Rafael Radi

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Rafael Radi Harry Ischiropoulos John Crow Luis
Barbieto Alvaro Estévez Patricia Cassina
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The Linus Pauling Institute andEnvironmental
Health Sciences CenterOregon State University
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O2--gtO2.-
Cu,Zn SOD
Arg--gtNO--gtcGMP
H2O2 -gt HO.
Measure with aminotriazole/catalase J Appl
Physiol. 198763 353-8
H2O2 ki/kcat
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Superoxide Nitric Oxide Fact or Fiction
1.6 x 1010 M-1s-1
(W. Koppenol)
ONOO- CO2
O2.- .NO
.017 s-1
Virtually every collisionproduces
cis-peroxynitrite Peroxynitrite is kinetically
sluggish in its subsequentreactions, which
makes itmore selective than HO.
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SOD-catalyzedtyrosine nitration
Ischiropoulos et al. Peroxynitrite formation
from macrophage-derived nitric oxide. Arch.
Biochem. Biophys. 298446-451 1992.
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Lower motor neurons make muscles to contract.
We have about than500,000 for life
The neuromuscular junction
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Motor Neurons Degenerating early in ALS in The
Ventral Horn of the Spinal Cord
After 1-3 months
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SOD in Reverse Making ONOO- from NO and Oxygen
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Estevez, A.G. Spear, N. Manuel, S.M. Radi, R.
Henderson, C.E. Barbeito, L. Beckman, J.S.
Nitric oxide and superoxide contribute to motor
neuron apoptosis induced by trophic factor
deprivation. J. Neurosci. 18923-931 1998.
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Effects of SOD on nNOS Expression and
Nitrotyrosine
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Cu,Zn SOD keeps trophic factor deprived motor
neurons
Zn- SOD induces apoptosis in motor neurons
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Copper, SOD and ALS

• nNOS knockout not protective
• Mutant SODs still have high affinity for zinc
• Copper chaperone for SOD not protective (but 18
  residual activity and can accelerate disease in
  some mice -- Beckman et al, FRBM 1993)
• Double mutant to copper site still develops ALS
• Quad mutation to copper site still develops
  ALS (Binds copper and is redox active)

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Cu (61.5)
Cu (61.5)
Apo Quad
Cu (61.5)
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Aggregation of Zn(-)SOD (DC)
Estimated MW
Cu,Zn SOD31,820
Cu,(-) SOD31,140
Actual MW
Cu,Zn SOD31,856
Cu,(-) SOD31,650
evidence of larger radius
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Czapski and Goldstein The uniqueness of
superoxide dismutase (SOD) - why cannot most
copper compounds substitute for SOD in vivo?
Free Rad. Res. Comm. 4225-229 1988.

• The rate of Cu1 reoxidation is very slow in
  Cu,Zn SOD
• The reverse reaction is a billion times
  slower,but with high SOD (10 µM) and low fluxes
  of superoxide, the concentration of oxygen may
  approach a billion times that of superoxide

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Copper has no role in SOD toxicity in ALS!
Wang et al. (2002) Fibrillar inclusions and
motor neuron degeneration in transgenic mice
expressing superoxide dismutase 1 with a
disrupted copper-binding site. Neurobiol Dis 10,
128-138. Wang et al. (2003) Copper-binding-site-
null SOD1 causes ALS in transgenic mice
aggregates of non-native SOD1 delineate a common
feature. Hum Mol Genet 12, 2753-2764.
But no one asked the protein?
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Protein Aggregation and SOD

• Aggregation occurs and commonly thought to be
  toxic
• 8-13 of soluble protein is SOD in the ALS mouse
• Very little evidence of cell death in neurons
  overexpressing mutant SOD in cell culture
• Large aggregates may be protective
• SOD aggregates in mitochondria

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Nitrotyrosine Staining in G93A Transgenic Rat
Spinal Cord Root(x 5)
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Reactive Astrocytes revealed by
GFAPImmunoreactivity in ALS
Symptomatic G93A SOD Transgenic Rat
Nontransgenic littermate
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Reactive Astrocyte Kill Motor Neuronsby
Secreting Nerve Growth Factor!

• The NGF is secreted in a pro-form
• p75 neurotrophic receptor expressed on the
  motor neuron cell surface is required for
  toxicity
• Nitric oxide is required for the motor neurons
  to die.

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5,000 U FGF1 vs1 U NGF/g in brain
Oxidative StressNeeded to release FGF1 from
neurons
G93A SOD
Activates Receptorsthat can cause cells
todivide, differentiateor apoptose
NON-TRANSGENIC
Tony Thompson, UAB Surgery
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Control
Nitroarg
FGF
urate
MnTBAP
FeTCPP
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FGF1
FGF1
FGF1
FGF1
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ALS Collaborators
Alvaro Estévez
Kristine Robinson
Luis Barbeito