Alessandra Baracca

Hypoxia and mitochondrial oxidative metabolism

It is now clear that mitochondrial defects are associated with a large variety of clinical phenotypes. This is the result of the mitochondrias central role in energy production, reactive oxygen species homeostasis, and cell death. These processes are interdependent and may occur under various stressing conditions, among which low oxygen levels (hypoxia) are certainly prominent. Cells exposed to hypoxia respond acutely with endogenous metabolites and proteins promptly regulating metabolic pathways, but if low oxygen levels are prolonged, cells activate adapting mechanisms, the master switch being the hypoxia-inducible factor 1 (HIF-1). Activation of this factor is strictly bound to the mitochondrial function, which in turn is related with the oxygen level. Therefore in hypoxia, mitochondria act as [O2] sensors, convey signals to HIF-1 directly or indirectly, and contribute to the cell redox potential, ion homeostasis, and energy production. Although over the last two decades cellular responses to low oxygen tension have been studied extensively, mechanisms underlying these functions are still indefinite. Here we review current knowledge of the mitochondrial role in hypoxia, focusing mainly on their role in cellular energy and reactive oxygen species homeostasis in relation with HIF-1 stabilization. In addition, we address the involvement of HIF-1 and the inhibitor protein of F1F0 ATPase in the hypoxia-induced mitochondrial autophagy.

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.

Oxidative phosphorylation in cancer cells.

Evidence suggests that mitochondrial metabolism may play a key role in controlling cancer cells life and proliferation. Recent evidence also indicates how the altered contribution of these organelles to metabolism and the resistance of cancer mitochondria against apoptosis-associated permeabilization are closely related. The hallmarks of cancer growth, increased glycolysis and lactate production in tumours, have raised attention due to recent observations suggesting a wide spectrum of oxidative phosphorylation deficit and decreased availability of ATP associated with malignancies and tumour cell expansion. More specifically, alteration in signal transduction pathways directly affects mitochondrial proteins playing critical roles in controlling the membrane potential as UCP2 and components of both MPTP and oxphos complexes, or in controlling cells life and death as the Bcl-2 proteins family. Moreover, since mitochondrial bioenergetics and dynamics, are also involved in processes of cells life and death, proper regulation of these mitochondrial functions is crucial for tumours to grow. Therefore a better understanding of the key pathophysiological differences between mitochondria in cancer cells and in their non-cancer surrounding tissue is crucial to the finding of tools interfering with these peculiar tumour mitochondrial functions and will disclose novel approaches for the prevention and treatment of malignant diseases. Here, we review the peculiarity of tumour mitochondrial bioenergetics and the mode it is linked to the cell metabolism, providing a short overview of the evidence accumulated so far, but highlighting the more recent advances.

Mitochondrial respiratory chain super-complex I–III in physiology and pathology

Recent investigations by native gel electrophoresis showed the existence of supramolecular associations of the respiratory complexes, confirmed by electron microscopy analysis and single particle image processing. Flux control analysis demonstrated that Complex I and Complex III in mammalian mitochondria kinetically behave as a single unit with control coefficients approaching unity for each component, suggesting the existence of substrate channeling within the super-complex. The formation of this supramolecular unit largely depends on the lipid content and composition of the inner mitochondrial membrane. The function of the super-complexes appears not to be restricted to kinetic advantages in electron transfer: we discuss evidence on their role in the stability and assembly of the individual complexes, particularly Complex I, and in preventing excess oxygen radical formation. There is increasing evidence that disruption of the super-complex organization leads to functional derangements responsible for pathological changes, as we have found in K-ras-transformed fibroblasts.

Mitochondrial Complex I decrease is responsible for bioenergetic dysfunction in K-ras transformed cells.

Many cancer cells are characterized by high rate of glycolysis and reduced rate of aerobic respiration, whose mechanism is still elusive. Here we investigate the down-regulation of oxidative phosphorylation (OXPHOS) in K-ras transformed mouse fibroblasts as compared to a control counterpart. Transcriptional analysis showed different expression levels of several OXPHOS nuclear genes in the two cell lines. In particular, during the exponential growth phase most genes encoding proteins of Complex I were expressed at lower levels in transformed cells. Consistently, a significant decrease of Complex I content was found in transformed cells. Moreover, analysis of NAD-dependent respiration and ATP synthesis indicated a strong decrease of Complex I activity in the mitochondria from neoplastic cells, that was confirmed by direct assay of the enzyme redox activity. At variance, succinate-dependent respiration and ATP synthesis were not significantly affected. Taken together, our results provide the new insight that the reduction of respiration observed in K-ras transformed cells is specifically due to a Complex I activity decrease.

Severe ultrastructural mitochondrial changes in lymphoblasts homozygous for Huntington disease mutation

Mutated huntingtin is expressed in nervous and non nervous system included lymphoblasts. Eneregetic metabolism is impaired in Huntingtons disease (HD) and other neurodegenerative diseases. Human HD lymphoblasts have provided clear-cut data on mitochondnal disruption. Here we report morphological, morphometric and membrane potential differences in mitochondria from lymphoblasts obtained from patients homozygous and heterozygous for the CAG mutation, and controls. Homozygotes, who despite a similar age at onset show a more aggressive phenotype than heterozygotes, had giant mitochondria and a reduced membrane potential. We argue that early mitochondrial impairment at basal level may affect the severity of HD progression in patients.

