α -Synuclein Interaction with Phospholipids: Possible Implications for Parkinson’s Disease

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 A2α-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)

medweb.bham.ac.uk(8/28/02) Normal vs. Diseased Brains

Lewy Bodies www.saigata-nh.go.jp/ (8/28/02)

www.sfn.org/images/brainbriefings/ august2001_big.jpg (8/28/02) Lewy Bodies Stain Positive for α-Synuclein

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

A30P A53T Amphipathicdomain NAC domain Acidic Tail pKTKEGVaxaA repeats Regions of α-Synuclein

Ile,Leu, Phe Lys, Arg Glu, Asp Class A2α-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” Segrest et al. J. Lipid Res. 1992

Phospholipid Protein α-Synuclein Preferentially Binds Brain Phospholipids α-Synuclein Ovalbumin Brain phospholipid extract POPC vesicles Davidson et al.J. Biol. Chem. 273 (16) 1998

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 Free Protein PC PC/PA PC/PS Davidson et al.J. Biol. Chem. 273 (16) 1998

Helical Wheel Analysis of α-Synuclein Hydrophobic Polar Charged * = 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

Full Length Exon Deletion Abolishes Binding to PS; PA Binding is Unaffected Perrin et al. J.Biol.Chem. 275(44) 2000

Single Exons Sufficient for Binding to PA Vesicles Full Length Perrin et al. J.Biol.Chem. 275(44) 2000

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 * = Biotinylation Perrin et al. J.Biol.Chem. 275(44) 2000

α-Synuclein Bound to PS is Less α-helical POPC/POPA POPC/POPS Free Protein A30P WT A53T Perrin et.al. J.Biol.Chem. 275(44) 2000

Conclusions • Entire hydrophobic region necessary for PS binding • Electrostatics not primary mediator of lipid binding • Lysine residues play a role in lipid binding

PC/PS PC/PA PE/PS PE/PA ----- Free protein ----- Free protein A30P Mutant Shows Defective Lipid Binding -θ 222 nm PS Binding PA Binding Jo et al. J. Mol. Biol. 315, 2002

A30P POPS Binding Abolished at High Ionic Strength WT A30P -θ 222 nm Jo et al. J. Mol. Biol. 315, 2002

pellet supernatant WT A30P Membrane Bound α-Synuclein is Dimeric α-syn ~ 14 kD Jo et al. J. Mol. Biol. 315, 2002

Conclusions • A30P binds acidic vesicles less than wild type • Binding improves with PE vs. PC • Membrane bound α-synuclein is a dimer

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 α β PLD1 PLD2 Jenco et al. Biochem. 37 (14), 1998

PIP2 Cannot Overcome α-Synuclein Inhibition of PLD2 PIP2 [μM] Jenco et al. Biochem. 37 (14), 1998

Lipid Concentration Affects Inhibition of PLD2 by α-Synuclein No Synuclein 100 nM α-synuclein Jenco et al. Biochem. 37 (14), 1998

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

Model of Dopamine Homeostasis Dopamine Neuron Dopamine Transporter VMAT2

Decreased DAT and VMAT in LV-A53T Cells Lotharius et al. J. Biol. Chem. In Press

A53T Cells Show Decreased Dopamine Uptake and Release GFP A53T Lotharius et al. J. Biol. Chem. In Press

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

Model for PD Pathogenesis Dopamine Dopamine Transporter VMAT2

Protofibrillar a-synuclein Model for PD Pathogenesis Dopamine Dopamine Transporter VMAT2

Thank You • Hilary Frase • Kaisa Ejendal • Ney Diop • Erin Seeley • Christa Feasley • Bindu Varghese • Erina Vlashi

α -Synuclein Interaction with Phospholipids: Possible Implications for Parkinson’s Disease