Transient Global Cerebral Ischemia Induces RNF213, a Moyamoya Disease Susceptibility Gene, in Vulnerable Neurons of the Rat Hippocampus CA1 Subregion and Ischemic Cortex

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The RING finger protein 213 (RNF213) is an important susceptibility gene for moyamoya disease (MMD) and is also implicated in other types of intracranial major artery stenosis/occlusion (ICAS); however, the role of RNF213 in the development of ICAS including MMD is unclear. The constitutive expression of the RNF213 gene is relatively weak in brain tissue, while information regarding the expression patterns of the RNF213 gene under cerebral ischemia, which is one of characteristic pathologies associated with ICAS, is currently limited. Our objective was to address this critical issue, and we investigated Rnf213 mRNA expression in rat brains after 5 minutes of transient global cerebral ischemia (tGCI) by occluding the common carotid arteries coupled with severe hypotension. Rnf213 gene expression patterns were investigated with in situ RNA hybridization and a real-time polymerase chain reaction (PCR) from 1 to 72 hours after tGCI. In situ RNA hybridization revealed a significant increase in Rnf213 mRNA levels in the hippocampus CA1 sub-region 48 hours after tGCI. The significant induction of the Rnf213 gene was also evident in the ischemic cortex. Double staining of Rnf213 mRNA with NeuN immunohistochemistry revealed Rnf213 hybridization signal expression exclusively in neurons. The real-time PCR analysis confirmed the induction of the Rnf213 gene after tGCI. The up-regulation of the Rnf213 gene in vulnerable neurons in the hippocampus CA1 after tGCI suggests its involvement in forebrain ischemia, which is an underlying pathology of MMD. Further investigations are needed to elucidate its exact role in the pathophysiology of ICAS including MMD.

Introduction

The RING finger protein 213 (RNF213) gene is located in the 17q25-ter region, and encodes a protein with RING finger domain which plays critical roles in modulating protein levels of mammalian tissues by E3 ubiquitin-protein ligase activity.1 Although the exact role of this gene is still undetermined, recent in vitro study suggested that RNF213 gene plays an important role in cell death and/or survival.2 Regarding cerebrovascular diseases, RNF213 gene is implicated in various types of the occlusive cerebrovascular diseases such as moyamoya disease (MMD) and atherosclerotic intracranial major artery stenosis/occlusion.3, 4, 5, 6 Particularly in MMD, recent genome-wide and locus-specific association studies identified RNF213 gene as an important susceptibility gene among East Asian patients.4, 5 Further studies demonstrated that patients with the homozygous mutation pattern of a single nucleotide polymorphism in the RNF213 gene had a significantly earlier disease onset and higher incidence of cerebral infarction and/or cognitive impairment, suggesting that an RNF213 polymorphism leads to a more severe clinical condition for MMD.7, 8 However, the exact mechanisms by which genetic abnormalities in the RNF213 gene result in the development of intracranial major artery stenosis/occlusion including MMD have not yet been clarified. A quantitative polymerase chain reaction (PCR) analysis revealed that the constitutive expression of the RNF213 gene was very weak in adult and embryonic brain tissue4; however, information regarding the temporal and spatial expression patterns of the RNF213 gene under chronic cerebral ischemia, which is one of the major underlying pathologies of intracranial major artery stenosis/occlusion including MMD, is currently limited.3 To address this important issue, we herein investigated Rnf213 mRNA expression in rat brains subjected to transient global cerebral ischemia (tGCI).

Section snippets

Animal Model of tGCI and Reperfusion

Male Sprague-Dawley rats weighing 280-320 g (7 weeks) were subjected to tGCI using the 2-vessel occlusion method coupled with severe hypotension.9 The femoral artery was cannulated and connected to a blood pressure monitor, and blood was aspirated through the external jugular vein until blood pressure decreased to approximately 25 mmHg. Both of the common carotid arteries were occluded using aneurysm clips for 5 minutes while maintaining blood pressure at between 25–30 mmHg (mean 28 mmHg). The

Rnf213 mRNA Was Upregulated in the Ischemic Brain after tGCI

Previous studies reported that the human RNF213 gene is normally expressed at markedly lower levels in brain tissue than in other tissues such as the spleen and leukocytes.4, 5 We initially examined the relative expression levels of the rat Rnf213 gene (Rnf213) in the intact tissues of each organ. A semiquantitative real-time PCR analysis with primers for Rnf213 confirmed that the Rnf213 gene was expressed in intact tissues; however, its expression levels were markedly lower in the cerebral

Discussion

The present study provided novel results showing that the mRNA of RNF213, a susceptibility gene for MMD that is implicated in various types of intracranial major artery stenosis/occlusion, was significantly upregulated in the vulnerable neurons of the rat hippocampus CA1 subregion and ischemic cortex after tGCI. Based on the relatively weaker expression of Rnf213 in the primate brain under normal physiological conditions,3, 4 the significant induction of Rnf213 after tGCI is apparently unique.

Conclusion

Rnf213 was upregulated in the vulnerable neurons of the hippocampus CA1 subregion and ischemic cortex after tGCI. Furthermore, increases in the expression of Rnf213 in neurons that are destined to show apoptosis appear to be unique. These results may provide insights into the role of Rnf213 in cerebral ischemia, which is an underlying pathology of MMD. Further investigations are needed to clarify its exact role in the pathophysiology of MMD.

Acknowledgments

This study was supported by JSPS KAKENHI Grant Number K426462150 and by AMED J160001258. We would like to thank Ms. Maki Sugawara for her technical assistance with in situ hybridization and immunohistochemistry.

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Grant support: This study was supported by JSPS KAKENHI Grant Number K426462150 and by AMED J160001258.

Conflict of interest: The authors declare that they have no conflicts of interest related to this manuscript.

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