Anna Ghelli

Defective oxidative phosphorylation in thyroid oncocytic carcinoma is associated with pathogenic mitochondrial DNA mutations affecting complexes I and III

Oncocytic tumors are characterized by cells with an aberrant accumulation of mitochondria. To assess mitochondrial function in neoplastic oncocytic cells, we studied the thyroid oncocytic cell line XTC.UC1 and compared it with other thyroid non-oncocytic cell lines. Only XTC.UC1 cells were unable to survive in galactose, a condition forcing cells to rely solely on mitochondria for energy production. The rate of respiration and mitochondrial ATP synthesis driven by complex I substrates was severely reduced in XTC.UC1 cells. Furthermore, the enzymatic activity of complexes I and III was dramatically decreased in these cells compared with controls, in conjunction with a strongly enhanced production of reactive oxygen species. Osteosarcoma-derived transmitochondrial cell hybrids (cybrids) carrying XTC.UC1 mitochondrial DNA (mtDNA) were generated to discriminate whether the energetic failure depended on mitochondrial or nuclear DNA mutations. In galactose medium, XTC.UC1 cybrid clones showed reduced viability and ATP content, similarly to the parental XTC.UC1, clearly pointing to the existence of mtDNA alterations. Sequencing of XTC.UC1 mtDNA identified a frameshift mutation in ND1 and a nonconservative substitution in cytochrome b, two mutations with a clear pathogenic potential. In conclusion, this is the first demonstration that mitochondrial dysfunction of XTC.UC1 is due to a combined complex I/III defect associated with mtDNA mutations, as proven by the transfer of the defective energetic phenotype with the mitochondrial genome into the cybrids.

Oestrogens ameliorate mitochondrial dysfunction in Leber’s hereditary optic neuropathy

Lebers hereditary optic neuropathy, the most frequent mitochondrial disease due to mitochondrial DNA point mutations in complex I, is characterized by the selective degeneration of retinal ganglion cells, leading to optic atrophy and loss of central vision prevalently in young males. The current study investigated the reasons for the higher prevalence of Lebers hereditary optic neuropathy in males, exploring the potential compensatory effects of oestrogens on mutant cell metabolism. Control and Lebers hereditary optic neuropathy osteosarcoma-derived cybrids (11778/ND4, 3460/ND1 and 14484/ND6) were grown in glucose or glucose-free, galactose-supplemented medium. After having shown the nuclear and mitochondrial localization of oestrogen receptors in cybrids, experiments were carried out by adding 100 nM of 17β-oestradiol. In a set of experiments, cells were pre-incubated with the oestrogen receptor antagonist ICI 182780. Lebers hereditary optic neuropathy cybrids in galactose medium presented overproduction of reactive oxygen species, which led to decrease in mitochondrial membrane potential, increased apoptotic rate, loss of cell viability and hyper-fragmented mitochondrial morphology compared with control cybrids. Treatment with 17β-oestradiol significantly rescued these pathological features and led to the activation of the antioxidant enzyme superoxide dismutase 2. In addition, 17β-oestradiol induced a general activation of mitochondrial biogenesis and a small although significant improvement in energetic competence. All these effects were oestrogen receptor mediated. Finally, we showed that the oestrogen receptor β localizes to the mitochondrial network of human retinal ganglion cells. Our results strongly support a metabolic basis for the unexplained male prevalence in Lebers hereditary optic neuropathy and hold promises for a therapeutic use for oestrogen-like molecules.

Caspase-independent death of Leber’s hereditary optic neuropathy cybrids is driven by energetic failure and mediated by AIF and Endonuclease G

Leber’s hereditary optic neuropathy (LHON) is associated with mitochondrial DNA point mutations affecting different subunits of complex I. By replacing glucose with galactose in the medium, cybrids harboring each of the three LHON pathogenic mutations (11778/ND4, 3460/ND1, 14484/ND6) suffered a profound ATP depletion over a few hours and underwent apoptotic cell death, which was caspase-independent. Control cybrids were unaffected. In addition to cytochrome c, apoptosis inducing factor (AIF) and endonuclease G (EndoG) were also released from the mitochondria into the cytosol in LHON cybrids, but not in control cells. Exposure of isolated nuclei to cytosolic fractions from LHON cybrids maintained in galactose medium caused nuclear fragmentation, which was strongly reduced by immuno-depletion with anti-AIF and anti-EndoG antibodies. In conclusion, the caspase-independent death of LHON cybrids incubated in galactose medium is triggered by rapid ATP depletion and mediated by AIF and EndoG.

