Connar Stanley James Westgate

Background: Idiopathic intracranial hypertension (IIH) is a neurometabolic disorder most commonly affecting young females with obesity. The disease presents with daily headaches and papilledema caused by raised intracranial pressure (ICP). Although hormonal dysfunction has been implicated in IIH, the connection between altered steroid hormones and choroid plexus (CP) physiology is unclear. The objective of this translational study was to define the steroid hormone profile in newly diagnosed, treatment naïve patients with IIH and identify the influence of steroid hormones on CP functionality. Methods: A case-control study of prospectively recruited cohort of female patients with newly diagnosed treatment naïve IIH ( n = 56) and age, sex and body mass index matched healthy controls ( n = 34) had comprehensive steroid profiling of paired serum and cerebrospinal fluid (CSF) via liquid chromatography tandem mass-spectrometry, assessing 18 steroids. Steroid profiles in 10 patients in remission, with matching samples at time of diagnosis, were also assessed. Complementary preclinical studies in rat and mice were performed to examine the effects of sex steroid hormones on CP function. In CP, mRNA-sequencing and calcium dynamics were assessed after sex steroid hormone exposure. Results: Patients displayed a distinct steroid profile, with six and three differential steroids in the CSF and serum, respectively. The progestins, pregnenolone (q < 0.01) and progesterone (q < 0.05), as well as the androgens testosterone (q < 0.01) and dehydroepiandrosterone-sulphate (q < 0.001), were higher in the CSF of patients compared to controls. In the serum, progesterone (q < 0.01) was higher, with the androgens, dehydroepiandrosterone (q < 0.01) and dihydrotestosterone (q < 0.001) lower. At remission, CSF pregnenolone (q < 0.05) and testosterone (q < 0.05) normalized. Progesterone ( P < 0.0001 ) , and testosterone ( P < 0.0001) increased intracellular calcium levels. Conclusions: We demonstrate profound steroid hormone deviation in patients where steroid dysfunction normalizes at remission. Our findings suggest that progesterone and testosterone can activate CP. This potential link between steroid hormones and CP dynamics could influence CSF secretion and contribute to a raised intracranial pressure, making it an interesting future study target in IIH pathophysiology.

Objectives: The pathophysiology of idiopathic intracranial hypertension (IIH) is poorly understood and disease‐specific biomarkers are lacking. We aimed to shed light on IIH pathophysiology and identify disease‐specific biomarkers. Methods: This prospective cross‐sectional cohort study included patients with new‐onset IIH and age‐, body mass index‐, and sex‐matched healthy controls from 2 tertiary Danish Headache Centers (discovery cohort). Liquid chromatography coupled to tandem mass spectrometry analysis was used to measure reproducible proteomic profiles for paired serum and cerebrospinal fluid (CSF) samples. Results were validated using 3 validation cohorts comprising patients with IIH (pwIIH), cerebral sinus venous thrombosis (pwCSVT), and normal pressure hydrocephalus (pwNPH). Results: We included 53 pwIIH and 35 controls. PwIIH presented with papilledema and increased median lumbar puncture opening pressure (interquartile range [IQR]) (38.0 [33–46] vs 19.0 [17.0–23.0] cmH ₂ O; p < 0.001) compared to controls. Machine learning analysis identified 20 IIH‐predicting proteins for serum and CSF segregating pwIIH, but neither pwCSVT nor pwNPH, from controls with areas under the curve (AUC) of 0.84 and 0.92 (pwIIH, discovery cohort), 0.99 and 0.90 (pwIIH, validation cohort 1), 0.63 (plasma; pwCSVT), and 0.67 (CSF; pwNPH), respectively. Serum carbonic anhydrases (CA) 1 and 2 were upregulated and among the most IIH‐predicting proteins as were amyloid precursor protein (APP), S100P, and S100A12. Interpretation: We identified a panel of independently validated IIH‐specific candidate biomarkers. The serum candidate biomarkers pointed toward CA2 driven CSF hypersecretion in IIH providing a possible explanation for the therapeutic efficacy of CA inhibitors. The regulation of markers associated with neuronal impairment (APP, S100P/S100A12) confirmed the non‐benign character of IIH. ANN NEUROL 2026

