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CRITICAL APPRAISAL Bob Lightowlers Mitochondrial Research Group Institute of Neuroscience

CRITICAL APPRAISAL Bob Lightowlers Mitochondrial Research Group Institute of Neuroscience. NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!!. NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!! ONLY 15% OF PUBLICATIONS ARE TRUSTWORTHY. NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!!

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CRITICAL APPRAISAL Bob Lightowlers Mitochondrial Research Group Institute of Neuroscience

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  1. CRITICAL APPRAISAL Bob Lightowlers Mitochondrial Research Group Institute of Neuroscience

  2. NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!!

  3. NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!! ONLY 15% OF PUBLICATIONS ARE TRUSTWORTHY

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

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

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

  7. Why ? • Lack of FH is a negative risk factor

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

  9. Human mtDNA • An autosomally replicating genome • Found in mitochondrial matrix • Circular genome with short (1.2knt) • noncoding region (D-loop) 16,569 bp • 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

  10. 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

  11. 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

  12. 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

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

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

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

  16. 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)

  17. Biopsy EthBr Enucleation Generation of transmitochondrial cybrids

  18. 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

  19. 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

  20. Results 506 Patients and 95 controls

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

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

  23. 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

  24. 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

  25. 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

  26. 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

  27. 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 ?

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