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⁺/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.

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 ([Ca²⁺]_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 "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.

Conclusions: Collectively, these findings indicate that hypoxia-induced SIAH2-E3 ligase activation influences mitochondrial fusion and fission, as well as function, by inducing NCX3 degradation. Video Abstract.

Keywords: Hypoxia; Mitochondria; NCX3, cortical neurons; siah2.

Fig. 1

Mitochondrial morphology in primary cortical neurons obtained from siah2+/+ and siah2−/− mice exposed to OGD and OGD/Reoxygenation. (a- left) Imaging mitochondrial morphology in siah+/+ and siah−/− cortical neurons by confocal microscopy and MitoTracker Red (20 nM) (left panel). N: neurons, scale bars: 10 μm. (a- right) quantification of the changes in mitochondrial morphology by Image J software. Form factor (FF) and Aspect ratio (AR) in siah+/+ and siah−/− neurons. b Western Blot analysis of Mfn1 and DRP1 protein expression in siah2+/+ and siah2−/−cortical neurons exposed to OGD and OGD/Reoxygenation. Each bar represents the mean + S.E.M. of the percentage of different experimental values obtained in three independent experimental sessions. *P < 0.05 vs siah2+/+ CTL; **P < 0.05 vs siah2+/+ OGD; ^P < 0.05 vs siah2+/+ OGD/Reoxy

Fig. 2

Mitochondrial function in primary cortical neurons obtained from siah2+/+ and siah2−/− mice exposed to OGD and OGD/Reoxygenation. Quantification of mitochondrial parameters in primary cortical neurons obtained from siah2+/+ and siah2−/− mice exposed to OGD and OGD followed by reoxygenation: a Mitochondrial calcium concentration, b Mitochondrial membrane potential, c Mitochondrial oxidative capacity and d ATP production. Each bar represents the mean + S.E.M. of the percentage values of at least 20–30 neurons recorded in three independent experimental sessions. *P < 0.05 vs siah2+/+ CTL; **P < 0.05 vs siah2+/+ OGD; ^P < 0.05 vs siah2+/+ OGD/Reoxy

Fig. 3

Biochemical interaction between SIAH2 and NCX3. a Immunoprecipitation assay in siah2+/+ neurons. b NCX3 protein expression in siah2+/+ and siah2−/− cortical neurons exposed to OGD and OGD/Reoxygenation. c Immunoprecipitation assay in siah2+/+ mice treated with the proteasome inhibitor MG132 (1 μl was administered icv from a 40 mM stock twice, 3 h before the induction of transient ischemia and immediately after filament withdrawn). d Mfn1, Mfn2, and Drp1 protein expression in siah2+/+ neurons exposed to OGD in the presence and in the absence of MG132 (40 μM). Each bar represents the mean +/– S.E.M. of the percentage of different experimental values obtained in three independent experimental sessions. *P < 0.05 vs siah2+/+ CTL; **P < 0.05 vs siah2+/+ OGD; ^P < 0.05 vs siah2+/+ OGD/Reoxy

Fig. 4

Mitochondrial morphology and function in primary cortical neurons obtained from ncx3+/+ and ncx3−/− mice. (a-left), Imaging mitochondrial morphology in ncx3+/+ and ncx3−/− cortical neurons by confocal microscopy and MitoTracker Red (20 nM), N: neurons; scale bars: 10 μm. (a-right), quantification of the changes in mitochondrial morphology by Image J software. Form Factor (FF) and Aspect ratio (AR) in ncx3−/− neurons. b Confocal analysis of mitochondrial membrane potential and mitochondrial calcium concentration in ncx3+/+ and ncx3−/− cortical neurons. Each bar represents the mean + S.E.M. of the percentage of different experimental values obtained in three independent experimental sessions. *P < 0.05 vs ncx3+/+ and CTL; **P < 0.05 vs OGD