1. An Investigation into Zinc Transporter Expression in an Animal Model of Amyotrophic Lateral Sclerosis By Thomas Lew Mentor: Dr. Joe Beckman Linus Pauling Institute

2. Amyotrophic Lateral Sclerosis Results from the death of motor neurons Muscle degeneration Paralysis Death http://starklab.slu.edu/signal/Growth.htm

3. Amyotrophic Lateral Sclerosis Majority of ALS cases are sporadic but approximately 10% of all cases are familial Of these familial cases, 20% of individuals inherit dominant autosomal mutations in the SOD1 gene SOD1 gene codes for copper-zinc superoxide dismutase (SOD) Superoxide Oxygen Superoxide Hydrogen Peroxide e-e- e-e-

4. SOD Mutations and ALS Over 100 different ALS causing mutations have been discovered dispersed throughout the SOD1 gene However, the toxicity of these mutations is not due to reduced superoxide scavenging ability Something about these mutations causes them to become toxic to cells

5. Mutant SOD and Familial ALS Mutant SODs have a reduced affinity for binding zinc. Copper atom in zinc-deficient SOD is much more reactive.

6. Zinc-deficient SOD and ALS: Supporting Evidence More Cu,Zn(-)SOD in Ventral Region The question is - Why? Spinal cord Cross-section

7. Objective The objective of this research is to investigate if a dysregulation of zinc transport pathways could account for the increased levels of zinc-deficient SOD in the ventral spinal cord

8. Hypothesis Levels of zinc-deficient SOD are increased in the ventral grey matter as a consequence of zinc transporter dysregulation

9. Methods Utilize real-time Polymerase Chain Reaction (real- time PCR) to quantify the expression of three zinc transporter genes in the dorsal and ventral grey matter of the spinal cord: i) ZnT-1 ii) ZnT-3 iii) ZnT-4

10. Methods Real Time Polymerase Chain Reaction - can be used to quantify the expression level of a specific gene. Fluorescence Time or cycle number Add Taq polymerase, dGTP, dCTP, dATP, dTTP to synthesize complimentary strand. Add SYBR green for fluorescence

11. Results Zinc Transporter 3 (ZnT-3) –Facilitates zinc transport from the cytosol into synaptic vesicles.

12. Results Zinc Transporter 4 (ZnT-4) –Facilitates zinc transport from the cytosol to the Golgi apparatus and endoplasmic reticulum

13. Results Zinc Transporter 1 (ZnT-1) –Facilitates zinc export from the cytosol into the extracellular space

14. Summary ZnT1: decline in expression in ventral spinal cord of G93A vs. NTG control –Indicative of dysregulation? Neither ZnT-3 or ZnT-4 exhibit any change in expression level when comparing the dorsal and ventral region of the spinal cord. However, these results were found in 40 day old rats, and it should be noted that we have been unable to detect zinc-deficient SOD in rats younger than 50 days.

15. Acknowledgements Howard Hughes Medical Institute Dr. Kevin Ahern Dr. Joe Beckman Dr. Mark Levy The Beckman Lab