1. Developing an in vitro cellular model for Fabry Disease Part II Project Emma Brewer 31.01.2007

2. Topics covered in this presentation: Introduction to Fabry Disease – a glycolipid storage disease. Using an α-galactosidase inhibitor, DGJ to achieve a cellular model for the disease: - do we see storage of the glycolipid? - are there any other changes, eg glycoprotein inhibition? Chemical chaperoning in Fabry mutant cells. - do we see a reduction in glycolipid storage? Questions

3. FABRY DISEASE Deficiency of α-galactosidase A due to point mutations Responsible for biodegradation of CTH Result: insufficient breakdown of CTH CTH builds up in cells i.e. glycolipid storage disorder. CTH:Ceramide-Glc-Galβ-Galα α-Galactosidase cleaves here α-Galactosidase cleaves here

4. Glycolipid Storage Disorders

5. Symptoms of Fabry Disease Fatigue, inability to sweat, vomitting and diarrhoea Impaired arterial circulation + increased risk of early heart attack or stroke Cloudiness of the cornea Small, painless, raised red-purple blemishes on the skin Burning sensation in hands and feet Renal failure is usually the cause of death, due to proteinuria induced hypertension

6. More about Fabry Disease It is an X-linked disease, and so is far more common in men, although women carriers often show some symptoms Long term accumulation of the glycolipid means that severity of symptoms increases over time Patients typically live into their early forties Therapy Enzyme replacement therapy can be successful – involves an intravenous infusion to replace the deficient enzyme – up to twice weekly Substrate Reduction Therapy (SRT) Molecular Chaperone Therapy / Chaperone-Mediated Therapy (CMT)

7. X-ray Crystal Structure of α-galactosidase A Garman and Garboczi JMB 337, 2, 319-335 (2004)

8. The DGJ Experiment – using an inhibitor of α-galactosidase A to produce the Fabry phenotype in healthy cells Aim of this experiment – to create an in vitro, cellular model for Fabry disease by addition of an α-galactosidase A inhibitor to healthy cell lines I assessed samples of these cells at different time points of treatment between 0 days and 70 days, and at different concentrations of the inhibitor

9. HPLC Profiles: oligosaccharides from GSLs extracted from cell samples

10. Change in Gb3 levels (relative to GM3) over time. Blue=untreated, Pink=100uM DGJ

11. Plotting Gb3 levels against log conc of DGJ at different time points

12. Reason for very little storage – slow turnover of the glycolipid? Determining CTH half life: Treat cells with an inhibitor of glycolipid synthesis, NB-DNJ. Take and analyse cell samples each day Monitor the levels of CTH against control cells. Amount of CTH per unit mass of protein should be constant in the control, and should fall in the treated cells. Control was not constant, results from treated cells were variable Plot a difference graph…

13. Using peak areas for CTH relative to the amount of protein:

14. FOS expressed in DGJ treated cells (10720 22d)

15. Looking at FB17 cells FB17 cells have a Fabry mutation, results in stored CTH Cell fractionation showed this CTH to be in the lysosome Does DGJ treatment decrease the amount of stored CTH? Molecular Chaperone Therapy Chaperone vs inhibitor - concentration is crucial! Incubate FB17 cells with different concentrations of DGJ and collect samples at various time points Analyse samples for GSLs and FOS

17. Conclusions: At earlier time points, up to 20 days, DGJ treatment does result in a clear rise in the levels of CTH in MDA-MB-231 cells, and to a lesser degree in 10720 cells. Treating FB17 Fabry mutant cells with DGJ for up to 16 days does not result in any decrease in the amount of stored CTH.

18. References: Butters, T.D., Dwek R.A., Platt F.M. (2005) Imino sugar inhibitors for treating lysosomal glycosphingolipidoses. Glycobiology, 15, 43-52. Garman, S.C., Garboczi, D.N. (2004) The molecular defect leading to Fabry disease: structure of human α-galactosidase. JMB 337, 319-335. Asano, N., Ishii, S., Kizu, H. (2000) In vitro inhibition and intracellular enhancement of lysosomal α-galactosidase A activity in Fabry lymphoblasts by 1-deoxygalactonojirimycin and its derivatives. Eur. J. Biochem, 267, 4179-4186. Brady, R. O. (2006) Emerging strategies for the treatment of hereditary metabolic storage disorders. Rejuvenation Research, 9, 237-244. Yam, G., Bosshard, N., Zuber, C. Pharmacological chaperone corrects lysosomal storage in Fabry disease caused by trafficking- incompetent variants. (2005) Am J Physiol Cell Physiol 290. Online Mendelian Inheritance in Man http://www.mssm.edu/genetics/fabry/

19. With Thanks To: Dr. Terry Butters Miss Stephanie Boomkamp Dr. David Neville Dr. Gabrielle Reinkensmeier Prof. Raymond Dwek

20. Thanks for listening! Any questions?