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Biomedical subjects

Pierre Rustin

Publications and source records attributed to Pierre Rustin.

At least 37 records · Page 2Linked to original sources
PubMed2004

Respiratory chain defects: what do we know for sure about their consequences in vivo?

The function and the structure of mitochondria have been the subject of intensive research since the discovery of these organelles. Yet, the investigation of patients with mitochondrial disease reveals that we do not understand a large part of the underlying pathogenic processes. This has disastrous consequences in terms of the therapy possibly proposed to the patients and their family. An attempt is made in this short review to question our present ideas on the potential consequences of mitochondrial dysfunctions and to enlighten new observations which might be valuable in the understanding of the physiopathology of these diseases.

Adenosine Triphosphate↗
PubMed2004

Glutathione precursors replenish decreased glutathione pool in cystinotic cell lines.

Cystinosis is an inherited disorder due to mutations in the CTNS gene which encodes cystinosin, a lysosomal transmembrane protein involved in cystine export to the cytosol. Both accumulation of cystine in the lysosome and decreased cystine in the cytosol may participate in the pathogenic mechanism underlying the disease. We observed that cystinotic cell lines have moderate decrease of glutathione content during exponential growth phase. This resulted in increased solicitation of oxidative defences of the cell denoted by concurrent superoxide dismutase induction, although without major oxidative insult under our experimental conditions. Finally, decreased glutathione content in cystinotic cell lines could be counterbalanced by a series of exogenous precursors of cysteine, denoting that lysosomal cystine export is a natural source of cellular cysteine in the studied cell lines.

Adolescent↗
PubMed2004

AIF deficiency compromises oxidative phosphorylation.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.

Adenosine Triphosphate↗
PubMed2004

Quinone analogues regulate mitochondrial substrate competitive oxidation.

Quinone derivatives are among the rare compounds successfully used as therapeutic reagents to fight mitochondrial diseases. However, their beneficial effect appears to depend on their side chain which presumably governs their interaction with the respiratory chain. The effect of four quinone derivatives was comparatively studied on NADH- and succinate-competitive oxidation by a sub-mitochondrial fraction. Under our experimental conditions, the less hydrophobic derivatives (menadione, duroquinone) poorly affected electron flow from either NADH or succinate to oxygen, yet readily diverting electrons from isolated complex I. This latter effect was abolished by succinate addition. More hydrophobic derivatives (idebenone, decylubiquinone) stimulated oxygen uptake from succinate. But while NADH oxidation was slightly inhibited by idebenone, it was somewhat increased by decylubiquinone. As a result, idebenone strongly favoured succinate over NADH oxidation. This study therefore suggests that any therapeutic use of quinone analogues should take into account their specific effect on each respiratory chain dehydrogenase.

Animals↗
PubMed2004

Idebenone delays the onset of cardiac functional alteration without correction of Fe-S enzymes deficit in a mouse model for Friedreich ataxia.

Friedreich ataxia (FRDA), a progressive neurodegenerative disorder associated with cardiomyopathy, is caused by severely reduced frataxin, a mitochondrial protein involved in Fe-S cluster assembly. We have recently generated mouse models that reproduce important progressive pathological and biochemical features of the human disease. Our frataxin-deficient mouse models initially demonstrate time-dependent intramitochondrial iron accumulation, which occurs after onset of the pathology and after inactivation of the Fe-S dependent enzymes. Here, we report a more detailed pathophysiological characterization of our mouse model with isolated cardiac disease by echocardiographic, biochemical and histological studies and its use for placebo-controlled therapeutic trial with Idebenone. The Fe-S enzyme deficiency occurs at 4 weeks of age, prior to cardiac dilatation and concomitant development of left ventricular hypertrophy, while the mitochondrial iron accumulation occurs at a terminal stage. From 7 weeks onward, Fe-S enzyme activities are strongly decreased and are associated with lower levels of oxidative stress markers, as a consequence of reduced respiratory chain activity. Furthermore, we demonstrate that the antioxidant Idebenone delays the cardiac disease onset, progression and death of frataxin deficient animals by 1 week, but does not correct the Fe-S enzyme deficiency. Our results support the view that frataxin is a necessary, albeit non-essential, component of the Fe-S cluster biogenesis, and indicate that Idebenone acts downstream of the primary Fe-S enzyme deficit. Furthermore, our results demonstrate that Idebenone is cardioprotective even in the context of a complete lack of frataxin, which further supports its utilization for the treatment of FRDA.

