Developing an Developing an in vitroin vitro cellular model for cellular model for

Fabry DiseaseFabry Disease

Part II ProjectPart II ProjectEmma BrewerEmma Brewer

31.01.200731.01.2007

Topics covered in this presentation:Topics covered in this presentation:

Introduction to Fabry Disease – a glycolipid storage Introduction to Fabry Disease – a glycolipid storage disease.disease.

Using an Using an αα--galactosidase inhibitor, DGJ to achieve a galactosidase inhibitor, DGJ to achieve a cellular model for the disease:cellular model for the disease:

- do we see storage of the glycolipid?- do we see storage of the glycolipid?- are there any other changes, eg glycoprotein - are there any other changes, eg glycoprotein inhibition? inhibition?

Chemical chaperoning in Fabry mutant cells. Chemical chaperoning in Fabry mutant cells. - do we see a reduction in glycolipid storage?- do we see a reduction in glycolipid storage?

QuestionsQuestions

FABRY DISEASEFABRY DISEASE

Deficiency of Deficiency of αα--galactosidase A due to point galactosidase A due to point mutationsmutationsResponsible for biodegradation of CTHResponsible for biodegradation of CTHResult: insufficient breakdown of CTHResult: insufficient breakdown of CTHCTH builds up in cells i.e. glycolipid storage CTH builds up in cells i.e. glycolipid storage disorder.disorder.

CTH:CTH: Ceramide-Glc-Galβ-GalαCeramide-Glc-Galβ-Galα α-Galactosidase cleaves hereα-Galactosidase cleaves here

Glycolipid Storage DisordersGlycolipid Storage Disorders

LysosomeEnzyme degradtion of glycolipids occur

Cell membrane Glycolipids performingtheir function at the cellsurface

Synthesis

Symptoms of Fabry DiseaseSymptoms of Fabry Disease

Fatigue, inability to sweat, Fatigue, inability to sweat, vomitting and diarrhoeavomitting and diarrhoea

Impaired arterial circulation + Impaired arterial circulation + increased risk of early heart attack increased risk of early heart attack or strokeor stroke

Cloudiness of the corneaCloudiness of the cornea

Small, painless, raised red-purple Small, painless, raised red-purple blemishes on the skinblemishes on the skin

Burning sensation in hands and Burning sensation in hands and feetfeet

Renal failure is usually the cause Renal failure is usually the cause of death, due to proteinuria of death, due to proteinuria induced hypertensioninduced hypertension

More about Fabry DiseaseMore about Fabry DiseaseIt is an X-linked disease, and so is far more common in It is an X-linked disease, and so is far more common in men, although women carriers often show some men, although women carriers often show some symptomssymptoms

Long term accumulation of the glycolipid means that Long term accumulation of the glycolipid means that severity of symptoms increases over timeseverity of symptoms increases over time

Patients typically live into their early fortiesPatients typically live into their early forties

TherapyTherapyEnzyme replacement therapy can be successful – Enzyme replacement therapy can be successful – involves an intravenous infusion to replace the deficient involves an intravenous infusion to replace the deficient enzyme – up to twice weeklyenzyme – up to twice weekly

Substrate Reduction Therapy (SRT)Substrate Reduction Therapy (SRT)

Molecular Chaperone Therapy / Chaperone-Mediated Molecular Chaperone Therapy / Chaperone-Mediated Therapy (CMT)Therapy (CMT)

X-ray Crystal Structure of X-ray Crystal Structure of αα--galactosidase Agalactosidase A Garman and Garboczi JMBGarman and Garboczi JMB 337, 2, 319-335 (2004)337, 2, 319-335 (2004)

