Search

International audience

Down syndrome (DS) is a common birth defect characterized by an extra copy of chromosome 21 that for approximately 95% of observed cases is caused by a meiotic non-disjunction event dur- ing gametogenesis. Individuals with trisomy 21 have typical phys- ical phenotypes besides mental retardation, early form of Alzheimer’s disease, pre-mature aging, congenital heart disease and immune hematologic anomalies. Several observations suggest that mitochondrial dysfunction may play an important role in the pathogenesis of DS[1]. In the present study, we have investigated some bioenergetic parameters of mitochondria from livers and brains of middle aged Ts65Dn mice, an animal model of DS with three copies of human chromosome 21 orthologs [2]. Our results indicate that the respiratory activities are decreased in the brain of Ts65Dn mice; in particular NADH oxidation in permeabilized mitochon- dria is reduced; flux control analysis of NADH oxidation shows that control by Complex I is slightly decreased indicating that some other step may have become more rate-controlling. Two- dimensional polyacrylamide gel electrophoresis (2D BN/SDS PAGE) suggests disruption of the supramolecular association of Complex III with Complex IV in the DS mice offering a possible explanation of the decrease of NADH oxidation accompanied by lower control by Complex I. A slight decrease of NADH-Coen- zyme Q reductase activity, however, suggests that Complex I is also partially affected. The most interesting result up to now has been the activation of the permeability transition observed in freshly isolated liver mitochondria of Ts65Dn mice. The results indicate that mtPTP responsiveness to stepwise calcium additions to mitochondria is dramatically increased in mitochondria of the mouse model Ts65Dn, evidencing an enhanced propensity to the permeability transition and thus to apoptotic cell death. References: 1. Coskun, PE et al. J Alzheimers Dis 2010; 20 s2: s293–310. 2. Moore CS, Roper RJ. Mamm Genome 2007; 18 : 431–443.

There are several experimental evidences of the supramolecular organization of the mitochondrial respiratory chain in supercom- plexes [1-2], but only few works deal with its functional implica- tion. Indeed supercomplex organization provides a kinetic advantage through the direct channeling of electrons between the enzyme components and it can be postulated that the presence of supercomplexes may limit the formation of radical oxygen species (ROS) from the respiratory chain [3]. A methodological approach by metabolic flux control analysis used in our laboratory allowed to demonstrate that Complex I and Complex III act as one enzy- matic unit in mammalian mitochondria [4]. Starting from these previous observations, our aim is to investi- gate the characteristics of supercomplex I+III and to evaluate the functional implications of its disassembling. For this purpose we analyzed the structure, the kinetic properties and the produc- tion of ROS after inducing supercomplex I+III dissociation in two experimental models: (i) in proteoliposomes enriched in Complex I and Complex III with different protein to lipid ratios, we observed that supercomplex I+III dissociation occurs in the presence of an excess of phospholipids or if lipid peroxidation is chemically induced; (ii) in mitochondrial membranes, the I+III super-assembly is loss after treatment with chaotropic agents. Kinetic analysis showed lower efficiency with loss of channelling; on the other hand preliminary results on ROS production showed changes in the amount and sensitivity to Complex I inhibitors. References: 1. Schägger H. IUBMB Life 2001; 52: 119–28. 2. Schäfer E et al. Biochemistry 2007; 46:12579–85. 3. Lenaz G et al. Antioxidants & Redox Signaling 2010; 12: 961–1008. 4. Bianchi C et al. J Biol Chem 2004; 279: 36562–9.

We have exploited metabolic flux control analysis to differentiate between randomly distributed respiratory complexes and substrate channelling in supercomplexes in mitochondria. Blue-native gel electrophoresis shows supercomplexes comprising Complexes I, III and IV in most systems examined, however our flux control studies demonstrated that electron channelling occurs only between Complexes I and III in mammalian mitochondria, whereas Complex IV is functionally distinct and is fed electrons by the pool behaviour of cytochrome c. This is at difference with plant mitochondria where Complexes I, III and IV represent a single functional unit. The stability of supercomplexes is dictated by the lipid content and is strongly decreased by lipid peroxidation.

BRCA testing is recommended in all Ovarian Cancer (OC) patients, but the optimal approach is debated. The landscape of BRCA alterations was explored in 30 consecutive OC patients: 6 (20.0%) carried germline pathogenic variants, 1 (3.3%) a somatic mutation of BRCA2, 2 (6.7%) unclassified germline variants in BRCA1, and 5 (16.7%) hypermethylation of the BRCA1 promoter. Overall, 12 patients (40.0%) showed BRCA deficit (BD), due to inactivation of both alleles of either BRCA1 or BRCA2, while 18 (60.0%) had undetected/unclear BRCA deficit (BU). Regarding sequence changes, analysis performed on Formalin-Fixed-Paraffin-Embedded tissue through a validated diagnostic protocol showed 100% accuracy, compared with 96.3% for Snap-Frozen tissue and 77.8% for the pre-diagnostic Formalin-Fixed-Paraffin-Embedded protocol. BD tumors, compared to BU, showed a significantly higher rate of small genomic rearrangements. After a median follow-up of 60.3 months, the mean PFS was 54.9 ± 27.2 months in BD patients and 34.6 ± 26.7 months in BU patients (p = 0.055). The analysis of other cancer genes in BU patients identified a carrier of a pathogenic germline variant in RAD51C. Thus, BRCA sequencing alone may miss tumors potentially responsive to specific treatments (due to BRCA1 promoter methylation or mutations in other genes) while unvalidated FFPE approaches may yield false-positive results.