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CRITICAL APPRAISAL
Bob LightowlersMitochondrial Research GroupInstitute 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!!
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 transmitochondrialcybrids
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 ?
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