The DGJ Experiment – using an inhibitor of The DGJ Experiment – using an inhibitor of αα--galactosidase A to produce the Fabry galactosidase A to produce the Fabry

phenotype in healthy cellsphenotype in healthy cells

Aim of this experiment – Aim of this experiment – to create an in vitro, to create an in vitro, cellular model for Fabry cellular model for Fabry disease by addition of an disease by addition of an αα--galactosidase A inhibitor galactosidase A inhibitor to healthy cell linesto healthy cell linesI assessed samples of I assessed samples of these cells at different these cells at different time points of treatment time points of treatment between 0 days and 70 between 0 days and 70 days, and at different days, and at different concentrations of the concentrations of the inhibitorinhibitor

HO

OH

OH

H

H

H

H

H

CHHOH

NH HO

OH

OH

H

H

H

H

H

CHHOHO OH

OH

OHOH

OH

NH

OH

OHOH

OHO

OH

HCl

HPLC Profiles: oligosaccharides from GSLs extracted from cell samplesHPLC Profiles: oligosaccharides from GSLs extracted from cell samples

23days untreated

23 days 100uM DGJ

Gb3

GM3

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

Gb3:GM3 ratio vs Time (MDA-MB-231)

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50 60 70

Time (days)

Gb

3 p

eak

area

/ G

M3

pea

k ar

ea

Gb3:GM3 ratio vs Time (10720)

0

0.5

1

1.5

2

2.5

3

3.5

4

0 10 20 30 40 50 60 70

Time (days)G

b3

pea

k ar

ea /

GM

3 p

eak

area

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

Gb3/GM3 vs log Concentration of DGJ at 7 days (MDA-MB-231)

1.4

1.6

1.8

2

2.2

2.4

2.6

0 0.5 1 1.5 2 2.5 3 3.5

log (Concentration of DGJ)

Gb

3 p

eak

area

/ G

M3

pea

k ar

ea

Gb3/GM3 vs log Concentration of DGJ at 14 days (MDA-MB-231)

1.5

1.7

1.9

2.1

2.3

2.5

0 0.5 1 1.5 2 2.5

log (Concentration of DGJ)

Gb

3 p

eak

area

/ G

M3

pea

k ar

ea

Gb3/GM3 vs log Concentration of DGJ at 27 days (MDA-MB-231)

1.5

1.7

1.9

2.1

2.3

0 0.5 1 1.5 2 2.5

log (Concentration DGJ)

Gb

3 p

eak

area

/ G

M3

pea

k ar

ea

Gb3/GM3 vs log Concentration of DGJ at 31 days (MDA-MB-231)

0.8

1

1.2

1.4

1.6

1.8

2

2.2

0 0.5 1 1.5 2 2.5

log (Concentration of DGJ)

Gb

3 p

eak

area

/ G

M3

pea

k ar

ea

Reason for very little storage – slow turnover Reason for very little storage – slow turnover of the glycolipid? Determining CTH half life:of the glycolipid? Determining CTH half life:

Treat cells with an inhibitor of glycolipid synthesis, Treat cells with an inhibitor of glycolipid synthesis, NB-DNJ. NB-DNJ.

Take and analyse cell samples each dayTake and analyse cell samples each day

Monitor the levels of CTH against control cells.Monitor the levels of CTH against control cells.

Amount of CTH per unit mass of protein should be Amount of CTH per unit mass of protein should be constant in the control, and should fall in the constant in the control, and should fall in the treated cells.treated cells.

Control was not constant, results from treated Control was not constant, results from treated cells were variablecells were variablePlot a difference graph…Plot a difference graph…

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

Control CTH - NB-DNJ Treated CTH Levels

-3000

-2000

-1000

0

1000

2000

3000

4000

5000

6000

0 50 100 150 200 250 300

Time (hours)

Contr

ol -

Tre

ated

(Pea

k ar

ea/P

rote

in)

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

100uM

50uM

Retention time= 32.5 mins

Looking at FB17 cellsLooking at FB17 cells

FB17 cells have a Fabry mutation, results in stored CTHFB17 cells have a Fabry mutation, results in stored CTH

Cell fractionation showed this CTH to be in the lysosomeCell fractionation showed this CTH to be in the lysosome

Does DGJ treatment decrease the amount of stored Does DGJ treatment decrease the amount of stored CTH?CTH?

