Part II project by Katherine Warre-Cornish Inhibition of ganglioside biosynthesis by imino sugars reduces binding of Guillain-Barré Syndrome autoantibodies Part II project by Katherine Warre-Cornish

Guillain Barré syndrome: Auto-immune disease Presence of anti-ganglioside antibodies Causes immune system to attack neurons Symptoms: limb-weakness and pain, impaired reflexes, coordination and respiration 85% of patients make a full recovery within 6 to 12 months Mortality rate of approximately 5% and 7-15% of patients left with persistent minor symptoms Incidence:1-3 cases in 100 000 people per year Current therapies: plasma exchange (total exchange of about 5 plasma volumes) high dose intravenous IgG

Causes of GBS Antecedent bacterial infection (1-2 weeks before GBS onset) Most commonly associated with Campylobacter jejuni Haemophilus influenzae? Cytomegalovirus? Epstein-Barr? Molecular mimicry Lipo-oligosaccharide (LOS) is the principal cell surface structure that is recognised by the host. Forms of LOS have been isolated with identical terminal sugar residues to various human gangliosides

Human gangliosides and C.jejuni lipo-oligo saccharides Bacterial Human GM1-like LOS Ceramide GM1 Core-lipid A GD1a-like LOS Core-lipid A Ceramide GD1a GD1c-like LOS Core-lipid A Ceramide GQ1b Glucose Galactose N-Acetylneuraminic acid N-Acetylgalactosamine

Immune system attacks neurons with high levels of ganglioside Due to cross reactivity with LOS, antibodies against self ganglioside are present in patients. IgG antibodies to GM1 and GD1a strongly associated with GBS IgG antibodies to GQ1b associated with Miller-Fischer syndrome (MFS) (caused similarly to GBS, different neurological symptoms) Neurological features of the disease and sites of neurological damage are determined by the antibody specificities.

Is it possible to treat an auto-immune disease by metabolically decreasing levels of auto-epitope?

N-alkylated imino-sugars: inhibitors of ceramide specific glucosyltransferase Glycolipid Synthesis NB-DNJ and NB-DGJ are glucosyl transferase inhibitors NB-DNJ: IC50 of 20.4uM NB-DGJ: IC50 of 30uM NB-DNJ/ NB-DGJ Inhibition Butters et al, 2000, Chemical Reviews V100, 12, pp4683-4696

Results

ELISA – determination of the degree of patient vs control sera antibody binding to gangliosides GM1 GM2 GD1a GQ1b GD1b 12 patient sera (black dots) and 4 control sera (white dots) tested against 5 GSLs Most significant differences between patient and control Ab binding observed with GM1 and GQ1b

ELISAs carried out using GM1 and GQ1b with increasing dilution factors of selected patient sera

Patient sera binding curves to GM1

Patient sera binding curve to GQ1b

Does inhibition of ganglioside biosynthesis reduce antibody binding? Thin Layer Chromatography: demonstrate decrease in levels of antibody binding to GM1 in extract from drug treated cells Glycolipids were extracted from RAW cells (high GM1 content) treated with a range of concentrations of drugs: NB-DNJ and NB-DGJ Components were separated by TLC. Extracts were run parallel to GM1 standard.

Immuno-overlays: detection of patient antibody binding to TLC plate Analogous to a western blot TLC plates were incubated a 1:1000 dilution of patient sera Plates were then incubated a 1:1000 dilution of secondary anti-human antibody, labelled with horse-radish peroxidase Bands were detected by ECL Horse-radish peroxidase Goat anti-human antibody Patient sera IgG GM1

TLC - results Orcinol staining (proportional to carbohydrate concentration) confirms high levels of GM1 in RAW cells Conc(uM) GM1 Standard 10 20 50 100 1000 10 50 100 1000 Drug: NB-DNJ NB-DGJ

TLC - results Patient sera Control sera Drug: NB-DGJ NB-DNJ Conc(uM) GM1 Standard 10 20 50 100 1000 NB-DNJ Conc(uM) NB-DGJ

Metabolic inhibition reduces patient serum IgG binding. Conclusions from TLC Decrease in patient antibody binding as concentrations of NB-DNJ or NB-DGJ are increased. Effect appears to be more pronounced with NB-DGJ than NB-DNJ Physiologically, IgG binding is enhanced by glycolipid clustering. Relatively low concentrations of drug could disrupt this clustering (TLC- glycolipids are artificially clustered together) Lower drug concentrations may be required than results suggest.

Current work: Can this approach be extended to other cell lines…? Glycosphingolipids were extracted from PC12 cells treated with NB-DNJ and NB-DGJ Extracted GSLs fluorescently labelled Separated by HPLC Spectra obtained Levels of gangliosides quantified by measuring peak areas

HPLC results NB-DNJ GQ1b GD1b GD1c GM1a Gb3

HPLC results NB-DGJ GQ1b GD1b GD1c GM1a Gb3

General Conclusions Use of a metabolic inhibitor of GSL synthesis could potentially be used as a treatment for Guillain-Barré syndrome This depends on: Levels of dosage required for decreased antibody binding – can these be tolerated physiologically Turnover rate of gangliosides in the affected neurons? NBDGJ appears better than NBDNJ Further research: additional, eg neuronal cell lines Animal models – (GBS mice)

Acknowledgements Dr Chris Scanlan Dr Terry Butters