nuclear encoded ncx3 and akap121 two novel modulators of mitochondrial calcium efflux in normoxic and hypoxic neurons

Abstract Mitochondria are highly dynamic organelles extremely important for cell survival. Their structure resembles that of prokaryotic cells since they are composed with two membranes, the inner (IMM) and the outer mitochondrial membrane (OMM) delimitating the intermembrane space (IMS) and the matrix which contains mitochondrial DNA (mtDNA). This structure is strictly related to mitochondrial function since they produce the most of the cellular ATP through the oxidative phosphorylation which generate the electrochemical gradient at the two sides of the inner mitochondrial membrane an essential requirement for mitochondrial function. Cells of highly metabolic demand like those composing muscle, liver and brain, are particularly dependent on mitochondria for their activities. Mitochondria undergo to continual changes in morphology since, they fuse and divide, branch and fragment, swell and extend. Importantly, they move throughout the cell to deliver ATP and other metabolites where they are mostly required. Along with the capability to control energy metabolism, mitochondria play a critical role in the regulation of many physiological processes such as programmed cell death, autophagy, redox signalling, and stem cells reprogramming. All these phenomena are regulated by Ca2+ ions within this organelle. This review will discuss the molecular mechanisms regulating mitochondrial calcium cycling in physiological and pathological conditions with particular regard to their impact on mitochondrial dynamics and function during ischemia. Particular emphasis will be devoted to the role played by NCX3 and AKAP121 as new molecular targets for mitochondrial function and dysfunction

Pharmacological improvement of mitochondrial function as strategy to prevent neuronal damage in parkinson's disease

Parkinson’s disease is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta causing a deficiency of dopamine in the striatum. Specific neuropathological hallmarks of disease are represented by the presence of intraneuronal protein aggregates called Lewy bodies and Lewy neurites. The exact mechanism that leads to the selective loss of dopaminergic neurons is not yet completely clear. Some of the proposed cellular mechanisms include α-synuclein accumulation, oxidative stress, alterations of mitochondrial calcium homeostasis and mitochondrial dysfunctions. The present study was adresssed to investigate the intracellular pathways affected by ornithine treatment in in vitro and in vivo models of PD with particular regards to cellular metabolism and mitochondrial function in order to identify new and selective therapeutic strategies to prevent neuronal dysfunction occurring in PD and consequently to slow down disease progression. This aspect is extremely relevant considering the key role played by mitochondria in regulating synaptic activity. Therefore, a therapeutic strategy able to promote mitochondrial function might results useful to counteract the early synaptic dysfunction and the functional and pathological changes occurring in the brain of PD-affected patients. To this aim in vitro experiments have been performed to evaluate the effect of L-Ornithine L-Aspartate (LOLA) on mitochondrial function in two cellular models of PD represented by SH-SY5Y cells treated with rotenone (ROT), an inhibitor of mitochondrial complex I, and 6-hydroxydopamine (6-OHDA), an inducer of oxidative stress. Parallel in vivo experiments have been performed in mice expressing the mutation A53T of human α-synuclein (A53T-α-Syn) during aging, to evaluate the relationship between α-synuclein accumulation, mitochondrial dysfunction and behavioral phenotype of A53T-α-Syn-mice compared to WT, with particular regard to the motor and non-motor symptoms. These experiments will allow to identify an animal model recapitulating the prodromal phase of PD potentially useful to investigate the effect of drugs able to improve mitochondria function in animal models of PD progression

L-Ornithine L-Aspartate Restores Mitochondrial Function and Modulates Intracellular Calcium Homeostasis in Parkinson's Disease Models

The altered crosstalk between mitochondrial dysfunction, intracellular Ca2+ homeostasis, and oxidative stress has a central role in the dopaminergic neurodegeneration. In the present study, we investigated the hypothesis that pharmacological strategies able to improve mitochondrial functions might prevent neuronal dysfunction in in vitro models of Parkinson's disease. To this aim, the attention was focused on the amino acid ornithine due to its ability to cross the blood-brain barrier, to selectively reach and penetrate the mitochondria through the ornithine transporter 1, and to control mitochondrial function. To pursue this issue, experiments were performed in human neuroblastoma cells SH-SY5Y treated with rotenone and 6-hydroxydopamine to investigate the pharmacological profile of the compound L-Ornithine-L-Aspartate (LOLA) as a new potential therapeutic strategy to prevent dopaminergic neurons' death. In these models, confocal microscopy experiments with fluorescent dyes measuring mitochondrial calcium content, mitochondrial membrane potential, and mitochondrial ROS production, demonstrated that LOLA improved mitochondrial functions. Moreover, by increasing NCXs expression and activity, LOLA also reduced cytosolic [Ca2+] thanks to its ability to modulate NO production. Collectively, these results indicate that LOLA, by interfering with those mitochondrial mechanisms related to ROS and RNS production, promotes mitochondrial functional recovery, thus confirming the tight relationship existing between cytosolic ionic homeostasis and cellular metabolism depending on the type of insult applied

