Golubinskaya Veronika, Vontell Regina, Supramaniam Veena, Wyatt-Ashmead Josephine, Gustafsson Helena, Mallard Carina, Nilsson Holger
Department of Physiology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
Division of Imaging Sciences & Biomedical Engineering, Centre for the Developing Brain, King's College London, King's Health Partners, St Thomas' Hospital, London, United Kingdom.
Front Physiol. 2019 Jan 29;10:23. doi: 10.3389/fphys.2019.00023. eCollection 2019.
Bestrophin-3, a potential candidate for a calcium-activated chloride channel, recently was suggested to have cell-protective functions. We studied the expression and alternative splicing of bestrophin-3 in neonatal mouse brain and after hypoxic-ischemic (HI) injury and in human neonatal brain samples. HI brain injury was induced in 9-day old mice by unilateral permanent common carotid artery occlusion in combination with exposure to 10% oxygen for 50 min. Endoplasmic reticulum stress was induced by thapsigargin treatment in primary culture of mouse brain astrocytes. We also investigated expression of bestrophin-3 protein in a sample of human neonatal brain tissue. Bestrophin-3 protein expression was detected with immunohistochemical methods and western blot; mRNA expression and splicing were analyzed by RT-PCR. HI induced a brain tissue infarct and a pronounced increase in the endoplasmic reticulum-associated marker CHOP. Three days after HI a population of astrocytes co-expressed bestrophin-3 and nestin in a penumbra-like area of the injured hemisphere. However, total levels of Bestrophin-3 protein in mouse cortex were reduced after injury. Mouse astrocytes in primary culture also expressed bestrophin-3 protein, the amount of which was reduced by endoplasmic reticulum stress. Bestrophin-3 protein was detected in astrocytes in the hippocampal region of the human neonatal brain which had patchy white matter gliosis and neuronal loss in the Sommer's sector of the Ammon's horn (CA1). Analysis of bestrophin-3 mRNA in mouse brain with and without injury showed the presence of two truncated spliced variants, but no full-length mRNA. Total amount of bestrophin-3 mRNA increased after HI, but showed only minor injury-related change. However, the splice variants of bestrophin-3 mRNA were differentially regulated after HI depending on the presence of tissue injury. Our results show that bestrophin-3 is expressed in neonatal mouse brain after injury and in the human neonatal brain with pathology. In mouse brain bestrophin-3 protein is upregulated in a specific astrocyte population after injury and is co-expressed with nestin. Splice variants of bestrophin-3 mRNA respond differently to HI, which might indicate their different roles in tissue injury.
Bestrophin-3作为一种钙激活氯离子通道的潜在候选蛋白，最近被认为具有细胞保护功能。我们研究了Bestrophin-3在新生小鼠脑内、缺氧缺血（HI）损伤后以及人类新生儿脑样本中的表达和可变剪接情况。通过单侧永久性颈总动脉闭塞并暴露于10%氧气中50分钟，诱导9日龄小鼠发生HI脑损伤。在小鼠脑星形胶质细胞原代培养中，通过毒胡萝卜素处理诱导内质网应激。我们还研究了Bestrophin-3蛋白在人类新生儿脑组织样本中的表达。采用免疫组织化学方法和蛋白质印迹法检测Bestrophin-3蛋白表达；通过逆转录聚合酶链反应（RT-PCR）分析mRNA表达和剪接情况。HI诱导脑组织梗死，并使内质网相关标志物CHOP显著增加。HI损伤后3天，一群星形胶质细胞在损伤半球的半暗带样区域共表达Bestrophin-3和巢蛋白。然而，损伤后小鼠皮质中Bestrophin-3蛋白的总水平降低。原代培养的小鼠星形胶质细胞也表达Bestrophin-3蛋白，内质网应激使其表达量减少。在人类新生儿脑的海马区星形胶质细胞中检测到Bestrophin-3蛋白，该区域存在斑片状白质胶质增生以及海马角（CA1） Sommer扇区的神经元丢失。对有损伤和无损伤的小鼠脑内Bestrophin-3 mRNA进行分析，发现存在两种截短的剪接变体，但无全长mRNA。HI损伤后Bestrophin-3 mRNA总量增加，但仅显示出与损伤相关的微小变化。然而，HI损伤后Bestrophin-3 mRNA的剪接变体根据组织损伤的存在情况受到不同调节。我们的结果表明，Bestrophin-3在损伤后的新生小鼠脑以及有病理改变的人类新生儿脑中表达。在小鼠脑中，Bestrophin-3蛋白在损伤后的特定星形胶质细胞群体中上调，并与巢蛋白共表达。Bestrophin-3 mRNA的剪接变体对HI的反应不同，这可能表明它们在组织损伤中发挥不同作用。
