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Superoxide Dismutase and Superoxide Dismutase and Amyotrophic Lateral SclerosisAmyotrophic Lateral Sclerosis
Dan Zollinger
PI: Dr. Joe Beckman
Linus Pauling Institute
Oxidative Stress and Familial Oxidative Stress and Familial ALSALS
Motor Neuron Death– Progressive Neurodegeneration– Muscle Wasting– Paralysis – Death
Mutations to CuZn Superoxide Dismutase (SOD)
CuZn Superoxide DismutaseCuZn Superoxide Dismutase
Cu2+ + O2- O2 + Cu1+
G85R and ALSG85R and ALS
Does G85R bind copper and show superoxide scavenging activity?
Can copper play a role in the disease?
G85R SOD ActivityG85R SOD Activity
Nitro Blue Formazan
Bruijn, Et. al., Neuron 1997
900 ng 600 ng 300 ng
Mass Spec Data: Recombinant G85R SOD
0.00E + 00
2.00E + 03
4.00E + 03
6.00E + 03
8.00E + 03
1.00E + 04
1.20E + 04
1.40E + 04
1.60E + 04
1.80E + 04
2.00E + 04
15800 15850 15900 15950 16000 16050 16100 16150 16200
15904
15964
16027
Apo
CuZn
Metal def.
C679H1081N203O224S4CuZn
G85R SOD Crystal Structure
Hart, J. Biol. Chem, 2008
G85R G85R in vivoin vivo
Identify metal status of SOD in diseased transgenic mice– Focus on motor neurons in ventral horn of
spinal cord
Tissue Samples for MS SOD
Assay
Dorsal
Ventral
ZipTip® Method
Western Results of Mouse Spinal Cord
Recom
binant G85R
(300 ng)
G85R
mouse tissue
Non transgenic m
ouse tissue
S PP S
5 punches (~250 g/punch) suspended in 25 L of 50mM Ammonium Acetate pH 5.1
G85R Mouse Tissue
Apo G85R
Mouse SOD
Metal def. G85R
Apo G85R
Metal def. G85R
Toxicity of G85R SOD
Why is G85R more toxic than other fALS linked mutants?
Hypothesis:– G85R SOD as a lower affinity for zinc than
other ALS mutants.
Method– Test the metal release of the mutant using metal
chelators and compare to other mutants
Acknowledgements
Joe Beckman Keith Nylin Nathan Lopez Tim Rhoads Kevin Ahern Howard Hughes Medical Institute Biochemistry and Biophysics Department