Ncx3-Induced Mitochondrial Dysfunction in Midbrain Leads to Neuroinflammation in Striatum of A53t-α-Synuclein Transgenic Old Mice

The exact mechanism underlying selective dopaminergic neurodegeneration is not completely understood. The complex interplay among toxic alpha-synuclein aggregates, oxidative stress, altered intracellular Ca2+-homeostasis, mitochondrial dysfunction and disruption of mitochondrial integrity is considered among the pathogenic mechanisms leading to dopaminergic neuronal loss. We herein investigated the molecular mechanisms leading to mitochondrial dysfunction and its relationship with activation of the neuroinflammatory process occurring in Parkinson’s disease. To address these issues, experiments were performed in vitro and in vivo in mice carrying the human mutation of α-synuclein A53T under the prion murine promoter. In these models, the expression and activity of NCX isoforms, a family of important transporters regulating ionic homeostasis in mammalian cells working in a bidirectional way, were evaluated in neurons and glial cells. Mitochondrial function was monitored with confocal microscopy and fluorescent dyes to measure mitochondrial calcium content and mitochondrial membrane potential. Parallel experiments were performed in 4 and 16-month-old A53T-α-synuclein Tg mice to correlate the functional data obtained in vitro with mitochondrial dysfunction and neuroinflammation through biochemical analysis. The results obtained demonstrated: 1. in A53T mice mitochondrial dysfunction occurs early in midbrain and later in striatum; 2. mitochondrial dysfunction occurring in the midbrain is mediated by the impairment of NCX3 protein expression in neurons and astrocytes; 3. mitochondrial dysfunction occurring early in midbrain triggers neuroinflammation later into the striatum, thus contributing to PD progression during mice aging

Gender Differences in Neurodegeneration, Neuroinflammation and Na+-Ca2+ Exchangers in the Female A53T Transgenic Mouse Model of Parkinson’s Disease

Twelve-month-old male mice expressing the human A53T variant of α-synuclein (A53T) develop dopamine neuron degeneration, neuroinflammation, and motor deficits, along with dysfunctions of the mitochondrial Na+-Ca2+ exchanger (NCX) isoforms 1 (NCX1) and 3 (NCX3) in the nigrostriatal system. Since gender is thought to play a role in the etiology of Parkinson’s disease (PD), we characterized neurochemical and behavioral alterations in 12-month-old female A53T transgenic mice. We investigated the presence of dopaminergic degeneration, astrogliosis and microgliosis using immunohistochemistry for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1 (IBA-1) in both the substantia nigra pars compacta (SNc) and striatum. In the same regions, we also evaluated the co-localization of NCX1 in cells positive for IBA-1 and the co-localization of NCX3 in TH-positive neurons and fibers. Furthermore, in both male and female mice, we performed motor (beam walking and pole tests) and memory [novel object recognition (NOR) and spontaneous alternation] tasks, together with tests to evaluate peripheral deficits (olfactory and stool collection tests). Female A53T transgenic mice displayed degeneration of nigral dopaminergic neurons, but neither microgliosis nor astrogliosis in the SNc and striatum. Moreover, female A53T transgenic mice displayed co-localization between NCX1 and IBA-1 positive cells in the striatum but not SNc, whereas NCX3 did not co-localize with either TH-positive terminals or neuronal bodies in the nigrostriatal system. Furthermore, female A53T transgenic mice showed increased crossing time in the beam walking test, but no impairments in the pole or memory tests, and in tests that evaluated peripheral deficits, whereas male A53T transgenic mice displayed motor, memory and peripheral deficits. Immunohistochemical and behavioral results obtained here in the female mice differ from those previously observed in males, and suggest a dissimilar influence of NCX1 and NCX3 on dopaminergic function in female and male A53T transgenic mice, strengthening the validity of these mice as a model for studying the etiological factors of PD