Background: Cluster headache is associated with compensated hypogonadism in males, suggesting impaired testicular steroidogenesis. It is unknown if adrenal steroidogenesis is dysregulated and how this is linked to cluster headache pathophysiology. We therefore aimed to define the adrenal steroid profile in cluster headache and define the differences between the phases of episodic cluster headache. We secondarily aimed to assess whether the steroid profile could distinguish between chronic cluster headache and episodic cluster headache. Methods: A prospective case-control study containing adult males with chronic cluster headache ( n = 60), paired episodic cluster headache in and out of bout ( n = 60) and healthy controls ( n = 60). A fasted serum steroid profile for 16 steroid hormones was assessed via liquid chromatography tandem mass-spectrometry, assessing mineralocorticoids, glucocorticoids, and androgens. Explorative machine learning was used to segregate the cluster headache states. Results: The mineralocorticoids 11-deoxycorticosterone ( P < 0.05), corticosterone ( P < 0.05) and aldosterone ( P < 0.05) were lower in those with episodic cluster headache compared to controls. 11-deoxycorticosterone ( P < 0.01), corticosterone ( P < 0.01), and the glucocorticoids 11-deoxycortisol ( P < 0.01) and cortisol ( P < 0.05) and cortisol/DHEAS ratio ( P < 0.001) are lower in episodic in bout compared to their paired remission state. Those with chronic cluster headache had lower concentrations of 17-OH pregnenolone ( P < 0.05) and DHEAS ( P < 0.001) and a higher cortisol/DHEAS ratio ( P < 0.01). The steroid hormone profile distinguished between chronic and episodic cluster in bout with a specificity of 86% (95%CI: 69%-90%), sensitivity of 81% (95%CI: 73%-93%) and AUC of 0.92 ( P < 0.0001). Attack frequency and time from last attack correlated with the degree of steroid dysfunction in episodic cluster headache but not in chronic cluster headache. Conclusion: We identify distinct steroid hormone profiles in episodic and chronic cluster headache. Episodic in bout was associated with reduced adrenal steroid synthesis that resolves in remission. Chronic cluster headache was associated with stress induced androgen suppression. Together, these differences are likely secondary to cluster headache and may reflect distinct neurobiological bases underlying episodic and chronic cluster headache. Clinical trail number: Not applicable.

Background: The transient receptor potential melastatin 3 (TRPM3) ion channel has been implicated in sensory modulation and pain transmission and may contribute to migraine pathophysiology through calcitonin gene‐related peptide (CGRP) release in the trigeminovascular system. This study aimed to investigate TRPM3 activation and its role in CGRP release, vasodilatory responses, and migraine‐relevant behaviors using preclinical models. Methods: Male and female Sprague‐Dawley rats were used to evaluate CGRP release from trigeminal ganglia (TG) and dura mater following stimulation with the TRPM3 agonist CIM0216. CGRP levels were quantified using ELISA. Myograph studies assessed vasodilation in the middle cerebral artery (MCA) and middle meningeal artery. Immunohistochemistry was used to examine TRPM3 and CGRP localization in TG, dura mater, MCA, and human dura mater. Potential behavioral responses to subcutaneous CIM0216 administration were assessed via mechanical sensitivity tests. Calcium responses to CIM0216 were investigated on CGRP neurons in the TG of transgenic female mice. Results: TRPM3 channel activation with CIM0216 triggered CGRP release from TG at 100 μM, with indications of enhanced release in female tissues. Immunohistochemistry confirmed colocalization of the TRPM3 channel and CGRP in TG neurons. Additionally, TRPM3 expression was detected in arterial structures, indicating potential involvement in vascular regulation. Although CIM0216 induced CGRP release ex vivo, subcutaneous CIM0216 administration was unable to induce allodynia‐like symptoms in rats. Application of CIM0216 induced an increase of cytosolic calcium in trigeminal CGRP neurons. Conclusions: TRPM3 activation triggers CGRP release and vasodilatation. The findings that TRPM3 induced CGRP release support further investigation of TRPM3 as a therapeutic target for migraine.

