Sandro Sonnino

Department of Medical Biotechnology and Translational Medicine, Centre of Excellence on Neurodegenerative Diseases,

University of Milan, Italy

Plasma membrane associated glycohydrolasesin Gaucher disease fibroblasts

Sphingolid metabolic pathways

(?)

MUB-substrate solubilized in appropriate cell culture medium

Free MUB

1 hrs at 37°CCell medium

Fluorimeter

MUB-β-D-galactopyranoside b-galactosidase

MUB- β-D-glucopyranoside + AMP-DNM b-glucosidase GBA1

MUB- β-D-glucopyranoside + CBE b-glucosidase GBA2

High Throughput Assay for the detection in living cells of hydrolase activities associated to the external site of the plasma membrane by the use of artificial substrates solubilized into the cell culture medium. (Aureli M. et al. (2011) J. Neurochem)

MUB-N-acetyl- β-D-Glucosaminide-6-sulfate b-Hexosaminidase

6-hexadecanoylamino-4-MUB-phosphorylcholine SMase

PM associated hydrolytic activities were found in all cell lines tested

The fluorescence detected in the cell medium was not due to a release of the enzymes in the medium during the assay and this means that the measured activity was really due to cell surface enzymes

No fluorescence was found associated to cell homogenates: the artificial substrates do not enter into the cells.

P group< 0.0035

Experimental design

18 lines of human fibroblasts derived from patients affected by Gaucher disease (6 for each of the three GD types) displaying several distinct genotypes.

Cell Lines:

3 lines of human fibroblasts derived from healthy donors (CTRL)

Experimental tools:

Evaluation of the total cell associated glycohydrolase activities

Evaluation of the glycohydrolase activities associated to the cell surface in living cells

b-Glucosidase residual activity in GD fibroblastsnm

oles

/(106 c

ells

*h)

* p<0,0023 vs CTRL° p<0,03 vs GD1# p<0,003 vs GD2

nmol

es/(1

06 cel

ls *h

)

* p<0,0001 vs CTRL

A possible genic cross-talk between GBA1 and GBA2.

GBA2 total cell activity

° p<0,003 vs GD1

nmol

es/(1

06 cel

ls *h

)

GB

A2/

GA

PDH

GBA2 mRNA expression level

What about the other PM glycohydrolases?

* p<0,004 vs CTRL° p<0,0035 vs GD1 nm

oles

/(106 c

ells

*h)

Total cell activities

b-galactosidaseb-hexosaminidase PM activities

nmol

es/(1

06 cel

ls *h

)β-gal activity is up-regulated in GD2

β-hex activity is up- regulated depending on the clinical phenotype considered.

No significant changes are found for both enzymes in the total cell associated activity

@ p<0,0035 vs GD2

We found significant differences in the plasma membrane enzymes activity among the three clinical types of the disease that could provide a cell “fingerprint” specific for each of the clinical types thus representing a potential new tool useful for a better clinical typing of this pathology.

We found significant differences of the plasma membrane enzyme activities among the three clinical types of the disease, that could provide a cell “fingerprint” for each of the clinical types thus representing a potential new tool useful for a better clinical typing of this pathology

In the PM of fibroblasts derived from patients affected by Gaucher disease type 2, the form of the disease characterized by a severe impairment of the central nervous system, we found the major increase of two enzymes: β-galactosidase and GBA2.

Is the increased activity of the PM β-galactosidase and GBA2 correlated with the

neuronal impairment?

GBA2 b-galactosidase

Changes of PM glycohydrolase activities in neuronal differentiation of rat granule cells in culture

*p< 0.003

2 8 17Days in culture (DIC)

Initial stage of neuronal differentiation

Morphologically and biochemically fully

differentiated neuronsLate stage of neuronal development (aging)

Analyzing the PM glychohydrolases activity during in vitro neuronal differentiation, we found that the PM associated activities of GBA2 and β-galactosidase strongly increase in senescent neurons with respect to the fully differentiated neurons.

Neu3β-GalGBA1 and

GBA2

PM ceramide production

Apoptosis

GlcCer LacCer GM3

++

+

PM sphingolipid enzyme activities and the neuronal impairment of Gaucher disease

As already demonstrated for other cells, also in neurons the increased activity of the PM associated glycohydrolases leads to the ectopic production of ceramide that determines the onset of apoptotic phenotype.

PM associated activities in living fibroblasts at different pHs

4.5 5 5.5 6 6.5 7 7.5 8 8.5

2000

400600

1200

8001000

GBA2

pmol

es/m

g ce

ll pr

ot* h

4.5 5 5.5 6 6.5 7 7.5 8 8.5

1000

0

2000

3000

4000GBA1

pH

pH4.5 5 5.5 6 6.5 7 7.5 8 8.50

400

800

1200

1600

2000 b-Hexosaminidase

4.5 5 5.5 6 6.5 7 7.5 8 8.50

1000

2000

3000

4000 b-Galactosidase

The PM associated β-hexosaminidase, β-galactosidase, and β-glucosidase GBA1 and GBA2, detected at the cell surface of living cell, are characterized by a strictly dependence on the pH showing the best working condition at acidic pH.