A novel deletion in the GTPase domain of OPA1 causes defects in mitochondrial morphology and distribution, but not in function

Autosomal dominant optic atrophy (ADOA), the commonest cause of inherited optic atrophy, is caused by mutations in the ubiquitously expressed gene optic atrophy 1 (OPA1), involved in fusion and biogenesis of the inner membrane of mitochondria. Bioenergetic failure, mitochondrial network abnormalities and increased apoptosis have all been proposed as possible causal factors. However, their relative contribution to pathogenesis as well as the prominent susceptibility of the retinal ganglion cell (RGC) in this disease remains uncertain. Here we identify a novel deletion of OPA1 gene in the GTPase domain in three patients affected by ADOA. Muscle biopsy of the patients showed neurogenic atrophy and abnormal morphology and distribution of mitochondria. Confocal microscopy revealed increased mitochondrial fragmentation in fibroblasts as well as in myotubes, where mitochondria were also unevenly distributed, with clustered organelles alternating with areas where mitochondria were sparse. These abnormalities were not associated with altered bioenergetics or increased susceptibility to pro-apoptotic stimuli. Therefore, changes in mitochondrial shape and distribution can be independent of other reported effects of OPA1 mutations, and therefore may be the primary cause of the disease. The arrangement of mitochondria in RGCs, which degenerate in ADOA, may be exquisitely sensitive to disturbance, and this may lead to bioenergetic crisis and/or induction of apoptosis. Our results highlight the importance of mitochondrial dynamics in the disease per se, and point to the loss of the fine positioning of mitochondria in the axons of RGCs as a possible explanation for their predominant degeneration in ADOA.

Inefficient coupling between proton transport and ATP synthesis may be the pathogenic mechanism for NARP and Leigh syndrome resulting from the T8993G mutation in mtDNA

Mutations in the ATP6 gene of mtDNA (mitochondrial DNA) have been shown to cause several different neurological disorders. The product of this gene is ATPase 6, an essential component of the F1F0-ATPase. In the present study we show that the function of the F1F0-ATPase is impaired in lymphocytes from ten individuals harbouring the mtDNA T8993G point mutation associated with NARP (neuropathy, ataxia and retinitis pigmentosa) and Leigh syndrome. We show that the impaired function of both the ATP synthase and the proton transport activity of the enzyme correlates with the amount of the mtDNA that is mutated, ranging from 13-94%. The fluorescent dye RH-123 (Rhodamine-123) was used as a probe to determine whether or not passive proton flux (i.e. from the intermembrane space to the matrix) is affected by the mutation. Under state 3 respiratory conditions, a slight difference in RH-123 fluorescence quenching kinetics was observed between mutant and control mitochondria that suggests a marginally lower F0 proton flux capacity in cells from patients. Moreover, independent of the cellular mutant load the specific inhibitor oligomycin induced a marked enhancement of the RH-123 quenching rate, which is associated with a block in proton conductivity through F0 [Linnett and Beechey (1979) Inhibitors of the ATP synthethase system. Methods Enzymol. 55, 472-518]. Overall, the results rule out the previously proposed proton block as the basis of the pathogenicity of the mtDNA T8993G mutation. Since the ATP synthesis rate was decreased by 70% in NARP patients compared with controls, we suggest that the T8993G mutation affects the coupling between proton translocation through F0 and ATP synthesis on F1. We discuss our findings in view of the current knowledge regarding the rotary mechanism of catalysis of the enzyme.

Biochemical analysis of respiratory function in cybrid cell lines harbouring mitochondrial DNA mutations

We analysed key biochemical features that reflect the balance between glycolysis and glucose oxidation in cybrids (cytoplasmic hybrids) harbouring a representative sample of mitochondrial DNA point mutations and deletions. The cybrids analysed had the same 143B cell nuclear background and were isogenic for the mitochondrial background. The 143B cell line and its rho(0) counterpart were used as controls. All cells analysed were in a dynamic state, and cell number, time of plating, culture medium, extracellular volume and time of harvest and assay were strictly controlled. Intra- and extra-cellular lactate and pyruvate levels were measured in homoplasmic wild-type and mutant cells, and correlated with rates of ATP synthesis and O2 consumption. In all mutant cell lines, except those with the T8993C mutation in the ATPase 6 gene, glycolysis was increased even under conditions of low glucose, as demonstrated by increased levels of extracellular lactate and pyruvate. Extracellular lactate levels were strictly and inversely correlated with rates of ATP synthesis and O2 consumption. These results show increased glycolysis and defective oxidative phosphorylation, irrespective of the type or site of the point mutation or deletion in the mitochondrial genome. The different biochemical consequences of the T8993C mutation suggest a uniquely different pathogenic mechanism for this mutation. However, the distinct clinical features associated with some of these mutations still remain to be elucidated.

The effect of dietary lipid changes on the fatty acid composition and function of liver, heart and brain mitochondria in the rat at different ages

A correlation between dietary lipids and cellular enzyme activities is a problem that has only been partially addressed by nutritionists. Therefore, changes in the fatty acid composition and the activities of some key metabolic enzymes (ubiquinol-2-cytochrome c reductase (EC 1.10.2.2), cytochrome oxidase (EC 1.9.3.1) and ATPase (EC 3.6.1.3)) in the mitochondria of liver, heart and brain of rats fed on diets differing extensively in their polyunsaturated fatty acid compositions have been investigated. The results showed that fatty acid compositional changes brought about by the dietary differences were associated with extensive changes in the activities of these key enzymes in the mitochondria. The extent of the influence differed considerably with the period over which the diets were fed. The role of dietary lipids to effect changes through the preservation of membrane structural integrity is discussed.