Animals↗
PubMed2004

Mitochondrial transcription factor A regulates mtDNA copy number in mammals.

Mitochondrial DNA (mtDNA) copy number regulation is altered in several human mtDNA-mutation diseases and it is also important in a variety of normal physiological processes. Mitochondrial transcription factor A (TFAM) is essential for human mtDNA transcription and we demonstrate here that it is also a key regulator of mtDNA copy number. We initially performed in vitro transcription studies and determined that the human TFAM protein is a poor activator of mouse mtDNA transcription, despite its high capacity for unspecific DNA binding. Next, we generated P1 artificial chromosome (PAC) transgenic mice ubiquitously expressing human TFAM. The introduced human TFAM gene was regulated in a similar fashion as the endogenous mouse Tfam gene and expression of the human TFAM protein in the mouse did not result in down-regulation of the endogenous expression. The PAC-TFAM mice thus had a net overexpression of TFAM protein and this resulted in a general increase of mtDNA copy number. We used a combination of mice with TFAM overexpression and TFAM knockout and demonstrated that mtDNA copy number is directly proportional to the total TFAM protein levels also in mouse embryos. Interestingly, the expression of human TFAM in the mouse results in up-regulation of mtDNA copy number without increasing respiratory chain capacity or mitochondrial mass. It is thus possible to experimentally dissociate mtDNA copy number regulation from mtDNA expression and mitochondrial biogenesis in mammals in vivo. In conclusion, our results provide genetic evidence for a novel role for TFAM in direct regulation of mtDNA copy number in mammals.

Animals↗
PubMed2004

Real-time flow cytometry analysis of permeability transition in isolated mitochondria.

Mitochondrial membrane permeabilization (MMP) is a key event in necrotic and (intrinsic) apoptotic processes. MMP is controlled by a few major rate-limiting events, one of which is opening of the permeability transition pore (PTP). Here we develop a flow cytometry (FC)-based approach to screen and study inducers and blockers of MMP in isolated mitochondria. Fixed-time and real-time FC permits to co-evaluate and order modifications of mitochondrial size, structure and inner membrane (IM) electrochemical potential (DeltaPsi(m)) during MMP. Calcium, a major PTP opener, and alamethicin, a PTP-independent MMP inducer, trigger significant mitochondrial forward scatter (FSC) increase and side scatter (SSC) decrease, correlating with spectrophotometrically detected swelling. FC-based fluorescence detection of the DeltaPsi(m)-sensitive cationic lipophilic dye JC-1 permits to detect DeltaPsi(m) variations induced by PTP openers or specific inducers of inner MMP such as carbonylcyanide m-chlorophenylhydrazone (mClCCP). These simple, highly sensitive and quantitative FC-based methods will be pertinent to evaluate compounds for their ability to control MMP.

Alamethicin↗
PubMed2004

Friedreich ataxia mouse models with progressive cerebellar and sensory ataxia reveal autophagic neurodegeneration in dorsal root ganglia.

Friedreich ataxia (FRDA), the most common recessive ataxia, is characterized by degeneration of the large sensory neurons of the spinal cord and cardiomyopathy. It is caused by severely reduced levels of frataxin, a mitochondrial protein involved in iron-sulfur cluster (ISC) biosynthesis. Through a spatiotemporally controlled conditional gene-targeting approach, we have generated two mouse models for FRDA that specifically develop progressive mixed cerebellar and sensory ataxia, the most prominent neurological features of FRDA. Histological studies showed both spinal cord and dorsal root ganglia (DRG) anomalies with absence of motor neuropathy, a hallmark of the human disease. In addition, one line revealed a cerebellar granule cell loss, whereas both lines had Purkinje cell arborization defects. These lines represent the first FRDA models with a slowly progressive neurological degeneration. We identified an autophagic process as the causative pathological mechanism in the DRG, leading to removal of mitochondrial debris and apparition of lipofuscin deposits. These mice therefore represent excellent models for FRDA to unravel the pathological cascade and to test compounds that interfere with the degenerative process.

