1. A mitochondrial quality control pathway revealed from studies of familial Parkinson’s Disease genes Leo Pallanck Department of Genome Sciences

2. OUTLINE I.Overview of Parkinson’s disease (PD) -characteristics -influence of environment -influence of genetics II. Studies of the familial PD genes PINK1 & Parkin reveal a mitochondrial QC pathway -Part I: contributions from work in flies -Part II: contributions from work in cell culture

3. Parkinson’s disease (PD) -Motor dysfunction (rigidity, tremor, bradykinesia, etc.). Composed of: -  -synuclein -ubiquitin -synphilin-1 -neurofillaments -etc. -Lewy body pathology (cytoplasmic proteinaceous inclusions). -Dopamine neuron degeneration in midbrain. -Highly prevalent (~1% over age 65).

4. What causes Parkinson’s disease? Environmental Toxins OR Genetic Factors

5. MPTPMPP+ 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Dopamine transporter The potential role of environmental factors

6. MPTPMPP+ Dopamine transporter Mitochondrial Complex I MPP+ 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) The potential role of environmental factors

7. MPTPMPP+ Dopamine transporter Mitochondrial Complex I MPP+ CELL DEATH Oxidative Stress 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) The potential role of environmental factors -What are mitochondria? -What does mitochondrial complex I do? -Why does inhibition of complex I kill neurons?

8. Roles: -ATP synthesis -Ca 2+ -buffering -apoptosis -metabolite synthesis Textbook view of mitochondria cristae matrix inner membrane outer membrane intermembrane space Some background information on mitochondrial biology

9. HeLa cell yeast cell mouse embryonic fibroblast The secret lives of mitochondria

10. Mitochondria undergo continual fission and fusion events Video: Lab of David Chan

11. Fission and fusion are mediated by evolutionarily conserved factors FISSION: Drp1

12. FISSION: Drp1 FUSION: Mitofusin (Mfn) Fission and fusion are mediated by evolutionarily conserved factors

13. FISSION: Drp1 FUSION: Mitofusin (Mfn) fission and fusion are dynamic processes and their rates influence mitochondrial morphology Drp1 Mfn Fission and fusion are mediated by evolutionarily conserved factors

14. FISSION: Drp1 FUSION: Mitofusin (Mfn) fission and fusion are dynamic processes and their rates influence mitochondrial morphology Drp1 Mfn Fission and fusion are mediated by evolutionarily conserved factors fission or fusion Untreated HeLa cells Legros S, et al., (2003) MBC 13, 4343-54 fusion or fission For example… Why?

15. Mitochondrial anatomy outer membrane inner membrane matrix intermembrane space respiratory chain ~1,000 mitochondrial proteins encoded in the nucleus and imported post-translationally mitochondrial genome (~40 genes) Complex I Complex III Complex IV Complex V Complex II intermembrane space matrix inner membrane O2-O2-O2-O2- O2-O2-O2-O2- H2O2H2O2H2O2H2O2 O2-O2-O2-O2- H2O2H2O2H2O2H2O2

16. O2-O2- O2-O2- O2-O2- O2-O2- H2O2H2O2 OH Damage lipids, proteins & DNA Proximity to ROS makes mitochondria a particularly vulnerable target The vicious cycle theory: The progressive accumulation of ROS-induced mitochondrial damage leads to cell death and aging O2-O2- OH O2-O2- O2-O2- H2O2H2O2 H2O2H2O2 H2O2H2O2 H2O2H2O2 H2O2H2O2 O2-O2- The ‘Vicious Cycle’ theory of aging Harman D. (1972) Journal of the American Geriatrics Society 20: 145.

17. MPTPMPP+ Dopamine transporter Mitochondrial Complex I MPP+ CELL DEATH 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) The potential role of environmental factors O2-O2- OH O2-O2- O2-O2- H2O2H2O2

18. Additional evidence for environmental factors in PD Rotenone (a pesticide) produces Parkinsonism in rats Rotenone is a mitochondrial complex I inhibitor

19. Rotenone (a pesticide) produces Parkinsonism in rats Rotenone is a mitochondrial complex I inhibitor (complex I activity is decreased in many cases of idiopathic PD) Additional evidence for environmental factors in PD Muftuoglu M, et al. (2004) Mov Disord 19, 544-8. controlsParkinson’s disease

20. Rotenone (a pesticide) produces Parkinsonism in rats. Rotenone is a mitochondrial complex I inhibitor. (complex I activity is decreased in many cases of idiopathic PD) Epidemiological studies: -associations with altered risk: farming (herbicides? pesticides?), welding (Fe?Mg?), well water (toxins?), smoking, coffee, etc. Additional evidence for environmental factors in PD ≥40 years 20-39 years 0-19 years nonsmoker Relative Risk 010.5 Smoking: ≥28 oz/day 20-24 oz/day 12-16 oz/day 4-8 oz/day nondrinker Relative Risk 010.5 Coffee consumption:

