1. Human GM3 Synthase Deficiency: A Novel Form of Hereditary Childhood Epilepsy David A Priestman Glycobiology Institute Department of Biochemistry University of Oxford

2. Glycosphingolipids and Human Disease A Disorders of glycosphingolipid catabolism: Gaucher Types 1, 2 and 3 Fabry Tay-Sachs Sandhoff GM1 gangliosidosis Krabbe Metachromatic leukodystrophy B Disorders of glycosphingolipid biosynthesis: No confirmed inherited diseases known

3. Autosomal Recessive Infantile-Onset Symptomatic Epilepsy Syndrome No overt abnormalities at birth During first two months, irritability and poor feeding Extreme developmental retardation and blindness Onset of seizures occurs in first year (multifocal)

4. Extended Pedigree of Two Old Order Amish Families with Infantile-Onset Epilepsy Syndrome

5. GM3 Synthase (EC 2.4.99.9) CMP-NeuAc: Lactosylceramide  -2,3 sialyltransferase Patients have a nonsense mutation in SIAT9 leading to premature termination predicted to result in a non-functional protein product CMP-NeuAc + Lac-Cer GM3 GM3 synthase (Gal  4GlcCer)(NeuAc  3Gal  4GlcCer)

6. Glycosphingolipid Biosynthesis NeuAc (  2,3)paragloboside NeuAc (  2,6)paragloboside Ceramide Glc-Cer Lac-Cer GA2 GA1 GM1b GD1c GD1 GM3 GD3 Gb3 Lc3 GM2 GT1a GM1a GD1a GD2 GQ1b GT1b GD1b Gb4 paragloboside o-seriesa-seriesb-seriesglobo-seriesneolacto-series Gangliosides Ceramide Glucosyl Transferase GalNAcT Lactosyl Ceramide Synthase GlcNAcT GM3S GalT

7. Glycosphingolipid Biosynthesis NeuAc (  2,3)paragloboside NeuAc (  2,6)paragloboside Ceramide Glc-Cer Lac-Cer GA2 GA1 GM1b GD1c GD1 GM3 GD3 Gb3Lc3 GM2 GT1a GM1a GD1a GD2 GQ1b GT1b GD1b Gb4 paragloboside o-seriesa-seriesb-seriesglobo-seriesneolacto-series Gangliosides Ceramide Glucosyl Transferase GalNAcT Lactosyl Ceramide Synthase GlcNAcT GM3S GalT

8. Gangliosides Sub-family of Glycosphingolipids with one or more sialic acids Synthesized in the distal/trans Golgi Located on external leaflet of plasma membrane Implicated in growth, differentiation and adhesion Particularly abundant in CNS Involved in formation of lipid rafts Modulate ion channel function and membrane signalling

9. GM3 Synthase Knockout Mouse Has GM1b, GD1 and GD1c No premature death Fertile Normal CNS function OGTT results in more rapid clearance of glucose Increased autophosphorylation of the insulin receptor leading to increased signalling response GM3 suppresses tyrosine phosphorylation of receptor and IRS1 Ganglioside GM3 participates in the pathological conditions of insulin resistance. Tagami S, Inokuchi Ji J, Kabayama K, Yoshimura H, Kitamura F, Uemura S, Ogawa C, Ishii A, Saito M, Ohtsuka Y, Sakaue S, Igarashi Y. J Biol Chem. 2002 Feb 1;277(5):3085-92.

10. Control Plasma HPLC Minutes 81012141618202224262830 Fluorescence (arbitrary units) Lac Gb3 GM3 GD3 Gb4 GT1b pGb GM1a GA2 GA1 GM2 GD1a GD1b 2-6spG Le b Le x Lc3 2-3spG

11. Peak Assignment GM3 GD3 GT1b GM1a GM2 GD1a GD1b 2-6spG ? 2-3spG Gb4 pGb Gb3 Lac Le b Le x

12. Sialidase Digest to Confirm Peak Assignment Lac GM3 GD3 GT1b GM1a GM2 GD1a GD1b 2-6spG ? 2-3spG Minutes 8101214161820222426283032 Overnight digest with Arthrobacter Ureafaciens Sialidase Gb4 pGb Gb3 Lac

