASMs, they have so far failed in eliminating drug resistance and in delivering disease‐modifying treatments. This emphasizes the need for new drug discovery strategies in epilepsy. RNA‐based drugs have recently shown promise as a new modality with the potential of providing disease modification and counteracting drug resistance in epilepsy. RNA therapeutics can be directed either toward noncoding RNAs, such as microRNAs, long noncoding RNAs (ncRNAs), and circular RNAs, or toward messenger RNAs. The former show promise in sporadic, nongenetic epilepsies, as interference with ncRNAs allows for modulation of entire disease pathways, whereas the latter seem more promising in monogenic childhood epilepsies. Here, we describe therapeutic strategies for modulating disease‐associated RNA molecules and highlight the potential of RNA therapeutics for the treatment of different patient populations such as sporadic, drug‐resistant epilepsy, and childhood monogenic epilepsies.

Endothelin-1 (ET-1) overactivity has been implicated as a factor contributing to glaucomatous neuropathy, and it has been utilized in animal models of retinal ischemia. The functional effects of long-term ET-1 exposure and possible compensatory mechanisms have, however, not been investigated. This was therefore the purpose of our study. ET-1 was delivered into rat eyes via a single intravitreal injection of 500 µM or via transgene delivery using an adeno-associated viral (AAV) vector. Retinal function was assessed using electroretinography (ERG) and the retinal expression of potentially compensatory genes was evaluated by means of qRT-PCR. Acute ET-1 delivery led to vasoconstriction and a significant reduction in the ERG response. AAV–ET-1 resulted in substantial transgene expression and ERG results similar to the acute ET-1 injections and comparable to other models of retinal ischemia. Compensatory changes were observed, including an increase in calcitonin gene-related peptide (CGRP) gene expression, which may both counterbalance the vasoconstrictive effects of ET-1 and provide neuroprotection. This chronic ET-1 ischemia model might be especially relevant to glaucoma research, mimicking the mild and repeated ischemic events in patients with long-term vascular dysfunction. The compensatory mechanisms, and particularly the role of vasodilatory CGRP in mitigating the retinal damage, warrant further investigation with the aim of evaluating new therapeutic strategies.

We are delighted to share with you our twelfth Journal Club and highlight some of the most interesting papers published recently

Circular RNAs (circRNAs) constitute an abundant class of covalently closed noncoding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys, as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. In this study, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We found that, while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes. In summary, our results demonstrate that LNA-modified ASOs complementary to backsplice-junction sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.

Significance: The hypocretin (Hcrt, also known as orexin) neuropeptides regulate sleep and wake stability, and disturbances of Hcrt can lead to sleep disorders. MicroRNAs (miRNAs) are short noncoding RNAs that fine-tune protein expression levels, and miRNA-based therapeutics are emerging. We report a functional interaction between miRNA (miR-137) and Hcrt . We demonstrate that intracellular miR-137 levels in Hcrt neurons regulate Hcrt expression with downstream effects on wakefulness. Specifically, lowering of miR-137 levels increased wakefulness in mice. We further show that the miR-137:Hcrt interaction is conserved across mice and humans, that miR-137 also regulates sleep–wake balance in zebrafish, and that the MIR137 locus is genetically associated with sleep duration in humans. Together, our findings reveal an evolutionarily conserved sleep–wake regulatory role of miR-137.