21. Twin studies : Dizygotic twins (~50% genome in common) Monozygotic twins (100% genome in common) Evidence for genetic factors in PD No difference in concordance rate between monozygotic and dizygotic twins with onset after age 51 However… -Positron emission tomography revealed high concordance of preclinical neuronal dysfunction in monozygotic twins, regardless of age -Concordance was 2X greater for monozygotic twins in PD individuals with onset before 51 years-justified search for genes among early- onset cases -May provide insight into common forms of PD -May help distinguish cause and consequence

22. The identification of rare PD-causing gene mutations has been a major advance AD=autosomal dominantAR=autosomal recessive Gene PARK1 PARK2 PARK3 PARK5 PARK6 PARK7 PARK8 PARK9 PARK10 Mode of Inheritance AD AR AD AD(?) AR AD AR ? Age of Onset ~45yrs 3-64yrs ~60yrs ~50yrs ~40yrs ~30yrs ~50yrs teens >50yrs (  -synuclein) (parkin) (UCH-L1?) (PINK1) (DJ-1) (LRRK2) (ATP13A2)

23. The identification of rare PD-causing gene mutations has been a major advance AD=autosomal dominantAR=autosomal recessive Gene PARK1 PARK2 PARK3 PARK5 PARK6 PARK7 PARK8 PARK9 PARK10 Mode of Inheritance AD AR AD AD(?) AR AD AR ? Age of Onset ~45yrs 3-64yrs ~60yrs ~50yrs ~40yrs ~30yrs ~50yrs teens >50yrs (  -synuclein) (parkin) (UCH-L1?) (PINK1) (DJ-1) (LRRK2) (ATP13A2)

24. A little more about  -synuclein  -synuclein: Increased gene dosage Missense alleles that reduce  -synuclein turnover Suggests that excess  -synuclein may be a pathogenic factor in most cases of Parkinson’s disease -A major component of Lewy Body inclusions -Normal cellular function unknown -Phenotype inherited in autosomal dominant fashion: Major questions: -why is  synuclein toxic? -how does it become toxic in sporadic cases? -does it influence mitochondrial integrity?

25. The identification of rare PD-causing gene mutations has been a major advance AD=autosomal dominantAR=autosomal recessive Gene PARK1 PARK2 PARK3 PARK5 PARK6 PARK7 PARK8 PARK9 PARK10 Mode of Inheritance AD AR AD AD(?) AR AD AR ? Age of Onset ~45yrs 3-64yrs ~60yrs ~50yrs ~40yrs ~30yrs ~50yrs teens >50yrs (  -synuclein) (parkin) (UCH-L1?) (PINK1) (DJ-1) (LRRK2) (ATP13A2) (parkin) (PINK1)

26. Background info on Parkin & PINK1 -loss-of-function mutations cause autosomal recessive Parkinson’s disease -encodes a cytoplasmic E3 ubiquitin protein ligase Parkin:

27. The ubiquitin proteasome degradation pathway E3 E1 E2 E3 X X E2 E3 X X Degradation by proteasome Ubiquitin (parkin) Target protein +

28. -loss-of-function mutations cause autosomal recessive Parkinson’s disease -encodes a Ser/Thr protein kinase with a mitochondrial targeting sequence PINK1: -loss-of-function mutations cause autosomal recessive Parkinson’s disease -encodes a cytoplasmic E3 ubiquitin protein ligase Parkin: Background info on Parkin & PINK1

29. OUTLINE I.Overview of Parkinson’s disease (PD) -characteristics -influence of environment -influence of genetics II. Studies of the familial PD genes PINK1 & Parkin reveal a mitochondrial QC pathway -Part I: contributions from work in flies -Part II: contributions from work in cell culture

31. Part I: Genetic studies of PINK1 and Parkin homologs in fruit flies

32. parkin & PINK1 mutants exhibit mitochondrial pathology Transverse sections from flight muscle wild-typepark-/- mitochondria myofibrils PINK1-/- Data from Park, J et al. (2006) Nature 441, 1157. parkin - wild-type Mitochondrial defects also seen in other affected tissues (e.g., germline, neurons)

33. PINK1 and Parkin act in a common pathway PINK1 Parkin mitochondrial integrity PINK1 -/- WT …but, PINK1 over-expression does not rescue the parkin phenotypes also, PINK1 parkin double mutants are indistinguishable from the single mutants PINK1 -/-  Parkin ? Park, J. et al. (2006) Nature 441, 1157; Clark, I., et al. (2006) Nature 441, 1162; Yang, Y et al. (2006) PNAS 103, 10793; Poole, A., et al. (2008) PNAS 105, 1638.

