Forty five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol c), cause the early onset mitochondrial DNA (mtDNA) depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal poly- merase region of pol c revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542 g ? a; T851A, c.2551 a ? g; R852C, c.2554 c ? t; R853Q, c.2558 g ? a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637 g ? t and T885S, c.2653 a ? t). Biochemical characterization of purified, recombinant forms of pol c revealed that Alpers mutations in the thumb subdomain reduced polymerase activity more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50–70% of wild-type polymerase activity. All six mutant enzymes retained physical and functional interaction with pol c’s accessory subunit (p55), and none of the six mutants exhibited defects in the misinser- tion fidelity in vitro. However, differential DNA binding by these mutants suggest a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study repre- sents the first structure-function analysis of the thumb subdomain in pol c and examines the consequences of mitochondrial disease mutations in this region. doi:10.1016/j.mito.2009.12.085 94 Cardiac, hepatic and renal mitochondrial toxicity of doxoru- bicin in a sub-chronic in vivo model Goncalo C. Pereira a , Susana S. Pereira a , Maria S. Santos a , António J. Moreno b , Paulo J. Oliveira b,* a Centre for Neurosciences and Cell Biology, Department of Zoology, Coimbra, Portugal; b Institute for Marine Research (IMAR), Department of Zoology, Coimbra, Portugal Nowadays, doxorubicin (DOX) is used to treat several types of human tumors. However, treatment is accompanied by a cumulative cardiotoxicity with a strong mitochondrial dysfunction component but which full mechanism remains to be elucidated. The aim of this work was to evaluate and characterize the DOX-induced toxicity in a sub-chronic animal model at the mitochondrial level. Eight weeks old male Wistar rats were weekly injected with saline solution or 2 mg/Kg DOX, during 7 weeks. Heart mitochondria appear to be mostly affected, presenting lower membrane potential together with a decrease in both state 3 and state 4 respiration without differences in ADP/O ratio or RCR regardless of the substrate used. Hepatic mito- chondria present only differences at the complex I level (membrane potential, lag phase and state 3) contrarily to renal mitochondria which have increased lag phase and decreased ADP/O ratio, however only when complex II substrates were used. Mitochondrial calcium loading capacity results indicate that cardiac and renal mitochondria from DOX-treated rats have increased calcium-release rate and decreased retention time, respectively. Hydrogen peroxide produc- tion by the MRC shows that heart and liver mitochondria from DOX-treated rats produce more hydrogen peroxide in the presence of complex I substrates. The present work is the first to show a direct comparison of in vivo DOX mitochondrial effects in three distinct organs from the same animal. In conclusion, our data supports the notion that DOX in vivo treatment causes mitochondrial alterations, which are more evident in the heart and that contribute for DOX- induced cardiomyopathy. This work is supported by the Portuguese Foundation for Science and Technology (FCT): Ph.D. Fellowship (SFRH/BD/36938/2007) to GS1GCP and research Grant PTDC-SAU-OSM-64084-2006 to PJO. doi:10.1016/j.mito.2009.12.086 95 A mouse model of Parkinson’s Disease based on CoQ deficiency David L. Gasser a,* , Marni J. Falk b , Harry Ischiropoulos b , Dana Bakalar c , Julie A. Blendy c a Department of Genetics, University of Pennsylvania, USA; b Department of Pediatrics, Children’s Hospital of Philadelphia, PA, USA; c Department of Pharmacology, University of Pennsylvania, Philadelphia, PA, USA Coenzyme Q is composed of a benzoquinone ring with a polyi- soprenoid side chain that is synthesized by a heterotetramer consist- ing of two prenyl diphosphate synthase subunits encoded by Pdss1 and Pdss2. Mutations in each of these genes are known to cause se- vere neuromuscular disease in humans. A mouse model with a mis- sense mutation in Pdss2 develops a lethal kidney disease, which is recapitulated in conditional knockouts in which the Pdss2 gene was targeted to glomerular podocytes (Peng et al., PLoS Genetics 4: e1000061, 2008). The kidney disease phenotype can be ameliorated to a large extent in missense mutants by CoQ 10 supplementation (Saiki et al., Am J Physiol Renal Physiol 295: F1535, 2008). Because CoQ 10 supplementation has been reported to have beneficial effects in Parkinson’s disease, we generated conditional Pdss2 knockouts targeted to dopaminergic neurons by a cre gene under the control of the dopamine transporter (DAT) promoter. Pdss2 loxP =loxP , DAT/cre- positive mice appear to be completely normal for the first 6 weeks of life, but then develop motor difficulties as assessed both by home cage locomotor activity and a rotorod test. The rotorod test consists of a wheel turning at constant speed, and requires that the animal make postural adjustments in order to maintain equilibrium. While Pdss2 loxP =loxP , DAT/cre-positive mouse mutants behave normally on the learning component of the rotorod, their fall latencies are signif- icantly less than those for wildtype controls. This deficiency is evi- dent at 6 weeks old, and worsens as the animal ages. These findings are consistent with what is observed in Parkinson’s Disease, and thus indicate an important role for Pdss2 in motor coordination. This work demonstrates that Pdss2-related primary CoQ deficiency offers a mouse model for studying mechanisms and potential thera- pies of Parkinson’s Disease. doi:10.1016/j.mito.2009.12.087 96 Biochemical analysis of POLG2 variants associated with mitochondrial disease Matthew J. Young a,* , Matthew J. Longley a , Rajesh Kasiviswanathan a , Lee-Jun Wong b , William C. Copeland c a Mitochondrial DNA Replication Group, Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, USA; b Department of Molecular and Human Genetics, Mitochondrial Diagnostic Laboratory, Baylor College of Medicine, USA; c Mitochondrial DNA Replication Group, Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, USA Human mitochondrial DNA (mtDNA) is replicated by the poly- merase c holoenzyme consisting of p140, the 140-kDa catalytic sub- unit, and p55 the 55-kDa accessory subunit. The p55 accessory protein is a DNA binding protein encoded by POLG2 on chromosome 17q that confers processivity on the mitochondrial DNA polymerase holoenzyme. In vitro analysis has demonstrated that the p55 G451E variant is unable to stimulate p140 due to a defect in holoenzyme subunit interaction. This mutation was found in a patient diagnosed with autosomal dominant progressive external ophthalmoplegia (adPEO). Another POLG2 mutant (c.1247G>C/p.G416A) was also characterized biochemically but did not behave differently from the wild-type protein. In a genetic search of mitochondrial disease patients suspected of conferring mutations in nuclear genes seven novel POLG2 mutations were identified. These seven mutations map throughout the protein including the p55 dimer interface as well as the C-terminal domain that is known to interact with the 226 Abstracts / Mitochondrion 10 (2010) 200–242