Mitochondrial Calcium Homeostasis in the Pathology and Therapeutic Application in Friedreich's Ataxia

Friedreich’s ataxia (FRDA) is the most common neurodegenerative disease caused by an autosomal recessive single-gene mutation, affecting 2–4 per 100,000 Caucasians [1]. The causative gene is frataxin (FXN), located on chromosome 9q13, and it encodes a 210-amino-acid mitochondrial matrix protein. When the GAA triplet repeats expand to >200 in the first intron of the gene, it promotes the formation of DNA heterochromatin and transcriptional silencing, resulting in reduced FXN expression in all tested tissues (review in [2]).

The currently recognized primary function of mitochondrial FXN is to participate in the biosynthesis of iron-sulfur (Fe–S) clusters via interaction with two core components, ISCU as a scaffold protein and NFS1 as a cysteine desulfurase [3]. Fe–S clusters are not only important for the electron transport chain but also for aconitase in mitochondria. Therefore, FRDA is considered to be a nuclear gene-encoded atypical mitochondrial disease.

Maintenance of Mitochondrial Ca²⁺ Homeostasis

Different compartments of the cell, such as mitochondria, endoplasmic reticulum (ER), and plasma membrane, contribute to Ca²⁺ homeostasis. Therefore, the focus on mitochondria in this review unfortunately excludes a broad view of Ca²⁺ handling, but this has been intensively reviewed elsewhere [4–7].

Mitochondria and ER contacts form microdomains, called mitochondria-associated membranes (MAMs), which provide a platform that is fundamental for several cellular functions, such as Ca²⁺ trafficking. At MAMs, the distance between the ER and the outer mitochondrial membrane is 10–20 nm. GRP75 is an essential tethering protein maintaining MAM structure through the IP3R–GRP75–VDAC1 complex (Fig. 1). The Ca²⁺ released from the ER can quickly enter the mitochondria through voltage-dependent anion channels (VDACs) in the outer mitochondrial membrane. Ca²⁺ enters the inner mitochondrial membrane mainly through the mitochondrial Ca²⁺ uniporter (MCU), which depends on the concentration of Ca²⁺ ions in the cytoplasm. When this concentration increases, the MCU channel opens to let Ca²⁺ in. Mitochondria release Ca²⁺ mainly through three pathways: the Na⁺–Ca²⁺ pump (Na⁺/Ca²⁺ exchanger, NCLX), the H⁺–Ca²⁺ pump (H⁺/Ca²⁺ exchanger), and the mitochondrial permeability transition pore (mPTP) (Fig. 1), reviewed in [5]. Na⁺/Ca²⁺ exchange occurs in excitatory tissues (such as brain and heart), while H⁺/Ca²⁺ exchange occurs in non-excitatory tissues (such as liver). The mPTP is a group of protein complexes between the mitochondrial inner and outer membranes. It is a nonspecific Ca²⁺ ion channel associated with the apoptosis caused by disrupted Ca²⁺ homeostasis [6].

Fig. 1.

Ca²⁺ channels in the mitochondria-associated membrane (MAM) where FXN is involved. FXN is considered to be mainly located in the mitochondrial matrix and is involved in synthesizing Fe–S clusters (1). It has been demonstrated that FXN is a member of the MAM network and interacts with the MAM components GRP75 and IP3R (2). In FRDA models, the ryanodine receptor (RyR) is open (3); the mitochondrial permeability transition pore (mPTP) is leaky (4); the expression of the Na⁺-Ca²⁺ pump (NCLX) decreases (5); and the expression of the mitochondrial uniporter (MCU) remains unchanged (6), but the ability of mitochondria to buffer Ca²⁺ is reduced (see text). Cardiolipin (CL) is believed to be related to the stability of MCU conformation and opening of the mPTP, and the reduction of cardiolipin leads to a decrease in FXN expression (7).

Disturbed Mitochondrial Ca²⁺ Homeostasis in FRDA–Possibly One of the Primary Effects of FXN-Deficiency

The first recognition of Ca²⁺ involvement in FRDA came from Cortopassi, Ristow, and colleagues [8, 9]. They used the intracellular Ca²⁺ chelator BAPTA-AM to treat fibroblasts derived from FRDA patients following oxidant (H₂O₂) treatment and found that the chelator significantly rescued the fibroblasts [9]. FXN has been found to increase mitochondrial Ca²⁺ uptake [8]. A very recent study by Gonzalez-Cabo et al. found a few interesting phenomena: that the number of interactions between glucose-regulated protein 75 (GRP75) and inositol trisphosphate receptor (IP3R) and between VDAC1 and GRP75 were significantly reduced compared with control cells using proximity ligation assays, indicating disturbed mitochondria–ER interactions and dysregulation of mitochondrial Ca²⁺ homeostasis in FXN-deficient cells [10]. Further study found that FXN functions directly as a member of the MAM protein network, interacting with GRP75 and IP3R, the major proteins associated with the mitochondria–ER connection. These results indicate that FXN plays a vital role in the VDAC–GRP75–IP3R tethering bridge, in accord with the finding that GRP75 overexpression rescues FXN deficiency [11]. These studies also support the existence of extra-mitochondrial FXN [12–14], suggesting novel physiological functions of FXN.