Animals↗
PubMed2004

The deleterious G15498A mutation in mitochondrial DNA-encoded cytochrome b may remain clinically silent in homoplasmic carriers.

We report on a patient with severe growth retardation and IgF1 deficiency, in which a mitochondrial abnormality was suspected. An isolated mitochondrial respiratory chain complex III deficiency was found in blood lymphocytes and skin fibroblasts. Sequence analysis of the cytochrome b, which is the only mitochondrial DNA-encoded subunit of complex III, revealed a homoplasmic G15498A mutation, resulting in the substitution of a highly conserved amino acid (glycine 251 into an aspartic acid). The mutation was found to be homoplasmic in all tissues examined from the mother and her brother (lymphocytes, fibroblasts, hair roots and buccal cells). Complex III deficiency was also demonstrated in these cells. Nevertheless, the mother and the brother were asymptomatic. This mutation had been considered as a cardiomyopathy-generating mutation in a previously reported case, and its pathogenicity has been demonstrated recently in yeast. However, it seems not to fulfil the classical criteria for pathogenicity of a mitochondrial DNA mutation, especially the heteroplasmic status, and to be clinically silent, albeit present, in nonaffected relatives. We suggest that other factors are contributing to the clinical variability expression of the G15498A mtDNA mutation.

Amino Acid Sequence↗
PubMed2004

Processing of the 24 kDa subunit mitochondrial import signal is not required for assembly of functional complex I in Yarrowia lipolytica.

A small deletion in the second intron of human NDUFV2 (IVS2+5_+8delGTAA) has been shown to cause hypertrophic cardiomyopathy and encephalomyopathy [Bénit, P., Beugnot, R., Chretien, D., Giurgea, I., de Lonlay-Debeney, P., Issartel, J.P., Kerscher, S., Rustin, P., Rötig, A. & Munnich, A. (2003) Human Mutat.21, 582-586]. Skipping of exon 2 results in a partial deletion of the mitochondrial targeting sequence of the precursor for the 24 kDa subunit of respiratory chain complex I. Immunoreactivity of the 24 kDa subunit and complex I activity, both present at 30-50% of normal levels in patient mitochondria, raised the question of how the mutant 24 kDa subunit precursor can be imported and assembled into functional complex I. In the present study, we have remodelled the human NDUFV2 mutation by deleting codons 17-32 from the orthologous NUHM gene of the obligate aerobic yeast Yarrowia lipolytica. The resulting mutant enzyme was indistinguishable from parental complex I with regard to activity, inhibitor sensitivity and EPR signature. Size, isoelectric point and presumably also N-terminal acetylation were altered, indicating that the residual targeting sequence was retained on the mature 24 kDa protein. Complete removal of the NUHM presequence resulted in the absence of complex I activity, strongly arguing against the presence of an internal mitochondrial targeting sequence within the 24 kDa protein.

Amino Acid Sequence↗
PubMed2004

Revisiting pitfalls, problems and tentative solutions for assaying mitochondrial respiratory chain complex III in human samples.

The assessment of mitochondrial respiratory chain enzyme activity in human samples is a difficult task due to both the small amount of tissue generally available and the frequent need to perform enzyme activity measurement in crude mitochondrial fraction. This is particularly true for the measurement of complex III activity which partial deficiency can be easily overlooked. In this review, we first consider the several interfering reactions occurring when measuring this activity. We subsequently describe the use of an alkyl glycoside detergent, lauryl maltoside, to keep these interfering reactions to a very low level. Next, we quantify the effect of the detergent on the actual measurement of complex III in various human tissue samples and cells. Finally, we also demonstrate that the use of the detergent allows (i) a better detection of an inherited partial defect affecting cytochrome b, a catalytic subunit of the mitochondrial complex III, (ii) to possibly discriminate decreased complex III activity resulting from an abnormal complex III assembly (BCS1 gene mutation) from an hampered catalytic activity originating from a cytochrome b mutation. This detailed review of the problems associated with complex III assessment and of their tentative solution highlights the difficulties still encountered in the measurements of mitochondrial respiratory chain in humans.

