SOD1ALS link:

1
Molecular Mechanisms of CuZnSOD-Linked ALS
Lecture 1 SOD1-ALS link Gain of
function mechanism Protein aggregation
vs. oxidative damage Chemistry of superoxide
and other ROS Lecture 2 ALS Clinical
aspects SODs and SOD mechanisms Lecture 3
Model studies in cell culture and ALS Tg mice
and rats WT and mutant SOD1
structures Lecture 4 Protein aggregation and
disease Oxidative stress in ALS? Lecture 5
Biophysical properties of WT and mutant SOD1s
2
Location of Known ALS Mutations
SOD1 monomer
3
Location of Known ALS Mutations
Each black sphere represents a different amino
acid where a mutation is known to cause ALS
4
Two Classes of Pathogenic SOD1
Wild type- like mutants includes ?-barrel and
subunit interface
Metal-binding region mutants includes
metal-binding ligands, zinc loop and
electrostatic loop
5
Metal Binding Region Mutants
Asn 139
Ser 134
Asp 125
Examples H46R, H48Q, G85R, D124V, D125H, S134N
6
Half lives of the mutant SOD1 polypeptides differ
greatly in cell culture
Metabolically radio-labeled COS-1 cells
7
SOD1 Zinc and Electrostatic Loops are Linked
180o
Disruption of linkage between loops affects metal
binding properties
8
Cell culture studies shows differences between
the different mutants
SOD1 activity in cell lysates from transiently
transfected COS1 cells
Some mutants show activity while others do not
Native activity gel
Western blot
Human Molecular Genetics, 1999
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Assay for SOD activity Rescue of SOD-null yeast
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The biophysical properties of FALS mutant SOD1 do
not correlate with disease severity
ND, not determined.aData taken from Cudkowicz et
al. (60), Radunovic et al. (17) and Juneja et al.
(18).bData taken from Cleveland et al. (16). It
is of note that this work described an Australian
G37R kindred with short-duration disease
however, a second examination of this family has
demonstrated that this kindred actually carries
the H43R mutation (61). cData taken from
previous estimates by assay gel analysis of
extracts from transiently transfected COS-1 cells
(7).dData on duration of illness taken from Aoki
et al. (20).eEstimated from assay gel analysis
of extracts from transiently transfected COS-1
cells from Figure 2 of this study. fData taken
from Enayat et al. (21).gThis mutation shows
some variation among European kindreds
(17).hData published by Pramatarova et al. (25).

Human Molecular Genetics, 1999
11
G85R presents an interesting case low affinity
for copper?
Yeast rescue studies Yeast G85R fails to rescue
but human G85R does rescue SOD1-null yeast
12
G85R presents an interesting case low affinity
for copper?
Human G85R is active in COS1 lysates But
human G85R shows no activity when lysates are run
on an SOD activity gel Does the mutant have
decreased copper affinity such that it loses the
metal upon electrophoresis?
Human Molecular Genetics, 1999
13
Mouse (and rat) models of SOD1 linked ALS
G37R G85R mG85R G93A mouse and rat G37R I113T D90A
G93R L126X G127Z L84V mouse H46R rat Designed
ALS mutant mice H46R/H48Q H46R/H48Q/H63G/H120G G3
7R/CCS- G93A/CCS- G85R/CCS-
G37R Mouse
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Abstract Cu/Zn superoxide dismutase (SOD1), a
crucial cellular antioxidant, can in certain
settings mediate toxic chemistry through its Cu
cofactor. Whether this latter property explains
why mutations in SOD1 cause FALS has been
debated. Here, we demonstrate motor neuron
disease in transgenic mice expressing a SOD1
variant that mutates the four histidine residues
that coordinately bind Cu. In-depth analyses of
this new mouse model, previously characterized
models and FALS human tissues revealed that the
accumulation of detergent-insoluble forms of SOD1
is a common feature of the disease. These
insoluble species include full length SOD1
proteins, peptide fragments, stable oligomers and
ubiquitinated entities. Moreover, chaperones
Hsp25 and aB-crystallin specifically
co-fractionated with insoluble SOD1. In cultured
cells, all 11 of the FALS variants tested
produced insoluble forms of mutant SOD1.
Importantly, expression of recombinant peptide
fragments of wild-type SOD1 in cultured cells
also produced insoluble species, suggesting that
SOD1 possesses elements with an intrinsic
propensity to aggregate. Thus, modifications to
the protein, such as FALS mutations,
fragmentation and possibly covalent modification,
may simply act to augment a natural, but
potentially toxic, propensity to aggregate.
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Metal Binding Region Mutants
Asn 139
Ser 134
Asp 125
Examples H46R, H48Q, G85R, D124V, D125H, S134N
16
Is copper binding to the copper site of SOD1 a
necessity for fALS?
To address this question a fALS mouse with a
copper site knockout was made.
17
Several mice show Thioflavin-S positive fibrillar
inclusions
Neurobiol Dis. 2002 Jul10(2)128-38
18
The quad mouse shows all the same biophysical
hallmarks of fALS
Ub-immuno staining
Thioflavin-S
Quad
Non-tg
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Abnormal inclusions and fibrils in spinal cord
and brain stem of symptomatic SOD1-Quad mice
stained with Thioflavin-S. (A and B) Low power
view of spinal cord cross-sections stained with
Thioflavin-S. Fluorescent inclusions and fibrils
were observed only in the mutant mice A as
compared to controls B. Scale bar 200 µm. (C
and D) In sagittal sections of the brain stem
region just below the cerebellum, both scattered
inclusions and selected cell bodies in a subset
of neuronal populations were positive for
Thioflavin-S in the SOD1-Quad mice as compared to
nontransgenic controls D. Scale bar 400 µm.
20
Typically, aggregates from ALS Tg mice show high
immuno reactivity To ubiquitin but low to
SOD1 The arrows point to inclusion bodies
Neurobiol Dis. 2002 Jul10(2)128-38
21
H46R/H48Q mice do have high molecular weight
species that contain SOD1
serial dilutions