pmol

es/m

g ce

ll pr

ot* h

Na+, H+ antiporter

Na+

PKC

+

PIP2

IP3

DAG

+

extracellular signals

Model of the possible modulation of the SL metabolism at the cell surface trough the control of the extracellular pH

b-gal

H+

LacCer+

GBA2

GlcCer

Cer

intracellular signals

H+

phospholipase C

Na+

Ca++

Na+, H+ antiporterNa+

Model of the possible modulation of the SL metabolism at the cell surface through the control of the extracellular pH

b-gal

H+

LacCer

GBA2

GlcCer

H+

Na+

It is reasonable to hypothesize that, the activity of the proton transporters might be responsible for the creation of a transient local acidic microenvironment close to the cell surface, enough to cause the activation of PM glycohydrolases and thus triggering a local change in the cell surface glycosphingolipid compositionCer

Na+, H+ antiporter

Na+

b-gal

H+

LacCer

GBA2

H+

Na+

-

- 5-(N-Ethyl-N-isopropyl)- amiloride (EIPA)

The use of inhibitors of the proton transporters could block or reduce the activity of the PM glycohydrolases

Proton transporter activators/inhibitors

ΔpH* in fibroblasts ΔpH* in glioma cells ΔpH* in neuroblastoma cells

acetazolamide -0.35±0.01 -0.39±0.01 -0.29±0.04

EGTA -0.28±0.03 -0.38±0.07 -0.31±0.02

EIPA +0.38±0.07 +0.34±0.02 +0.28±0.09

esomeprazole +0.31±0.05 +0.29±0.09 +0.36±0.03

EGTA: cell surface acidification by removal of extracellular Ca++ induces a marked spike in O2 consumption associated to the reduction of mitochondrial and cytosolic Ca++, membrane depolarization and influx of extracellular Na+ with release of protons

Acetazolamide: cell surface acidification by inhibition of the carboxy-anhydrase

EIPA: cell surface alkalinization by inhibition of the NHEs

Esomeprazole: cell surface alkalinization by inhibition of the K+ proton pump

Changes in the plasma membrane pH measured in living cells after drug treatments

*The reported ΔpH respect to the physiological pH 7.4 represent the mean ± S.D. obtained by 3 independent experiments each consisting of a minimum of 6-9 wells stained with pH sensitive fluorescent probe DHPE in presence of different drugs able to modulate the activity of the PM proton exchanger. The fluoresce was detected by the microplate fluorescence reader

CTRL 0,1mM 1mM

pmol

es/(1

06 cel

ls*h

)

* *

GBA1 GBA2

CTRL 0,1mM 1mM0

2000

4000

6000

0

200

400

600

0

100

200

300

400β-galactosidase

CTRL 0,1mM 1mM

** + * *

CTRL 0,1mM 1mM

β-Hexosaminidase

0

150

300

450

*GBA1

CTRL 1mM 2mM

*

GBA2

CTRL 1mM 2mM

*

β-Hexosaminidase

CTRL 1mM 2mM

*

CTRL 1mM 2mM

β-galactosidase

0

150

300

450

600

750

900

pmol

es/(1

06 cel

ls*h

)

EIPA: inhibitors of the NHEs proton pump, alkalinization of the cell surface

EGTA: Removal of extracellular Ca++ induces a marked spike in O2 consumption associated to reduction of mitochondrial and cytosolic Ca++, membrane depolarization and influx of extracellular Na+ with release of protons

Cell surface proton pump modulation. A new opportunity for changes of PM glycosidase activities.

Dr Massimo Aureli

Prof. Alessandro PrinettiProf. Vanna ChigornoDr. Nicoletta LobertoDr. Rosaria BassiDr. Maura SamaraniDr. Valentina MurdicaDr. Elena Chiricozzi

Thanks to:

Dr. Mirella FilocamoDr. Stefano Regis

Prof. Johannes M. Aerts Dr. Rolf G. Boot

Department of Medical Chemistry, Biochemistry and Biotechnology The Medical School University of Milan

"Diagnosi Pre-Postnatale Malattie Metaboliche" Laboratory, G. Gaslini Institute

Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam

Telethon Genetic Biobank Network “Cell Line and DNA Biobank from Patients affected by Genetic Disease” (G. Gaslini Institute).

y=y0*e (K*X)

y0= 0.01484K= 0.4982X= (Ln y - Ln 0.01484)/0.4982Fl

uor p

H X

/ flu

or p

H7.

4

R2= 0,99

CTRL: pH 7,33 ±0.05 EIPA: pH 7,81 ±0.07 EGTA: pH 7,05±0.03

Calibration curve

Fluorimetric determination of the pH at the cell surface after the use of NHEs modulators

EIPA: inhibitors of the NHEs proton pump, alkalinization of the cell surfaceEGTA: Removal of extracellular Ca++ induces a marked spike in O2 consumption associated to reduction of mitochondrial and cytosolic Ca++, membrane depolarization and influx of extracellular Na+ with release of protons

Work in progress

Apply the method to amniocytes or chorionic villous for the prenatal diagnosis and prognosis of the Gaucher Disease

Preliminary results on human chorionic villousCell surface associated activities on human chorionic villous cells derived from normal and subject affected by Gaucher disease type 2.

GBA1 GBA2 β-Hex β-Gal * p<0,05 vs CTRL

*

*

*

*

The cell surface enzymatic pattern is similar to that found in fibroblasts

pmol

es/(1

06 cel

ls *h

)