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Leucine-rich repeat containing 8A contributes to the expansion of brain ventricles in zebrafish embryos.

ABSTRACT: The sodium osmotic gradient is necessary for the initiation of brain ventricle inflation, but a previous study predicted that organic and inorganic osmolytes play equivalently important roles in osmotic homeostasis in astrocytes. To test whether organic osmoregulation also plays a role in brain ventricle inflation, the core component for volume-regulated anion and organic osmolyte channel, lrrc8a, was investigated in the zebrafish model. RT-PCR and whole-mount in situ hybridization indicated that both genes were ubiquitously expressed through to 12?hpf, and around the ventricular layer of neural tubes and the cardiogenic region at 24?hpf. Knocking down either one lrrc8a paralog with morpholino oligos resulted in abnormalities in circulation at 32?hpf. Morpholino oligos or CRISPR interference against either paralog led to smaller brain ventricles at 24?hpf. Either lrrc8aa or lrrc8ab mRNA rescued the phenotypic penetrance in both lrrc8aa and lrrc8ab morphants. Supplementation of taurine in the E3 medium and overexpression csad mRNA also rescued lrrc8aa and lrrc8ab morphants. Our results indicate that the two zebrafish lrrc8a paralogs are maternal message genes and are ubiquitously expressed in early embryos. The two genes play redundant roles in the expansion of brain ventricles and the circulatory system and taurine contributes to brain ventricle expansion via the volume-regulated anion and organic osmolyte channels.

SUBMITTER: Tseng YT

PROVIDER: S-EPMC6994961 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Leucine-rich repeat containing 8A contributes to the expansion of brain ventricles in zebrafish embryos.

Tseng Yen-Tzu YT Ko Chun-Lin CL Chang Chia-Teng CT Lee Yen-Hua YH Huang Fu Wei-Chun WC Liu I-Hsuan IH

Biology open 20200129 1

The sodium osmotic gradient is necessary for the initiation of brain ventricle inflation, but a previous study predicted that organic and inorganic osmolytes play equivalently important roles in osmotic homeostasis in astrocytes. To test whether organic osmoregulation also plays a role in brain ventricle inflation, the core component for volume-regulated anion and organic osmolyte channel, <i>lrrc8a</i>, was investigated in the zebrafish model. RT-PCR and whole-mount <i>in situ</i> hybridization ...[more]

PMID: 31941702

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Project description:BackgroundGlioblastoma (GBM) is the most common primary malignant brain tumor in adults. Ubiquitously expressed volume-regulated anion channels (VRAC) are thought to play a role in cell proliferation, migration, and apoptosis. VRAC are heteromeric channel complexes assembled from proteins belonging to the leucine-rich repeat-containing 8A (LRRC8A through E), among which LRRC8A plays an indispensable role. In the present work, we used an RNAi approach to test potential significance of VRAC and LRRC8A in GBM survival and sensitivity to chemotherapeutic agents.MethodsPrimary GBM cells were derived from a human surgical tissue sample. LRRC8A expression was determined with quantitative RT-PCR and downregulated using siRNA. The effects of LRRC8A knockdown on GBM cell viability, proliferation, and sensitivity to chemotherapeutic agents were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and Coulter counter assays. Cell cycle progression was further explored using fluorescence-activated cell sorting analysis of propidium iodide-stained cells.ResultsTemozolomide (TMZ), carmustine, and cisplatin reduced GBM cell survival with the IC50 values of ~1,250, 320, and 30?µM, respectively. Two of three tested gene-specific siRNA constructs, siLRRC8A_3 and siLRRC8A_6, downregulated LRRC8A expression by >80% and significantly reduced GBM cell numbers. The most potent siLRRC8A_3 itself reduced viable cell numbers by ?50%, and significantly increased toxicity of the sub-IC50 concentrations of TMZ (570?µM) and carmustine (167?µM). In contrast, the effects of siLRRC8A_3 and cisplatin (32?µM) were not additive, most likely because cisplatin uptake is VRAC-dependent. The results obtained in primary GBM cells were qualitatively recapitulated in U251 human GBM cell line.ConclusionDownregulation of LRRC8A expression reduces GBM cell proliferation and increases sensitivity to the clinically used TMZ and carmustine. These findings indicate that VRAC represents a potential target for the treatment of GBM, alone or in combination with the current standard-of-care.

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Leucine-rich repeat containing 8A contributes to the expansion of brain ventricles in zebrafish embryos.

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Leucine-rich repeat containing 8A contributes to the expansion of brain ventricles in zebrafish embryos.

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