Electron Transport↗
PubMed2003

Synthesis and preliminary biological evaluations of ionic and nonionic amphiphilic alpha-phenyl-N-tert-butylnitrone derivatives.

In this study we report the synthesis of a series of new amphiphilic compounds derived from alpha-phenyl-N-tert-butylnitrone (PBN). The nitrone function was fitted into the core of the molecule between its polar and apolar groups. The polar head consisted of a lactobionamide, an ammonium, or a carboxylate group. The hydrophobic part consisted of a hydro- or a perfluorocarbon chain. The hydrophobic chain was linked to the tert-butyl group of the PBN derivatives using an urethane, a thioether, or an amide bond. The impact of these different parameters on the hydrophilic lipophilic balance of these compounds and their spin trap activity were studied. The various ESR measurements indicated that the aromatic and tert-butyl functional groups of PBN did not affect its spin trap properties. Moreover, these compounds were found to increase the viability of cultured human skin fibroblasts harboring the neurogenic ataxia retinitis pigmentosa mutation and presenting a severe ATPase deficiency.

Adenosine Triphosphatases↗
PubMed2003

Cytochrome c oxidase subassemblies in fibroblast cultures from patients carrying mutations in COX10, SCO1, or SURF1.

Cytochrome c oxidase contains two redox-active copper centers (Cu(A) and Cu(B)) and two redox-active heme A moieties. Assembly of the enzyme relies on several assembly factors in addition to the constituent subunits and prosthetic groups. We studied fibroblast cultures from patients carrying mutations in the assembly factors COX10, SCO1, or SURF1. COX10 is involved in heme A biosynthesis. SCO1 is required for formation of the Cu(A) center. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated severely decreased levels of holoenzyme in the patient cultures compared with controls. In addition, the blots revealed the presence of five subassemblies: three subassemblies involving the core subunit MTCO1 but apparently no other subunits; a subassembly containing subunits MTCO1, COX4, and COX5A; and a subassembly containing at least subunits MTCO1, MTCO2, MTCO3, COX4, and COX5A. As some of the subassemblies correspond to known assembly intermediates of human cytochrome c oxidase, we think that these subassemblies are probably assembly intermediates that accumulate in patient cells. The MTCO1.COX4.COX5A subassembly was not detected in COX10-deficient cells, which suggests that heme A incorporation into MTCO1 occurs prior to association of MTCO1 with COX4 and COX5A. SCO1-deficient cells contained accumulated levels of the MTCO1.COX4.COX5A subassembly, suggesting that MTCO2 associates with the MTCO1.COX4.COX5A subassembly after the Cu(A) center of MTCO2 is formed. Assembly in SURF1-deficient cells appears to stall at the same stage as in SCO1-deficient cells, pointing to a role for SURF1 in promoting the association of MTCO2 with the MTCO1.COX4.COX5A subassembly.

Alkyl and Aryl Transferases↗
PubMed2003

Mitochondrial activities in human cultured skin fibroblasts contaminated by Mycoplasma hyorhinis.

BACKGROUND: Mycoplasma contaminations are a recurrent problem in the use of cultured cells, including human cells, especially as it has been shown to impede cell cycle, triggering cell death under various conditions. More specific consequences on cell metabolism are poorly known. RESULTS: Here we report the lack of significant consequence of a heavy contamination by the frequently encountered mycoplasma strain, M. hyorhinis, on the determination of respiratory chain activities, but the potential interference when assaying citrate synthase. Contamination by M. hyorhinis was detected by fluorescent imaging and further quantified by the determination of the mycoplasma-specific phosphate acetyltransferase activity. Noticeably, this latter activity was not found equally distributed in various mycoplasma types, being exceptionally high in M. hyorhinis. CONCLUSION: While we observed a trend for respiration reduction in heavily contaminated cells, no significant and specific targeting of any respiratory chain components could be identified. This suggested a potential interference with cell metabolism rather than direct interaction with respiratory chain components.

