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  • br Transparency document br Introduction Friedreich ataxia F


    Transparency document
    Introduction Friedreich ataxia (FA) is caused by decreased Tigecycline of the mitochondrial protein frataxin due to large expansions of GAA triplet repeats in the first intron of the gene [1]. Patients with FA suffer progressive limb and gait ataxia, dysarthria, reduced tendon reflex, extensor plantar responses and loss of position and vibration senses [2]. The pathologic changes occur first in dorsal root ganglia (DRG) with loss of large sensory neurons, followed by degeneration of the spinocerebellar and corticospinal tracts [3]. DRG neurons express the highest levels of frataxin and display a high vulnerability to frataxin down-regulation. Specific deleterious effects of frataxin depletion have been studied in DRGs using conditional knockout mice [4] and samples from patients with FA [5]. In frataxin-deficient DRG primary cultures we observed markers of apoptosis and decreased cell viability [6]. Such phenotype would rely in an alteration of calcium homeostasis which triggered, among others, Bax induction, CREB activation, fodrin cleavage by calpain and caspase 3, and increased protein nitration. Cell survival was rescued either by the BH4 antiapoptotic domain of Bcl-xL or by calcium chelators such as BAPTA. In primary cultures of frataxin-deficient cardiomyocytes we observed an increase of disarranged, swollen mitochondria and lipid droplets accumulation [7]. Such perturbations can be associated to mitochondrial permeability transition pore (MPTP) formation [8]. Proteins involved in MPTP formation have been controversial for many years but recent data indicate that it is mainly composed by dimers of ATP synthase and the mitochondrial phosphate carrier (PiC) [9], [10]. Pore opening is activated by cyclophilin D in response to changes in mitochondrial calcium concentrations. When opened, it contributes to the mitochondrial swelling and consequently to mitochondrial dysfunctions [11]. MPTP opening can also contribute to release of calcium from mitochondria to cytosol that, in turn, can activate Ca-dependent pathways [8], [12]. Inhibition of MPTP opening can be achieved by cyclosporin A (CsA), which, by binding to CypD, avoids its interaction with the other members of the pore [13], [14]. Besides cyclosporin A, other compounds such as NIM811, Debio025 or olesoxime (TRO19622) can also act as pore modulators [15], [16]. The mitochondrial sodium-calcium exchanger, NCLX, is the key transporter for calcium efflux from mitochondria to cytosol and its activity should be in balance with MCU, the mitochondrial calcium uniporter [17]. The relevance of NCLX function has been highlighted by the fact that calcium efflux from mitochondria is blocked if NCLX is knocked down [18]. It is also relevant its role in B lymphocytes chemotaxis by modulating calcium and in regulating the beating automaticity in HL-1, a spontaneously beating cardiac cell line [19]. Consequently, reduction of NCLX levels results in higher mitochondrial calcium concentrations and altering mitochondrial functions. The NFAT (Nuclear Factor of Activated T cells) is a family of transcription factors [20] that were first discovered on T lymphocytes but, in fact, they are expressed in many other cell types including cardiac tissue [21] and neurons [22], [23]. In the basal state, NFAT remains in the cytosol as a hyperphosphorylated, inactive protein. Its activation is triggered by Ca-activated phosphatases, such as calcineurin, allowing the dephosphorylated form to be imported into the nucleus. When normal calcium levels are restored, protein kinases, such as GSK3β, rephosphorylate NFAT and transported back to the cytosol [24].
    Discussion In this paper we have highlighted the importance of MPTP opening in frataxin-depleted cells and that CsA can prevent deleterious effects occurring after reduction of frataxin levels. The specificity of CsA on interacting with the mitochondrial protein CypD, allows unequivocally involve the MPTP in the events occurring after frataxin depletion. This has been complemented by calcein-cobalt quenching assays shown in Fig. 1. The contribution of MPTP to cell dysfunction has been described in many pathophysiological situations [11], [33], [34] related to toxic effects after ischemia-reperfusion. The ability of CsA to inhibit MPTP opening has also ben related to neuroprotection [35], [36], [37], protecting from heart injury [13], [34], [38] and able to restore NADH oxidation and normal calcium efflux in guinea pig cardiac mitochondria [39]. Mitochondrial impairment is of special importance for cardiac cells since energy metabolism depends, to a great extent, on oxidative catabolism of fatty acids. It is worth mentioning that neonatal rat ventricular myocytes are able to consume fatty acids [40]. In our cardiac cell model, we already showed that fatty acid supplementation to these cultures increase very long-chain acyl-CoA dehydrogenase (VLCAD) levels, indicative of the active β-oxidation pathway [7]. In this context, altered lipid metabolism as a consequence of frataxin deficiency has been reported in lymphoblast cells derived from FA patients [41]. DRG neurons are high energy demanding cells and the decreased mitochondrial membrane potential in frataxin-depleted DRGs [6], can be contributed by MPTP opening. Improvement of cell parameters exerted by CsA in DRGs (Fig. 5) is in line with the findings in cardiac cells since, once treated with CsA, frataxin-depleted DRG neurons survival is increased and fodrin cleavage reduced, both indicative of attenuated neurotoxicity. These results are in good agreement to the protecting role of CsA in neuronal cell models of ischemia–reperfusion situations [42]. In addition, although more clinical trials will be needed, experimental approaches and in phase II clinical research data suggest that the use of CsA as a neuroprotective compound should be considered [37], [43], [44].