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

44 Bioenergetics

BK: bradykinin; COX: cytchrome oxidase Htt: Huntingtin; NC: non communicated; ND: not determined; ROS: reactive oxygen species; SOC: store operated Ca2+ entry; PDH: Pyruvate

Insertion; (2) Deletion; (3) repeat.

Table 1. Calcium deregulation in OXPHOS diseases

dehydrogenase; KO: knock out; [Ca2+]cyt, cytosolic calcium-concentration; [Ca2+]er, endoplasmic reticulum calcium-concentration; [Ca2+]mt, mitochondrial calcium-concentration; Ca2+, calcium. (1) 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) were normal in both cell types (Brini et al., 1999).

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 (Szczepanowska et al., 2004).

Abnormal Ca2+ homeostasis and mitochondrial polarization was also reported in fibroblasts from patients with MELAS syndrome. These cells showed an increased Ca2+ influx associated to a decreased mitochondrial potential (Moudy, 1995).

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

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

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 membrane or reduced Ca2+ uptake capacity by the mitochondria.

#### **5.2 Calcium deregulation in Complex I deficiency**

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

Mitochondrial Calcium Signalling: Role in Oxidative Phosphorylation Diseases 47

The data obtained upon complex II inhibition by 3NP are in accordance with those obtained from Huntington's patients and transgenic mice. Mitochondria isolated from lymphoblasts of individuals with HD showed reduced mitochondrial potential and increased sensitivity to depolarization upon Ca2+ addition. Similar results were obtained in transgenic HD mice expressing mutated huntingtin (Panov et al., 2002). In addition, mitochondria from HD mice showed lower Ca2+ retention capacity. These mitochondrial abnormalities preceded the onset of pathological or behavioural tract by months, suggesting that mitochondrial Ca2+ deregulation occurs early in HD (Panov et al., 2002). In a recent study, Lim and collaborators explore Ca2+ homeostasis and mitochondrial dysfunction in clonal striatal cell lines established from a transgenic HD mouse model and showed transcriptional changes in the components of the phosphatidylinositol cycle and in receptors for myo-inositol triphosphate-linked agonist. The overall result of such changes is to decrease basal Ca2+ in mutant cells. Mitochondria from mutant cells failed to handle large Ca2+ loads and this seems to be due to increased Ca2+ sensitivity of the permeability transition. This study reveals a compensatory attempt to prevent the Ca2+ stress that would exacerbate

Our group was the first to investigate Ca2+ homeostasis in human fibroblasts isolated from a patient with Leigh's syndrome harbouring a homozygous R554W substitution in the flavoprotein subunit of the complex II (SDHA). Our study was conducted in parallel in control fibroblasts and in neuroblastoma SH-SY5Y cells upon inhibition of complex II with 3NP or Atpenin A5 at doses which did not induce cell death, thus affording to study complex II deficiency independently from cell death. We showed that mutation or chronic inhibition of complex II determined a large increase in basal and agonist-evoked Ca2+ signals in the cytosol and mitochondria, in parallel with mitochondrial dysfunction (membrane potential loss, ATP reduction and increased ROS). Cytosolic and mitochondrial Ca2+ overload are linked to increased ER Ca2+ leakage, and to PMCA and SERCA2b proteasome-dependent degradation. Increased mitochondrial Ca2+ load is also contributed by decreased mitochondrial motility and increased ER-mitochondrial contacts. These findings are interesting since they link for the first time OXPHOS-related mitochondrial pathology to the regulation of the stability of two major actors in Ca2+ signalling regulation, namely PMCA and SERCA. We postulate that SERCA2b and PMCA degradation is predictably related to a decrease of mitochondrial ATP production, since SERCA2b and PMCA degradation was also observed upon ATP synthase inhibition by rotenone. This phenomenon could be interpreted as an adaptation response to ATP demise in OXPHOS diseases. Our study revealed also the activation of a compensatory attempt to restore total ATP level through the activation of anaerobic glycolysis in a Ca2+-dependent manner (M'Baya et al., 2010). This study revealed a double hint of Ca2+ signalling deregulation in complex II deficiency. On the one hand Ca2+ overload may favour the activation of glycolytic ATP production and on the other hand favoured Ca2+-mediated mitochondrial

**5.4 Calcium deregulation in OXPHOS diseases linked to defects in OXPHOS assembly** 

Leigh's syndrome associated with COX deficiency is usually caused by mutations of SURF1, a gene coding a putative COX assembly factor. Fibroblasts isolated from patients harboring SURF1 mutation displayed a low Ca2+ influx through SOC (store operated Ca2+ channels) as

mitochondrial damage in HD (Lim et al., 2008).

pathology (M'Baya et al., 2010).