Adolescent↗
PubMed2003

Mutations in the SDHB gene are associated with extra-adrenal and/or malignant phaeochromocytomas.

Germ-line mutations in the genes encoding succinate dehydrogenase complex subunits B (SDHB) and D (SDHD) have been reported in familial paragangliomas and apparently sporadic phaeochromocytomas (ASP), but the genotype-phenotype relationships of these mutations are unknown. Eighty-four patients (all but 2 followed up for 8.8 +/- 5.7 years) with ASP (57 with adrenal tumors, 27 with extra-adrenal, multiple, malignant, or recurrent tumors) were screened for the major susceptibility genes for phaeochromocytoma (RET, VHL, SDHD, and SDHB). Thirty-three tumors were available for molecular analysis, enzyme assays, and immunohistochemistry. No (0%) RET and 2 (2.4%) VHL mutations were detected. Only two coding single nucleotide polymorphisms in the SDHD gene (G12S and H50R) were found in 6 patients (7%). Conversely, six deleterious mutations in the SDHB gene were identified in 8 patients (9.5%). Ectopic site and recurrence or malignancy were strongly associated with SDHB mutations (7 of 8, 87%, versus 20 of 76, 26%; P = 0.001). Somatic DNA analysis indicated a loss of heterozygosity at chromosome 1p36 (SDHB locus) in 16 of 33 cases (48%). A loss of heterozygosity at the SDHB locus was found in all tumors with SDHB mutation, and assays of respiratory chain enzymes showed a complete loss of complex II catalytic activity. The vascular architecture of tumors with SDHB mutations displayed features typical of malignancy. These data strongly suggest that SDHB gene is a tumor suppressor gene and that the identification of germ-line mutations in SDHB gene in patients with ASPs should be considered as a high-risk factor for malignancy or recurrence.

Adrenal Gland Neoplasms↗
PubMed2003

Persistent mitochondrial dysfunction in HIV-1-exposed but uninfected infants: clinical screening in a large prospective cohort.

BACKGROUND: Antiretroviral prevention of mother to child HIV-1 is established but tolerance remains to be assessed. AIM To determine the risk for persistent mitochondrial dysfunction in HIV-uninfected children born to seropositive mothers. METHOD: An exhaustive study in a large prospective cohort with predetermined algorithm of the unexplained symptoms compatible with mitochondrial dysfunction. A total of 2644 of 4392 children were exposed to antiretrovirals. Complementary investigations were carried out on a case-by-case basis using classification with a diagnostic probability scale, based on experience with constitutional diseases. A spontaneous notification register for children not included in the cohort was created. RESULTS: Good circumstantial evidence of mitochondrial dysfunction was found for twelve children. Seven were from the cohort. All presented neurological symptoms, often associated with abnormal magnetic resonance image (10 of 12) and/or a significant episode of hyperlactatemia (seven of 12). All had either a profound deficit in one of the respiratory chain complexes (11 of 12) and/or a typical histological pattern (two of 12). All were perinatally exposed to antiretrovirals. None of them had perinatal morbidity that could explain this symptomatology. The 18-month incidence was 0.26% (95% confidence interval, 0.10-0.54) in exposed children, in comparison with the general figure of 0.01% for paediatric neuro-mitochondrial diseases in the general population. Fourteen other children in the cohort, all exposed to antiretrovirals, had unexplained symptoms, mostly neurological, for which one of the possible differential diagnoses was mitochondrial dysfunction. Close similarities in clinical, neuroradiological and histological findings strongly suggest a common pathological process in all these 26 children. CONCLUSION: Children exposed to nucleoside analogues during the perinatal period are at risk of a neurological syndrome associated with persistent mitochondrial dysfunction.

Anti-HIV Agents↗