Parkinson’s Disease: From genetics to pathogenesis

1. Parkinson’s Disease: From genetics to pathogenesis alpha-Synuclein in Parkinson’s Disease: From genetics to pathogenesis Leonidas Stefanis Laboratory of Neurodegenerative Diseases Biomedical Research Foundation Academy of Athens Second Department of Neurology University of Athens Medical School

2. Parkinson’s disease • Cardinal clinical signs: bradykinesia, tremor, rigidity, postural instability • Progressive neurodegenerative disorder • Pathological hallmark: degeneration of dopaminergic nigral neurons in SNpc and formation of cytoplasmic ubiquitinated inclusions, termed Lewy bodies Modified from Gowers, 1893

3. Pathological hallmarks of Parkinson’s Disease Dauer and Przedborski, 2003

4. Gene Mode of inheritance Locus Chromosomal location a-synuclein Autosomal dominant PARK1 4q21 [PDB] -q23 Parkin Autosomal recessive PARK2 6q25.2-27 Unknown Autosomal dominant PARK3 2p13 a-synuclein Autosomal dominant PARK4 4q UchL1 Autosomal dominant PARK5 4p14 PINK-1 Autosomal recessive PARK6 1p35 [PDB] -p36 DJ-1 Autosomal recessive PARK7 1p36 LRKK2 Autosomal dominant PARK8 12p11q13.1 ATP13A2 Autosomal recessive PARK9 1p36 Unknown Late-onset susceptibility gene PARK10 1p32 [PDB] NR4A2 Susceptibility gene NA 2q22-23 Synphilin-1 Susceptibility gene NA 5q23.1-23.3 Tau Susceptibility gene NA 17q21 Genes and genetic loci associated with Parkinson’s disease

5. Gene Mode of inheritance Locus Chromosomal location a-synuclein Autosomal dominant PARK1 4q21 [PDB] -q23 Parkin Autosomal recessive PARK2 6q25.2-27 Unknown Autosomal dominant PARK3 2p13 a-synuclein Autosomal dominant PARK4 4q UchL1 Autosomal dominant PARK5 4p14 PINK-1 Autosomal recessive PARK6 1p35 [PDB] -p36 DJ-1 Autosomal recessive PARK7 1p36 LRKK2 Autosomal dominant PARK8 12p11q13.1 ATP13A2 Autosomal recessive PARK9 1p36 Unknown Late-onset susceptibility gene PARK10 1p32 [PDB] NR4A2 Susceptibility gene NA 2q22-23 Synphilin-1 Susceptibility gene NA 5q23.1-23.3 Tau Susceptibility gene NA 17q21 Genes and genetic loci associated with Parkinson’s disease

6. α-synuclein is a major factor leading to Parkinson’s Disease • Point mutations in SNCA, the gene encoding for α-synuclein(A30P, A53T, E46K)lead to PD orPDD (PARK1 locus) • Expansion (duplication or triplication) of the SNCA locus leads to PD (PARK4 locus)

7. α-synuclein is a major factor leading to Parkinson’s Disease • Point mutations in SNCA, the gene encoding for α-synuclein(A30P, A53T, E46K)lead to PD orPDD (PARK1 locus) • Expansion (duplication or triplication) of the SNCA locus leads to PD (PARK4 locus) • Many studies, including a large meta-analysis, report that a polymorphism within the 5’promoter region of alpha-synuclein leads to an increased risk of developing sporadic PD; others report disease association with other parts of the gene

8. α-synuclein is a major factor leading to Parkinson’s Disease • Point mutations in SNCA, the gene encoding for α-synuclein(A30P, A53T, E46K)lead to PD orPDD (PARK1 locus) • Expansion (duplication or triplication) of the SNCA locus leads to PD (PARK4 locus) • Many studies, including a large meta-analysis, report that a polymorphism within the 5’promoter region of alpha-synuclein leads to an increased risk of developing sporadic PD; others report disease association with other parts of the gene …therefore ASYN may also be genetically involved in sporadic PD

9. α-synuclein is a major factor leading to Parkinson’s Disease • Point mutations in SNCA, the gene encoding for α-synuclein(A30P, A53T, E46K)lead to PD orPDD (PARK1 locus) • Expansion (duplication or triplication) of the SNCA locus leads to PD (PARK4 locus) • Genetic involvement in sporadic PD

10. Braak et al., 2003

11. α-synuclein is a major factor leading to Parkinson’s Disease • Point mutations in SNCA, the gene encoding for α-synuclein(A30P, A53T, E46K)lead to PD orPDD (PARK1 locus) • Expansion (duplication or triplication) of the SNCA locus leads to PD (PARK4 locus) • Genetic involvement in sporadic PD • It is a basic and early component of LBs

12. Schematic representation of alpha-synuclein structure Apolipoprotein motif Acidic tail Repeat 6 Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5 140 1 N C 61 95 A30P A53T E46K Mutations Genetically Linked to Familial Parkinson’s Disease NAC region necessary for aggregation