Respiratory Complex I Dysfunction Due to Mitochondrial DNA Mutations Shifts the Voltage Threshold for Opening of the Permeability Transition Pore toward Resting Levels

We have studied mitochondrial bioenergetics in HL180 cells (a cybrid line harboring the T14484C/ND6 and G14279A/ND6 mtDNA mutations of Leber hereditary optic neuropathy, leading to an ∼50% decrease of ATP synthesis) and XTC.UC1 cells (derived from a thyroid oncocytoma bearing a disruptive frameshift mutation in MT-ND1, which impairs complex I assembly). The addition of rotenone to HL180 cells and of antimycin A to XTC.UC1 cells caused fast mitochondrial membrane depolarization that was prevented by treatment with cyclosporin A, intracellular Ca2+ chelators, and antioxidant. Both cell lines also displayed an anomalous response to oligomycin, with rapid onset of depolarization that was prevented by cyclosporin A and by overexpression of Bcl-2. These findings indicate that depolarization by respiratory chain inhibitors and oligomycin was due to opening of the mitochondrial permeability transition pore (PTP). A shift of the threshold voltage for PTP opening close to the resting potential may therefore be the underlying cause facilitating cell death in diseases affecting complex I activity. This study provides a unifying reading frame for previous observations on mitochondrial dysfunction, bioenergetic defects, and Ca2+ deregulation in mitochondrial diseases. Therapeutic strategies aimed at normalizing the PTP voltage threshold may be instrumental in ameliorating the course of complex I-dependent mitochondrial diseases.

The ND1 gene of complex I is a mutational hot spot for Leber's hereditary optic neuropathy

A novel mitochondrial DNA (mtDNA) transition (3733G→A) inducing the E143 K amino acid change at a very conserved site of the NADH dehydrogenase subunit 1 (ND1) was identified in a family with six maternally related individuals with Lebers hereditary optic neuropathy (LHON) and in an unrelated sporadic case, all negative for known mutations and presenting with the canonical phenotype. The transition was not detected in 1,082 control mtDNAs and was heteroplasmic in several individuals from both pedigrees. In addition, the mtDNAs of the two families were found to belong to different haplogroups (H and X), thus confirming that the 3733G→A mutation occurred twice independently. Phosphorus magnetic resonance spectroscopy disclosed an in vivo brain and skeletal muscle energy metabolism deficit in the four examined patients. Muscle biopsy from two patients showed slight mitochondrial proliferation with abnormal mitochondria. Biochemical investigations in platelets showed partially insensitive complex I to rotenone inhibition. We conclude that the 3733G→A transition is a novel cause of LHON and, after those at positions 3460 and 4171, is the third ND1 mutation to be identified in multiple unrelated families. This finding shows that, in addition to ND6, the ND1 subunit gene is also a mutational hot spot for LHON. Ann Neurol 2004;56:631–641

The effects of idebenone on mitochondrial bioenergetics

We have studied the effects of idebenone on mitochondrial function in cybrids derived from one normal donor (HQB17) and one patient harboring the G3460A/MT-ND1 mutation of Lebers Hereditary Optic Neuropathy (RJ206); and in XTC.UC1 cells bearing a premature stop codon at aminoacid 101 of MT-ND1 that hampers complex I assembly. Addition of idebenone to HQB17 cells caused mitochondrial depolarization and NADH depletion, which were inhibited by cyclosporin (Cs) A and decylubiquinone, suggesting an involvement of the permeability transition pore (PTP). On the other hand, addition of dithiothreitol together with idebenone did not cause PTP opening and allowed maintenance of the mitochondrial membrane potential even in the presence of rotenone. Addition of dithiothreitol plus idebenone, or of idebenol, to HQB17, RJ206 and XTC.UC1 cells sustained membrane potential in intact cells and ATP synthesis in permeabilized cells even in the presence of rotenone and malonate, and restored a good level of coupled respiration in complex I-deficient XTC.UC1 cells. These findings demonstrate that idebenol can feed electrons at complex III. If the quinone is maintained in the reduced state, a task that in some cell types appears to be performed by dicoumarol-sensitive NAD(P)H:quinone oxidoreductase 1 [Haefeli et al. (2011) PLoS One 6, e17963], electron transfer to complex III may allow reoxidation of NADH in complex I deficiencies.

A Mutation Threshold Distinguishes the Antitumorigenic Effects of the Mitochondrial Gene MTND1, an Oncojanus Function

The oncogenic versus suppressor roles of mitochondrial genes have long been debated. Peculiar features of mitochondrial genetics such as hetero/homoplasmy and mutation threshold are seldom taken into account in this debate. Mitochondrial DNA (mtDNA) mutations generally have been claimed to be protumorigenic, but they are also hallmarks of mostly benign oncocytic tumors wherein they help reduce adaptation to hypoxia by destabilizing hypoxia-inducible factor-1α (HIF1α). To determine the influence of a disassembling mtDNA mutation and its hetero/homoplasmy on tumorigenic and metastatic potential, we injected mice with tumor cells harboring different loads of the gene MTND1 m.3571insC. Cell cultures obtained from tumor xenografts were then analyzed to correlate energetic competence, apoptosis, α-ketoglutarate (α-KG)/succinate (SA) ratio, and HIF1α stabilization with the mutation load. A threshold level for the antitumorigenic effect of MTND1 m.3571insC mutation was defined, above which tumor growth and invasiveness were reduced significantly. Notably, HIF1α destabilization and downregulation of HIF1α-dependent genes occurred in cells and tumors lacking complex I (CI), where there was an associated imbalance of α-KG/SA despite the presence of an actual hypoxic environment. These results strongly implicate mtDNA mutations as a cause of oncocytic transformation. Thus, the antitumorigenic and antimetastatic effects of high loads of MTND1 m.3571insC, following CI disassembly, define a novel threshold-regulated class of cancer genes. We suggest these genes be termed oncojanus genes to recognize their ability to contribute either oncogenic or suppressive functions in mitochondrial settings during tumorigenesis.

