Background

Ischemia and reperfusion (I/R) injury exacerbate the prognosis of ischemic diseases. The cause of this exacerbation is partly a mitochondrial cell death pathway. Mitochondrial calpain-5 is proteolyzed/autolyzed under endoplasmic reticulum stress, resulting in inflammatory caspase-4 activation. However, the role of calpain-5 in I/R injury remains unclear. We hypothesized that calpain-5 is involved in ischemic brain disease.

Methods

Mitochondria from C57BL/6J mice were extracted via centrifugation with/without proteinase K treatment. The expression and proteolysis/autolysis of calpain-5 were determined using western blotting. The mouse and human brains with I/R injury were analyzed using hematoxylin and eosin staining and immunohistochemistry. HT22 cells were treated with tunicamycin and CAPN5 siRNA.

Results

Calpain-5 was expressed in the mitochondria of mouse tissues. Mitochondrial calpain-5 in mouse brains was responsive to calcium earlier than cytosolic calpain-5 in vitro calcium assays and in vivo bilateral common carotid artery occlusion model mice. Immunohistochemistry revealed that neurons were positive for calpain-5 in the normal brains of mice and humans. The expression of calpain-5 was increased in reactive astrocytes at human infarction sites. The knockdown of calpain-5 suppressed of cleaved caspase-11.

Conclusions

The neurons of human and mouse brains express calpain-5, which is proteolyzed/autolyzed in the mitochondria in the early stage of I/R injury and upregulated in reactive astrocytes in the end-stage.

General significance

Our results provide a comprehensive understanding of the mechanisms underlying I/R injury. Targeting the expression or activity of mitochondrial calpain-5 may suppress the inflammation during I/R injuries such as cerebrovascular diseases.

中文翻译：

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calpain-5在脑缺血再灌注损伤中的作用

背景

缺血和再灌注（I/R）损伤会加剧缺血性疾病的预后。这种恶化的部分原因是线粒体细胞死亡途径。线粒体 calpain-5 在内质网应激下被蛋白水解/自溶，导致炎症 caspase-4 激活。然而，calpain-5 在 I/R 损伤中的作用仍不清楚。我们假设 calpain-5 与缺血性脑病有关。

方法

通过离心提取 C57BL/6J 小鼠的线粒体，使用/不使用蛋白酶 K 处理。使用蛋白质印迹法测定 calpain-5 的表达和蛋白水解/自溶。使用苏木精和伊红染色以及免疫组织化学分析患有 I/R 损伤的小鼠和人脑。用衣霉素和 CAPN5 siRNA处理 HT22 细胞。

结果

Calpain-5 在小鼠组织的线粒体中表达。在体外钙测定和体内双侧颈总动脉闭塞模型小鼠中，小鼠大脑中的线粒体 calpain-5 对钙的反应早于胞质 calpain-5。免疫组织化学显示，小鼠和人类正常大脑中的神经元钙蛋白酶 5 呈阳性。人梗塞部位的反应性星形胶质细胞中 calpain-5 的表达增加。calpain-5 的敲低抑制了 caspase-11 的裂解。

结论

人和小鼠大脑的神经元表达 calpain-5，它在 I/R 损伤的早期阶段在线粒体中蛋白水解/自溶，并在终末阶段在反应性星形胶质细胞中上调。

一般意义

我们的结果提供了对 I/R 损伤机制的全面了解。靶向线粒体 calpain-5 的表达或活性可能会抑制脑血管疾病等 I/R 损伤期间的炎症。