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By Thomas Lew Mentor: Dr. Joe Beckman Linus Pauling Institute

An Investigation into Zinc Transporter Expression in an Animal Model of Amyotrophic Lateral Sclerosis. By Thomas Lew Mentor: Dr. Joe Beckman Linus Pauling Institute. Amyotrophic Lateral Sclerosis. Results from the death of motor neurons Muscle degeneration Paralysis Death.

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By Thomas Lew Mentor: Dr. Joe Beckman Linus Pauling Institute

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  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. Superoxide e- Superoxide e- Oxygen Hydrogen Peroxide 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)

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

  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

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