**and iron homeostasis: COX and frataxin deficiencies** 

revealed also a decrease in mutated cells as compared to controls. Regression analysis revealed that the increase in [ATP]mit was directly proportional to the increases in [Ca2+]cyt and [Ca2+]mit and to the ER Ca2+ content. This was the first report showing a pathological ER Ca2+ homeostasis in OXPHOS disease models. The authors postulated that the reduced ER Ca2+ content could be the direct cause of the impaired agonist-induced increase in [ATP]mit in human complex I deficiency (Visch et al., 2006). However, the molecular mechanisms underlying ER Ca2+ deregulation were not revealed.

Another key cellular feature that was extensively investigated in patient fibroblasts harboring complex I deficiency is mitochondrial morphology. The quantification of mitochondrial morphology in a cohort of 14 patients fibroblast cell lines revealed two distinct classes of patient fibroblasts, one in which the cells mainly contained short circular fragmented mitochondria, and one in which the cells displayed a normal filamentous mitochondrial morphology (Koopman et al., 2007). Authors postulated that these differences are linked to ROS levels (Koopman et al., 2007). In a second report, the authors analyzed the relationship between mitochondrial dynamics and structure and Ca2+/ATP handling in the same cohort. Regression analysis of the agonist-induced Ca2+/ATP handling and mitochondrial morphology shows that increased mitochondrial number is associated to reduced Ca2+-stimulated mitochondrial ATP and reduced stimulation of cytosolic Ca2+ removal rate (Willems et al., 2009).

#### **5.3 Calcium deregulation in Complex II deficiency**

The investigation of Ca2+ deregulation linked to complex II deficiency were largely performed upon complex II inhibition by 3-nitropropionic acid (3NP) . The inhibition of complex II by 3NP is related to neuronal death, anatomic and neurochemical changes similar to those occurring in Huntington's disease (HD).

In primary cultures of rodent central nervous system, 3NP elicits an early increase in neuronal [Ca2+]cyt, and both apoptotic and necrotic neuronal death (Greene et al., 1998). 3NP treatment produces a long term potentiation of the NMDA-mediated synaptic excitation in striatal spiny neurons. This also involves increased intracellular Ca2+ (Calabresi et al., 2001).

To the mechanisms underlying increased [Ca2+]cyt upon 3NP treatment, it was shown that short treatment-induced [Ca2+]cyt increase occurs through NMDA-GLUR (Glutamate receptor) and VGCC and implicates also internal stores (Lee et al., 2002). In astrocyte cultures, Tatiani, R. Rosenstock and collaborators showed that 3NP is also able to release mitochondrial Ca2+ independently from internal stores and from Ca2+ entry through the plasma membrane (Rosenstock et al., 2004). Another group showed that 3NP-induced necrosis in primary hippocampal neurons is associated with an increase in both cytosolic and mitochondrial [Ca2+], decreased ATP and rapid mitochondrial potential depolarization. In this context, the increased [Ca2+] was shown to result from Ca2+ influx through NMDA receptors (Nasr et al., 2003).

The occurrence of mitochondrial permeability transition (PT) was shown to be the cause of the loss of neuronal viability induced by complex II inhibition (Maciel et al., 2004). This is in line with studies showing increased susceptibility of striatal mitochondria to Ca2+- induced PT (Brustovetsky et al., 2003) and that cyclosporine A (inhibitor of PT) protects against 3NP toxicity in striatal neurons (Leventhal et al., 2000) and astrocytes (Rosenstock et al., 2004). Accordingly, inhibition of mitochondrial Ca2+ influx by ruthenium red significantly reduces 3NP-induced cell death (Ruan et al., 2004).