The background of mitochondrial DNA haplogroup J increases the sensitivity of Leber's hereditary optic neuropathy cells to 2,5-hexanedione toxicity.

Lebers hereditary optic neuropathy (LHON) is a maternally inherited blinding disease due to mitochondrial DNA (mtDNA) point mutations in complex I subunit genes, whose incomplete penetrance has been attributed to both genetic and environmental factors. Indeed, the mtDNA background defined as haplogroup J is known to increase the penetrance of the 11778/ND4 and 14484/ND6 mutations. Recently it was also documented that the professional exposure to n-hexane might act as an exogenous trigger for LHON. Therefore, we here investigate the effect of the n-hexane neurotoxic metabolite 2,5-hexanedione (2,5-HD) on cell viability and mitochondrial function of different cell models (cybrids and fibroblasts) carrying the LHON mutations on different mtDNA haplogroups. The viability of control and LHON cybrids and fibroblasts, whose mtDNAs were completely sequenced, was assessed using the MTT assay. Mitochondrial ATP synthesis rate driven by complex I substrates was determined with the luciferine/luciferase method. Incubation with 2,5-HD caused the maximal loss of viability in control and LHON cells. The toxic effect of this compound was similar in control cells irrespective of the mtDNA background. On the contrary, sensitivity to 2,5-HD induced cell death was greatly increased in LHON cells carrying the 11778/ND4 or the 14484/ND6 mutation on haplogroup J, whereas the 11778/ND4 mutation in association with haplogroups U and H significantly improved cell survival. The 11778/ND4 mutation on haplogroup U was also more resistant to inhibition of complex I dependent ATP synthesis by 2,5-HD. In conclusion, this study shows that mtDNA haplogroups modulate the response of LHON cells to 2,5-HD. In particular, haplogroup J makes cells more sensitive to its toxic effect. This is the first evidence that an mtDNA background plays a role by interacting with an environmental factor and that 2,5-HD may be a risk element for visual loss in LHON. This proof of principle has broad implications for other neurodegenerative disorders such as Parkinsons disease.

Protection against oxidant-induced apoptosis by exogenous glutathione in Leber hereditary optic neuropathy cybrids.

PURPOSEnTo use different paradigms of oxidative and metabolic stress in a cellular model of Leber hereditary optic neuropathy (LHON), with the aim of evaluating the efficacy of potentially therapeutic molecules for the treatment of this disease.nnnMETHODSnCybrids bearing one of the three most common LHON pathogenic mutations (11778/ND4, 3460/ND1, 14484/ND6) were incubated with two compounds known to induce oxidative injury, tert-butyl hydroperoxide (t-BH) and rotenone. To mimic metabolic stress, cells were incubated in a glucose-free medium containing galactose. Cell viability was determined using the MTT assay. To identify the apoptotic type of cell death, nuclear morphology was examined after cell loading with Hoechst. Cellular glutathione (GSH), and oxidized glutathione (GSSG) levels were measured enzymatically.nnnRESULTSnIncubation with t-BH caused apoptotic cell death of control and LHON cybrids, whereas only LHON cybrids were damaged by rotenone concentrations up to 2.5 muM. Both types of stress caused a marked imbalance in the glutathione levels, but an increase in the GSSG/GSH+GSSG ratio was detected only after rotenone treatment. The efficacy of several antioxidant and antiapoptotic compounds was then assessed in cells exposed to these two oxidative paradigms. Only exogenous GSH remarkably protected the t-BH- and rotenone-treated cybrids from cell death. In contrast, GSH was unable to increase the viability of cybrids exposed to metabolic stress.nnnCONCLUSIONSnThese results suggest that GSH is an effective antioxidant compound to be tested as a potential treatment for LHON.

Apoptosis induced by staurosporine in ECV304 cells requires cell shrinkage and upregulation of Cl conductance

AbstractWe show that dysregulation of the Cl− homeostasis mediates the staurosporine-induced apoptotic cell death in human ECV304 cells. A pronounced apoptotic volume decrease (AVD), and an increase in plasma membrane Cl− conductance were early (<1u2009h) events following staurosporine challenge. Both processes were involved in apoptotic death, as demonstrated by the observation that the Cl− channel blocker phloretin inhibited both the staurosporine-evoked Cl− current and AVD, and preserved cell viability. Prolonged incubation (>2u2009h) with staurosporine caused a decrease in intracellular pH, which, however, was not required for the progression of the apoptotic process, because inhibitors of proton extrusion pathways, which lowered cytoplasmic pH, failed to inhibit both caspase-3 activation and DNA laddering. Moreover, clamping the cytosolic pH to an alkaline value did not prevent the apoptotic cell death. Collectively, these data demonstrate that staurosporine-mediated apoptosis of ECV304 cells is caused by the upregulation of Cl− channel activity and subsequent AVD, but is independent of intracellular acidification.