Elevated Levels of Cholesterol Metabolites in NPC Phenotype Cells Daniel Witter Scripps/ Oxford Lab Glycobiology Institute Oxford University

Niemann-Pick C Disease GSL storage disease Cholesterol accumulation Intracellular mistrafficking Neurodegeneration Upregulated microglia and astrocyte activity

Cholesterol oxidation

Reactive Oxygen Species >Superoxide O 2 - >Hydrogen Peroxide H 2 O 2 >Hydroxyl radical OH >Singlet Molecular Oxygen 1 O 2 >Ozone? O 3

Atheronals 5-6 secosterol oxidation products Steroid nucleus is cleaved Ketoaldehyde 1 Aldol 2 Produced by singlet molecular oxygen and possibly ozone

Where are atheronals? Found in serum Found in atherosclerotic plaques Found in brain tissue Need cholesterol and inflammation together

Biological significance of atheronals Macrophage recruitment Upregulation of SR-A Monocyte differentiation ß-amyloid misfolding

Cholesterol accumulation in NPC disease Wild type NPC1 null Filipin labeled B-lymphoblasts bar=5µm

Finding Atheronals in NPC Cells ROS generation--light activated hematoporphyrin IX Lipid extraction Derivatization with 2,4-DNPH HPLC

mAU minutes Aldol Isocratic gradient--85% acetonitrile; 12%water w/ 0.1%TFA; 3%methanol Untreated NPC cells

HPLC Wild type NPC mAU minutes aldol Isocratic gradient--85% acetonitrile; 12%water w/ 0.1%TFA; 3%methanol

Microscopy Fluorescent dansyl amide derivatized atheronal

ATHERONAL LOCALIZATION RAW macrophages treated with 50 µM DA aldol for 60 minutes, followed by 15 minute paraformaldehyde fix, 60 minute block, and 60 minute stain with FITC anti-GM130.

ATHERONAL LOCALIZATION RAW macrophages treated with 50 µM DA aldol for 60 minutes, followed by 15 minute paraformaldehyde fix, 60 minute block, and 60 minute stain with FITC anti-calnexin.

ATHERONAL LOCALIZATION RAW macrophages treated with 50 µM DA aldol for 60 minutes, followed by 15 minute paraformaldehyde fix, 60 minute block, and 10 minute stain with DiI.

ATHERONAL LOCALIZATION Live RAW macrophage treated with 50 µM DA aldol for 1 hr. LiveRAW macrophage treated with 50 µM DA aldol for 2 hr.

Conclusions Atheronal levels are higher in NPC cells ROS raise atheronal levels Atheronals appear to localize in the ER

Future Work IF microscopy Lipid raft isolation Glutathione depletion Other cell lines

Acknowledgements Paul Wentworth/ Raymond Dwek John Offer Jorge Nieva Emyr Lloyd-Evans GBI State of Florida