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

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

CuZn Superoxide Dismutase Cu2+ + O2- O2 + Cu1+

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

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

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

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

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

ZipTip® Method

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

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

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