α-Synuclein Interaction with Phospholipids:
Possible Implications for Parkinson’s Disease
Literature Seminar
by
Jessica L. Anderson
Outline• Parkinson’s Disease (PD)• α-Synuclein involvement in PD• Class A2 α-helical model
• Defective interaction of mutant α-synuclein• Inhibition of phospholipase D2 (PLD2)• α-Synuclein role in dopamine homeostasis• Model for PD pathogenesis
Parkinson’s Disease• Affects 1-1.5 million Americans• Symptoms
– Bradykinesia (Slowness of movement)– Rigidity– Tremor– Problems Walking– Poor Balance
• Caused by loss of dopamine in the nigrostriatal pathway
Nigrostriatal Pathway
http://www.aafp.org/afp/990415ap/2155.html (8/28/02)
Normal vs. Diseased Brains
medweb.bham.ac.uk (8/28/02)
Outline• Parkinson’s Disease (PD)• α-Synuclein involvement in PD• Class A2 α-helical model
• Defective interaction of mutant α-synuclein
• Inhibition of phospholipase D2 (PLD2)• α-Synuclein role in dopamine
homeostasis• Model for PD pathogenesis
Lewy Bodies
www.saigata-nh.go.jp/ (8/28/02)
Lewy Bodies Stain Positive for α-Synuclein
www.sfn.org/images/brainbriefings/ august2001_big.jpg (8/28/02)
α-Synuclein
• 14-kD protein with “random coil” secondary structure
• Localized to presynaptic vesicles• Function of protein remains unknown• Up to 1% of total protein from soluble brain
fractions• Two other family members, b- and g-
synuclein
α-Synuclein Function
• Under oxidative stress–Non-dopaminergic cell Neuroprotective–Dopaminergic cell Neurotoxic
• Potent in vivo inhibitor of phospholipase D2
• Regulates the size of the synaptic vesicle pool
α-Synuclein Fibrillization
Volles et al. Biochemistry 2001
Mutant α-Synuclein• Two disease causing mutations exist
–A30P–A53T
• Lead to early onset PD
• Form fibrils faster (A53T) or at about the same rate (A30P) as wild type
• Form fibrils at lower protein concentration than wild type
Regions of α-Synuclein
Amphipathic domain NAC domain Acidic Tail
A53TA30P
pKTKEGVaxaA repeats
Outline
• Parkinson’s Disease (PD)• α-Synuclein involvement in PD• Class A2 α-helical model• Defective interaction of mutant α-
synuclein• Inhibition of phospholipase D2 (PLD2)• α-Synuclein role in dopamine
homeostasis• Model for PD pathogenesis
Class A2 α-Helical Model• 11 or 22-mer tandem repeats• Clustering of Lys at polar/nonpolar face• Clustering of Glu on polar face• High Lys:Arg ratio• Lysine “snorkeling”
Ile,Leu, PheLys, ArgGlu, Asp
Segrest et al. J. Lipid Res. 1992
α-Synuclein Ovalbumin
Brain phospholipid extract
POPC vesicles
Davidson et al. J. Biol. Chem. 273 (16) 1998
α-Synuclein Preferentially Binds Brain Phospholipids
PhospholipidProtein
Lipid Head Group Structure
Phosphatidylcholine (PC) Phosphatidylethanolamine (PE)
Phosphatidic Acid (PA)
Phosphatidylserine (PS)Phosphatidylinositol (PI)
www.lipid.co.uk (9/04/02)
α-Synuclein Selectively Binds Acidic Phospholipids
Davidson et al. J. Biol. Chem. 273 (16) 1998
Lipid Binding Causes Conformation Change
Davidson et al. J. Biol. Chem. 273 (16) 1998
Free ProteinPC/PA
PCPC/PS
Helical Wheel Analysis of α-Synuclein
HydrophobicPolarCharged
* = Helix Breaker
Davidson et al. J. Biol. Chem. 273 (16) 1998
Conclusions
• α-Synuclein binds phospholipid vesicles with a net negative charge
• Lipid binding increases α-helical character
Exon Deletion Abolishes Binding to PS; PA Binding is Unaffected
Perrin et al. J.Biol.Chem. 275(44) 2000
Full Length
Single Exons Sufficient for Binding to PA Vesicles
Perrin et al. J.Biol.Chem. 275(44) 2000
Full Length
Charged Residues on Hydrophobic Face Alter Phospholipid Binding
