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Human primary (hpBMEC) and induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial-like cells (hiBMEC) are interchangeably used in blood-brain barrier models to study neurological diseases and drug delivery. Both hpBMEC and hiBMEC use glutamine as a source of carbon and nitrogen to produce metabolites and build proteins essential to cell function and communication. We used metabolomic, transcriptomic, and computational methods to examine how hpBMEC and hiBMEC metabolize glutamine, which may impact their utility in modeling the blood-brain barrier. We found that glutamine metabolism was systemically different between the two cell types. hpBMEC had a higher metabolic rate and produced more glutamate and GABA, while hiBMEC rerouted glutamine to produce more glutathione, fatty acids, and asparagine. Higher glutathione production in hiBMEC correlated with higher oxidative stress compared to hpBMEC. α-ketoglutarate (α-KG) supplementation increased glutamate secretion from hiBMEC to match that of hpBMEC; however, α-KG also decreased hiBMEC glycolytic rate. These fundamental metabolic differences between BMEC types may impactin vitroblood-brain barrier model function, particularly communication between BMEC and surrounding cells, and emphasize the importance of evaluating the metabolic impacts of iPSC-derived cells in disease models.
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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 thatLrrc8adisruption in the brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated brain‐wide conditional LRRC8A knockout mice (LRRC8A bKO) usingNestinCre‐drivenLrrc8aflox/floxexcision 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 deletion leads to a lethal phenotype involving progressive astrogliosis and dysregulation of astrocytic uptake and supply of amino acid neurotransmitters and their precursors.
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- Award ID(s):
- 1655365
- PAR ID:
- 10644790
- Publisher / Repository:
- DOI PREFIX: 10.1096
- Date Published:
- Journal Name:
- The FASEB Journal
- Volume:
- 35
- Issue:
- 10
- ISSN:
- 0892-6638
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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