1. Superoxide Dismutase and Amyotrophic Lateral Sclerosis
Dan Zollinger
PI: Dr. Joe Beckman
Linus Pauling Institute

2. Oxidative Stress and Familial ALS
Motor Neuron Death
Progressive Neurodegeneration
Muscle Wasting
Paralysis
Death
Mutations to CuZn Superoxide Dismutase (SOD)

3. CuZn Superoxide Dismutase
Cu2+ + O O2 + Cu1+

4. G85R and ALS
Does G85R bind copper and show superoxide scavenging activity?
Can copper play a role in the disease?

5. G85R SOD Activity Nitro Blue Formazan 900 ng 600 ng 300 ng
Bruijn, Et. al., Neuron 1997
Nitro Blue Formazan

6. Mass Spec Data: Recombinant G85R SOD
CuZn
Metal def.
C679H1081N203O224S4CuZn
Apo

7. G85R SOD Crystal Structure
Hart, J. Biol. Chem, 2008

8. Tissue Samples for MS SOD Assay
G85R in 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

9. ZipTip® Method

10. Western Results of Mouse Spinal Cord
Recombinant G85R (300 ng)
G85R mouse tissue
Non transgenic mouse tissue
5 punches (~250 g/punch) suspended in 25 L of 50mM Ammonium Acetate pH 5.1

11. G85R Mouse Tissue Apo G85R Apo G85R Metal def. G85R Mouse SOD

12. 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

13. Acknowledgements Joe Beckman Keith Nylin Nathan Lopez Tim Rhoads
Kevin Ahern
Howard Hughes Medical Institute
Biochemistry and Biophysics Department