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Using Drosophila to Study the PINK1-Parkin Mitochondrial Quality C ontrol P athway

Using Drosophila to Study the PINK1-Parkin Mitochondrial Quality C ontrol P athway Leo Pallanck University of Washington. The PINK1-Parkin Pathway. Mitofusins. Drp1. Mitofusins. Parkin. PINK1. The PINK1-Parkin Pathway. Mitofusins. Drp1. Mitofusins. Parkin. PINK1. Lysosome.

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Using Drosophila to Study the PINK1-Parkin Mitochondrial Quality C ontrol P athway

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  1. Using Drosophila to Study the PINK1-Parkin Mitochondrial Quality Control Pathway Leo Pallanck University of Washington

  2. The PINK1-Parkin Pathway Mitofusins Drp1 Mitofusins Parkin PINK1

  3. The PINK1-Parkin Pathway Mitofusins Drp1 Mitofusins Parkin PINK1 Lysosome

  4. The PINK1-Parkin Pathway Mitofusins Drp1 Mitofusins Parkin Contributions of fly genetics to this model: =>PINK1 & Parkin mutants accumulate enlarged defective mitochondria =>PINK1 & Parkin act in a common pathway =>Parkinubiquitinatesmitofusin to promote mt fragmentation PINK1 Lysosome

  5. The PINK1-Parkin Pathway Mitofusins Some important remaining questions: =>Do PINK1 & Parkin promote mitochondrial turnover in vivo? Drp1 Mitofusins Parkin PINK1 =>Do PINK1 and Parkin influence mitochondrial quality control in Dopaminergic neurons? =>What other factors act in the PINK1-Parkin pathway? Lysosome

  6. Do PINK1& Parkin promote mitochondrial turnover in vivo? Our approach: stable isotope labeling and mass spectrometry (collaboration with the laboratory of Mike MacCoss) CD3 D3-leucine (mass: +3) D3-leucine % of leucine in peptides D3 trypsin digest total proteins unlabeled leucine D3 D3 D3 D3 5 & 10 days time

  7. Technical challenges with this approach: 1. Quantifying the amount of D3-leucine incorporation into protein 2. Estimating the D3-leucine “precursor pool” to allow an accurate measurement of turnover Solution: TOPOGRAPH; an algorithm that we helped developed in collaboration with the MacCoss laboratory

  8. 1. Quantifying the amount of D3-leucine incorporation into protein Topograph finds the “best fit” (natural isotope distribution + n D3-leucines) actual amount of isotope Fractional Abundance predicted % 0 D3-leucines % 1 D3-leucine isotope (additional mass units)

  9. Technical challenges with this approach: 1. Quantifying the amount of D3-leucine incorporation into protein 2. Estimating the D3-leucine “precursor pool” to allow an accurate measurement of turnover Solution: TOPOGRAPH; an algorithm that we developed in collaboration with the MacCoss laboratory

  10. 2. Estimating the D3-leucine “precursor pool” to allow an accurate measurement of turnover precursor pool 100% D3-leucine Requires multiple Leu containing peptides 25% turnover e.g., AIGLPEDLIQK (2 leucines) “Old” “New” precursor pool 50% D3-leucine 50% turnover “Old” D3-leucine content after X hours of labeling: 25% “New”

  11. 2. Estimating the D3-leucine “precursor pool” to allow an accurate measurement of turnover “Old”: TOPOGRAPH determines this distribution = 50% turnover “New”: and uses probability-based calculations to estimate the D3-leucine content of the precursor pool enables an estimate of turnover

  12. Predictions of the PINK1-Parkin mitochondrial QC model: Mitochondrial proteins Non-mitochondrial proteins PINK1 or Parkin overexpression PINK1 or Parkin overexpression control control PINK1 or parkinmutants % D3-leucine % D3-leucine PINK1 or parkinmutants time time

  13. mitochondrial protein turnover is decreased in parkin mutants *p<0.0001 mean decreasein half-life: ~31%

  14. nonmitochondrialprotein turnover is similarly decreased in parkin mutants *p<0.02

  15. Why are nonmitochondrial proteins affected? • Parkin regulates protein turnover more broadly than anticipated? • The accumulation of defective mitochondria has a general effect on mitochondrial turnover?

  16. PINK1 overexpression increases mitochondrial protein turnover *p<0.0001 mean decreasein half-life: ~10%

  17. Importantly, mitochondrial proteins are selectively affected by PINK1 overexpression *p < 0.0001

  18. The PINK1-Parkin Pathway Mitofusins Important remaining questions: =>Do PINK1 & Parkin promote mitochondrial turnover in vivo? Drp1 Mitofusins Parkin PINK1 =>Do PINK1 and Parkin influence mitochondrial quality control in Dopaminergic neurons? =>What other factors act in the PINK1-Parkin pathway? Lysosome

  19. Do PINK1 & Parkin influence mitochondrial QC in dopamine neurons? First needed to develop a simple method to purify specific dopaminergic neurons from the fly brain: -Uses the UAS/GAL4 system to mark the neurons of interest Examples Dopaminergic neurons Cholinergic neurons Etc. GFP (Brand and Perrimon, 1993) -Uses FACS to collect the marked neurons following mechanical and enzymatic brain dissociation

  20. FACS purification of dopaminergic neurons from the adult Drosophila brain: TH-GAL4; UAS-GFP Non-transgenic flies 0.02% of events 2% of events DAPI DAPI FITC (GFP) FITC (GFP) FACS-purified dopaminergic neurons also express appropriate markers (e.g., TH, VMAT, etc.)

