**5. Calcium deregulation in OXPHOS diseases**

42 Bioenergetics

Stefani et al., 2011). Mutations of conserved acidic residues within the short sequence linking the two transmembrane domains abrogated the ability of MCU to reconstitute mitochondrial Ca2+ uptake, whereas mutation of a nearby serine residue (S259) conferred resistance to Ru360, indicating that the acidic residues are required for Ca2+ uptake and that S259 is critical for MCU sensitivity to ruthenium red (Baughman et al., 2011). Finally, and most convincingly, expression of the purified protein in planar lipid bilayers was sufficient to reconstitute ion channel activity in solutions containing only Ca2+ (De Stefani et al., 2011). The currents were carried by a channel of small conductance (6–7 pS), fast opening/closing kinetics, and low opening probability, and were inhibited by ruthenium red, as expected for the MCU. Proteins mutated at two of the conserved acidic residues failed to generate Ca2+ currents when inserted into bilayers and acted as dominant negative when expressed in HeLa cells. These data clearly identified MCU as mitochondrial Ca2+ uniporter. In accordance to the notion that mitochondrial Ca2+ overload enhances the sensitivity to apoptosis, it was also demonstrated that cells overexpressing MCU were more sensitive to apoptosis after treatment with ceramide and

Compared to the MCU, the proteins that catalyze the efflux of Ca2+ from mitochondria have received much less attention. The extrusion of Ca2+ from mitochondria is coupled to the entry of Na+ across an electrogenic 1Ca+:3Na+ exchanger (Dash & Beard, 2008) that is inhibited by the benzothiazepine derivative CGP37157 ((Cox et al., 1993), and reviewed in (Bernardi, 1999)). The subsequent efflux of sodium ions by the mitochondrial 1Na+:1H+ exchanger (mNHE) eventually results in the entry of three protons into the matrix for each Ca2+ ion that leaves mitochondria. Ca2+ extrusion thus has a high energetic cost, as it dissipates the proton gradient generated by the respiratory chain (reviewed in (Bernardi, 1999)). The molecule catalyzing mitochondrial Na+/Ca2+ exchange has been recently identified as NCLX/NCKX6, a protein localized in mitochondrial cristae (Palty et al., 2010), whereas stomatin-like protein 2 (SLP-2), an inner membrane protein, was shown to negatively modulate the activity of the mitochondrial Na+/Ca2+ exchanger (Da Cruz et al., 2010). Functional evidence from knock-down and overexpression studies indicate that NCLX is an essential part of the mitochondrial sodium Ca2+ exchanger whereas SLP-2 is an

accessory protein that negatively regulates mitochondrial Ca2+ extrusion (Figure 1B).

**4.2.3 Mitochondrial calcium overload: Activation of the permeability transition pore**  When mitochondrial Ca2+ loads exceed the buffering capacity of inner membrane exchangers, an additional pathway for Ca2+ efflux from mitochondria may exist through opening of the permeability transition pore (PTP). The PTP is a voltage-dependent, cyclosporin A (CsA)-sensitive, high-conductance channel of the inner mitochondrial membrane (for reviews, see (Bernardi et al., 2006; Rasola & Bernardi, 2007)). Indeed, the interplay between the rate of mitochondrial Ca2+ influx and efflux modulates mitochondrial matrix Ca2+, which in turn is widely considered to be a key factor for the regulation of the PTP open–closed transitions (Bernardi et al., 1999). Although opening of the PTP in response to Ca2+ has been documented in isolated mitochondria and permeabilized cells (Bernardi et al., 2006; Rasola & Bernardi, 2007), assessing opening of the PTP in intact neurons and other

H2O2 (De Stefani et al., 2011) (Figure 1B).

**4.2.2 Mechanisms of mitochondrial calcium efflux** 

The direct consequences of OXPHOS defects include alteration of mitochondrial membrane potential, ATP/ADP ratio, ROS production and mitochondrial Ca2+ homeostasis. The varied biochemical changes that occur in cases of OXPHOS deficiencies have a direct effect on cellular functions. Yet, they are also key underlying mediators of the (retrograde) communication between the mitochondrion and the nucleus, which results in specific gene expression of both nuclear and mitochondrial genomes (see review (Reinecke et al., 2009)).

We will review in this chapter only Ca2+ deregulation in OXPHOS. We will discuss the consequences of such deregulation on mitochondrial function and the cross regulation between Ca2+ and bioenergetics in the development of cellular pathology. We summarized in Table 1 the alterations of subcellular Ca2+ signals in OXPHOS related diseases (Table 1).

Decreased proton pumping due to respiratory chain defects can result in reduced mitochondrial membrane potential and proton gradient, which are used to generate ATP. Deregulation of the membrane potential secondary to a deficiency in the respiratory chain may modify the kinetics and/or accumulation capacity of Ca2+ in the mitochondria, with possible consequences not only at the level of respiratory chain function (loop effect) and of the mitochondria in general, but also at the level of the ER function, which is largely dependent on Ca2+ concentrations, and at the level of cytosolic Ca2+ signalling, which plays a major role in regulating cell functions. Deficiencies of OXPHOS also result in other immediate and downstream metabolic, structural, and functional effects. These effects are closely associated with mitochondrial dysfunction. The nicotinamide dinucleotide (NAD) redox balance, which is converted to the reduced state in OXPHOS deficiencies, is a fundamental mediator of several biological processes, such as energy metabolism, Ca2+ homeostasis, cellular redox balance, immunological function, and gene expression (Munnich & Rustin, 2001; Ying, 2008).

