Biology subjects

Toft-Bertelsen, T. L.

Publications and source records attributed to Toft-Bertelsen, T. L..

9 recordsLinked to original sources

bioRxiv2026

SPAK as a candidate mediator of TRPV4-induced NKCC1 activation and a possible pharmacological entry point in posthemorrhagic hydrocephalus

Hydrocephalus arises from pathological disturbances in cerebrospinal fluid (CSF) homeostasis, yet current treatment relies almost exclusively on neurosurgical diversion procedures that frequently require surgical revision. No specific and efficient pharmacological alternative is available as a complement to the invasive neurosurgery due to our lack of understanding of the molecular regulators of CSF secretion. By in vivo determination of CSF dynamics in rats and in vitro quantification of choroid plexus transporter activity, we demonstrate that the STE20-proline-alanine-rich kinase (SPAK) is a critical modulator of CSF secretion via its regulation of the Na+/K+/2Cl- cotransporter 1 (NKCC1) and the Na/K-ATPase. Systemic administration of a SPAK inhibitor after a mimicked hemorrhagic event attenuated posthemorrhagic hydrocephalus formation 24h post-hemorrhage. Activation of the choroid plexus transient receptor potential vanilloid 4 (TRPV4) ion channel induced hydrocephalus through CSF hypersecretion. This TRPV4-mediated hypersecretion occurred via activation of NKCC1, not the Na+/K+-ATPase, and required SPAK activity as a molecular link. Together, these findings identify SPAK as a central integrator of TRPV4-dependent signaling and choroid plexus transporter activity, positioning the TRPV4-SPAK axis as a potential pharmacological target for modulating CSF dynamics in hydrocephalus and other pressure-related pathologies.

neuroscience↗

bioRxiv2026

Discovery of Membrane Channel Modulators via DNA-Encoded Library Screening Using Native-Like Membrane Protein Nanoparticles

Developing novel drugs against membrane proteins is a major challenge in drug discovery due to the difficulty of stabilizing these targets for high-throughput screenings. Pannexin 1 (PANX1) is a membrane channel protein involved in various physiological and pathological processes, making it a promising target for drug discovery. However, efforts to develop PANX1-targeting therapeutics have been hindered by the inherent challenges of stabilizing the protein channel and conducting effective pharmacological screening. Here, we report a proof-of-concept workflow that integrates the Salipro lipid nanoparticle platform with DNA-Encoded Library screenings in a detergent-free format. In this case study, the Salipro DirectMX method was used to generate functional PANX1 nanoparticles for drug discovery and characterisation. Using a high-stringency selection strategy and computational approaches, we identified a specific set of candidate compounds with selective PANX1 enrichment. Surface Plasmon Resonance analysis confirmed the identification of hit compounds. Cryo-Electron Microscopy of the Salipro-PANX1-Compound complex provided structural insights into a potential compound binding site. Electrophysiological recordings in PANX1-expressing Xenopus laevis oocytes demonstrated dose-dependent inhibition of PANX1-mediated ion conductance by the compounds. These findings establish a robust workflow for ligand discovery against challenging membrane protein targets and provide novel chemical starting points for the development of PANX1 modulators.

biochemistry↗

bioRxiv2024

The SH Protein of Mumps Virus is a Druggable Pentameric Viroporin

Viral infections are on the rise and drugs targeting viral proteins are needed. Viroporins constitute a growing group of virus-encoded transmembrane oligomeric proteins that allow passage of small molecules across the membrane. Despite sparsity in viroporin structures, recent work has revealed diversity in both the number of transmembrane helices and oligomeric states. Here we provide evidence that the small hydrophobic protein (SH) from mumps virus is a pentameric viroporin. From extensive biophysical data, a HADDOCK model of full-length SH shows its intracellular C-terminal region to form an extended structure crucial to stabilization of the pentamer. Heterologous expression of wild type SH and variants in Xenopus laevis oocytes reveals the viroporin as a chloride channel, facilitated by conserved hydroxyl-carrying residues lining the pore. The channel function of SH is inhibited by the small-molecule BIT225, highlighting the potential for antiviral targeting through SH.

