Human CASPR2 Antibodies Reversibly Alter Memory and the CASPR2 Protein Complex.

OBJECTIVE: The encephalitis associated with antibodies against contactin-associated proteinlike 2 (CASPR2) is presumably antibody-mediated, but the antibody effects and whether they cause behavioral alterations are not well known. Here, we used a mouse model of patients' immunoglobulin G (IgG) transfer and super-resolution microscopy to demonstrate the antibody pathogenicity. METHODS: IgG from patients with anti-CASPR2 encephalitis or healthy controls was infused into the cerebroventricular system of mice. The levels and colocalization of CASPR2 with transient axonal glycoprotein 1 (TAG1) were determined with stimulated emission depletion microscopy (40-70µm lateral resolution). Hippocampal clusters of Kv1.1 voltage-gated potassium channels (VGKCs) and GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were quantified with confocal microscopy. Behavioral alterations were assessed with standard behavioral paradigms. Cultured neurons were used to determine the levels of intracellular CASPR2 and TAG1 after exposure to patients' IgG. RESULTS: Infusion of patients' IgG, but not controls' IgG, caused memory impairment along with hippocampal reduction of surface CASPR2 clusters and decreased CASPR2/TAG1 colocalization. In cultured neurons, patients' IgG led to an increase of intracellular CASPR2 without affecting TAG1, suggesting selective CASPR2 internalization. Additionally, mice infused with patients' IgG showed decreased levels of Kv1.1 and GluA1 (two CASPR2-regulated proteins). All these alterations and the memory deficit reverted to normal after removing patients' IgG. INTERPRETATION: IgG from patients with anti-CASPR2 encephalitis causes reversible memory impairment, inhibits the interaction of CASPR2/TAG1, and decreases the levels of CASPR2 and related proteins (VGKC, AMPAR). These findings fulfill the postulates of antibody-mediated disease and provide a biological basis for antibody-removing treatment approaches. ANN NEUROL 2022;91:801-813.

The Background K+ Channel TRESK in Sensory Physiology and Pain.

TRESK belongs to the K2P family of potassium channels, also known as background or leak potassium channels due to their biophysical properties and their role regulating membrane potential of cells. Several studies to date have highlighted the role of TRESK in regulating the excitability of specific subtypes of sensory neurons. These findings suggest TRESK could be involved in pain sensitivity. Here, we review the different evidence available that involves the channel in pain and sensory perception, from studies knocking out the channel or overexpressing it to identified mutations that link the channel to migraine pain. In addition, the therapeutic possibilities are discussed, as targeting the channel seems an interesting therapeutic approach to reduce nociceptor activation and to decrease pain.

Author Correction: The LRRC8-mediated volume-regulated anion channel is altered in glaucoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TRESK background K+ channel deletion selectively uncovers enhanced mechanical and cold sensitivity.

KEY POINTS: TRESK background K+ channel is expressed in sensory neurons and acts as a brake to reduce neuronal activation. Deletion of the channel enhances the excitability of nociceptors. Skin nociceptive C-fibres show an enhanced activation by cold and mechanical stimulation in TRESK knockout animals. Channel deletion selectively enhances mechanical and cold sensitivity in mice, without altering sensitivity to heat. These results indicate that the channel regulates the excitability of specific neuronal subpopulations involved in mechanosensitivity and cold-sensing. ABSTRACT: Background potassium-permeable ion channels play a critical role in tuning the excitability of nociceptors, yet the precise role played by different subsets of channels is not fully understood. Decreases in TRESK (TWIK-related spinal cord K+ channel) expression/function enhance excitability of sensory neurons, but its role in somatosensory perception and nociception is poorly understood. Here, we used a TRESK knockout (KO) mouse to address these questions. We show that TRESK regulates the sensitivity of sensory neurons in a modality-specific manner, contributing to mechanical and cold sensitivity but without any effect on heat sensitivity. Nociceptive neurons isolated from TRESK KO mice show a decreased threshold for activation and skin nociceptive C-fibres show an enhanced activation by cold and mechanical stimulation that was also observed in behavioural tests in vivo. TRESK is also involved in osmotic pain and in early phases of formalin-induced inflammatory pain, but not in the development of mechanical and heat hyperalgesia during chronic pain. In contrast, mice lacking TRESK present cold allodynia that is not further enhanced by oxaliplatin. In summary, genetic removal of TRESK uncovers enhanced mechanical and cold sensitivity, indicating that the channel regulates the excitability of specific neuronal subpopulations involved in mechanosensitivity and cold-sensing, acting as a brake to prevent activation by innocuous stimuli.

