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

Download Report

Transcript Superoxide Dismutase and Amyotrophic Lateral Sclerosis Dan Zollinger PI: Dr. Joe Beckman

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
900 ng
600 ng 300 ng
Bruijn, Et. al., Neuron 1997
Nitro Blue Formazan
Mass Spec Data:
Recombinant G85R SOD
2.00E + 04
CuZn
1.80E + 04
16027
1.60E + 04
Metal def.
15964
1.40E + 04
C679H1081N203O224S4CuZn
1.20E + 04
1.00E + 04
8.00E + 03
6.00E + 03
4.00E + 03
Apo
15904
2.00E + 03
0.00E + 00
15800
15850
15900
15950
16000
16050
16100
16150
16200
G85R SOD Crystal Structure
Hart, J. Biol. Chem, 2008
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
S
P
S
P
Non transgenic mouse tissue
G85R mouse tissue
Recombinant G85R (300 ng)
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
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