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
e-
Superoxide e-
Oxygen
Hydrogen
Peroxide
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.
Normal
nerve fibre
Normal
nerve fibre
Zinc-deficient SOD and ALS:
Supporting Evidence
• More Cu,Zn(-)SOD in Ventral Region
• The question is - Why?
Spinal cord
Cross-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 (realtime 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
Methods
Add Taq polymerase,
dGTP, dCTP, dATP, dTTP to
synthesize complimentary
strand. Add SYBR green for
fluorescence
Fluorescence
Real Time Polymerase Chain Reaction
- can be used to quantify the expression level of
a specific gene.
Time or cycle number
Results
• Zinc Transporter 3 (ZnT-3)
– Facilitates zinc transport from the cytosol into synaptic
vesicles.
ZnT-3 Gene Expression Level
(per 1000 -actin transcripts)
ZnT-3 Gene Copy Number
15
10
5
0
Ntg Dorsal
Ntg Ventral
G93A Dorsal
Spinal Cord Region
G93A Ventral
Results
• Zinc Transporter 4 (ZnT-4)
– Facilitates zinc transport from the cytosol to the Golgi
apparatus and endoplasmic reticulum
ZnT-4 Gene Expression Level
(per 10,00 b-actin transcripts)
ZnT-4 Gene Copy Number
50
40
30
20
10
0
Ntg Dorsal
Ntg Ventral
G93A Dorsal
Spinal Cord Region
G93A Ventral
Results
• Zinc Transporter 1 (ZnT-1)
– Facilitates zinc export from the cytosol into the
extracellular space
80
(per -actin transcript)
ZnT-1 Gene Copy Number
ZnT-1 Gene Expression Level
60
40
20
0
Ntg Dorsal
Ntg Ventral
G93A Dorsal
Spinal Cord Region
G93A Ventral
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