Knocking-out the Siah2 E3 ubiquitin ligase prevents mitochondrial NCX3 degradation, regulates mitochondrial fission and fusion, and restores mitochondrial function in hypoxic neurons

Abstract

Background: Na + /Ca 2+ exchanger isoform 3 (NCX3) regulates mitochondrial Ca 2+ 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. Methods: 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 ([Ca 2+ ] m) by using specific fluorescent probes, confocal microscopy, and Western Blot analysis. Results: 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 "lengthto-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 [Ca 2+ ] m was reduced, ΔΨm, mitochondrial oxidative capacity and ATP production were improved. Interestingly, our immunoprecipitation assay revealed that Siah2 interacted with NCX3. Indeed, siah2 knockout prevented NCX3 degradation in neurons exposed to OGD. Finally, when siah2−/− neurons were exposed to OGD/reoxygenation, FF, AR,

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“…Besides SIAH1, the other members of the SIAH family have been found to regulate different aspects of mitochondrial biology. For example, SIAH2 has been shown to regulate mitochondrial dynamics by controlling the degradation of Mfn1 and Drp1 in cortical neurons under hypoxia [ 106 ]. Moreover, SIAH2 negatively regulates mitochondrial biogenesis by ubiquitination and subsequent proteasomal degradation of nuclear respiratory factor 1 (NRF1), a crucial factor for mitochondrial biogenesis [ 107 ] under hypoxic microenvironments [ 108 ].…”

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