biophysics↗

bioRxiv2023

Towards new AQP4 inhibitors: ORI-TRN-002

Cerebral edema is a life-threatening condition that can cause permanent brain damage or death if left untreated. Existing therapies aim at mitigating the associated elevated intracranial pressure, yet they primarily alleviate pressure rather than preventing edema formation. Prophylactic anti-edema therapy necessitates novel drugs targeting edema formation. Aquaporin 4 (AQP4), an abundantly expressed water pore in mammalian glia and ependymal cells, has been proposed to be involved in cerebral edema formation. A series of novel compounds have been tested for their potential inhibitory effects on AQP4. However, selectivity, toxicity, functional inhibition, sustained therapeutic concentration, and delivery into the central nervous system are major challenges. Employing extensive DFT calculations, we identified a previously unreported thermodynamically stable tautomer of the recently identified AQP4-specific inhibitor TGN-020. This novel form, featuring a distinct hydrogen bonding pattern, served as a template for a COSMOsim-3D based virtual screen of proprietary compounds from Origenis. The screening identified ORI-TRN-002, an electronic homologue of TGN-020, demonstrating high solubility and low protein binding. Evaluating ORI-TRN-002 on AQP4-expressing Xenopus laevis oocytes using a high-resolution volume recording system revealed an IC50 of 2.9 {+/-} 0.6 M, establishing it as a novel AQP4 inhibitor. ORI-TRN-002 exhibits superior solubility and free fraction limitations compared to other reported AQP4 inhibitors, suggesting its potential as a promising antiedema therapy for treating cerebral edema in the future.

pharmacology and toxicology↗

bioRxiv2022

Transcriptional profiling of transport mechanisms and regulatory pathways in rat choroid plexus

BackgroundDysregulation of brain fluid homeostasis associates with brain pathologies in which fluid accumulation leads to elevated intracranial pressure. Surgical intervention remains standard care, since specific and efficient pharmacological treatment options are limited for pathologies with disturbed brain fluid homeostasis. Such lack of therapeutic targets originates, in part, from the incomplete map of the molecular mechanisms underlying cerebrospinal fluid (CSF) secretion by the choroid plexus. MethodsThe transcriptomic profile of rat choroid plexus was generated by RNA Sequencing (RNAseq) of whole tissue and epithelial cells captured by fluorescence-activated cell sorting (FACS), and compared to proximal tubules. The bioinformatic analysis comprised mapping to reference genome followed by filtering for type, location, and association with alias and protein function. The transporters and associated regulatory modules were arranged in discovery tables according to their transcriptional abundance and tied together in association network analysis. ResultsThe transcriptomic profile of choroid plexus displays high similarity between sex and species (human, rat, and mouse) and lesser similarity to another secretory epithelium, the proximal tubules. The discovery tables provide lists of transport mechanisms that could participate in CSF secretion and suggest regulatory candidates. ConclusionsWith quantification of the transport protein abundance in choroid plexus and their potentially linked regulatory modules, we envision a molecular tool to devise rational hypotheses regarding future delineation of choroidal transport proteins involved in CSF secretion and their regulation. Our vision is to obtain pharmaceutical targets towards modulation of CSF production in pathologies involving disturbed brain water dynamics.

neuroscience↗

bioRxiv2022

Lysophosphatidic acid, a CSF marker in posthemorrhagic hydrocephalus that drives CSF accumulation via TRPV4-induced hyperactivation of NKCC1

A range of neurological pathologies can cause secondary hydrocephalus. For decades, treatment has been limited to surgical CSF diversion, as still no pharmacological options exist due to the elusive molecular nature of the CSF secretion apparatus and its regulatory properties. We have now identified phospholipid lysophosphatidic acid (LPA) as a biomarker for posthemorrhagic hydrocephalus (PHH) in patients with subarachnoid hemorrhage (SAH) and mimicked our results in an animal model of intraventricular hemorrhage (IVH). Intraventricular administration of LPA caused elevated brain water content and ventriculomegaly in experimental rats, via its action as an agonist of the choroidal transient receptor potential vanilloid 4 (TRPV4) channel. TRPV4 was revealed as a novel regulator of ICP in experimental rats via its ability to modulate the CSF secretion rate through its direct activation of the Na+, K+, 2Cl- cotransporter (NKCC1) implicated in CSF secretion. Together, our data reveal that a biomarker present in brain pathologies with hemorrhagic events promotes CSF hypersecretion and ensuing brain water accumulation via its direct action on TRPV4 and its downstream regulation of NKCC1. TRPV4 may therefore be a promising future pharmacological target for pathologies involving disturbed brain fluid dynamics.

