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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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slide1

CRITICAL APPRAISAL

Bob Lightowlers

Mitochondrial Research Group

Institute of Neuroscience

slide3

NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!!

ONLY 15% OF PUBLICATIONS ARE TRUSTWORTHY

slide4

NOT EVERYTHING THAT IS PUBLISHED IS CORRECT!!

ONLY 15% OF PUBLICATIONS ARE TRUSTWORTHY

GUILTY UNTIL PROVEN INNOCENT

slide5

Mutations in mitochondrial cytochrome c oxidase

genes segregate with late-onset Alzheimer Disease

slide6

Hypothesis:

Alzheimers Disease could be caused by defects in activity

of the respiratory chain complex cytochrome c oxidase

slide7

Why ?

  • Lack of FH is a negative risk factor
slide8

Why ?

  • Lack of FH is a negative risk factor
  • Risk of AD increases with affected maternal relative (mtDNA?)
slide10

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
slide11

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
slide12

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
slide13

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
slide14

Methods used

  • MtDNA isolation and sequencing in patients,
  • asymptomatic relatives and controls
slide15

Methods used

  • MtDNA isolation and sequencing in patients,
  • asymptomatic relatives and controls
  • All three COX genes sequenced
slide16

Methods used

  • MtDNA isolation and sequencing in patients,
  • asymptomatic relatives and controls
  • All three COX genes sequenced
  • Quantification of mutations in all samples
slide17

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)
slide18

Biopsy

EthBr

Enucleation

Generation of transmitochondrial

cybrids

slide19

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
slide20

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
slide21

Results

506 Patients and 95 controls

slide22

Results

506 Patients and 95 controls

10 clones of all three COX genes sequence

slide23

Results

506 Patients and 95 controls

10 clones of all three COX genes sequence

6 mutations found in COI and COII

slide24

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

slide27

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

slide28

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

slide30

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

slide32

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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