13. Sialidase Digest to Confirm Peak Assignment Lac GM3 GD3 GT1b GM1a GM2 GD1a GD1b 2-6spG ? 2-3spG Minutes 8101214161820222426283032 Gb3 Gb4 pGb GA2 GA1 Le b Overnight digest with Arthrobacter Ureafaciens Sialidase Le x ? Gb4 pGb Gb3 Lac

14. Glycosphingolipids in Human Plasma CTH (Gb3) (Gal  4Gal  4GlcCer) Amino CTH (GlcNAc   Gal  4GlcCer) Globo-series  1-4 galactosyl transferase Ganglio Series Lacto (neo)-series  -3 N-acetyl glucosaminyl transferase (a, b, c series)  2-3 sialyl transferase (GM3 Synthase) (o-series)  -4 N-acetylglucosaminyl transferase Ceramide Glucosyl-Ceramide Lactosyl-Ceramide (Gal  4GlcCer)  -4 galactosyl transferase Paragloboside (pGb) (Gal  4GlcNAc   Gal  4GlcCer) Sialyl(  2-3)paragloboside (NeuAc   3Gal  4GlcNAc   Gal  4GlcCer) Sialyl(  2-6)paragloboside (NeuAc   6Gal  4GlcNAc   Gal  4GlcCer) NeuAc   3(Gal  4GlcNAc   ) 2 Gal  4GlcCer NeuAc   3 (Gal  4GlcNAc   ) 2 [Gal  4GlcNAc  6]Gal  4GlcCer H 1 / LNFPI (Fuc  2Gal  4GlcNAc   Gal  4GlcCer) Le a / LNFPII (Gal  3[Fuc  4]GlcNAc   Gal  4GlcCer) Le x / LNFPIIII (Gal  4[Fuc  3]GlcNAc   Gal  4GlcCer) Le b / LNDFHI (Fuc  2Gal  3[Fuc  4]GlcNAc   Gal  4GlcCer) GM3 (NeuAc   3Gal  4GlcCer) GD3 (NeuAc  8NeuAc  3Gal  4GlcCer) GM2 (GalNac  4[NeuAc  3]Gal  4GlcCer) GD1a (NeuAc  3Gal  3GalNac  4[NeuAc  3]Gal  4GlcCer) GD1b (Gal  3GalNAc  4[NeuAc  8NeuAc  3]Gal  4GlcCer) GT1b (NeuAc  8NeuAc  3Gal  3GalNAc  4[NeuAc  3]Gal  4GlcCer) GQ1b (NeuAc  8NeuAc  3Gal  3GalNAc  4[NeuAc  8NeuAc  3]Gal  4GlcCer) GA2 (GlcNAc  4Gal  4GlcCer) GA1 (Gal  3GlcNAc  4Gal  4GlcCer) Globoside (Gb4) (GalNAc  3Gal  4Gal  4GlcCer) More complex glycosphingolipids found in human plasma from the the above precursors

15. HPLC Profiles for Control and GM3 Synthase Deficiency Plasmas 0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 600 Minutes 1213141516171819202122232425 GM3 Gb3 GM2 Gb4 pGb GM1aGD3 -2,6 spGb GD1a -2,3 spGb Control Father A Mother A Child A1 Child A2 Father B Mother B Child B1 Child B2 Child B3  -2,6 spGb Minutes 1213141516171819202122232425 GM3 Gb3 GM2 Gb4 pGb GM1aGD3GD1a -2,3 spGb Le b

16. GSLs in Plasma of Patients and Controls LacGM3GD3GD1aGb3Gb4pGb 0 5 10 15 20 25 30 GSL Concentration (nmol/ml) Controls (n=8) Patients (n=5)

17. GalT Milder Phenotype NeuAc (  2,3)paragloboside NeuAc (  2,6)paragloboside Ceramide Glc-Cer Lac-Cer GA2 GA1 GM1b GD1c GD1 GM3 GD3 Gb3Lc3 GM2 GT1a GM1a GD1a GD2 GQ1b GT1b GD1b Gb4 paragloboside o-seriesa-seriesb-seriesglobo-seriesneolacto-series Gangliosides CGT GalNAcT LCS GlcNAcT GM3S Build up of precursor Lac-Cer or flux through alternative pathways?

