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

http://starklab.slu.edu/signal/Growth.htm

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

HydrogenPeroxide

e-

e-

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

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

reactive.

Normalnerve fibre Normal

nerve fibre

Zinc-deficient SOD and ALS:Supporting Evidence

• More Cu,Zn(-)SOD in Ventral Region

• The question is - Why?

Spinal cordCross-section

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

Hypothesis• Levels of zinc-deficient SOD are increased

in the ventral grey matter as a consequence of zinc transporter dysregulation

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

MethodsReal Time Polymerase Chain Reaction

- can be used to quantify the expression level of a specific gene.

Flu

ores

cenc

e

Time or cycle number

Add Taq polymerase, dGTP, dCTP, dATP, dTTP to synthesize complimentary strand. Add SYBR green for fluorescence

Results• Zinc Transporter 3 (ZnT-3)

– Facilitates zinc transport from the cytosol into synaptic vesicles.

ZnT-3 Gene Expression Level

0

5

10

15

Ntg Dorsal Ntg Ventral G93A Dorsal G93A Ventral

Spinal Cord Region

ZnT-3 Gene Copy Number (per 1000

_- )actin transcripts

Results• Zinc Transporter 4 (ZnT-4)

– Facilitates zinc transport from the cytosol to the Golgi apparatus and endoplasmic reticulum

ZnT-4 Gene Expression Level

0

10

20

30

40

50

Ntg Dorsal Ntg Ventral G93A Dorsal G93A Ventral

Spinal Cord Region

ZnT-4 Gene Copy Number (per 10,00 b-actin transcripts)

Results• Zinc Transporter 1 (ZnT-1)

– Facilitates zinc export from the cytosol into the extracellular space

ZnT-1 Gene Expression Level

0

20

40

60

80

Ntg Dorsal Ntg Ventral G93A Dorsal G93A Ventral

Spinal Cord Region

ZnT-1 Gene Copy Number

(per

_- )actin transcript

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.

Acknowledgements

• Howard Hughes Medical Institute

• Dr. Kevin Ahern

• Dr. Joe Beckman

• Dr. Mark Levy

• The Beckman Lab