Disruption of mitochondrial Ca²⁺ homeostasis has been reported in many FRDA models with low FXN expression. For example, in the human neuroma cell line SH-SY5Y, after artificially disrupting Ca²⁺ homeostasis, cells lost the capacity to maintain a proper ER, cytosolic, and mitochondrial Ca²⁺ concentrations when FXN was knocked down [15]. Ca²⁺ deposits in the form of scattered concretions have been found in the myocardium of FRDA patients [16]. Although no change in MCU expression was found, the overexpression of MCU in frataxin-deficient fly glia completely reversed the locomotor impairment [17], suggesting less Ca²⁺ influx into mitochondria in FRDA. Interestingly, FXN-knockdown cells show a decrease of NCLX, an essential carrier of mitochondrial Ca²⁺ efflux to the cytoplasm, and blocking mPTP or Ca²⁺ chelation recovers the level of NCLX [18]. The above results imply that mitochondrial Ca²⁺ buffering capacity is reduced, and artificially accelerating Ca²⁺ flow can improve mitochondrial function.

Oxidative Stress and Abnormal Ca²⁺ Homeostasis–a Vicious Cycle in FRDA

Iron overload has been shown to upregulate Ca²⁺ flow through L-type Ca²⁺ channels, which are also the primary means of iron entry into cardiomyocytes, resulting in further accumulation of cardiac iron and thus further increased oxidative stress. Due to oxidative stress in cardiomyocytes, the key participants in Ca²⁺ homeostasis are dysregulated. For example, the ryanodine receptor (RyR) contains many free cysteine residues sensitive to redox. Oxidative stress may increase the probability and duration of the RyR channel open state by influencing the redox state of the RyR, thus leading to Ca²⁺ leakage from the sarcoplasmic reticulum.

Antioxidant treatment can improve mitochondrial Ca²⁺ uptake by increasing the contact between the ER and mitochondria [10]. Vitamin E restores mitochondrial Ca²⁺ uptake in cerebellar granule neurons and cardiomyocytes lacking FXN. In neuroblastoma models with low FXN expression, both an increase of the VDAC1–IP3R interaction and enhanced mitochondrial Ca²⁺ uptake are improved by either Trolox or N-acetylcysteine treatment. The antioxidant therapies and defense against oxidative stress in FRDA have been intensively reviewed [19, 20]. The Phase III drug Omaveloxolone, an Nrf2 activator, was evaluated for the safety and efficacy in patients with FRDA and was found to significantly improve neurological function with acceptable safety and tolerance, representing a potential therapeutic agent [21].

Effect of Cardiolipin on Mitochondrial Ca²⁺ Homeostasis in FRDA

Cardiolipin is a negatively-charged lipid found predominantly within the inner mitochondrial membrane and is thought to be closely associated with the mitochondrial bioenergy process. Due to its high content of unsaturated fatty acids and its proximity to reactive oxygen species, cardiolipin is prone to peroxidation, which can affect the biochemical function of the mitochondrial inner membrane.

A direct link between cardiolipin and Ca²⁺ homeostasis has been reported [22]. We detected all three types of anti-cardiolipin antibodies (IgG, IgA, and IgM) in the serum and tissues of Y47 mice (FXN wild-type) and YG8R mice (FXN-deficient) by ELISA and found significant increases in YG8R mice (unpublished). Whether the abnormal rise in cardiolipin antibody plays a role in the development of FRDA remains unclear.