18. Summary Plasma HPLC profiling shows a complete lack of GM3 and its downstream derivatives First confirmed human genetic disorder resulting from disruption in ganglioside biosynthesis Implicates a critical role for gangliosides in the CNS Decreased flux through GM3 synthase leads to increased Lac-Cer and other GSL species (Simpson et al. Nature Genetics 36(11) 2004)

19. Options for Therapy Gene Therapy Dietary GSLs Plasma/CSF GM3 infusion Substrate Reduction Therapy (Miglustat)

20. Ongoing and Future Studies Enzyme Assays GSL profiling in tissues/organs of KO mice Patient samples CSF

21. 20 30 40 50 60 70 80 90 100 110 120 Minutes 14161820222426283032343638 GA2GM2 GM1a GD1a GD1b GT1b GA1 GM3 Gb3 GD3 HPLC profile of 2-AA labeled oligosaccharides derived from ceramide glycanase digestion of glycosphingolipids from a sample of cerebrospinal fluid from a patient with Sandhoff disease. Glycosphingolipids in Cerebrospinal Fluid

22. Ongoing and Future Studies Enzyme Assays GSL profiling in tissues/organs of KO mice Patient samples CSF Other epilepsies

23. Yu, R.K. & Glaser, G.H. Possible role of gangliosides in epilepsy: effects of epileptic seizures on cerebral gangliosides. Trans. Am. Neurol. Assoc. 100, 261−263 (1975) Yu, R.K., Holley, J.A., Macala, L.J. & Spencer, D.D. Ganglioside changes associated with temporal lobe epilepsy in the human hippocampus. Yale J. Biol. Med. 60, 107−117 (1987) Izumi, T., Ogawa, T., Koizumi, H. & Fukuyama, Y. Low levels of CSF gangliotetraose-series gangliosides in West syndrome: implication of brain maturation disturbance. Pediatr. Neurol. 9, 293−296 (1993)

24. 0 1 2 3 4 5 6 7 LacGA2CTHGM3GM2Gb4pGbGA1GM1aGD3  2,3 spg  2,6 spg 4.88 GU GD1aLebGD1bGT1b Control Patient 1 Patient 2 * GSLs with elevated levels in patient 1 and/or patient 2 plasma Total GSL concentration Control: 16.07 ± 2.81, patient 1: 23.1, patient 2: 20.2 nmol/ml ******* GSL concentration (nmol/ml plasma Glycosphingolipid concentrations in plasmas from controls (n=21) and two epileptic patients with ‘unusual’ GSL profiles.

25. Ongoing and Future Studies Enzyme Assays GSL profiling in tissues/organs of KO mice Patient samples CSF Other epilepsies T-Cells

26. Minutes HPLC profile of 2-AA labeled oligosaccharides derived from ceramide glycanase digestion of glycosphingolipids from a sample of human T-cells. GSLs have been provisionally assigned according to their Glucose Unit values. 0 20 40 60 80 100 120 140 160 14161820222426283032343638 GA2 Gb4 pGb GM3 Gb3 GD3 Lc3 Glycosphingolipids in T-cells

27. Ongoing and Future Studies Enzyme Assays GSL profiling in tissues/organs of KO mice Patient samples CSF Other epilepsies T-Cells Fibroblasts Cord blood Autopsy tissues

28. Acknowledgements Harold Cross Department of Opthalmology, University of Arizona School of Medicine, Tucson, USA Oxford Glycobiology Institute Department of Biochemistry University of Oxford David C. A. Neville Gabriele Reinkensmeier Raymond A. Dwek Terry D. Butters Frances M. Platt Department of Medical Genetics St George’s Hospital Medical School University of London Michael A. Simpson Christos Proukakis Argyro Verganelaki Anna Pryde Michael A. Patton Andrew H. Crosby Kay Gurtz ‘ Windows of Hope’ Genetic Studies, Baltic, Ohio, USA Max Wiznitzer Department of Pediatrics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio, USA.

29. Lipid Rafts