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Analysis of L-leucine amino acid transporter species activity and gene expression by human blood brain barrier hCMEC/D3 model reveal potential LAT1, LAT4, B⁰AT2 and y⁺LAT1 functional cooperation.

ABSTRACT: In the CNS, amino acid (AA) neurotransmitters and neurotransmitter precursors are subject to tight homeostatic control mediated by blood-brain barrier (BBB) solute carrier amino acid transporters (AATs). Since the BBB is composed of multiple closely apposed cell types and opportunities for human in vivo studies are limited, we used in vitro and computational approaches to investigate human BBB AAT activity and regulation. Quantitative real-time PCR (qPCR) of the human BBB endothelial cell model hCMEC/D3 (D3) was used to determine expression of selected AAT, tight junction (TJ), and signal transduction (ST) genes under various culture conditions. L-leucine uptake data were interrogated with a computational model developed by our group for calculating AAT activity in complex cell cultures. This approach is potentially applicable to in vitro cell culture drug studies where multiple "receptors" may mediate observed responses. Of 7 Leu AAT genes expressed by D3 only the activity of SLC7A5-SLC3A2/LAT1-4F2HC (LAT1), SLC43A2/LAT4 (LAT4) and sodium-dependent AATs, SLC6A15/B⁰AT2 (B⁰AT2), and SLC7A7/y⁺LAT1 (y⁺LAT1) were calculated to be required for Leu uptake. Therefore, D3 Leu transport may be mediated by a potentially physiologically relevant functional cooperation between the known BBB AAT, LAT1 and obligatory exchange (y⁺LAT1), facilitative diffusion (LAT4), and sodium symporter (B⁰AT2) transporters.

SUBMITTER: Taslimifar M

PROVIDER: S-EPMC8721536 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ischemic stroke (IS) is a major neurological disease with high fatality and residual disability burdens. Long noncoding RNAs (lncRNAs) have been found to play an important role in IS. However, the roles and significance of most lncRNAs in IS are still unknown. This study was performed to identify differentially expressed (DE) lncRNAs using a lncRNA microarray in whole blood samples of patients suffering from acute cerebral ischemia. Bioinformatics analyses, including GO, KEGG pathway enrichment analysis, and proximity to putative stroke risk location analysis were performed. The novel lncRNA, ENST00000530525, significantly decreased after IS. Furthermore, we evaluated lncRNA ENST00000530525 expression in cultured hCMEC/D3 cells under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions using fluorescent in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (RT-qPCR) analysis. To investigate the function of lncRNA ENST00000530525, its over-expression (OE) and negative control (NC) plasmids were transfected into hCMEC/D3 cells, and cell viability was detected by a cell counting kit-8 (CCK-8) assay after OGD/R. LncRNA ENST00000530525 and ANO1 expression were investigated using RT-qPCR and immunofluorescence. For blood-brain barrier (BBB) permeability, FITC-dextran transendothelial permeability assay and tight junction (TJ) protein immunofluorescence assays were performed. There were 3352 DE lncRNAs in the blood samples of acute IS patients. The validation results were consistent with the gene chip data. The GO and KEGG results showed that these lncRNAs were mainly related to oxygen and glucose metabolism, leukocyte transendothelial migration, mitophagy and cellular senescence. Among these, lncRNA ENST00000530525 was the most highly downregulated lncRNA and it was mapped within the IS-associated gene anoctamin-1 (ANO1). We further found that lncRNA ENST00000530525 was downregulated in hCMEC/D3 cells under 4 h OGD and 20 h reoxygenation (OGD4/R20) conditions. Upregulating lncRNA ENST00000530525 by plasmid transfection decreased cell viability while increasing ANO1 expression and it contributed to BBB injury in hCMEC/D3 cells after OGD4/R20. The lncRNA ENST00000530525 might play deleterious roles in post-stroke pathogenesis. These results show that some DE lncRNAs in humans participate through characteristic roles in post-stroke pathogenesis; thus, the roles and significance of some novel lncRNAs in IS warrant further study.

Dataset Information

Analysis of L-leucine amino acid transporter species activity and gene expression by human blood brain barrier hCMEC/D3 model reveal potential LAT1, LAT4, B⁰AT2 and y⁺LAT1 functional cooperation.

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Dataset Information

Analysis of L-leucine amino acid transporter species activity and gene expression by human blood brain barrier hCMEC/D3 model reveal potential LAT1, LAT4, B0AT2 and y+LAT1 functional cooperation.

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OmicsDI is part of the ELIXIR infrastructure

Tweets

Analysis of L-leucine amino acid transporter species activity and gene expression by human blood brain barrier hCMEC/D3 model reveal potential LAT1, LAT4, B⁰AT2 and y⁺LAT1 functional cooperation.