Perrin et al. J.Biol.Chem. 275(44) 2000
Biotinylation of Lysine and A30P Mutation Decrease Lipid Binding
Perrin et al. J.Biol.Chem. 275(44) 2000
* = Biotinylation
α-Synuclein Bound to PS is Less α-helical
Perrin et.al. J.Biol.Chem. 275(44) 2000
POPC/POPA POPC/POPSFree Protein
WT
A30P
A53T
Conclusions• Entire hydrophobic region
necessary for PS binding
• Electrostatics not primary mediator of lipid binding
• Lysine residues play a role in lipid binding
Outline
• Parkinson’s Disease (PD)• α-Synuclein involvement in PD• Class A2 α-helical model• Defective interaction of mutant α-
synuclein• Inhibition of phospholipase D2 (PLD2)• α-Synuclein role in dopamine
homeostasis• Model for PD pathogenesis
A30P Mutant Shows Defective Lipid Binding
PS Binding PA Binding
Jo et al. J. Mol. Biol. 315, 2002
PC/PS
PE/PS----- Free protein
-θ 222 nm
PC/PA
PE/PA----- Free protein
A30P POPS Binding Abolished at High Ionic Strength
Jo et al. J. Mol. Biol. 315, 2002
WTA30P
-θ 222 nm
Membrane Bound α-Synuclein is Dimeric
Jo et al. J. Mol. Biol. 315, 2002
pellet supernatant
WT A30P
α-syn ~ 14 kD
Conclusions
• A30P binds acidic vesicles less than wild type
• Binding improves with PE vs. PC
• Membrane bound α-synuclein is a dimer
Outline• Parkinson’s Disease (PD)• α-Synuclein involvement in PD• Class A2 α-helical model• Defective interaction of mutant α-
synuclein• Inhibition of phospholipase D2 (PLD2)• α-Synuclein role in dopamine
homeostasis• Model for PD pathogenesis
Phospholipase D2
• Hydrolyzes PC to PA and choline
• Localized to plasma membrane and possibly early endosomes
• Involved in vesicle recycling
• Constituitively active in vitro but constantly under (-) regulation in vivo
Regulation of Vesicle Budding
Liscovitch et al. Biochem J. 345, 2000
α-Synuclein Inhibits Phospholipase D2
Jenco et al. Biochem. 37 (14), 1998
PLD2
PLD1
β
α
PIP2 Cannot Overcome α-Synuclein Inhibition of PLD2
Jenco et al. Biochem. 37 (14), 1998
PIP2 [μM]
Lipid Concentration Affects Inhibition of PLD2 by α-Synuclein
Jenco et al. Biochem. 37 (14), 1998
No Synuclein100 nM α-synuclein
Conclusions
• α- and β-Synucleins are inhibitors of PLD2
• Inhibition is selective for PLD2
• Inhibition is independent of PIP2
• Lipid concentration can reduce
α-synuclein inhibition
Outline
• Parkinson’s Disease (PD)• α-Synuclein involvement in PD• Class A2 α-helical model• Defective interaction of mutant α-
synuclein• Inhibition of phospholipase D2 (PLD2)• α-Synuclein role in dopamine
homeostasis• Model for PD pathogenesis
Model of Dopamine HomeostasisDopamine
Dopamine Transporter
VMAT2
Neuron
Decreased DAT and VMAT in LV-A53T Cells
Lotharius et al. J. Biol. Chem. In Press
A53T Cells Show Decreased Dopamine Uptake and Release
Lotharius et al. J. Biol. Chem. In Press
GFP
A53T
Dopamine Redistributed to Cytosol in A53T Cells
Lotharius et al. J. Biol. Chem. In Press
Conclusions
• A53T α-synuclein expression in MESC2.10 cells
–Reduces the number of synaptic vesicles
–Redistributes dopamine from synaptic vesicles to the cytosol
Outline
• Parkinson’s Disease (PD)• α-Synuclein involvement in PD• Class A2 α-helical model
• Defective interaction of mutant α-synuclein
• Inhibition of phospholipase D2 (PLD2)• α-Synuclein role in dopamine
homeostasis• Model for PD pathogenesis
Model for PD PathogenesisDopamine
Dopamine Transporter
VMAT2
Model for PD PathogenesisDopamine
Dopamine Transporter
VMAT2
Protofibrillar a-synuclein
Thank You
Hilary Frase
Kaisa Ejendal
Ney Diop
Erin Seeley
Christa Feasley
Bindu Varghese
Erina Vlashi