critical appraisal bob lightowlers mitochondrial research group institute of neuroscience
DESCRIPTION
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!! - PowerPoint PPT PresentationTRANSCRIPT
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CRITICAL APPRAISAL
Bob LightowlersMitochondrial Research GroupInstitute of Neuroscience
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NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!!
![Page 3: CRITICAL APPRAISAL Bob Lightowlers Mitochondrial Research Group Institute of Neuroscience](https://reader035.vdocuments.net/reader035/viewer/2022070418/568159a3550346895dc6f9c3/html5/thumbnails/3.jpg)
NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!!
ONLY 15% OF PUBLICATIONS ARE TRUSTWORTHY
![Page 4: CRITICAL APPRAISAL Bob Lightowlers Mitochondrial Research Group Institute of Neuroscience](https://reader035.vdocuments.net/reader035/viewer/2022070418/568159a3550346895dc6f9c3/html5/thumbnails/4.jpg)
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
![Page 20: CRITICAL APPRAISAL Bob Lightowlers Mitochondrial Research Group Institute of Neuroscience](https://reader035.vdocuments.net/reader035/viewer/2022070418/568159a3550346895dc6f9c3/html5/thumbnails/20.jpg)
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.vdocuments.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
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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 ?