CRITICAL APPRAISAL Bob Lightowlers Mitochondrial Research Group Institute of Neuroscience
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CRITICAL APPRAISAL
Bob LightowlersMitochondrial Research GroupInstitute of Neuroscience
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NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!!
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NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!!
ONLY 15% OF PUBLICATIONS ARE TRUSTWORTHY
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NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!!
ONLY 15% OF PUBLICATIONS ARE TRUSTWORTHY
GUILTY UNTIL PROVEN INNOCENT
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Mutations in mitochondrial cytochrome c oxidase
genes segregate with late-onset Alzheimer Disease
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Hypothesis:
Alzheimers Disease could be caused by defects in activity of the respiratory chain complex cytochrome c oxidase
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Why ?
• Lack of FH is a negative risk factor
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Why ?
• Lack of FH is a negative risk factor • Risk of AD increases with affected maternal relative (mtDNA?)
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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
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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
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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
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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
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Methods used
• MtDNA isolation and sequencing in patients, asymptomatic relatives and controls
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Methods used
• MtDNA isolation and sequencing in patients, asymptomatic relatives and controls
• All three COX genes sequenced
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Methods used
• MtDNA isolation and sequencing in patients, asymptomatic relatives and controls
• All three COX genes sequenced
• Quantification of mutations in all samples
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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)
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Generation of transmitochondrialcybrids
Biopsy
EthBr Enucleation
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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
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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
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Results
506 Patients and 95 controls
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Results
506 Patients and 95 controls
10 clones of all three COX genes sequence
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Results
506 Patients and 95 controls
10 clones of all three COX genes sequence
6 mutations found in COI and COII
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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
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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
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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
![Page 29: CRITICAL APPRAISAL Bob Lightowlers Mitochondrial Research Group Institute of Neuroscience](https://reader035.fdocuments.net/reader035/viewer/2022070418/568159a3550346895dc6f9c3/html5/thumbnails/29.jpg)
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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
![Page 31: CRITICAL APPRAISAL Bob Lightowlers Mitochondrial Research Group Institute of Neuroscience](https://reader035.fdocuments.net/reader035/viewer/2022070418/568159a3550346895dc6f9c3/html5/thumbnails/31.jpg)
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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 ?