revealed also a decrease in mutated cells as compared to controls. Regression analysis revealed that the increase in [ATP]mit was directly proportional to the increases in [Ca2+]cyt and [Ca2+]mit and to the ER Ca2+ content. This was the first report showing a pathological ER Ca2+ homeostasis in OXPHOS disease models. The authors postulated that the reduced ER Ca2+ content could be the direct cause of the impaired agonist-induced increase in [ATP]mit in human complex I deficiency (Visch et al., 2006). However, the molecular mechanisms

Another key cellular feature that was extensively investigated in patient fibroblasts harboring complex I deficiency is mitochondrial morphology. The quantification of mitochondrial morphology in a cohort of 14 patients fibroblast cell lines revealed two distinct classes of patient fibroblasts, one in which the cells mainly contained short circular fragmented mitochondria, and one in which the cells displayed a normal filamentous mitochondrial morphology (Koopman et al., 2007). Authors postulated that these differences are linked to ROS levels (Koopman et al., 2007). In a second report, the authors analyzed the relationship between mitochondrial dynamics and structure and Ca2+/ATP handling in the same cohort. Regression analysis of the agonist-induced Ca2+/ATP handling and mitochondrial morphology shows that increased mitochondrial number is associated to reduced Ca2+-stimulated mitochondrial ATP and reduced stimulation of cytosolic Ca2+

The investigation of Ca2+ deregulation linked to complex II deficiency were largely performed upon complex II inhibition by 3-nitropropionic acid (3NP) . The inhibition of complex II by 3NP is related to neuronal death, anatomic and neurochemical changes

In primary cultures of rodent central nervous system, 3NP elicits an early increase in neuronal [Ca2+]cyt, and both apoptotic and necrotic neuronal death (Greene et al., 1998). 3NP treatment produces a long term potentiation of the NMDA-mediated synaptic excitation in striatal spiny neurons. This also involves increased intracellular Ca2+ (Calabresi et al., 2001). To the mechanisms underlying increased [Ca2+]cyt upon 3NP treatment, it was shown that short treatment-induced [Ca2+]cyt increase occurs through NMDA-GLUR (Glutamate receptor) and VGCC and implicates also internal stores (Lee et al., 2002). In astrocyte cultures, Tatiani, R. Rosenstock and collaborators showed that 3NP is also able to release mitochondrial Ca2+ independently from internal stores and from Ca2+ entry through the plasma membrane (Rosenstock et al., 2004). Another group showed that 3NP-induced necrosis in primary hippocampal neurons is associated with an increase in both cytosolic and mitochondrial [Ca2+], decreased ATP and rapid mitochondrial potential depolarization. In this context, the increased [Ca2+] was shown to result from Ca2+ influx through NMDA

The occurrence of mitochondrial permeability transition (PT) was shown to be the cause of the loss of neuronal viability induced by complex II inhibition (Maciel et al., 2004). This is in line with studies showing increased susceptibility of striatal mitochondria to Ca2+- induced PT (Brustovetsky et al., 2003) and that cyclosporine A (inhibitor of PT) protects against 3NP toxicity in striatal neurons (Leventhal et al., 2000) and astrocytes (Rosenstock et al., 2004). Accordingly, inhibition of mitochondrial Ca2+ influx by ruthenium red significantly reduces

underlying ER Ca2+ deregulation were not revealed.

**5.3 Calcium deregulation in Complex II deficiency** 

similar to those occurring in Huntington's disease (HD).

removal rate (Willems et al., 2009).

receptors (Nasr et al., 2003).

3NP-induced cell death (Ruan et al., 2004).

The data obtained upon complex II inhibition by 3NP are in accordance with those obtained from Huntington's patients and transgenic mice. Mitochondria isolated from lymphoblasts of individuals with HD showed reduced mitochondrial potential and increased sensitivity to depolarization upon Ca2+ addition. Similar results were obtained in transgenic HD mice expressing mutated huntingtin (Panov et al., 2002). In addition, mitochondria from HD mice showed lower Ca2+ retention capacity. These mitochondrial abnormalities preceded the onset of pathological or behavioural tract by months, suggesting that mitochondrial Ca2+ deregulation occurs early in HD (Panov et al., 2002). In a recent study, Lim and collaborators explore Ca2+ homeostasis and mitochondrial dysfunction in clonal striatal cell lines established from a transgenic HD mouse model and showed transcriptional changes in the components of the phosphatidylinositol cycle and in receptors for myo-inositol triphosphate-linked agonist. The overall result of such changes is to decrease basal Ca2+ in mutant cells. Mitochondria from mutant cells failed to handle large Ca2+ loads and this seems to be due to increased Ca2+ sensitivity of the permeability transition. This study reveals a compensatory attempt to prevent the Ca2+ stress that would exacerbate mitochondrial damage in HD (Lim et al., 2008).

