Establishing a Causative Link between Lipid Peroxidation and MBP Misfolding in Multiple Sclerosis Natalie K. Cygan Scripps-Oxford Laboratory June 13, 2007

Multiple Sclerosis (MS) ◘Inflammatory, demyelinating disease of the CNS ◘Autoimmune disease ◘Affects over 1 million people worldwide ◘Symptoms include: ◘changes in sensation ◘visual problems ◘muscle weakness ◘difficulties with coordination and speech ◘severe fatigue ◘cognitive impairment ◘problems with balance Myelin Sheath

Myelin Membrane ◘Lipids constitute 70% of myelin dry weight ◘Lipids contribute significantly to myelin structure and adhesion ◘25% of total lipid content is cholesterol ◘Cholesterol influences: ◘Membrane thickness ◘Membrane fluidity ◘Ion leakage

Myelin Basic Protein (MBP) ◘18.5 kDa protein ◘Intrinsically unstructured protein ◘Extensive post-translational modifications ◘Primary role: stabilization of the myelin sheath ◘Contains an immunodominant epitope ◘Exposure of epitope leads to breakdown of myelin

Myelinated Neuron MBP epitope exposure Antibody Response Demyelinated Neuron

Cholesterol Oxidation Cholesterol Atheronal B Atheronal A ROS Aldolization Schiff Base

MBP MBP in the presence of lipid derived aldehydes Epitope

MBP immunodominant epitope exposure in the presence of lipid derived aldehydes DIRECTINDIRECT Schiff base formationMembrane effects 1. Enzymatic assay 2. Antibody assays 1.Vesicle aggregation 2. Calcein leakage

Cathepsin D digestion Cathepsin D PE 27% Chol 44% PC 11% PS 13% Sphingo 3% PI 2% Buffer Extrusion MBP Epitope F42-F43F86-F87 169aa [18.5kDa] [14kDa] [4.5kDa] [5kDa] [8.6kDa]

18.5kDa Time (h) MBP digestion in liposomes by Cathepsin D MBP digestion in liposomes containing 4.4% atheronal by Cathepsin D 14kDa 4.5kDa 5kDa 46kDa ◘ Atheronals appear to inhibit Cathepsin D digestion ◘ Peptide aldehydes have been shown to inhibit Cathepsin D

Membrane Effects ◘Schiff base formation between atheronals and phosphatidylethanolamine (PE) alters the biophysical membrane properties (E. Wachtel, D. Bach, R. F. Epand et al., Biochemistry 45 (4), 1345 (2006). ◘Ongoing experiments: ◘Turbidity assay ◘Calcein encapsulated liposomes

TurbidityCalcein encapsulation Add MBP AggregationFusion PE 27% Chol 39.6% PC 11% PS 13% Sphingo 3% PI 2% Ath 4.4% PE 27% Chol 39.6% PC 11% PS 13% Sphingo 3% PI 2% Ath 4.4% + Calcein

0% atheronal 4.4% atheronal 20% atheronal Time (sec) A 400 /A 400int Turbidity ◘ Atheronals present in liposomes decreases turbidity ◘ Less interaction between membranes when atheronals are present

0% atheronal 4.4% atheronal 20% atheronal Calcein Encapsulated Liposomes Time (sec) % Calcein Released ◘ Atheronals present in liposomes decrease % Calcein released ◘ Less fusion of membranes when atheronals are present

Conclusions ◘Cathepsin D digestion- atheronals appear to inhibit Cathepsin D digestion ◘Turbidity- less interaction between membranes when atheronals are present ◘Calcein leakage- less fusion between membranes when atheronals are present

Future Work ◘Detect conformational change in MBP ◘Enzymatic assay ◘Stromelysin-1 (MMP-3) ◘Epitope antibody ◘ELISA ◘BIACORE ◘Determine atheronal concentration in NAWM from healthy patients and MS patients

Acknowledgements Johanna Scheinost Dan Witter Sarah Tully Kanlaya Prapainop Jason Chang Michael Cole Alicia Izharuddin Kavitha Baruah Adria Lombardo John Offer Professor Paul Wentworth Scripps-Oxford Lab Sally Latham