Objective: Investigate the immune system's role in cluster headache by analyzing cytokines in people with cluster headache and headache‐free controls, and explore if certain cytokines could predict a specific phenotype.Methods: We measured 45 cytokines in adult participants from the Danish Cluster Headache Biobank in a prospective case–control setup. People with cluster headache were diagnosed according to the International Classification of Headache Disorders third‐edition. Controls were matched for age and sex.Results: A total of 412 were analyzed deriving from 99 with chronic cluster headache, 108 with episodic cluster headache (ECH) in bout, 105 with ECH in remission, and 100 successfully matched controls. Compared with controls, 13 cytokines were altered for ECH in bout (p < 0.05), 3 in remission (p < 0.05), and 10 for chronic cluster headache (p < 0.05). Oncostatin m was significantly elevated in all 3 disease states compared with controls (p < 0.05). Overall, the investigated cytokines showed distinct patterns of alterations between chronic cluster headache and episodic cluster headache in bout and, interestingly, IL‐1β was significantly associated with having chronic cluster headache rather than episodic in bout in a logistic regression model adjusting for potential confounders (p < 0.05).Interpretation: Findings show that the immune system is altered in all 3 states of cluster headache compared with controls. Oncostatin m was elevated, constituting a promising target for future studies. The distinct alterations between episodic and chronic cluster headache are striking and urges further research of the immune system in cluster headache to better understand its potential role in prediction of disease activity and treatment response. ANN NEUROL 2025;98:4–18

Objective: Idiopathic intracranial hypertension (IIH) is a neurometabolic disease with an increasing incidence. The pathophysiology is unknown, but improvement of diagnosis and management requires discovery of novel biomarkers. Our objective was to identify such candidate biomarkers in IIH, and secondarily, test for associations between identified metabolites and disease severity.Methods: This is a prospective case–control study with collection of cerebrospinal fluid (CSF), serum, and clinical data from new‐onset, treatment‐naïve patients with IIH (n = 60). Patients were included consecutively from 2 tertiary headache centers in Denmark, and age, sex, and body mass index (BMI) ‐matched healthy controls (n = 35) were recruited. Clinical data were retrieved at ocular remission (n = 55). Samples were analyzed using non‐targeted mass spectrometry.Results: Serum sphingosine 1‐phosphate (S1P), adenosine, and glutamate were 0.46‐fold (q < 0.0001), 0.25‐fold (q = 0.0048), and 0.44‐fold (q < 0.0001) lower, respectively, in IIH. CSF stearoyl‐lysophosphatidylcholine (LysoPC‐18) and 2‐palmitoyl‐lysophosphatidylcholine (LysoPC‐16) were 0.42 (q = 0.0025) and 0.37 (q < 0.001) ‐fold lower. LysoPC‐18 was higher in patients with moderate–severe versus mild papilledema (p = 0.022). LysoPC‐18 correlated positively with retinal nerve fiber layer thickness (p = 0.0012, r = 0.42) and inversely with mean deviation on automated perimetry (p = 0.01, r = −0.35). Higher baseline serum S1P (p = 0.018) and lower CSF LysoPC‐16 (p = 0.003) were associated with optic nerve atrophy at ocular remission. Pathway analysis suggests dysregulated lipid metabolism and redox disturbances in new‐onset IIH.Interpretation: We identify perturbed metabolism in new‐onset IIH. S1P and LysoPC‐16 demonstrate potential prognostic value due to association with subsequent optic nerve atrophy. This association between specific, differential metabolites and outcome provides substantial evidence for novel biomarkers of clinical significance that should be the focus of further targeted studies. ANN NEUROL 2024;96:595–607