Our group was the first to investigate Ca2+ homeostasis in human fibroblasts isolated from a patient with Leigh's syndrome harbouring a homozygous R554W substitution in the flavoprotein subunit of the complex II (SDHA). Our study was conducted in parallel in control fibroblasts and in neuroblastoma SH-SY5Y cells upon inhibition of complex II with 3NP or Atpenin A5 at doses which did not induce cell death, thus affording to study complex II deficiency independently from cell death. We showed that mutation or chronic inhibition of complex II determined a large increase in basal and agonist-evoked Ca2+ signals in the cytosol and mitochondria, in parallel with mitochondrial dysfunction (membrane potential loss, ATP reduction and increased ROS). Cytosolic and mitochondrial Ca2+ overload are linked to increased ER Ca2+ leakage, and to PMCA and SERCA2b proteasome-dependent degradation. Increased mitochondrial Ca2+ load is also contributed by decreased mitochondrial motility and increased ER-mitochondrial contacts. These findings are interesting since they link for the first time OXPHOS-related mitochondrial pathology to the regulation of the stability of two major actors in Ca2+ signalling regulation, namely PMCA and SERCA. We postulate that SERCA2b and PMCA degradation is predictably related to a decrease of mitochondrial ATP production, since SERCA2b and PMCA degradation was also observed upon ATP synthase inhibition by rotenone. This phenomenon could be interpreted as an adaptation response to ATP demise in OXPHOS diseases. Our study revealed also the activation of a compensatory attempt to restore total ATP level through the activation of anaerobic glycolysis in a Ca2+-dependent manner (M'Baya et al., 2010). This study revealed a double hint of Ca2+ signalling deregulation in complex II deficiency. On the one hand Ca2+ overload may favour the activation of glycolytic ATP production and on the other hand favoured Ca2+-mediated mitochondrial pathology (M'Baya et al., 2010).

#### **5.4 Calcium deregulation in OXPHOS diseases linked to defects in OXPHOS assembly and iron homeostasis: COX and frataxin deficiencies**

Leigh's syndrome associated with COX deficiency is usually caused by mutations of SURF1, a gene coding a putative COX assembly factor. Fibroblasts isolated from patients harboring SURF1 mutation displayed a low Ca2+ influx through SOC (store operated Ca2+ channels) as

Mitochondrial Calcium Signalling: Role in Oxidative Phosphorylation Diseases 49

of mitochondrial polymorphisms in the pathology of neurodegenerative diseases by

Aerobic metabolism may also affect mitochondrial Ca2+ homeostasis. Thus, deregulation of Ca2+ handling was also reported in human fibroblasts from a patient with an inherited defect in pyruvate dehydrogenase (PDH). Indeed, these cells show a decrease ability to sequester cytosolic Ca2+ into mitochondria without affecting basal cytosolic and mitochondrial Ca2+ levels. It was postulated that reduced mitochondrial uptake is linked to

OXPHOS disorders are complex and heterogeneous group of multisystem diseases. The fact that they can result from mutations in hundreds of genes distributed across all of the chromosomes as well as the mtDNA, render the understanding of causative factors and the identification of common disease-related factors difficult. Accordingly effective therapeutic interventions are still not readily available. There are two main approaches to mitochondrial disease therapy: genetic and metabolic pharmacological (for recent review see (Roestenberg

New approaches for genetic therapies for nDNA-encoded mitochondrial diseases as well as for mtDNA diseases are beginning to offer alternatives for individuals suffering from these devastating disorders. For mtDNA, these approaches include: (*a*) import of normal mtDNA polypeptides into the mitochondrion to complement the mtDNA defect, (*b*) reduction of the proportion of mutant mtDNAs (heteroplasmy shifting), and (*c*) direct medication of the mtDNA. Researchers are focusing also on the possible use of stem cell as a medication of OXPHOS disorders. However, these approaches are not as likely to relieve the devastating

