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