13. α-synuclein • Member of family of synucleins (α-, β-, γ-) • Predominantly neuronal protein • Predominant localization to presynaptic terminals in the mature CNS • Closely apposed to synaptic vesicles • Developmentally regulated • Without structure in solution, but assumes an α-helical conformation upon binding to phospholipids or synthetic vesicles • In vitro, it has the propensity to form oligomers, fibrils, aggregates

14. α-synuclein oligomeric species

15. α-synuclein is a major factor leading to Parkinson’s Disease • Point mutations in SNCA, the gene encoding for α-synuclein(A30P, A53T, E46K)lead to PD orPDD (PARK1 locus) • Expansion (duplication or triplication) of the SNCA locus leads to PD (PARK4 locus) • Genetic involvement in sporadic PD • It is a basic and early component of LBs • ASYN has the propensity to misfold, form oligomers and aggregates that have a beta-sheet, amyloid conformation

16. Model of PD pathogenesis Eriksen et al., 2004

17. α-synuclein is a major factor leading to Parkinson’s Disease • Point mutations in SNCA, the gene encoding for α-synuclein(A30P, A53T, E46K)lead to PD orPDD (PARK1 locus) • Expansion (duplication or triplication) of the SNCA locus leads to PD (PARK4 locus) • Genetic involvement in sporadic PD • It is a basic and early component of LBs • ASYN has the propensity to misfold, form oligomers and aggregates that have a beta-sheet, amyloid conformation Process of AGGREGATION is thought to be the major determinant of toxicity

18. Questions • Is the process of ASYN aggregation a primary event in sporadic PD? • Is the toxicity conferred by aberrant ASYN due to aggregation? • What are the factors influencing ASYN aggregation? • Which are the “toxic species” of ASYN aggregates? • What are the cellular consequences of ASYN aggregation? • Could the link of ASYN with PD be independent of aggregation?

19. Questions • Is the process of ASYN aggregation a primary event in sporadic PD? • Is the toxicity conferred by aberrant ASYN due to aggregation? • What are the factors influencing ASYN aggregation? • Which are the “toxic species” of ASYN aggregates? • What are the cellular consequences of ASYN aggregation? • Could the link of ASYN with PD be independent of aggregation?

20. What are the factors influencing ASYN aggregation? • Mutations • Expression levels (regulated by transcription and degradation) • Oxidative/Nitrative Stress • Molecular crowding • Presence of other proteins (tau) • Binding to lipids, membranes and fatty acids • Phosphorylation • Truncation • Dopamine and its metabolites

21. What are the factors influencing ASYN aggregation? • Mutations • Expression levels (regulated by transcription and degradation) • Oxidative/Nitrative Stress • Molecular crowding • Presence of other proteins (tau) • Binding to lipids, membranes and fatty acids • Phosphorylation • Truncation • Dopamine and its metabolites

22. What is the route of degradation of ASYN? • This is controversial, and still the route of degradation of ASYN in an in vivo context is not known • Degradation of monomeric WT ASYN has been suggested to occur in cell systems via the proteasome system, macroautophagy, chaperone-mediated autophagy (CMA) and calpains • Impairment of this degradation may lead to more ASYN available for degradation • There may also be specific routes for degradation of oligomeric/fibrillar ASYN

23. Intracellular Protein degradation Ubiquitin-dependent Proteasomes Ubiquitin-independent Macroautophagy Lysosomes Microautophagy Chaperone-mediated autophagy

24. α-synuclein expression levels are not increased with proteasomal inhibition Rideout et al., 2001

25. lysosome endosome CRINOPHAGY lysosome autophagic vacuole Lysosomal Pathways for Protein Degradation ENDOCYTOSIS MICROAUTOPHAGY CHAPERONE- MEDIATED AUTOPHAGY Golgi endoplasmic reticulum MACROAUTOPHAGY

26. Chaperone-Mediated Autophagy (CMA) lysosome Lamp2a Ly-Hsc-73 KFERQ ASYN Hsc-73 Cuervo et al., 2004

27. 48h 72h kDa scr L1 scr L1 scr L1 scr L1 Lamp2a ASYN GFP ERK 48h 72h L1 scr L1 95 43 26 72 17 ASYN β-actin CMA dysfunction via lentivirus-mediated Lamp2a downregulation leads to ASYN accumulation in cortical neurons A. B. C. scr Vogiatzi T, Xilouri M, Vekrellis K, Stefanis L J Biol Chem (2008)

28. scr scr L1 L1 96 Lamp 2a 72 17 ASYN 43 ERK GAPDH 34 CMA dysfunction via lentivirus-mediated Lamp2a downregulation leads to ASYN accumulation in post-natal VM neurons M I D B R A I N V E N T R A L Vogiatzi T, Xilouri M, Vekrellis K, Stefanis L J Biol Chem (2008)