The pharmacological approach includes the use of: (a) cofactors that increase the production of ATP (coQ, Idebenone, and succinate), (b) vitamins and metabolic supplements (thiamine, riboflavine, carnitine and L-arginine), (c) reactive oxygen species scavengers and mitochondrial antioxidants (CoQ/Idebenone, Vitamin E and Vitamin C), (d) modulators of PTP (cyclosporin A), and (e) regulators of mitochondrial biogenesis (bezafibrate and sirtuin

Current interventions based on metabolic correction include the use of mitochondrialtargeted drugs (compounds and peptides targeted to the mitochondrial matrix) such as mitoquinone "MitoQ", a derivative of coenzyme Q10, and SS-peptides, Szesto-Schiller

Another alternative to rescue mitochondrial bioenergetics defects is the use the mitochondrial Na+/Ca2+ exchanger inhibitor benzothiazepine CGP37157 (Cox & Matlib, 1993). CGP37157 normalized aberrant mitochondrial Ca2+ handling during hormone stimulation of cybrid cells carrying the tRNALys mutation associated with MERRF syndrom (Brini et al., 1999). Short-term pre-treatment with CGP37157 (1 μM, 2 min) fully normalized the amplitude of the hormone-induced mitochondrial Ca2+ signal in fibroblasts from patients with isolated complex I deficiency (Visch et al., 2004), without altering this

affecting Ca2+ dynamics (Kazuno et al., 2006).

decreased mitochondrial potential (Padua et al., 1998).

et al., 2011) and (Wallace et al., 2010)).

analogs).

**5.6 Calcium deregulation in Pyruvate Dehydrogenase deficiency** 

**6. OXPHOS therapies: The place for Ca2+ modulating drugs** 

symptoms suffered by individuals with bioenergetic diseases.

peptides, a novel class of small cell permeable peptide antioxidants.

compared to control fibroblast (Wasniewska et al., 2001). The energy state of the mitochondrial membrane in mutated cells is naturally decreased. Accordingly, it was demonstrated that mitochondria can control SOC in a numerous cell types and that the collapse of mitochondrial membrane potential, either by an uncoupler or an inhibitor of the respiratory chain, greatly reduces the SOC (Makowska et al., 2000). In an earlier study, Handran and collaborators failed to document either mitochondrial morphology alteration or intracellular Ca2+ deregulation in COX-deficient human fibroblasts (Handran et al., 1997). This discrepancy between these results may be accounted on the partial recovery of COX enzyme activity in COX deficient fibroblasts. Fibroblasts are not a robust system for the study of mitochondrial dysfunction and cultured cells relays less on mitochondria for ATP production. It was thus concluded that this deficiency is not detrimental to fibroblast or that anaerobic respiration rescues the phenotype. In a strange manner, SURF1-/- KO mouse displayed mild reduction of COX activity in all tissues and did not show encephalopathy. These mice show a complete protection from in vivo neurodegeneration induced by exposure to high doses of kainic acid (a glutamatergic epiloptogenic agonist). Thus the ablation of SURF1 drastically reduces the glutamate-induced increase of Ca2+ both in the cytosol and the mitochondria. Authors postulate that reduced buffering capacity by SURF1- /- mitochondria in the contact sites between mitochondria and plasma membrane or the ER may promote the feedback closure of the Ca2+ channels thus inhibiting the cytosolic Ca2+ transient rise (Dell'agnello et al., 2007).

As introduced in chapter 2-2-2, Friedreich's ataxia (FA) is an autosomal recessive disease caused by decreased expression of the mitochondrial protein frataxin. The biological function of frataxin is unclear. The homologue of frataxin in yeast, YFH1, is required for cellular respiration and was suggested to regulate mitochondrial iron homeostasis. Patients suffering from FA exhibit decreased ATP production in skeletal muscle. Accordingly, overexpression of frataxin in mammalian cells causes a Ca2+-induced up-regulation of tricarboxylic acid cycle flux and respiration, which, in turn, leads to an increased mitochondrial membrane potential and results in an elevated cellular ATP content. Thus, frataxin appears to be a key activator of mitochondrial energy conversion and oxidative phosphorylation (Ristow et al., 2000).