Molecular Chaperone TherapyMolecular Chaperone Therapy

Chaperone vs inhibitor - concentration is crucial!Chaperone vs inhibitor - concentration is crucial!

Incubate FB17 cells with different concentrations of DGJ Incubate FB17 cells with different concentrations of DGJ and collect samples at various time pointsand collect samples at various time points

Analyse samples for GSLs and FOSAnalyse samples for GSLs and FOS

Gb3/GM3 vs Time: DGJ-treated (pink) and

untreated (blue) FB17 cells

0

0.5

1

1.5

2

2.5

3

3.5

0 5 10 15 20

Gb3/GM3

Tim

e (d

ays)

Conclusions:Conclusions:

At earlier time points, up to 20 days, DGJ At earlier time points, up to 20 days, DGJ treatment does result in a clear rise in the treatment does result in a clear rise in the levels of CTH in MDA-MB-231 cells, and levels of CTH in MDA-MB-231 cells, and to a lesser degree in 10720 cells. to a lesser degree in 10720 cells.

Treating FB17 Fabry mutant cells with Treating FB17 Fabry mutant cells with DGJ for up to 16 days does not result in DGJ for up to 16 days does not result in any decrease in the amount of stored any decrease in the amount of stored CTH.CTH.

References:References:

Butters, T.D., Dwek R.A., Platt F.M. (2005) Imino sugar inhibitors for Butters, T.D., Dwek R.A., Platt F.M. (2005) Imino sugar inhibitors for treating lysosomal glycosphingolipidoses. Glycobiology, 15, 43-52.treating lysosomal glycosphingolipidoses. Glycobiology, 15, 43-52.Garman, S.C., Garboczi, D.N. (2004) The molecular defect leading Garman, S.C., Garboczi, D.N. (2004) The molecular defect leading to Fabry disease: structure of human to Fabry disease: structure of human αα--galactosidase. JMB 337, galactosidase. JMB 337, 319-335.319-335.Asano, N., Ishii, S., Kizu, H. (2000) Asano, N., Ishii, S., Kizu, H. (2000) In vitroIn vitro inhibition and intracellular inhibition and intracellular enhancement of lysosomal enhancement of lysosomal αα--galactosidase A activity in Fabry galactosidase A activity in Fabry lymphoblasts by 1-deoxygalactonojirimycin and its derivatives. Eur. lymphoblasts by 1-deoxygalactonojirimycin and its derivatives. Eur. J. Biochem, 267, 4179-4186.J. Biochem, 267, 4179-4186.Brady, R. O. (2006) Emerging strategies for the treatment of Brady, R. O. (2006) Emerging strategies for the treatment of hereditary metabolic storage disorders. Rejuvenation Research, 9, hereditary metabolic storage disorders. Rejuvenation Research, 9, 237-244.237-244.Yam, G., Bosshard, N., Zuber, C. Pharmacological chaperone Yam, G., Bosshard, N., Zuber, C. Pharmacological chaperone corrects lysosomal storage in Fabry disease caused by trafficking-corrects lysosomal storage in Fabry disease caused by trafficking-incompetent variants. (2005) incompetent variants. (2005) Am J Physiol Cell Physiol 290.Am J Physiol Cell Physiol 290.Online Mendelian Inheritance in ManOnline Mendelian Inheritance in Manhttp://www.mssm.edu/genetics/fabry/http://www.mssm.edu/genetics/fabry/

With Thanks To:With Thanks To:

Dr. Terry ButtersDr. Terry Butters

Miss Stephanie BoomkampMiss Stephanie Boomkamp

Dr. David NevilleDr. David Neville

Dr. Gabrielle Dr. Gabrielle ReinkensmeierReinkensmeier

Prof. Raymond DwekProf. Raymond Dwek

Thanks for listening!Thanks for listening!

Any questions?Any questions?