Lecture 1 • SOD1-ALS link:

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

Molecular Mechanisms of CuZnSOD-Linked ALS

Location of Known ALS Mutations

SOD1 monomer

Electrostatic Loop

Zinc Loop

Location of Known ALS Mutations

Each black sphere represents a different amino acid where a

mutation is known to cause ALS

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

Metal Binding Region Mutants

Examples: H46R, H48Q, G85R, D124V, D125H, S134N

NH

O O-

Gly 85

Asp 124Conserved Water

N

N N

NHH

NNCuN

N

Zn O

N

O

N

N

N

His 46His 71

Asp 83

His 80

His 120

His 48

His 63

H

H

H

O

H

H

OH H

Ser 134

Asn 139

Asp 125

Half lives of the mutant SOD1 polypeptides differ greatly in cell culture

Metabolically radio-labeled COS-1 cells

SOD1 Zinc and Electrostatic Loops are Linked

Electrostatic Loop residues 121-144

Zinc Loop residues 65-82

180o

Disruption of linkage between loops affects metal binding properties

Cell culture studies shows differences between the different mutants

SOD1 activity in cell lysates from transiently transfected COS1 cells

Native activity gel

Western blot

Some mutants show activity while

others do not

Human Molecular Genetics, 1999

Assay for SOD activity: Rescue of SOD-null yeast

The biophysical properties of FALS mutant SOD1 do not correlate with disease severity

Mutation Duration of illness (years)

Rescue of null yeast

Specific activity (%)

Protein half-life (h)

Solubility Resistance to proteolysis

A4V 2a,b Yes ~45c ~7.5c High +

G37R 18a,d Yes ~100c ~13c High ++++

H46R 18bdf No ~0e >24 ND ND

H48Q 4a,f No ~0e >24 ND ND

G85R 6a,b Yes ~0c ~7.5c High +

I113Tg 2a,b Yes ~65c ~20c High ++++

FS126 3h ND ~0c ~5 ND ND

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

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

NH

O O-

Gly 85

Asp 124Conserved Water

N

N N

NHH

NNCuN

N

Zn O

N

O

N

N

N

His 46His 71

Asp 83

His 80

His 120

His 48

His 63

H

H

H

O

H

H

OH H

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

Mouse (and rat) models of SOD1 linked ALS

G37RG85RmG85RG93A mouse and ratG37RI113TD90AG93RL126XG127ZL84V mouseH46R rat

Designed ALS mutant miceH46R/H48QH46R/H48Q/H63G/H120G

G37R/CCS-

G93A/CCS-

G85R/CCS-

G37R Mouse

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.

Metal Binding Region Mutants

Examples: H46R, H48Q, G85R, D124V, D125H, S134N

NH

O O-

Gly 85

Asp 124Conserved Water

N

N N

NHH

NNCuN

N

Zn O

N

O

N

N

N

His 46His 71

Asp 83

His 80

His 120

His 48

His 63

H

H

H

O

H

H

OH H

Ser 134

Asn 139

Asp 125

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.

Several mice show Thioflavin-S positive fibrillar inclusions

Neurobiol Dis. 2002 Jul;10(2):128-38

The quad mouse shows all the same biophysical hallmarks of fALS

Quad

Non-tg

Ub-immunostaining

Thioflavin-S

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.

Typically, aggregates from ALSTg mice show high immuno reactivityTo ubiquitin but low to SOD1

The arrows point to inclusion bodies

Neurobiol Dis. 2002 Jul;10(2):128-38

H46R/H48Q mice do have high molecular weight species that contain SOD1

Filter Trap Experiment

• Tissue homogenatues were treated with1% SDS before filtered through a cellulose acetate membrane

• The membrane was then exposed to SOD1 antibodies

serial dilutions

Neurobiol Dis. 2002 Jul;10(2):128-38

These high molecular weight SOD1 containing species are only found in nervous tissue

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.

All mutants (tested) adopt detergent insoluble structures

to varying degrees

HEK 293 cells

WT

H46R/H48Q

H46R/H48Q

J Wang, G Xu, V Gonzales, M Coonfield, D Fromholt,

NG Copeland, NA Jenkins, DR Borchelt, Neurobiology of Disease 10, 128–138 (2002)

hSOD1 Tg mouse

Thioflavin-S-positive

inclusions

Vacuolar pathology*

G37R + +++

G93A + +++

G85R +++ 0

H46R/H48Q +++ 0

WT-likemutations

Metal bindingRegion mutations

*EM shows vacuoles are composed of swollen and distended mitochondrial and endoplasmic reticulum membranes (Dal Canto and Gurney, 1995; Wong et al., 1995).

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

Specificity of Motor Neuron Toxicity in ALS

Cleveland and Rothstein, Nature Rev Neurosci 2:806-19 (2001)