It is important to mention that analyses of Ca2+ signalling targeting OXPHOS diseases are sporadic, partial and incomplete. This situation can be explained by : 1) the recent development of new techniques permitting detailed and specific subcellular Ca2+ analyses such as recombinant "aequorin" probes developed by the group headed by Professors Rizzuto and Pozzan, and the latest generation of GFP-based Ca2+ probes (camgaroos, cameleons and pericams) characterized by a great potential to analyse Ca2+ dynamics in mitochondria at the single cell level; 2) Absence of suitable "easy" study models (see chapter 3); and 3) the difficulty in the characterization of OXPHOS deficiencies (see chapter 2-2).


BK: bradykinin; COX: cytchrome oxidase Htt: Huntingtin; NC: non communicated; ND: not determined; ROS: reactive oxygen species; SOC: store operated Ca2+ entry; PDH: Pyruvate dehydrogenase; KO: knock out; [Ca2+]cyt, cytosolic calcium-concentration; [Ca2+]er, endoplasmic reticulum calcium-concentration; [Ca2+]mt, mitochondrial calcium-concentration; Ca2+, calcium. (1) Insertion; (2) Deletion; (3) repeat.

Table 1. Calcium deregulation in OXPHOS diseases

Mitochondrial Calcium Signalling: Role in Oxidative Phosphorylation Diseases 45

Calcium deregulation was first reported in OXPHOS diseases linked to mitochondrial mutation. Brini and collaborators monitored subcellular Ca2+ signalling in cybrid cells with 0% and 100% of the MERRF (nt 8356 T/C) and NARP (nt 8993 T/G) mutations using cytosolic aequorin and aequorin probe targeted to the mitochondria. They showed a reduced mitochondrial [Ca2+] ([Ca2+]mit) transient in MERRF cells but not in NARP cells upon stimulation with IP3-generating agonist, whereas cytosolic Ca2+ responses ([Ca2+]cyt)

In another study, cybrid cells with 98 % of NARP mutation (nt 8993 T/G) and Rho0 cells show a disturbed mitochondrial network and actin cytoskeleton. These cells show also a slower Ca2+ influx rates in comparison to parental cells. Authors postulate that proper actin cytoskeletal organization is important for CCE (capacitative Ca2+ entry) in these cells

Abnormal Ca2+ homeostasis and mitochondrial polarization was also reported in fibroblasts from patients with MELAS syndrome. These cells showed an increased Ca2+ influx

A comparative study was performed to establish sensitivity to oxidant in cybrid cells bearing the LHON, MELAS, or MERRF. The order of sensitivity to H2O2 exposure was MELAS>LHON>MERRF>controls. Consistent with the hypothesis that death induced by oxidative stress is Ca2+ dependent, depletion of Ca2+ from the medium protected all cells from cell death. This study reveals indirectly that LHON as well as MELAS and MERRF

In 2007, another study performed on cybrid cells incorporating two pathogenic mitochondrial mutations (nt 3243 A/G, nt 3202 A/G) reveal that the decreased ATP production by oxidative phosphorylation was compensated by a rise in anaerobic glycolysis. Regarding Ca2+ homeostasis, these cells did not show any alteration of Ca2+ signals in the cytosol but take longer to clear up the histamine induced Ca2+ signal in the mitochondria

All over, these studies revealed a deranged Ca2+ homeostasis in OXPHOS diseases linked to mitochondrial mutations. These alteration are not solely at the level of mitochondria but were also observed in the cytosol. Depending on the study model and/or mutation, increased cytosolic Ca2+ levels are linked to increased Ca2+ influx through the plasma

The consequences of mitochondrial complex I deficiency on Ca2+ homeostasis was first studied in a genetically characterized human complex I deficient fibroblast cell lines harbouring nuclear NDUFS7 (nt 364G/A) mutation linked to Leigh's syndrome. These cells show a reduced mitochondrial Ca2+ accumulation and consequent ATP synthesis (Visch et al., 2004). In 2006, the same group investigated the mechanism(s) underlying this impaired response. The study was conducted in fibroblasts from 6 healthy subjects and 14 genetically characterized patients expressing mitochondria targeted luciferase. The results revealed that the agonist-induced increase in mitochondrial ATP ([ATP]mit) was significantly, but to a variable degree, decreased in 10 patients. They also reported a reduced agonist-evoked mitochondrial [Ca2+] signal, measured with mitochondria targeted aequorin, and cytosolic [Ca2+] signal, measured with Fura-2, AM. Measurement of Ca2+ content of the ER, calculated from the increase in [Ca2+]Cyt evoked by thapsigargin, an inhibitor of the ER Ca2+ ATPase

**5.1 Calcium deregulation in MELAS, MERRF, NARP and LHON** 

associated to a decreased mitochondrial potential (Moudy, 1995).

show an increased basal Ca2+ load (Wong & Cortopassi, 1997).

membrane or reduced Ca2+ uptake capacity by the mitochondria.

**5.2 Calcium deregulation in Complex I deficiency** 

were normal in both cell types (Brini et al., 1999).

(Szczepanowska et al., 2004).

(von Kleist-Retzow et al., 2007).