The LRRC8-mediated volume-regulated anion channel is altered in glaucoma.

Regulation of cellular volume is an essential process to balance volume changes during cell proliferation and migration or when intracellular osmolality increases due to transepithelial transport. We previously characterized the key role of volume-regulated anion channels (VRAC) in the modulation of the volume of trabecular meshwork (TM) cells and, in turn, the aqueous humour (AH) outflow from the eye. The balance between the secretion and the drainage of AH determines the intraocular pressure (IOP) that is the major casual risk factor for glaucoma. Glaucoma is an ocular disease that causes irreversible blindness due to the degeneration of retinal ganglion cells. The recent identification of Leucine-Rich Repeat-Containing 8 (LRRC8A-E) proteins as the molecular components of VRAC opens the field to elucidate their function in the physiology of TM and glaucoma. Human TM cells derived from non-glaucomatous donors and from open-angle glaucoma patients were used to determine the expression and the functional activity of LRRC8-mediated channels. Expression levels of LRRC8A-E subunits were decreased in HTM glaucomatous cells compared to normotensive HTM cells. Consequently, the activity of VRAC currents and volume regulation of TM cells were significantly affected. Impaired cell volume regulation will likely contribute to altered aqueous outflow and intraocular pressure.

Anionic Phospholipids Bind to and Modulate the Activity of Human TRESK Background K+ Channel.

The background K+ channel TRESK regulates sensory neuron excitability, and changes in its function/expression contribute to neuronal hyperexcitability after injury/inflammation, making it an attractive therapeutic target for pain-related disorders. Factors that change lipid bilayer composition/properties (including volatile anesthetics, chloroform, chlorpromazine, shear stress, and cell swelling/shrinkage) modify TRESK current, but despite the importance of anionic phospholipids (e.g., PIP2) in the regulation of many ion channels, it remains unknown if membrane lipids affect TRESK function. We describe that both human and rat TRESK contain potential anionic phospholipid binding sites (apbs) in the large cytoplasmic loop, but only the human channel is able to bind to multilamellar vesicles (MLVs), enriched with anionic phospholipids, suggesting an electrostatically mediated interaction. We mapped the apbs to a short stretch of 14 amino acids in the loop, located at the membrane-cytosol interface. Disruption of electrostatic lipid-TRESK interactions inhibited hTRESK currents, while subsequent application of Folch Fraction MLVs or a PIP2 analog activated hTRESK, an effect that was absent in the rat ortholog. Strikingly, channel activation by anionic phospholipids was conferred to rTRESK by replacing the equivalent rat sequence with the human apbs. Finally, in the presence of a calcineurin inhibitor, stimulation of a Gq/11-linked GPCR reduced hTRESK current, revealing a likely inhibitory effect of membrane lipid hydrolysis on hTRESK activity. This novel regulation of hTRESK by anionic phospholipids is a characteristic of the human channel that is not present in rodent orthologs. This must be considered when extrapolating results from animal models and may open the door to the development of novel channel modulators as analgesics.

Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK.

It is often unclear why some genetic mutations to a given gene contribute to neurological disorders and others do not. For instance, two mutations have previously been found to produce a dominant negative for TRESK, a two-pore-domain K+ channel implicated in migraine: TRESK-MT, a 2-bp frameshift mutation, and TRESK-C110R. Both mutants inhibit TRESK, but only TRESK-MT increases sensory neuron excitability and is linked to migraine. Here, we identify a new mechanism, termed frameshift mutation-induced alternative translation initiation (fsATI), that may explain why only TRESK-MT is associated with migraine. fsATI leads to the production of a second protein fragment, TRESK-MT2, which co-assembles with and inhibits TREK1 and TREK2, two other two-pore-domain K+ channels, to increase trigeminal sensory neuron excitability, leading to a migraine-like phenotype in rodents. These findings identify TREK1 and TREK2 as potential molecular targets in migraine and suggest that fsATI should be considered as a distinct class of mutations.