Archives

  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • One key finding in our results observed after

    2019-08-11

    One key finding in our results observed after frataxin depletion is the decrease in NCLX levels. This transporter was described as a mitochondrial Na+/Ca exchanger by the group of I. Sekler [18], [45], [46] after initial studies of E. Carafoli [47]. While NCLX is a key mitochondrial transporter for calcium efflux, MCU is specific for calcium import into the mitochondria [48]. As shown in Fig. 6, NCLX showed reduced levels in cardiac myocytes, neurons and lymphoblastoid cell lines. Although the regulation of NCLX expression is yet unknown, altered levels of this protein play a relevant role in calcium dynamics physiology of heart [49], Fosfomycin calcium [50] and pancreatic β-cells [51] which, interestingly, are the most affected tissues in FA. This transporter has also shown to be involved in regulating automaticity in HL-1, a cardiac cell model [19], in the crosstalk between mitochondrial and cytosolic calcium to regulate cytosolic functions [12] and needed for the correct B lymphocyte functions [52]. Of note, it has been recently published that in a tamoxifen-induced NCLX deletion, caused severe myocardial dysfunction and death in mice [53]. Interestingly, in this article the authors show that the deleterious effects caused by NCLX depletion can be relieved by deleting CypD, which, as already mentioned, activates MPTP opening upon mitochondrial calcium concentrations increase. As a general trait, cells with reduced NCLX protein levels display calcium alterations and, at the mitochondrial level, such imbalance contribute to MPTP opening [54]. How can frataxin contribute to NCLX reduction is already unknown. Nevertheless, previous results of our group demonstrated a substantial survival increase when the calcium-chelating agent BAPTA was used in the neuronal model [6]. For this reason we hypothesized that BAPTA treatment could increase NCLX protein level. The increase of NCLX in frataxin-deficient DRG treated with BAPTA, showed in Fig. 7A and B, suggests that NCLX up-regulation can play an important role increasing cell survival not only in cardiac tissue (as reported in [53]) but also in neurons. In fact, NCLX up-regulation would restore the mitochondrial calcium balance, limiting calcium accumulation and, consequently, inhibiting the MPTP opening. The results obtained with BAPTA, also give some mechanistic clues about how it is able to restore NCLX levels because a calcium-dependent proteolytic cleavage of another Na+/Ca exchanger, NCX, occurs in the ER [28]. Consequently, it is conceivable that such proteases could play a role in decreasing NCLX levels in mitochondria and BAPTA would inhibit the proteolytic process. Further experiments to precisely define which proteases takes place in this process will be performed. In addition, we have added a proof that the effects on NCLX caused by frataxin-depletion can be restored by exogenous addition of the protein using TAT-MTScs-frataxin fusion protein (Fig. 7D and E). TAT-MTScs-frataxin can be delivered to cells by supplementing it to cultures, provided the penetrating abilities of TAT peptide and the mitochondrial targeting sequence (MTS). We have recently shown [31] that TAT-MTScs-frataxin is processed, targeted to mitochondria and that is able to restore cell survival in frataxin-deficient DRG neurons and can extend lifespan of mice models of Friedreich ataxia. Although the results shown here indicate that low NCLX levels would play a crucial role on mitochondrial calcium alterations, we cannot exclude the role of MCU. The reason for this rely on the knowledge that, although MCU amounts remain stable (Fig. S1), its function as a transporter could be affected by the regulators of its activity such as MICU1 or EMRE [55], [56]. Mitochondrial calcium transporters dysfunction can contribute to MPTP opening and, as a consequence, mitochondrial calcium can flow to the cytosol [57], [58], [59]. Such increase can activate Ca-dependent proteins such as calcineurin, a calcium/calmodulin-dependent phosphatase acting on several substrate proteins [60]. One of these targets is NFAT, a member of transcription factor family [61] that in heart, the most abundant forms are NFATc1, NFATc2, NFATc3 and NFATc4. The existence of several isoforms is not clear but it seems to have redundant functions and the c2, c3 and c4 isoforms have been reported to play an important role in cardiac hypertrophy [62]. The hyperphosphorylated NFAT remains in the cytosol while, upon dephosphorylation, migrates to the nucleus and activates a set of genes related to cardiac hypertrophy [63]. FA patients develop hypertrophy and cardiac problems are one of the major causes of death [64]. The cardiomyopathy has been clearly reproduced in mice models of the disease [65] in which hypertrophic markers such as Bnp (b-type natriuretic peptide) or Myh7 (myosin heavy chain-β) are up-regulated and are downstream targets of NFAT activation. Interestingly, results reported in [53], highlights the key role of NCLX in developing cardiac hypertrophy. Alteration of both proteins in our cell models (Fig. 7, Fig. 8), completely agrees with the results mentioned above and with the importance of calcium alterations as one of the initial disarrangements leading to the progression of the disease.