  21. Predictions: -mt membrane potential should correlate with PINK1-Parkin activity -accumulation of enlarged mt in parkin mutants. DA neurons DA neurons P < 0.05 park+/- 1.2 -CCCP P < 0.01 45% 55% 0.8 Relative MMP 22% 78% 0.4 # of cells 0 WT Park-/- PINK1OE park-/- +CCCP P < 0.01 100 62% 38% 80 100% 0% 60 % fused mt # of cells 40 20 0 WT Park-/- Mitotracker fluorescence Mitotracker fluorescence

  22. Do manipulations that promote mt fragmentation and turnover influence the parkin neuronal phenotypes? Mitofusins 1.2 P < 0.01 0.8 Relative MMP 0.4 Drp1 Mitofusins 0 WT park-/- park-/- TgDRP1 park-/- MfnRnai park-/- TgAtg8a PINK1 1.2 P < 0.001 Relative # PPL1 neurons 0.8 0.4 0 WT park-/- park-/- TgDRP1 park-/- MfnRnai park-/- TgAtg8a Lysosome

  23. What explains the selective sensitivity of dopaminergic neurons to mutations in PINK1 and parkin? -An enhanced sensitivity of mitochondria? -An increased sensitivity of cell survival in response to a general mitochondrial defect? P < 0.05 P < 0.01 100 1.2 80 P < 0.01 0.8 60 Relative MMP % fused mt 40 0.4 20 0 0 WT Park-/- WT DA neurons Park-/- Cholinergic neurons Park-/- =>Mitochondria in dopaminergic neurons are more sensitive to perturbations of the PINK1-Parkin pathway

  24. The PINK1-Parkin Pathway Mitofusins Important remaining questions: =>Do PINK1 & Parkin promote mitochondrial turnover in vivo? Drp1 Mitofusins Parkin PINK1 =>Do PINK1 and Parkin influence mitochondrial quality control in Dopaminergic neurons? =>What other factors act in the PINK1-Parkin pathway? Lysosome

  25. PINK1 park-/- A screen for PINK1 overexpression modifiers: Can this phenotype be used to screen for novel components of the PINK1-Parkin pathway? -mutations of activators (like parkin) should suppress -mutations of inhibitors (like mitofusin) should enhance UAS-PINK1 ey-GAL4 WT + + Del ; ; X + + Cy Sb A deletion bearing chromosome UAS-PINK1 ey-GAL4 Del suppression or enhancement? ; + + PINK1 Will this work???

  26. PINK1 park-/- A screen for PINK1 overexpression modifiers: Can this phenotype be used to screen for novel components of the PINK1-Parkin pathway? -mutations of activators (like parkin) should suppress -mutations of inhibitors (like mitofusin) should enhance WT 6 PINK1 Eye severity score 4 2 0 WT PINK1OE PINK1OE Park-/- PINK1OE Park+/- PINK1OE Mfn+/- Will this work???

  27. PINK1 park-/- A screen for PINK1 overexpression modifiers: Can this phenotype be used to screen for novel components of the PINK1-Parkin pathway? -mutations of activators (like parkin) should suppress -mutations of inhibitors (like mitofusin) should enhance WT Enhancer 6 PINK1 Eye severity score 4 2 Suppressor 0 WT PINK1OE PINK1OE Park-/- PINK1OE Park+/- PINK1OE Mfn+/- Will this work???

  28. A screen for PINK1 overexpression modifiers: -We have thus far screened deletions covering >95% of the genes residing on two of the three major chromosomes in Drosophila 31 suppressors 53 enhancers Some suppressors: Parkin, p62 Some enhancers: mitofusin, Afg3L2 Some are stronger modifiers than Parkin and Mitofusin Ongoing efforts: -Testing the specificity of modifiers -mapping the modifier gene

  29. Summary -The PINK1-Parkin pathway promotes selective mitochondrial turnover in vivo -PINK1 & Parkin influence mitochondrial QC in dopaminergic neurons -Mitochondria in dopaminergic neurons are selectively sensitive to the loss of the PINK1-Parkin pathway (a toxic effect of dopamine?) -We’ve identified a large collection of candidate PINK1-Parkin pathway components

  30. Acknowledgements EvvieVincow Jonathon Burman Ruth Thomas Michael MacCoss Nick Shulman Department of Genome Sciences University of Washington Former Contributors: Angela Poole Cornell University Alex Whitworth University of Sheffield Jessica Greene Fred Hutchinson Cancer Research Center National Institutes of Health UMDF

  31. How accurately does Topograph estimate the precursor pool? -A validation experiment using yeast • Uniformly label yeast proteins for many generations in media containing a defined D3-leucine content • Subject the yeast proteins to mass spectrometry and use Topograph to measure the precursor pool Amount of D3-Leucine in media 33% 67% Topograph’s estimate of D3-Leucine precursor pool 33% 68% Pretty Accurately!!!

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