29. Chaperone-Mediated Autophagy (CMA) lysosome Lamp2a Ly-Hsc-73 DDQ ASYN KFERQ WT ASYN Hsc-73 Cuervo et al., 2004

30. WTΔDQ : lacks the recognition motif for CMA-dependent degradation In rat cortical neurons WTΔDQexhibits slower turnover compared to WT ASYN WT WTΔDQ 14 h 14 h 0 h 8 h 0 h 8 h ASYN (35S labeled) n=2 17 Maria Xilouri

31. A. kDa 95 17 E16 E18 P0 P3 P4 P7 P14 P21 Lamp2a 72 ASYN 43 ERK A53T midbrain WT midbrain rat cortex rat cortex 17 43 mice kDa ASYN IP ERK IP 98 Lamp2a ASYN ERK In vivo relevance of ASYN-Lamp2a interaction C. Vogiatzi T, Xilouri M, Vekrellis K, Stefanis L J Biol Chem (2008)

32. Routes of degradation of various ASYN species may be different CMA

33. Intracellular Protein degradation Macroautophagy Lysosomes Microautophagy Chaperone-mediated autophagy

34. 3MA Process of autophagosome formation

35. 6h 12h 24h 3MA 3MA 3MA kDa epx Ctrl epx epx 17 130 ubiq 72 ASYN 43 ERK Pharmacological inhibition of macroautophagy, but not the proteasome, also leads to ASYN accumulation Vogiatzi T, Xilouri M, Vekrellis K, Stefanis L J Biol Chem (2008)

36. Routes of degradation of various ASYN species may be different CMA Macroautophagy

37. Questions • Is the process of ASYN aggregation a primary event in sporadic PD? • Is the toxicity conferred by aberrant ASYN due to aggregation? • What are the factors influencing ASYN aggregation? • Which are the “toxic species” of ASYN aggregates? • What are the cellular consequences of ASYN aggregation? • Could the link of ASYN with PD be independent of aggregation?

38. Theories for toxic effects of a-synuclein • Formation of pores on cellular and organelle membranes • Formation of pores on vesicles and resultant increase of cytosolic dopamine • Impairment of axonal transport • ER stress • Disruption of ER-Golgi trafficking • Association with apoptotic machinery • Impairment of protein degradation • Sequestration within aggregates/inclusions of molecules important for neuronal function/survival • Nuclear binding to histones • Interference with dopamine biosynthesis Process of AGGREGATION is thought to be the major determinant

39. Theories for toxic effects of a-synuclein • Formation of pores on cellular and organelle membranes • Formation of pores on vesicles and resultant increase of cytosolic dopamine • Impairment of axonal transport • ER stress • Disruption of ER-Golgi trafficking • Association with apoptotic machinery • Impairment of protein degradation • Sequestration within aggregates/inclusions of molecules important for neuronal function/survival • Nuclear binding to histones • Interference with dopamine biosynthesis Process of AGGREGATION is thought to be the major determinant

40. Altered morphology and granular-vacuolar cellular degeneration in PC12 cell lines expressing A53T a-synuclein Stefanis et al., 2001

41. PC12 cells expresssing MT A53T a-synuclein show features of macro-autophagy a: empty vector b: WT a-syn c-f: MT a-syn Stefanis et al., 2001

42. A53T a-synuclein expression Proteasomal Dysfunction Lysosomal Dysfunction Ubiquitinated aggregates/ Autophagy Cell Death Loss of DCGs Loss of evoked Dopamine release Stefanis et al. , 2001

43. A53T a-synuclein expression Proteasomal Dysfunction Lysosomal Dysfunction Ubiquitinated aggregates/ Autophagy Cell Death Loss of DCGs Loss of evoked Dopamine release Stefanis et al. , 2001

44. Very select soluble oligomeric species of ASYN are targeted to the proteasome and impede its function Emmanouilidou et al., Neurob Aging, 2008

45. Model for effect of aberrant a-synuclein on proteasomal function B A C Aberrant a-synuclein D E (less than 1% of total) Emmanouilidou et al., Neurob Aging, 2008

46. Routes of degradation of various ASYN species may be different CMA Macroautophagy Proteasome

47. Two interrelated themes ASYN Impact on protein degradation ASYN degradation

48. A53T a-synuclein expression Proteasomal Dysfunction Lysosomal Dysfunction Ubiquitinated aggregates/ Autophagy Cell Death Loss of DCGs Loss of evoked Dopamine release Stefanis et al. , 2001

49. Mutant α-synucleinsbind more tightly to the CMA receptor LAMP2aand interfere with the degradation of other CMA substrates Cuervo et al., 2004

50. lysosome Hypothesis: The aberrant function of mutant α-synucleins (or excessively high levels or post-translational modifications of WT α-synuclein) is, at least in part, due to CMA dysfunction Aberrant α-synuclein Lamp-2a CMA substrate