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