Late adolescence mortality in mice with brain‐specific deletion of the volume‐regulated anion channel subunit LRRC8A

Abstract

The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is critical for cell volume control, but its broader physiological functions remain under investigation. Recent studies in the field indicate that Lrrc8a disruption in the brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated brainwide conditional LRRC8A knockout mice (LRRC8A bKO) using Nestin Cre -driven Lrrc8a flox/flox excision in neurons, astrocytes, and oligodendroglia. LRRC8A bKO animals were born close to the expected Mendelian ratio and developed without overt histological abnormalities, but, surprisingly, all died between 5 and 9 weeks of age with a seizure phenotype, which was confirmed by video and EEG recordings. Brain slice electrophysiology detected changes in the excitability of pyramidal cells and modified GABAergic inputs in the hippocampal CA1 region of LRRC8A bKO. LRRC8A-null hippocampi showed increased immunoreactivity of the astrocytic marker GFAP, indicating reactive astrogliosis. We also found decreased whole-brain protein levels of the GABA transporter GAT-1, the glutamate transporter GLT-1, and the astrocytic enzyme glutamine synthetase.Complementary HPLC assays identified reduction in the tissue levels of the glutamate and GABA precursor glutamine. Together, these findings suggest that VRAC provides vital control of brain excitability in mouse adolescence. VRAC

Properties, Structures, and Physiological Roles of Three Types of Anion Channels Molecularly Identified in the 2010’s

“…Also, a number of disorders are found in recent studies using specific cell- or tissue-targeted Lrrc8a knockout mice. For example, impaired glucose tolerance and insulin resistance are caused by conditional knockout of Lrrc8a expressing in adipocytes ( Zhang et al, 2017 ), pancreatic β cells ( Kang et al, 2018 ), and skeletal muscle cells ( Kumar et al, 2020 ); male infertility by germ cell-specific Lrrc8a disruption ( Lück et al, 2018 ); angiotensin-II-stimulated hypertension by endothelial-targeted Lrrc8a knockout ( Alghanem et al, 2021 ); and brain hyperexcitability coupled to astrogliosis by brain-specific Lrrc8a disruption ( Wilson et al, 2021 ). However, it is not known whether these dysfunctions are caused by disordered anion-conductive activities of LRRC8A or non-conductive protein-protein interactions.…”

Conditional deletion of LRRC8A in the brain reduces stroke damage independently of swelling-activated glutamate release

“…Previously, we found that the brain-wide deletion of LRRC8A reduced astrocytic expression of the glutamate transporter GLT-1. 31 Therefore, we additionally explored if there were changes in GLT-1 immunoreactivity in the iaLRRC8A KO but no differences were detected in any of the tested groups ( Figures 1 H and 1I; primary images in Figure S2 ).…”

“…This assay, which has been thoroughly validated using RNAi and pharmacological tools, quantifies the activity of the LRRC8A-containing VRAC in large populations of astrocytes and gives comparable results to electrophysiological recordings of VRAC Cl − currents. 31 , 36 , 37 Hypoosmotic challenge of cultured astrocytes stimulated glutamate (D-[ 3 H]aspartate) release via VRAC, and such release was reduced by approximately 70% in the tamoxifen-treated iaLRRC8A KO cultures as compared to the DMSO-treated controls from the same genotype ( Figure 2 D). Taken together, the results of our in vivo and in vitro experiments suggest that we successfully downregulated LRRC8A expression in brain astrocytes and that such downregulation was associated with a significant loss of VRAC activity.…”

“…Indeed, the brainwide LRRC8A deletion produces reactive astrogliosis, gives rise to spontaneous seizures, and results in 100% animal mortality within the first eight weeks of postnatal development. 30 , 31 Therefore, in the present work, to avoid developmental compensation we tested the impact of LRRC8A deletion on stroke outcomes using inducible deletion of LRRC8A in astrocytes. Because astrocytic ablation was not neuroprotective, we extended our efforts to the conditional brain-wide LRRC8A knockout.…”

GPR37 Receptors and Megalencephalic Leukoencephalopathy with Subcortical Cysts

“…Similarly, defects of connexins have also been linked to myelin vacuolization [ 39 ]. As LRRC8 proteins of VRAC channels are involved in cell volume regulation and their absence cause cell vacuolation in several tissues, it will be important to analyse whether a conditional (because the complete KO is deleterious [ 40 ]) astrocyte KO of LRRC8A [ 41 ] (the main constituent of the VRAC channel [ 42 , 43 ]) also displays astrocyte and myelin vacuolization.…”