The Interaction Between NCX3 and AKAP121 on the Outer Mitochondrial Membrane Controls Hypoxia-Induced Mitochondrial Dynamics in Neurons

Mitochondrial dysfunction is a common feature of many pathologic conditions, such as neurodegenerative disorders. Previous data obtained in our laboratory showed that NCX3, the only isoform of sodium/calcium exchanger found also at mitochondrial level, regulates the mitochondrial Ca2+ homeostasis and prevents the hypoxia-induced cell death through its interaction with the protein kinase A anchoring protein 121 (AKAP121). Considering that the expression levels of NCX3 and AKAP121 decrease during hypoxia and mitochondrial morphology is altered, the aim of the study was understanding the role played by NCX3 and AKAP121 in the regulation of mitochondrial morphology during hypoxia. Because hypoxia induces the proteasomal degradation of AKAP121 through the activation of the E3-ubiquitin ligase Seven in-absentia homolog 2, Siah2, all the experiments were performed in cortical neurons obtained from wild type, siah2-/- and ncx3-/- mice exposed to 3 hrs of oxygen and glucose deprivation (OGD) and OGD followed by 24 hrs of reoxygenation (OGD/Reoxy). In these models the expression levels of Drp1 and Mfn1, two proteins respectively considered markers of mitochondrial fission and fusion events, were evaluated. Furthermore, mitochondrial morphology in living neurons and mitochondrial functional parameters, such as mitochondrial Ca2+ concentration ([Ca2+]m), mitochondrial membrane potential, ATP production and mitochondrial oxidative capacity were also assessed. The study conducted by Western Blot and mitochondrial morphology analysis showed that the exposure of siah2+/+ neurons to OGD induced mitochondrial fission and a reduction in NCX3 expression, while [Ca2+]m was increased. Interestingly, in siah2-/- neurons exposed to OGD no changes in NCX3 expression and mitochondrial morphology occurred, whereas [Ca2+]m was lower compared to siah2+/+ neurons. Moreover, the exposure of siah2-/- neurons to OGD/Reoxy increased mitochondrial interconnectivity and reduced [Ca2+]m compared to siah2+/+ neurons. In addition, a drop in ATP production, mitochondrial membrane potential and oxidative capacity was observed in siah2+/+ neurons exposed to OGD and OGD/Reoxy, whereas it was not in siah2-/- neurons. Finally, the analysis conducted in ncx3+/+ and ncx3-/- neurons did not show any difference among these two neuronal models in ATP production and in Drp1 and Mfn1 expression, while, interestingly, [Ca2+]m was increased in ncx3-/- neurons compared to ncx3+/+ neurons in basal conditions. Collectively, these findings suggested that NCX3 and AKAP121 might influence mitochondrial dynamics through the regulation of mitochondrial calcium concentrations in response to hypoxia.