neuroscience↗

bioRxiv2022

Acetazolamide modulates intracranial pressure directly by its action on the cerebrospinal fluid secretion apparatus

Elevated intracranial pressure (ICP) is observed in many neurological pathologies, e.g. hydrocephalus and stroke. This condition is routinely relieved with neurosurgical approaches, since effective and targeted pharmacological tools are still lacking. The carbonic anhydrase inhibitor, acetazolamide (AZE), may be employed to treat elevated ICP. However, its effectiveness is questioned, its location of action unresolved, and its tolerability low. Here, we employed in vivo and ex vivo approaches to reveal the efficacy and mode of action of AZE in the rat brain. The drug effectively reduced the ICP, irrespective of the mode of drug administration and level of anaesthesia. The effect occurred via a direct action on the choroid plexus and an associated decrease in cerebrospinal fluid secretion, and not indirectly via the systemic action of AZE on renal and vascular processes. Upon a single administration, the reduced ICP endured for approximately 10 h post-AZE delivery with no long-term changes of brain water content or choroidal transporter expression. However, a persistent reduction of ICP was secured with repeated AZE administrations throughout the day. Future specific targeting of choroidal carbonic anhydrases may limit the systemic side effects, and therefore enhance the treatment tolerability and effectiveness in select patient groups experiencing elevated ICP.

neuroscience↗

bioRxiv2021

Cerebrospinal fluid formation is controlled by membrane transporters to modulate intracranial pressure

Disturbances in the brain fluid balance can lead to life-threatening elevation in the intracranial pressure (ICP), which represents a vast clinical challenge. Nevertheless, the molecular mechanisms governing cerebrospinal fluid (CSF) secretion are largely unresolved, thus preventing targeted and efficient pharmaceutical therapy of cerebral pathologies involving elevated ICP. Here, we employed experimental rats to demonstrate low osmotic water permeability of the choroid plexus, lack of an osmotic gradient across this tissue, and robust CSF secretion against osmotic gradients. Together, these results illustrate that CSF secretion occurs independently of conventional osmosis, which challenges the existing assumption that CSF production is driven entirely by bulk osmotic forces across the CSF-secreting choroid plexus. Instead, we reveal that the choroidal Na+/K+/Cl- cotransporter NKCC1, Na+/HCO3- cotransporter NBCe2, and Na+/K+-ATPase are actively involved in CSF production and propose a molecular mode of water transport supporting CSF production in this secretory tissue. Further, we demonstrate that inhibition of NKCC1 directly reduces the ICP, illustrating that altered CSF secretion may be employed as a strategy to modulate ICP. These insights identify new promising therapeutic targets against brain pathologies associated with elevated ICP.

neuroscience↗

bioRxiv2020

Membrane cholesterol regulates TRPV4 function, cytoskeletal expression and the cellular response to tension

Despite the association of cholesterol with debilitating pressure-related diseases, its role in mechanotransduction is not well understood. We investigated the relationship between mechanical strain, free membrane cholesterol, actin cytoskeleton, and activation of stretch- activated TRPV4 (transient receptor potential vanilloid isoform 4) channel in human trabecular meshwork (TM) cells. Physiological levels of cyclic stretch resulted in time- dependent decreases in membrane cholesterol/phosphatidylcholine ratio and upregulation of stress fibers. Depletion of free membrane cholesterol with m-{beta}-cyclodextrin (M{beta}CD) augmented TRPV4 activation by the agonist GSK1016790A, swelling and strain, with the effects reversed by cholesterol supplementation. M{beta}CD increased membrane expression of TRPV4, caveolin-1 and flotillin. Caveolin-1 antibody partially precipitated a truncated [~]75 kDa variant whereas the majority of TRPV4 did not colocalize or interact with caveolae or lipid rafts, indicating that TRPV4 is mainly localized outside of cholesterol-enrichedmembrane domains. M{beta}CD induced currents in TRPV4-expressing Xenopus laevis oocytes. Thus, while the membrane C/P ratio reflects the biomechanical milieu, trabecular transduction of mechanical information is modulated by the membrane cholesterol content. Diet, cholesterol metabolism and mechanical stress might modulate the conventional outflow pathway and intraocular pressure in glaucoma and diabetes.

cell biology↗