Transcript Developing an in vitro cellular model for Fabry Disease Part II Project Emma Brewer 31.01.2007 Topics covered in this presentation: Introduction to Fabry Disease – a glycolipid.

Developing an in vitro
cellular model for
Fabry Disease
Part II Project
Emma Brewer
31.01.2007
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
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
Glycolipid Storage Disorders
Glyc olipids performing
their function at the cell
surface
Cell mem brane
Synthesis
Lysosome
Enzym e degradtion
of glyc olipids oc cur
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
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)
X-ray Crystal Structure of α-galactosidase A
Garman and Garboczi JMB 337, 2, 319-335 (2004)
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
CHHOH
Cl
H
N
HO
H
H
OH H
H
H
H
OH
OH
OH
CHHOH
O
HO
H
OH H
H
H
OH
O
OH
OH
H
OH
NH
OH OH
OH
OH
OH
HPLC Profiles: oligosaccharides from GSLs extracted from cell samples
23d ays untrea ted
Gb3
GM3
23 d ays 100uM DGJ
Change in Gb3 levels (relative to GM3) over time.
Blue=untreated, Pink=100uM DGJ
Gb3:GM3 ratio vs Time (MDA-MB-231)
Gb3:GM3 ratio vs Time (10720)
4
Gb3 peak area / GM3 peak area
Gb3 peak area / GM3 peak area
3
2.5
2
1.5
1
0.5
0
0
10
20
30
40
Time (days)
50
60
70
3.5
3
2.5
2
1.5
1
0.5
0
0
10
20
30
40
Time (days)
50
60
70
Plotting Gb3 levels against log conc of DGJ
at different time points
Gb3/GM3 vs log Concentration of DGJ at 14 days (MDA-MB231)
2.6
Gb3 peak area / GM3
peak area
Gb3 peak area / GM3
peak area
Gb3/GM3 vs log Concentration of DGJ at 7 days (MDAMB-231)
2.4
2.2
2
1.8
1.6
1.4
0
0.5
1
1.5
2
2.5
3
2.5
2.3
2.1
1.9
1.7
1.5
0
3.5
0.5
1
Gb3 peak area / GM3
peak area
Gb3 peak area / GM3
peak area
2.3
2.1
1.9
1.7
1.5
1
1.5
log (Concentration DGJ)
2.5
Gb3/GM3 vs log Concentration of DGJ at 31 days
(MDA-MB-231)
Gb3/GM3 vs log Concentration of DGJ at 27 days (MDA-MB231)
0.5
2
log (Concentration of DGJ)
log (Concentration of DGJ)
0
1.5
2
2.5
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0
0.5
1
1.5
log (Concentration of DGJ)
2
2.5
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…
Using peak areas for CTH relative to the amount of protein:
Control CTH - NB-DNJ Treated CTH Levels
6000
Control - Treated (Peak
area/Protein)
5000
4000
3000
2000
1000
0
-1000
0
50
100
150
-2000
-3000
Tim e (hours)
200
250
300
FOS expressed in DGJ treated cells (10720 22d)
100uM
50uM
Retention time= 32.5 mins
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
Gb3/GM3 vs Time:
DGJ-treated (pink) and
untreated (blue) FB17 cells
3.5
Time (days)
3
2.5
2
1.5
1
0.5
0
0
5
10
Gb3/GM3
15
20
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.
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 traffickingincompetent variants. (2005) Am J Physiol Cell Physiol 290.
Online Mendelian Inheritance in Man
http://www.mssm.edu/genetics/fabry/
With Thanks To:
Dr. Terry Butters
Miss Stephanie Boomkamp
Dr. David Neville
Dr. Gabrielle Reinkensmeier
Prof. Raymond Dwek
Thanks for listening!
Any questions?