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Sandro Sonnino Department of Medical Biotechnology and Translational Medicine,

Plasma membrane associated glycohydrolases in Gaucher disease fibroblasts. Sandro Sonnino Department of Medical Biotechnology and Translational Medicine, Centre of Excellence on Neurodegenerative Diseases, University of Milan , Italy. Sphingolid metabolic pathways.

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Sandro Sonnino Department of Medical Biotechnology and Translational Medicine,

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  1. Plasma membrane associated glycohydrolases in Gaucher disease fibroblasts Sandro Sonnino Department of Medical Biotechnology and Translational Medicine, Centre of Excellence on Neurodegenerative Diseases, University of Milan, Italy

  2. Sphingolidmetabolicpathways

  3. 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) b-galactosidase MUB-β-D-galactopyranoside b-Hexosaminidase MUB-N-acetyl-β-D-Glucosaminide-6-sulfate b-glucosidase GBA1 MUB- β-D-glucopyranoside + AMP-DNM b-glucosidase GBA2 MUB- β-D-glucopyranoside + CBE 6-hexadecanoylamino-4-MUB-phosphorylcholine SMase Cell medium (?) 1 hrs at 37°C Fluorimeter Free MUB MUB-substratesolubilized in appropriate cell culture medium

  4. PM associated hydrolytic activities were found in all cell lines tested No fluorescence was found associated to cell homogenates: the artificial substrates do not enter into the cells. The fluorescence detected in the cell medium was not due to a release of the enzymes in the medium during the assayand this means that the measured activity was really due to cell surface enzymes P group< 0.0035

  5. Experimental design Cell Lines: 3 lines of human fibroblasts derived from healthy donors (CTRL) 18 lines of human fibroblasts derived from patients affected by Gaucher disease (6 for each of the three GD types) displaying several distinct genotypes. Experimental tools: Evaluation of the total cell associated glycohydrolase activities Evaluation of the glycohydrolase activities associated to the cell surface in living cells

  6. -Glucosidaseresidualactivity in GD fibroblasts * p<0,0023 vs CTRL ° p<0,03 vs GD1 nmoles/(106cells*h) # p<0,003 vs GD2 nmoles/(106cells*h)

  7. A possible genic cross-talk between GBA1 and GBA2. GBA2 mRNAexpressionlevel GBA2 total cellactivity nmoles/(106cells*h) GBA2/GAPDH * p<0,0001 vs CTRL ° p<0,003 vs GD1

  8. What about the other PM glycohydrolases? b-galactosidase b-hexosaminidase PM activities β-gal activity is up-regulated in GD2 β-hex activity is up- regulated depending on the clinical phenotype considered. nmoles/(106cells*h) Total cellactivities No significant changes are found for both enzymes in the total cell associated activity nmoles/(106cells*h) * p<0,004 vs CTRL ° p<0,0035 vs GD1 @ 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.

  9. 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. GBA2 b-galactosidase Is the increased activity of the PM β-galactosidase and GBA2 correlated with the neuronal impairment?

  10. Changes of PM glycohydrolase activities in neuronal differentiation of rat granule cells in culture Initial stage of neuronal differentiation Morphologically and biochemically fully differentiated neurons Late stage of neuronal development (aging) 2 8 17 Days in culture (DIC) *p< 0.003 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.

  11. PM sphingolipid enzyme activities and the neuronal impairment of Gaucher disease + + GBA1 and GBA2 Neu3 β-Gal LacCer GlcCer GM3 PM ceramide production + Apoptosis • 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.

