Glycoconjugate storage & pathogenesis in an in vitro cellular model of

Sandhoff disease

Stephanie Boomkamp

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.

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

Galβ(1-4)GlcCer

GA2

LacCer

Glycoprotein degradation & ß-hexosaminidase

β-hexosaminidase

GlcNAc Man

Man

ManGlcNAc

R

R

GlcNAc GlcNAc

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.

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.

murine RAW macrophages

0-50 µM SR1 30 days

Culture cells

OS GSL

2-AA

MS

Enzyme digests

NP-HPLC

+N

AcHN OH

OH

Experimental procedures

IC50

Cell line PNP-GlcNAc (µM) PNP-GalNAc (µM)

RAW 1.05 ± 0.10 2.89 ± 0.40

SR1 elevates GM2 and GA2 levels in RAW cells

RAW UT

RAW 50 µM SR1

GA2

GM2

GM1a

GcGM1a

GD1a

GM1a

GcGM1a

GD1a

mV

RAW UT

RAW 50 µM SR1

SR1 elevates GlcNAc-terminating OS levels in RAW cells

David Harvey

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

23

4

5678

91011

12

GA2 GM2 GM1a fraction

density

123456789101112

density

fraction

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?

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

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

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

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.

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

N-alkylated imino sugars deplete GSL biosynthesis

NB-DNJ and NB-DGJ reduce GSL storage levels

GA2 GM2 GM1aRAW 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

< 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

Iminosugars normalize inflammatory response

Iminosugar Concentration (µM) Effect

NB-DNJ 5 Normalization

50 + MCP1

500

500 µM NB-DNJ

500 µM NB-DGJ

NB-DGJ 5 Normalization

50

500 + MCP1

Iminosugars reduce TGF-β1 levels

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500

uM iminosugar

TG

FB

1/u

g p

rote

in

NB-DNJ

NB-DGJ

UT RAW

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

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?

Acknowledgments

Terry Butters Raymond Dwek

David Neville David Harvey Robin Antrobus

OGBI