Na+-Ca2+ exchanger (NCX) isoforms constitute the major cellular Ca2+ extruding system in neurons and microglia. We herein investigated the role of NCX isoforms in the pathophysiology of Parkinson’s disease (PD). Their expression and activity were evaluated in neurons and glia of mice expressing the human A53T variant of α-synuclein (A53T mice), an animal model mimicking a familial form of PD. Western blotting revealed that NCX3 expression in the midbrain of 12-month old A53T mice was lower than that of wild type (WT). Conversely, NCX1 expression increased in the striatum. Immunohistochemical studies showed that glial fibrillary acidic protein (GFAP)-positive astroglial cells significantly increased in the substantia nigra pars compacta (SNc) and in the striatum. However, the number and the density of tyrosine hydroxylase (TH)-positive neurons decreased in both brain regions. Interestingly, ionized calcium binding adaptor molecule 1 (IBA-1)-positive microglial cells increased only in the striatum of A53T mice compared to WT. Double immunostaining studies showed that in A53T mice, NCX1 was exclusively co-expressed in IBA-1-positive microglial cells in the striatum, whereas NCX3 was solely co-expressed in TH-positive neurons in SNc. Beam walking and pole tests revealed a reduction in motor performance for A53T mice compared to WT. In vitro experiments in midbrain neurons from A53T and WT mice demonstrated a reduction in NCX3 expression, which was accompanied by mitochondrial overload of Ca2+ ions, monitored with confocal microscopy by X-Rhod-1 fluorescent dye. Collectively, in vivo and in vitro findings suggest that the reduction in NCX3 expression and activity in A53T neurons from midbrain may cause mitochondrial dysfunction and neuronal death in this brain area, whereas NCX1 overexpression in microglial cells may promote their proliferation in the striatum.

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,