CRITICAL APPRAISAL Bob Lightowlers Mitochondrial Research Group Institute of Neuroscience

NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!!

ONLY 15% OF PUBLICATIONS ARE TRUSTWORTHY

NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!! ONLY 15% OF PUBLICATIONS ARE TRUSTWORTHY GUILTY UNTIL PROVEN INNOCENT

Mutations in mitochondrial cytochrome c oxidase genes segregate with late-onset Alzheimer Disease

Hypothesis: Alzheimers Disease could be caused by defects in activity of the respiratory chain complex cytochrome c oxidase

Why ? Lack of FH is a negative risk factor

Why ? Lack of FH is a negative risk factor Risk of AD increases with affected maternal relative (mtDNA?)

Human mtDNA An autosomally replicating genome Found in mitochondrial matrix Circular genome with short (1.2knt) noncoding region (D-loop) Comprises app. 0.1% of total cell DNA Varies enormously in copy number/cell Approx. 700 in fibroblasts to >200,000 in some mammalian oocytes Maternally inherited Often heteroplasmic in the diseased state 16,569 bp

Why ? Lack of FH is a negative risk factor Risk of AD increases with affected maternal relative (mtDNA?) Mutations in mtDNA can lead to defective OXPHOS

Why ? Lack of FH is a negative risk factor Risk of AD increases with affected maternal relative (mtDNA?) Mutations in mtDNA can lead to defective OXPHOS Neurons may be particularly susceptible to such defects

Why ? Lack of FH is a negative risk factor Risk of AD increases with affected maternal relative (mtDNA?) Mutations in mtDNA can lead to defective OXPHOS Neurons may be particularly susceptible to such defects COX activity reported to decrease in brain of AD patients

Methods used MtDNA isolation and sequencing in patients, asymptomatic relatives and controls

Methods used MtDNA isolation and sequencing in patients, asymptomatic relatives and controls All three COX genes sequenced

Methods used MtDNA isolation and sequencing in patients, asymptomatic relatives and controls All three COX genes sequenced Quantification of mutations in all samples

Methods used MtDNA isolation and sequencing in patients, asymptomatic relatives and controls All three COX genes sequenced Quantification of mutations in all samples Platelet fusion from AD patients to neuronal cells lacking mtDNA (rho0)

Generation of transmitochondrial cybrids Biopsy EthBr Enucleation

Methods used MtDNA isolation and sequencing in patients, asymptomatic relatives and controls All three COX genes sequenced Quantification of mutations in all samples Platelet fusion from AD patients to neuronal cells lacking mtDNA (rho0) Analysis of respiratory enzyme activity in the cybrids

Methods used MtDNA isolation and sequencing in patients, asymptomatic relatives and controls All three COX genes sequenced Quantification of mutations in all samples Platelet fusion from AD patients to neuronal cells lacking mtDNA (rho0) Analysis of respiratory enzyme activity in the cybrids Analysis of ROS production in cybrids

Results 506 Patients and 95 controls

Results 506 Patients and 95 controls 10 clones of all three COX genes sequence

Results 506 Patients and 95 controls 10 clones of all three COX genes sequence 6 mutations found in COI and COII

Results 506 Patients and 95 controls 10 clones of all three COX genes sequence 6 mutations found in COI and COII Different levels of heteroplasmy but levels significantly greater in the AD cohort

Results 506 Patients and 95 controls 10 clones of all three COX genes sequence 6 mutations found in COI and COII Different levels of heteroplasmy but levels significantly greater in the AD cohort No disease-associated mutations in COIII gene

Results 506 Patients and 95 controls 10 clones of all three COX genes sequence 6 mutations found in COI and COII Different levels of heteroplasmy but levels significantly greater in the AD cohort No disease-associated mutations in COIII gene AD cybrids but not controls had low COX activity

Results 506 Patients and 95 controls 10 clones of all three COX genes sequence 6 mutations found in COI and COII Different levels of heteroplasmy but levels significantly greater in the AD cohort No disease-associated mutations in COIII gene AD cybrids but not controls had low COX activity Increased production of ROS in AD cybrids

Critical evaluation: How appropriate and robust are the methods ? Is the data (and evaluation) robust ? Are the conclusions valid, based on the reported data ? How often do the authors refer to themselves ? How does the paper stand the test of time ? Is there any conflict of interest ?