  12. PM associated activities in living fibroblasts at different pHs b-Galactosidase b-Hexosaminidase 4000 2000 1600 3000 1200 2000 pmoles/mg cellprot* h 800 1000 400 0 0 4.5 5 5.5 6.5 7 7.5 8 8.5 6 4.5 5 5.5 6 6.5 7 7.5 8 8.5 pH GBA2 1200 1000 GBA1 800 pmoles/mg cellprot* h 4000 600 400 3000 200 2000 0 pH 4.5 5 5.5 6 6.5 7 7.5 8 8.5 1000 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. 0 4.5 5 5.5 6 6.5 7 7.5 8 8.5

  13. Model of the possible modulation of the SL metabolism at the cell surface trough the control of the extracellular pH Na+ Na+ intracellularsignals + Na+, H+ antiporter H+ H+ + LacCer PKC b-gal + GlcCer GBA2 DAG Ca++ Cer IP3 phospholipase C extracellularsignals PIP2

  14. Model of the possible modulation of the SL metabolism at the cell surface through the control of the extracellular pH Na+ Na+ Na+ Na+ Na+, H+ antiporter 5-(N-Ethyl-N-isopropyl)- amiloride (EIPA) Na+, H+ antiporter - H+ H+ H+ H+ LacCer LacCer 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 composition b-gal - b-gal The use of inhibitors of the proton transporters could block or reduce the activity of the PM glycohydrolases GlcCer GBA2 GBA2 Cer

  15. Proton transporter activators/inhibitors Acetazolamide: cellsurfaceacidificationbyinhibitionof the carboxy-anhydrase EGTA: cellsurfaceacidificationby 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 EIPA: cellsurfacealkalinizationbyinhibitionof the NHEs Esomeprazole: cellsurfacealkalinizationbyinhibitionof the K+protonpump Changes in the plasma membrane pH measured in living cells after drug treatments *Thereported 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

  16. Cell surface proton pump modulation. A new opportunity for changes of PM glycosidase activities. 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 β-Hexosaminidase β-galactosidase GBA1 GBA2 β-Hexosaminidase β-galactosidase GBA2 GBA1 * 900 400 450 6000 * 600 * 750 * 300 * + * 600 * 4000 * 300 400 pmoles/(106cells*h) pmoles/(106cells*h) 450 200 * 150 * 300 2000 200 100 150 0 0 0 0 0 0,1mM 1mM 0,1mM 1mM 0,1mM 1mM 0,1mM 1mM 1mM 2mM 1mM 2mM 1mM 2mM 1mM 2mM CTRL CTRL CTRL CTRL CTRL CTRL CTRL CTRL EIPA: inhibitorsof the NHEsprotonpump, alkalinizationof the cellsurface

  17. Thanksto: • "Diagnosi Pre-Postnatale Malattie Metaboliche" Laboratory, • G. Gaslini Institute Department of Medical Chemistry, Biochemistry and Biotechnology The Medical School Universityof Milan • Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam Dr. Mirella Filocamo Dr. Stefano Regis Prof. Johannes M. Aerts Dr. Rolf G. Boot Dr Massimo Aureli Prof. Alessandro Prinetti Prof. Vanna Chigorno Dr. Nicoletta Loberto Dr. Rosaria Bassi Dr. Maura Samarani Dr. Valentina Murdica Dr. Elena Chiricozzi Telethon Genetic Biobank Network “Cell Line and DNA Biobank from Patients affected by Genetic Disease” (G. Gaslini Institute).

  18. Fluorimetric determination of the pH at the cell surface after the use of NHEs modulators Calibration curve y=y0*e (K*X) y0= 0.01484 K= 0.4982 Fluor pH X/ fluor pH7.4 X= (Ln y - Ln 0.01484)/0.4982 R2= 0,99 EGTA: pH 7,05±0.03 EIPA: pH 7,81 ±0.07 CTRL: pH 7,33 ±0.05 EIPA: inhibitorsof the NHEsprotonpump, alkalinizationof the cellsurface 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

  19. 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 villous Cell surface associated activities on human chorionic villous cells derived from normal and subject affected by Gaucher disease type 2. * * pmoles/(106cells*h) The cell surface enzymatic pattern is similar to that found in fibroblasts * * GBA1 GBA2 β-Hex β-Gal * p<0,05 vs CTRL

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