Our previous work demonstrated that the peptide SS-31, by targeting mitochondrial cardiolipin, upregulates FXN expression and improves mitochondrial function [23, 24]. We propose that this effect depends on mitochondrial membrane potential and, directly or indirectly, leads to recovery of the Ca²⁺ flow. Recently, it has been reported that FXN expression is reduced in yeast lacking cardiolipin [25]. In addition to being a component of MAMs, FXN seemingly exhibits mutual-reinforcement with cardiolipin in preserving mitochondrial Ca²⁺ homeostasis. Taken together, we speculate that cardiolipin plays a bridging role between FXN-dependent Ca²⁺ homeostasis and mitochondrial function and that cardiolipin might be a promising new target for the treatment of FRDA.

mPTP Opening-Mediated Abnormal Ca²⁺ Homeostasis in FRDA

The identity of this extraordinary channel mPTP is still debated. The early idea was that mPTP spans the mitochondrial inner and outer membranes and is mainly composed of VDACs in the outer membrane and adenine nucleotide translocators (ANTs) in the inner membrane. In addition, hexokinase II in the cytoplasm, creatine kinase in the intermembrane space, and cyclophilin D in the mitochondrial matrix are associated with the mPTP. How does FXN deficiency trigger mPTP opening? One possible explanation is oxidative stress resulting from disturbed Ca²⁺ and iron metabolism. Peroxidized cardiolipin cannot stabilize the conformation of the ANT dimer and interferes with the interaction between ANT and cardiolipin, leading to opening of the mPTP.

The latter idea is derived from that Ca²⁺ reversibly converts F1F0-ATP synthase to mPTP. Cyclophilin D binds to F1F0-ATP synthase, and this binding is promoted by phosphate and inhibited by cyclosporine A (CsA), which response is very similar to the characteristics of the mPTP. In cardiomyocytes, the lack of FXN leads to opening of the mPTP, and CsA has a protective effect on mitochondria, reduces lipid droplets and mitochondrial swelling in cardiomyocytes, restores DRG neuron survival, and inhibits the dephosphorylation of NFAT (nuclear factor of activated T cells) [18], indicating that the mPTP dysregulation-mediated disruption of Ca²⁺ homeostasis is truly involved in the pathophysiology of FRDA.

Although more clinical trials are needed, data from preclinical and phase II clinical studies indicate that CsA should be considered a neuroprotective agent. In addition, other mPTP regulators, such as NIM811, DeBio025, and TRO196221, are also considered strong candidates for the treatment of FRDA.

Perspectives

The lack of FXN protein causes FRDA, and physiological functions of FXN inside and outside mitochondria have been proposed. Clinically, significant progress has been made, particularly the new hope from the Phase III drug Omaveloxolone. A few drugs targeting Ca²⁺ homeostasis have also shown good preclinical efficacy in the treatment of FRDA [9, 18].

However, the clinical Ca²⁺ data are very limited. The enhancement of mitochondrial Ca²⁺ flux, including influx and efflux, benefits FXN-deficient cells in vitro. Further, Ca²⁺ chelation or the use of CsA to block the opening of the mPTP improves mitochondrial function. Is there a point of convergence among these seemingly opposing views? One possible explanation is that the increase of mitochondrial Ca²⁺ is an early response to FXN deficiency, and the decrease is a later consequence. Therefore, the effects of interventions to decrease or increase the concentration of Ca²⁺ could depend on the phases of the disease. This notion needs to be further tested by studying the Ca²⁺ concentration changes in an FXN deficiency model over time. Another explanation is, possibly, that the mitochondrial capacity for Ca²⁺ influx and efflux is reduced, narrowing the mitochondrial Ca²⁺ range or amplitude. This aligns with the diminished cellular oxidative phosphorylation and mitochondria-dependent ATP production in FRDA.

Studying the distinct consequences mediated by FXN loss from Fe–S cluster deficiency and Ca²⁺-homeostasis disruption poses a significant challenge to experimental design because FXN loss inevitably causes an increase of oxidative stress. Therefore, follow-up studies need to design experimental methods to express extra-mitochondrial and intra-mitochondrial FXN individually in cells derived from FRDA patients or FXN-knockdown cells. The Ca²⁺ homeostasis and Fe–S cluster biogenesis under various challenging conditions are to be evaluated to further establish the precise function of FXN, particularly the FXN isoforms. The expression of extra-mitochondrial FXN benefits mitochondrial dynamics [26] and acts against oxidative stress [13] have been demonstrated. The protein structure of the FXN–GRP75 or FXN–IP3Rs complex needs to be determined to consolidate the function of extra-mitochondrial FXN.

In summary, the primary and secondary consequences of FXN deficiency point to turbulence of Ca²⁺ homeostasis and vulnerability to oxidative stress, which make a vicious cycle to contribute to the aggravation of FRDA pathology. In principle, blocking either one would improve the manifestations in FRDA patients, as demonstrated by the phase III drug as an Nrf2 activator (https://www.curefa.org/pipeline). A combination of Ca²⁺-flow tuner with enhanced capacity against oxidative stress would make a profound impact on the treatment of FRDA.

Conflict of interest

The authors declare that there are no conflicts of interest.