It was reported that mean mitochondrial iron content was increased in FA fibroblasts harboring expansion of intronic GAA repeat in frataxin leading to its reduced expression, and that staurosporine-induced caspase 3 activity was higher in FA fibroblasts than controls. Treatment of cells with BAPTA, AM rescued FA from oxidant-induced death. These data indirectly demonstrate that FA fibroblasts displayed an increased cytosolic Ca2+ content leading to increased sensitivity to oxidative stress (Wong & Cortopassi, 1997).

#### **5.5 Calcium deregulation linked to mitochondrial DNA polymorphism**

mtDNA is highly polymorphic and its variation in humans may contribute to individual differences in function as well as susceptibility to various diseases such as neurodegenerative diseases. Kazuno and collaborators searched for mtDNA polymorphisms that have mitochondrial functional significance using cybrid cells. Increased mitochondrial basal Ca2+ levels and increased agonist evoked cytosolic Ca2+ signals were observed in two closely linked nonsynonymous polymorphisms. Interestingly, these data highlight the role

compared to control fibroblast (Wasniewska et al., 2001). The energy state of the mitochondrial membrane in mutated cells is naturally decreased. Accordingly, it was demonstrated that mitochondria can control SOC in a numerous cell types and that the collapse of mitochondrial membrane potential, either by an uncoupler or an inhibitor of the respiratory chain, greatly reduces the SOC (Makowska et al., 2000). In an earlier study, Handran and collaborators failed to document either mitochondrial morphology alteration or intracellular Ca2+ deregulation in COX-deficient human fibroblasts (Handran et al., 1997). This discrepancy between these results may be accounted on the partial recovery of COX enzyme activity in COX deficient fibroblasts. Fibroblasts are not a robust system for the study of mitochondrial dysfunction and cultured cells relays less on mitochondria for ATP production. It was thus concluded that this deficiency is not detrimental to fibroblast or that anaerobic respiration rescues the phenotype. In a strange manner, SURF1-/- KO mouse displayed mild reduction of COX activity in all tissues and did not show encephalopathy. These mice show a complete protection from in vivo neurodegeneration induced by exposure to high doses of kainic acid (a glutamatergic epiloptogenic agonist). Thus the ablation of SURF1 drastically reduces the glutamate-induced increase of Ca2+ both in the cytosol and the mitochondria. Authors postulate that reduced buffering capacity by SURF1- /- mitochondria in the contact sites between mitochondria and plasma membrane or the ER may promote the feedback closure of the Ca2+ channels thus inhibiting the cytosolic Ca2+

As introduced in chapter 2-2-2, Friedreich's ataxia (FA) is an autosomal recessive disease caused by decreased expression of the mitochondrial protein frataxin. The biological function of frataxin is unclear. The homologue of frataxin in yeast, YFH1, is required for cellular respiration and was suggested to regulate mitochondrial iron homeostasis. Patients suffering from FA exhibit decreased ATP production in skeletal muscle. Accordingly, overexpression of frataxin in mammalian cells causes a Ca2+-induced up-regulation of tricarboxylic acid cycle flux and respiration, which, in turn, leads to an increased mitochondrial membrane potential and results in an elevated cellular ATP content. Thus, frataxin appears to be a key activator of mitochondrial energy conversion and oxidative

It was reported that mean mitochondrial iron content was increased in FA fibroblasts harboring expansion of intronic GAA repeat in frataxin leading to its reduced expression, and that staurosporine-induced caspase 3 activity was higher in FA fibroblasts than controls. Treatment of cells with BAPTA, AM rescued FA from oxidant-induced death. These data indirectly demonstrate that FA fibroblasts displayed an increased cytosolic Ca2+ content leading to increased sensitivity to oxidative stress (Wong & Cortopassi,

mtDNA is highly polymorphic and its variation in humans may contribute to individual differences in function as well as susceptibility to various diseases such as neurodegenerative diseases. Kazuno and collaborators searched for mtDNA polymorphisms that have mitochondrial functional significance using cybrid cells. Increased mitochondrial basal Ca2+ levels and increased agonist evoked cytosolic Ca2+ signals were observed in two closely linked nonsynonymous polymorphisms. Interestingly, these data highlight the role

**5.5 Calcium deregulation linked to mitochondrial DNA polymorphism** 

transient rise (Dell'agnello et al., 2007).

phosphorylation (Ristow et al., 2000).

1997).

of mitochondrial polymorphisms in the pathology of neurodegenerative diseases by affecting Ca2+ dynamics (Kazuno et al., 2006).