Objective: Androgens have been hypothesized to be involved in the pathophysiology of cluster headache due to the male predominance, but whether androgens are altered in patients with cluster headache remains unclear.Methods: We performed a prospective, case‐controlled study in adult males with cluster headache. Sera were measured for hormones including testosterone, luteinizing hormone (LH), and sex hormone‐binding globulin in 60 participants with episodic cluster headache (during a bout and in remission), 60 participants with chronic cluster headache, and 60 age‐ and sex‐matched healthy controls. Free testosterone (fT) was calculated according to the Vermeulen equation. Shared genetic risk variants were assessed between cluster headache and testosterone concentrations.Results: The mean fT/LH ratio was reduced by 35% (95% confidence interval [CI]: 21%–47%, p < 0.0001) in patients with chronic cluster headache and by 24% (95% CI: 9%–37%, p = 0.004) in patients with episodic cluster headache compared to controls after adjusting for age, sleep duration, and use of acute medication. Androgen concentrations did not differ between bouts and remissions. Furthermore, a shared genetic risk allele, rs112572874 (located in the intron of the microtubule associated protein tau (MAPT) gene on chromosome 17), between fT and cluster headache was identified.Interpretation: Our results demonstrate that the male endocrine system is altered in patients with cluster headache to a state of compensated hypogonadism, and this is not an epiphenomenon associated with sleep or the use of acute medication. Together with the identified shared genetic risk allele, this may suggest a pathophysiological link between cluster headache and fT. ANN NEUROL 2024;95:1149–1161

Background: Female sex is a known risk factor of brain disorders with raised intracranial pressure (ICP) and sex hormones have been suggested to alter cerebrospinal fluid (CSF) dynamics, thus impairing ICP regulation in CSF disorders such as idiopathic intracranial hypertension (IIH). The choroid plexus (CP) is the tissue producing CSF and it has been hypothesized that altered hormonal composition could affect the activity of transporters involved in CSF secretion, thus affecting ICP. Therefore, we aimed to investigate if expression of various transporters involved in CSF secretion at CP were different between males and females and between females in different estrous cycle states. Steroid levels in serum was also investigated. Methods: Female and male rats were used to determine sex-differences in the genes encoding for the transporters Aqp1 and 4, NKCC1, NBCe2, NCBE; carbonic anhydrase enzymes II and III (CA), subunits of the Na⁺/K⁺-ATPase including Atp1a1, Atp1b1 and Fxyd1 at CP. The estrous cycle stage metestrus (MET) and estrous (ES) were determined before euthanasia. Serum and CP were collected and subjected to RT-qPCR analysis and western blots. Serum was used to measure steroid levels using liquid chromatography tandem mass spectrometry (LC–MS/MS). Results: Significant differences in gene expression and steroid levels between males and ES females were found, while no differences were found between male and MET females. During ES, expression of Aqp1 was lower (p < 0.01) and NKCC1 was higher in females compared to males. CAII was lower while CAIII was higher in ES females (p < 0.0001). Gene expression of Atp1a1 was lower in ES compared to male (p = 0.0008). Several of these choroidal genes were also significantly different in MET compared to females in ES. Differences in gene expression during the estrus cycle were correlated to serum level of steroid hormones. Protein expression of AQP1 (p = 0.008) and CAII (p = 0.035) was reduced in ES females compared to males. Conclusions: This study demonstrates for the first time that expression at CP is sex-dependent and markedly affected by the estrous cycle in female rats. Further, expression was related to hormone levels in serum. This opens a completely new avenue for steroid regulation of the expression of CSF transporters and the close link to the understanding of CSF disorders such as IIH.