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Premature ageing in mice expressing defective mitochondrial DNA polymerase

Aleksandra Trifunovic, Anna Wredenberg, Maria Falkenberg, Johannes N. Spelbrink, Anja T. Rovio, Carl E. Bruder, Mohammad Bohlooly-Y, Sebastian Gidlöf, Anders Oldfors, Rolf Wibom, Jan Tornell, Howard

T. Jacobs & Nils- Göran Larsson

Presentation by Beril Kumcuoglu

04/20/2010

Background Information

• mtDNA is in close proximity to the mitochondrial respiratory chain, which causes oxidative damage by the production of reactive oxygen species (ROS).

• Accumulation of somatic mtDNA mutations were claimed to have a role in aging.

• The number of cytochrome c oxidase (COX) deficient cells progressively increases in postmitotic tissues.

• Decline in oxidative phosphorylation may lead to an elevated ROS production.

• Somatic mutations have capacity to cause a variety of aging phenotypes in mammals, but its relative importance in mammalian aging is not clear.

Creation of mtDNA-mutator mice

•Analysis with restriction enzymes (1.b.) shows the insertion in clones that are heterozygous for PolGAmutNeo

• Exon 3 ( yellow box) of the targeting vector encodes alanineinstead of aspartate.•Restriction with Bg/I shows Frt-flanked PGK-neo casetteexcision.

DNA Polymerase and exonuclease activities

• POLGAmut containing enzyme lacked exonuclease activity but did not show deficiency in DNA synthesis activity.

Survival Curve of mtDNA-mutator mice

•mtDNA-mutator mice seemed normal until the age of 25 weeks.•The median lifespan was 48 weeks.•All of them died before the age of 61 weeks.

•Aging Related Phenotypes were first observed at the age of 25 weeks.

Weight lossReduced subcutaneous fatAlopecia (hair loss)Kyphosis ( curvature of the spine)OsteoporosisAnaemiaReduced fertilityHeart enlargement

Body Weight and Subcutaneous Fat Reduction in mtDNA-mutator mice

• A reduction in the subcutaneous fat was observed in mtDNA-mutator mice

• Body weight gain started to decelerate from 15 weeks and weight loss was noted in 24 weeks for both male and female mice.

• The ratio of lean body mass to length was normal

Kyphosis and Osteoporosis in mtDNA-mutator mice

• Kyphosis and alopecia started to appear at the age of 25 weeks.

•X-ray densitometry revealed a reduction in bone mineral density ( BMD) and bone mineral content (BMC)/ length observed at the age of 40 weeks.

Body composition of the whole mouse

Body composition of dissected femur bones

Deficiencies in cardiomyocytes of mtDNA-mutatormice

•40 week-old mtDNA-mutator had left ventricle hypertrophy.

•Cytochrome c oxidase (COX) activity deficiency was observed in some cardiomyocytes ( shown in blue).

• Cardiomyocytes had a vacuolated appearance because of the accumulation of abnormal mitochondria.

Other premature aging phenotypes

• Anaemia was observed in mtDNA-mutated mice. • Spleen enlergement and extramedullary haematopoiesis in the liver were present.• Reduction in fertility of both sexes occurred.

Deletion of the arc between OH and OL

• The mean level of full-length mtDNA un mutated mice was 70 % of the one of wild-type mice.

• The reduced level of full-length mtDNA did not affect expression levels .

Point mutation distribution of mtDNA-mutator mice

• Increased number of somatic mtDNA point mutations were found in brain, heart and liver.

•PolgAmut mutation was recessive.

• Mutations were evenly distributed along the cytochrome b gene.

•Reduction of respiratory chain enzyme activities was observed.

Conclusions:

• An experimental link was established between increased levels of somatic mtDNA mutations, respiratory chain dysfunction and ageing phenotypes in mammals.

• mtDNA-mutator created a random set of point mutations.

• Kinetics of accumulation is to be investigated.

• Whether increased somatic mtDNA mutation can be counteracted by dietary interventions or exercise need to be investigated.