Investigating the role of LRRC8A in microglia and macrophage function

UoM administered thesis: Phd

  • Authors:
  • James Cook

Abstract

LRRC8A is the obligatory subunit necessary for the formation of the volume-regulated anion channel (VRAC), which is a key regulator of cell volume under hypotonic conditions, as well as following cell swelling. VRAC has been proposed as a drug target for treating ischaemic stroke due to its role in excitotoxic glutamate release from astrocytes, but has also been suggested to regulate numerous aspects of microglial physiology. Microglia, the principal immune cells of the brain, are critical players in determining the course of various neuropathologies, including stroke. The role of VRAC in microglia is therefore of relevance for its utility as a drug target in CNS disease. However, current knowledge regarding the role of VRAC in determining microglial activity is based solely on studies using poorly selective channel inhibitors and requires more specific validation using genetic models. There is also some evidence that LRRC8A is able to participate in intracellular signalling cascades independently of its function as a VRAC component, though to date this phenomenon remains poorly explored. Thus, this study addressed two key questions, using a novel microglia/macrophage-targeted conditional knockout mouse model. Firstly, the role of VRAC in microglial function was assessed. Contrary to previous studies, it was found that loss of VRAC function in microglia does not affect phagocytic capacity, migration, chemotaxis or morphology. Moreover, loss of microglial VRAC did not induce major transcriptomic alterations in microglia or affect infarct volume in an experimental stroke model. Thus, this study concludes that VRAC does not contribute to a variety of key microglial functions, and VRAC could therefore be targeted as a stroke therapy without detrimentally impairing microglial function. Secondly, RNA-Sequencing was used to interrogate novel signalling pathways affected by LRRC8A following hypotonic cell swelling. These results indicated that LRRC8A is capable of modifying type-I interferon responses elicited by cell swelling, which occurs independently of the VRAC channel via a novel signalling pathway. Moreover, LRRC8A was found to activate the PI3K-AKT pathway following hypotonic swelling via two separate mechanisms differentially dependent on protein kinase C and the Src-family kinases Fyn/Hck and independent of VRAC. Thus, this study adds considerable evidence of a VRAC-independent signalling role for LRRC8A in regulating cellular physiology, which is important for interpreting genetic studies targeting LRRC8A as a component of VRAC.

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Original languageEnglish
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Award date1 Aug 2022