• Filter Trap Experiment
• Tissue homogenatues were treated with1 SDS
  before filtered through a cellulose acetate
  membrane
• The membrane was then exposed to SOD1 antibodies

Neurobiol Dis. 2002 Jul10(2)128-38
22
These high molecular weight SOD1 containing
species are only found in nervous tissue
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Accumulation of detergent-insoluble SOD1
monomers, fragments, and high molecular weight
species in spinal cords of affected mice and
human patients
(A) Mutant SOD1 from the spinal cords of affected
mice fractionated in the detergent-insoluble
pellet fraction (P3). Spinal cords were
differentially extracted and sedimented in
non-ionic detergent and SDS. insoluble species of
SOD1 were abundant in symptomatic mutant mice.
SOD1 monomers (open arrow), fragments (solid
arrow), and high molecular weight species (solid
arrow head) are indicated. Apparent dimers
(asterisk) and trimers (double asterisk) are
noted.
(C) The spinal cord of a patient with the A4V
mutation contains SOD1 monomers, fragments and
oligomers that are insoluble in non-ionic
detergent (P2). A representative age-matched
non-ALS control case was examined in parallel.
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All mutants (tested) adopt detergent insoluble
structures to varying degrees
HEK 293 cells
25
WT
WT-like mutations
H46R/H48Q
Metal binding Region mutations
H46R/H48Q
J Wang, G Xu, V Gonzales, M Coonfield, D
Fromholt, NG Copeland, NA Jenkins, DR Borchelt,
Neurobiology of Disease 10, 128138 (2002)
EM shows vacuoles are composed of swollen and
distended mitochondrial and endoplasmic reticulum
membranes (Dal Canto and Gurney, 1995 Wong et
al., 1995).
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Noncell autonomous toxicity of ALS-causing SOD1
mutants. (A) Morula aggregation to produce
chimeric mice in which the wild-type neurons were
marked by a trace level of human neurofilament-L
(NF-L), and mutant neurons and nonneurons were
marked by mutant human SOD1. (B - C) Even though
30 of mutant motor neurons expressed SOD1G37R,
none were killed in one chimeric animal even six
months after all mice that expressed the mutant
systemically had died from motor neuron loss. (B)
Immunofluorescent localization of mutant SOD1
(green), all axons with an antibody specific for
human NF-L (red) and myelin (blue) in a lumbar
motor root. (C) Robust extension of the life span
of chimeric mice with a high proportion of mutant
neurons. Chimeras were constructed similar to the
scheme in (A) except using SOD1G93A mutant
morulas. (D) There were no signs of degeneration
or axon loss, with 978 axons present (normal
animals have 927, / 99, axons in this root).
Scale bar is 40 microns. From Clement AM et
al., Science, 2003
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Specificity of Motor Neuron Toxicity in ALS

• Cleveland and Rothstein, Nature Rev Neurosci
  2806-19 (2001)