34. How does the PINK1/Parkin pathway influence mitochondrial integrity? Do PINK1 and Parkin regulate mitochondrial morphology? A Clue: Mutations in PINK1 & parkin result in fewer, but larger mitochondria: wildtypeParkin-/- Flight muscle: Male germline:

35. Parkin & PINK1 inhibit mitochondrial fusion… Evidence? =>Promoting fragmentation (e.g., Drp1 overexpression, mitofusin KO, etc.) suppressed the PINK1 & parkin phenotypes =>Promoting fusion (e.g., Drp1 KO, Mitofusin overexpression, etc.) worsened the PINK1 & parkin phenotypes …and they do so by targeting Mitofusin for ubiquitin-mediated degradation WT PINK1 - park - Mfn anti-Mfn WB WT PINK1 - park - Ub-Mfn Anti-Mfn IP anti-Ub WB Poole, A., et al (2008) PNAS 105, 1638 Poole, A., et al (2010) PLoS ONE 5, e10054

36. A hypothesis: PINK1 and Parkin promote the fragmentation of damaged mitochondria, such that they can be degraded through autophagy Poole, A., et al (2008) PNAS 105, 1638 Whats autophagy????

37. Mitochondrial quality control O2–O2– I. damage prevention (e.g., antioxidant enzymes) II. local repair (e.g., proteases, UPS, DNA repair enzymes, etc.) Three general categories:

38. III. Complete degradation of damaged mitochondria Mitochondrial quality control I. damage prevention (e.g., antioxidant enzymes) II. local repair (e.g., proteases, UPS, DNA repair enzymes, etc.) Three general categories:

39. Mitochondrial degradation occurs through autophagy -individual steps mediated by conserved autophagy-promoting factors (Atg factors) -first recognized >50 years ago -evidence for selective targeting of damaged mitochondria only over the past 10-15 years Image from: http://www.smallerquestions.org/blog/2012/3/5/brucella- is-running-the-show-here.html

40. Evidence for selective autophagic degradation of damaged mitochondria (mitophagy) TMRM: a membrane potential-dependent dye LC3-GFP: an autophagosome marker Photodamaged portion of a hepatocyte Kim, et al (2007) Arch Biochem Biophys 462:245.

41. Mitochondrial turnover occurs through autophagy -individual steps mediated by conserved autophagy-promoting factors (Atg factors) -first recognized >50 years ago -evidence for selective targeting of damaged mitochondria only over the past 10-15 years

42. Mitochondrial turnover occurs through autophagy -individual steps mediated by conserved autophagy-promoting factors (Atg factors) -first recognized >50 years ago -evidence for selective targeting of damaged mitochondria only over the past 10-15 years

43. Mitochondrial turnover occurs through autophagy -individual steps mediated by conserved autophagy-promoting factors (Atg factors) -first recognized >50 years ago -evidence for selective targeting of damaged mitochondria only over the past 10-15 years

44. Part II: Cell biological studies of PINK1 and Parkin in vertebrate cell culture

45. Data from: Narendra D, et al. (2008) J Cell Biol 183:795. Parkin Tom20 Merge Parkin is recruited to depolarized mitochondria… DMSO CCCP …and promotes their turnover in an atg5-dependent manner CCCP 24h WTatg5-/-

46. ->provides a cell biological explanation for genetic data linking PINK1 & Parkin in a common pathway Data from: Narendra et al. (2010) PLoS Biol 26: e1000298. PINK1 accumulates upon mt depolarization PINK1 is selectively stabilized on depolarized MTR CCCP treated cells PINK1 PINK1 required for Parkin recruitment to depolarized mt & their subsequent turnover DMSO CCCP Parkin-YFP Tom20 merge PINK1 siRNAcontrol PINK1 siRNA Data from: Vives-Bauza C, et al (2010) PNAS 107:378

47. A model of how PINK1 and parkin target mitochondria for autophagy Parkin: a cytosolic ubiquitin- protein ligase PINK1: a mitochondrially localized kinase

48. A model of how PINK1 and parkin target mitochondria for autophagy 1. PINK1 accumulates on depolarized mitochondria 2. PINK1 recruits Parkin to mitochondria 3. Parkin degrades mitofusin (& other proteins)

49. 1. PINK1 accumulates on depolarized mitochondria 2. PINK1 recruits Parkin to mitochondria 3. Parkin degrades mitofusin (& other proteins) A model of how PINK1 and parkin target mitochondria for autophagy 4. Depolarized mitochondria are degraded in the lysosome

50. Summary -Environment and genetics contribute to PD -Mitochondrial dysfunction is strongly implicated in PD (mitochondrial toxins, decrease complex I activity in sporadic PD, genetic forms associated with defects in mitochondrial QC) -Studies of PINK1 and Parkin indicate that they promote the fragmentation and degradation of damaged mitochondria

51. There is still a lot left for you guys to do! -Why are only dopaminergic neurons affected by mutations in PINK1 and Parkin? -Does alpha-synuclein impinge on mitochondrial health? How much do the various cellular QC pathways contribute to mito QC? -Does accumulated mitochondrial damage contribute to aging? -Exactly how do PINK1 and Parkin promote mitophagy? -Why is the accumulation of damaged mitochondria bad for the cell? -etc.

52. Acknowledgements Evvie Vincow Nick Shulman Ruth Thomas Jonathon Burman Michael MacCoss 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 NIH (NINDS, NIGMS)