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 indispensable for cell volume regulation but its broader physiological functions remain under investigation. Astrocyte-targeted Lrrc8a deletion in the nervous system reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against ischemic damage. Here we show that deletion of LRRC8A in all brain cells, using Nestin Cre -driven Lrrc8a fl/fl excision, is lethal. Mice devoid of brain LRRC8A are born close to the expected Mendelian ratio and develop without overt histological abnormalities. Nevertheless, they all die between 5 to 8 weeks of age with a seizure-like phenotype. Consistent with seizures, we found disruptions in cell excitability, GABAergic signaling, and astrocytic production of the GABA precursor glutamine, all of which might contribute to mortality. This work provides the first evidence of a critical role for VRAC in control of brain excitability during maturation.

Conditional deletion of LRRC8A in the brain protects against stroke damage independently of effect on swelling-activated glutamate release

“…To definitively examine this hypothesis, we reevaluated stroke outcomes in the Nestin Cre -driven, brain-wide LRRC8A KO mice. These mice show the complete loss of brain LRRC8A immunoreactivity, electrophysiological VRAC currents, and swelling-activated glutamate release (Yang et al, 2019;Zhou et al, 2020;Wilson et al, 2021). Using this genetic strategy, Zhou and coworkers compared cerebral infarction in the two genotypes, Lrrc8a fl/fl controls and LRRC8A KO (Zhou et al, 2020).…”

“…The functional loss of VRAC activity in bLRRC8A KO has been extensively characterized in our prior work (Wilson et al, 2021) (reproduced with permission in Figure 4G). The critical finding of this latter study is nearly complete loss of swelling-activated glutamate (D-[ 3 H]aspartate) release in bLRRC8A KO astrocytes (Figure 4G).…”

“…Nestin is a type VI intermediate filament protein that is abundant in neuronal stem cells throughout development and adulthood; it is expressed in developing mouse embryos as early as E7.5 (Lagace et al, 2007). Complete elimination of LRRC8A in the brain using the Nestin Cre strategy has been extensively validated in our recent work (Wilson et al, 2021). In the present study, we used the same breeding strategy (Figure 4A) and additionally used Ai14 tdTomato reporter to ensure that there is no non-specific germline recombination.…”

“…Finally, to confirm functional loss of VRAC activity, we employed a radiotracer assay, measuring swelling-activated release of the non-metabolizable glutamate analog, D-[ 3 H]aspartate. 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 (Abdullaev et al, 2006;Hyzinski-Garcia et al, 2014;Wilson et al, 2021).…”

“…Another caveat is that conditional knockouts can lead to developmental adaptations. Indeed, the brain wide LRRC8A deletion produces reactive astrogliosis, gives rise to spontaneous seizures, and results in 100% animal mortality withing the first eight weeks of postnatal development (Zhou et al, 2020;Wilson et al, 2021). Therefore, in the present work, to avoid developmental compensation we tested the impact of LRRC8A deletion on stroke outcomes using an inducible deletion of LRRC8A in astrocytes.…”