1. Glycoconjugate storage & pathogenesis in an in vitro cellular model of Sandhoff disease Stephanie Boomkamp

2. Sandhoff disease  Loss motor skills  Seizures  Hearing and vision loss  Mental retardation  Paralysis  Cherry red macular spot in retina  Enlarged organs  Greatly reduced lifespan  Disease severity and onset dependent on residual β-hexosaminidase activity.

3. GSL metabolic pathway & ß-hexosaminidase NeuAcα(2-3) Galβ(1-4)GlcCer GalNAcβ(1-4) GM2 Galβ(1-4)GlcCer NeuAcα(2-3) GM3 GalNAcβ(1-4) Galβ(1-4)GlcCer GA2 LacCer

4. Glycoprotein degradation & ß-hexosaminidase β-hexosaminidase GlcNAcMan GlcNAc R R β-hexosaminidase Chitobiase R = NeuAcα(2-3/6)Galβ(1-4) Asn  Bidirectional degradation in the lysosome  Deficient ß-hexosaminidase termination and accumulation of glycan.

5. Project aims  Development of an in vitro cellular model of Sandhoff disease  Characterization and cellular localization storage products  Disease pathology  Validation of substrate reduction therapeutics NB- DNJ and NB-DGJ: effects on storage levels and disease pathology.

6. murine RAW macrophages 0-50 µM SR1 30 days Culture cells OSGSL 2-AA MS Enzyme digests NP-HPLC + Experimental procedures IC 50 Cell linePNP-GlcNAc (µM)PNP-GalNAc (µM) RAW1.05 ± 0.102.89 ± 0.40

7. SR1 elevates GM2 and GA2 levels in RAW cells RAW UT RAW 50 µM SR1 GA2 GM2 GM1a GcGM1a GD1a GM1a GcGM1a GD1a

8. mV RAW UT RAW 50 µM SR1 SR1 elevates GlcNAc-terminating OS levels in RAW cells David Harvey

9. Stored GSL are localized in the lysosome - GlcNAc-OS in light fractions 7 8 9 10 RAW UT RAW SR1  LAMP1 ~ 44kDa Cell fraction 1 2 3 4 5 6 7 8 9 10 11 12 GA2GM2GM1a fraction density 1 2 3 4 5 6 7 8 9 10 11 12 density fraction

10. Stored GSL are localized in the lysosome - GlcNAc-OS in light fractions 1 2 3 4 5 6 7 8 9 10 11 12 OS GSL Robin Antrobus Density  Protein markers  Novel cellular compartment OS?

11. Disease pathology Wada R., Tifft C.J., Proia R.L. (2000) PNAS 20:10954-10959. GSL accumulation Neuronal damage/death Microglial phagocytosis Microglial activation/expansion Production neurotoxic mediators +MIP-1 , TNF- , TGF-ß1, IL-1

12. SR1 down-regulates cytokine expression RAW UT RAW SR1 IL-1  IL-6 TNF-  Stimulation with LPS: similar intracellular signalling events  presence of feedback control loop in SR1-treated RAW cells

13. TGF-β1 deactivates macrophages  Immunosuppressive: antagonist of IL-1α, IL-6, TNF-α  In HEXB -/- mice TGF-β1 only elevated at terminal stages, possibly in attempt to down-regulate the inflammatory cascade  High glucosamine levels promote activation of TGF-β1  Twofold increase in SR1-treated RAW cells

14. Summary  SR1 induces storage of GM2, GA2 and GlcNAc-OS in RAW cells as seen in Sandhoff patients/mice  GM2 and GA2 are localized in the lysosome, whereas OS are present in light, buoyant cell compartment(s)  SR1 triggers an immunosuppressive response due to a twofold increase in TGF-ß1.

15. Therapy  Bone marrow transplantation (BMT)  Enzyme replacement therapy (ERT)  Chaperone-mediated therapy (CMT)  Substrate reduction therapy (SRT)  Best thus far is SRT & BMT or combination of therapies with use of anti-inflammatory drugs. Inaccessibility by BBB

16. N-alkylated imino sugars deplete GSL biosynthesis

17. NB-DNJ and NB-DGJ reduce GSL storage levels GA2 GM2GM1a RAW UT RAW SR1 RAW SR1 + 0.5µM NB-DNJ RAW SR1 + 5µM NB-DNJ RAW SR1 + 50µM NB-DNJ RAW SR1 + 500µM NB-DNJ RAW SR1 + 0.5µM NB-DGJ RAW SR1 + 5µM NB-DGJ RAW SR1 + 50µM NB-DGJ RAW SR1 + 500µM NB-DGJ

18. < 500 µM NB-DNJ and NB-DGJ do not reduce GlcNAc-OS levels RAW UT RAW SR1 RAW SR1 + 0.5µM NB-DNJ RAW SR1 + 5µM NB-DNJ RAW SR1 + 50µM NB-DNJ RAW SR1 + 500µM NB-DNJ RAW SR1 + 0.5µM NB-DGJ RAW SR1 + 5µM NB-DGJ RAW SR1 + 50µM NB-DGJ RAW SR1 + 500µM NB-DGJ

19. Iminosugars normalize inflammatory response IminosugarConcentration (µM)Effect NB-DNJ 5Normalization 50+ MCP1 500 500 µM NB-DNJ 500 µM NB-DGJ NB-DGJ 5Normalization 50 500+ MCP1

20. Iminosugars reduce TGF-β1 levels UT RAW

21. Summary  NB-DNJ and NB-DGJ reduce GSL storage levels but do not affect GlcNAc-OS levels at achievable therapeutic concentrations  At concentrations ≥ 50 µM  increase MCP1, due to:  Overall reduction in GSL levels?  Reduction in OS storage?  Glucosylated/galactosylated OS?  5 µM NB-DNJ or NB-DGJ potentially induces a non- pathological phenotype (Jeyakumar et al 1999).  GSL, not OS, play a role in the disease pathology of Sandhoff disease

22. Future work  MCP1 ELISA  Sandhoff patient-derived cell lines:  Why no GSL storage?  Treatment with GM2  Use of SR1 as chemical chaperone: enhancement β- hexosaminidase activity?

23. Acknowledgments  Terry Butters  Raymond Dwek  David Neville  David Harvey  Robin Antrobus  OGBI