Na+/Ca2+ exchanger isoform 3 (NCX3) regulates mitochondrial Ca2+ handling through the outer mitochondrial membrane (OMM) and promotes neuronal survival during oxygen and glucose deprivation (OGD). Conversely, Seven In-Absentia Homolog 2 (Siah2), an E3-ubiquitin ligase, which is activated under hypoxic conditions, causes proteolysis of mitochondrial and cellular proteins. In the present study, we investigated whether siah2, upon its activation during hypoxia, interacts with NCX3 and whether such interaction could regulate the molecular events underlying changes in mitochondrial morphology, i.e., fusion and fission, and function, in neurons exposed to anoxia and anoxia/reoxygenation. To answer these questions, after exposing cortical neurons from siah2 KO mice (siah2 −/−) to OGD and OGD/Reoxygenation, we monitored the changes in mitochondrial fusion and fission protein expression, mitochondrial membrane potential (ΔΨm), and mitochondrial calcium concentration ([Ca2+]m) by using specific fluorescent probes, confocal microscopy, and Western Blot analysis. As opposed to congenic wild-type neurons, in neurons from siah2−/− mice exposed to OGD, form factor (FF), an index of the complexity and branching aspect of mitochondria, and aspect ratio (AR), an index reflecting the “length-to-width ratio” of mitochondria, maintained low expression. In KO siah2 neurons exposed to OGD, downregulation of mitofusin 1 (Mfn1), a protein involved in mitochondrial fusion and upregulation of dynamin-related protein 1 (Drp1), a protein involved in the mitochondrial fission, were prevented. Furthermore, under OGD conditions, whereas [Ca2+]m was reduced, ΔΨm, mitochondrial oxidative capacity and ATP production were improved. Interestingly, our immunoprecipitation assay revealed that Siah2 interacted with NCX3. Indeed, siah2 knock-out prevented NCX3 degradation in neurons exposed to OGD. Finally, when siah2−/− neurons were exposed to OGD/reoxygenation, FF, AR, and Mfn1 expression increased, and mitochondrial function improved compared to siah2+/+ neurons. Collectively, these findings indicate that hypoxia-induced SIAH2-E3 ligase activation influences mitochondrial fusion and fission, as well as function, by inducing NCX3 degradation.

中文翻译：

---

敲除Siah2 E3泛素连接酶可防止线粒体NCX3降解，调节线粒体裂变和融合，并恢复缺氧神经元中的线粒体功能。

Na + / Ca2 +交换异构体3（NCX3）通过线粒体外膜（OMM）调节线粒体Ca2 +的处理，并在缺氧和缺糖（OGD）期间促进神经元的存活。相反，在缺氧条件下被激活的E3泛素连接酶七个In-Absentia Homolog 2（Siah2）引起线粒体和细胞蛋白的蛋白水解。在本研究中，我们调查了siah2在缺氧期间激活后是否与NCX3相互作用，以及这种相互作用是否可以调节暴露于缺氧和缺氧神经元的线粒体形态变化（即融合和裂变以及功能）的分子事件。 /复氧。为了回答这些问题，将siah2 KO小鼠（siah2-/-）的皮质神经元暴露于OGD和OGD /复氧后，我们通过使用特定的荧光探针，共聚焦显微镜和Western Blot分析监测了线粒体融合和裂变蛋白表达，线粒体膜电位（ΔΨm）和线粒体钙浓度（[Ca2 +] m）的变化。与同质野生型神经元相反，在暴露于OGD的siah2-/-小鼠的神经元中，形态因子（FF），线粒体的复杂性和分支方面的指数以及长宽比（AR）（反映“线粒体的长宽比”保持低表达。在暴露于OGD的KO siah2神经元中，防止了线粒体蛋白1（Mfn1）（一种参与线粒体融合的蛋白）的下调以及与动力蛋白相关的蛋白1（Drp1）（一种参与线粒体裂变的蛋白）的上调。此外，在OGD条件下，[Ca2 +] m降低，而ΔΨm，线粒体的氧化能力和ATP产量得到改善。有趣的是，我们的免疫沉淀分析表明Siah2与NCX3相互作用。实际上，siah2敲除可防止暴露于OGD的神经元中NCX3降解。最后，当siah2-/-神经元暴露于OGD /复氧时，与siah2 + / +神经元相比，FF，AR和Mfn1表达增加，线粒体功能得到改善。总体而言，这些发现表明，低氧诱导的SIAH2-E3连接酶激活通过诱导NCX3降解影响线粒体融合和裂变以及功能。与siah2 + / +神经元相比，当siah2-/-神经元暴露于OGD /复氧时，FF，AR和Mfn1表达增加，线粒体功能得到改善。总体而言，这些发现表明，低氧诱导的SIAH2-E3连接酶激活通过诱导NCX3降解而影响线粒体融合和裂变以及功能。与siah2 + / +神经元相比，当siah2-/-神经元暴露于OGD /复氧时，FF，AR和Mfn1表达增加，线粒体功能得到改善。总体而言，这些发现表明，低氧诱导的SIAH2-E3连接酶激活通过诱导NCX3降解而影响线粒体融合和裂变以及功能。