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

HO

OH

O

Cholesterol

Atheronal B

Atheronal A

ROS

Aldolization

HO

OH

H+

N

R

Schiff Base

NH2

R

OOH

4-hydroxy nonenal, 4-HNE (7)

O O

malondialdehyde, MDA (6)

OO

glyoxal, GLO (4)

O

acrolein, ACR (8)

MBPMBP in the presence of lipid derived aldehydes

Epitope

MBP immunodominant epitope exposurein the presence of lipid derived aldehydes

DIRECT INDIRECT

Schiff base formation Membrane effects

1. Enzymatic assay2. 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

MBPEpitope

F42-F43 F86-F87169aa

1-169

1-42

43-169

87-169

43-86

[18.5kDa]

[14kDa]

[4.5kDa]

[5kDa]

[8.6kDa]

18.5kDa 18.5kDa

0 1 2 3 4 5 6 7 24 0 1 2 3 4 5 6 7 24 Time (h)

MBP digestion in liposomes by Cathepsin D

MBP digestion in liposomes containing 4.4% atheronal

by Cathepsin D

14kDa14kDa

4.5kDa5kDa

46kDa46kDa

◘ 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

Turbidity Calcein encapsulation

Add MBP

Aggregation Fusion

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

Turbidity

0.9

0.95

1

1.05

1.1

1.15

1.2

0 50 100 150 200 250 300 350 400 450

Time (sec)

A40

0/A

400i

nt

Time (sec)

A4

00/A

400

int

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

0

1

2

3

4

5

6

7

8

9

10

0 50 100 150 200 250 300 350 400

Time (sec)

% R

ele

as

e

Calcein Encapsulated Liposomes

Time (sec)

% C

alce

in R

elea

sed

◘ 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 ScheinostDan WitterSarah TullyKanlaya PrapainopJason Chang

Michael ColeAlicia IzharuddinKavitha BaruahAdria LombardoJohn Offer

Professor Paul WentworthScripps-Oxford Lab

Sally Latham