Investigation of mutated Cu/Zn Superoxide Dismutase

Sam Schuberg

Beckman Laboratory

Howard Hughes Medical Institute: Summer 2007

Amyotrophic Lateral Sclerosis

• Neurodegenerative disease caused by the destruction of the motor neurons along the spinal cord

• Gradual loss of voluntary muscular function – Not only difficulty in

moving, but also in speaking and swallowing

• Eventually affects respiratory system

Types of ALS

• Two types of ALS– Sporadic and Familial– 98% of patients have

sporadic ALS– 2% have genetically

inherited the disease• Nature 1993• Common link in a mutation

in chromosome 21– The gene with the

mutations is for SOD1– Currently over 100

mutations identified Beckman et al. Superoxide dismutase and the death of motor nuerons in ALS. Trends Nueroscience, 2001.

Wild Type Superoxide Dismutase

• Scavenges superoxide produced by normal metabolism• The dimer interface is

stabilized by a disulfide bond

• A cytosolic disulfides

• Active site contains two metal ions

• Rate: 2 x 109 M-1s-1

QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.

Mutated SOD

•It is widely accepted that these mutations not only deactivate SOD1’s scavenging feature, but cause it to gain a toxic function •Debate is on what this toxic function is

•One theory is aggregation

The hypothesis of Zn deficient SOD

• Mutations destabilize the enzyme and cause it to lose its affinity for Zn• Alters coordination of the

neighboring Cu through a shared histidine ligand

• The exposed Cu is readily reduced by antioxidents

• Reduced Cu donates an electron to oxygen to produce superoxide

• O2.- react with NO to form

peroxynitrite – Peroxynitrite modifies important

biological molecules leading to apoptosis

Beckman et al. Superoxide dismutase and the death of motor nuerons in ALS. Trends Nueroscience, 2001.

• Dr. Wang and his research group created SOD-mutated mice deficient in four histidines

• These coordinate and hold Cu in its active site– Their quad mice still get

ALS like symptoms – Their in vitro data was

interpreted as consistent with aggregation

– Their data suggests that CuII is not important in disease pathology

Cu

Wang et al. Journal of Neuroscience. Copper-binding-site-null SOD1 causes ALS in transgenic mice: aggregates of non-native SOD1 delineate a common feature. 2003.

My project

• To further investigate the aggregation of mutant superoxide dismutases in spinal cord punches from transgenic mice.

Spinal cord punches

Tissue Samples for MS SOD Assay

Dorsal

Ventral

Do the Quad and G93A mutants aggregate in animals?

• Western Blot Analysis– Location of SOD1

• Supernatant versus pellet

• Mice– Mutated SOD1

• Quad and G93A• Punches from the spinal

cords • ventral and dorsal side are

taken

Mutated SOD is primarily located in the supernatant

63.1

2 ng

SO

D s

td31

.56

ng S

OD

std

15.7

8 ng

SO

D s

td

G93A80 days

Heterozygous Quad 190 days

Sup

erna

tant

Sup

erna

tant

Sup

erna

tant

Sup

erna

tant

Pel

let

Pel

let

Pel

let

Pel

let

15.7

8 ng

SO

D s

td

31.5

6 ng

SO

D s

td

63.1

2 ng

SO

D s

td

Homozygous Quad 210 days

Heterozygous Quad 210 days

Sup

erna

tant

Sup

erna

tant

Sup

erna

tant

Sup

erna

tant

Pel

let

Pel

let

Pel

let

Pel

let

ventral ventralventralventral dorsal

dorsaldorsal

dorsal

Conclusions

• My results are not consistent with aggregation

• Previous results could be artifacts of sonication and grinding of the spinal cord

Acknowledgments

• Howard Hughes Medical Institute

• Dr. Joseph Beckman, Nathan Lopez and the Beckman Lab

• Dr. Kevin Ahern

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.