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Umeå University Medical Dissertations, New Series No 1285
Mutant Superoxide Dismutase-1-caused
pathogenesis in Amyotrophic Lateral
Sclerosis
Daniel Bergemalm
Department of Medical Biosciences,
Clinical Chemistry and Pathology
Department of Pharmacology and Clinical Neurosciences
Umeå 2010
Responsible publisher under Swedish law: the Dean of the Medical Faculty
Copyright © 2010 Daniel Bergemalm
ISBN: 978-91-7264-838-8
ISSN: 0346-6612
Cover: Two hands with atrophy of intrinsic muscles reaching towards the diseasecausing SOD1 molecule. Painted by Per-Olof Bergemalm.
E-version available at http://umu.diva-portal.org/
Printed by: Print och Media
Umeå, Sweden 2010
To my beloved children Hanna and Ludvig
Man's conquest of Nature turns out, in the moment of its
consummation, to be Nature's conquest of Man
- C.S Lewis
Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating disease that affects people in their
late mid-life, with fatal outcome usually within a few years. The progressive
degeneration of neurons responsible for muscle movement (motor neurons) throughout
the central nervous system (CNS) leads to muscle wasting and paralysis, and eventually
affects respiratory function. Most cases have no familial background (sporadic) whereas
about 10% of cases have relatives affected by the disease. A substantial number of
familial cases are caused by mutations in the gene encoding superoxide dismutase-1
(SOD1). Since the initial discovery of this relationship about 17 years ago, numerous
workers have tried to identify the pathogenicity of mutant SOD1 but without any final
agreement or consensus regarding mechanism. The experiments in this thesis have been
aimed at finding common pathogenic mechanisms by analyzing transgenic mouse
models expressing mutant SOD1s with widely different properties.
Mitochondrial pathology and dysfunction have been reported in both ALS patients and
murine models. We used density gradient ultracentrifugation for comparison of
mitochondrial partitioning of SOD1 in our transgenic models. It was found that models
with high levels of mutant protein, overloaded mitochondria with high levels of SOD1protein whereas models with wild type-like levels of mutant protein did not. No
significant association of the truncation mutant G127X with mitochondria was found.
Thus, if mitochondrial dysfunction and pathology are fundamental for ALS pathogenesis
this is unlikely to be caused by physical association of mutant SOD1 with mitochondria.
Density gradient ultracentrifugation was used to study SOD1 inclusions in tissues
from an ALS patient with a mutant SOD1 (G127X). We found large amounts in the
ventral horns of the spinal cord but also in the liver and kidney, although at lower levels.
This showed that such signs of the disease can also be found outside the CNS.
This method was used further to characterize SOD1 inclusions with regard to the
properties of mutant SOD1 and the presence of other proteins. The inclusions were found
to be complex detergent-sensitive structures with mutant SOD1 reduced at disulfide C57C146 being the major inclusion protein, constituting at least 50% of the protein content.
Ten co-aggregating proteins were isolated, some of which were already known to be
present in cellular inclusions. Of great interest was the presence of several proteins that
normally reside in the endoplasmic reticulum (ER), which is in accordance with recent
data suggesting that the unfolded protein response (UPR) has a role in ALS.
To obtain unbiased information on the pathogenesis of mutant SOD1, we performed a
total proteome study on spinal cords from ALS transgenic mice. By multivariate analysis
of the 1,800 protein spots detected, 420 (23%) were found to significantly contribute to
the difference between transgenic and control mice. From 53 proteins finally identified,
we found pathways such as mitochondrial function, oxidative stress, and protein
degradation to be affected by the disease. We also identified a previously
uncharacterized covalent SOD1 dimer.
In conclusion, the work described in this thesis suggests that mutant SOD1 affects the
function of mitochondria, but not mainly through direct accumulation of SOD1 protein.
It also suggests that SOD1 inclusions, present in both the CNS and peripheral tissues,
mainly consist of SOD1 but they also trap proteins involved in the UPR. This might be
deleterious as motor neurons, unable to renew themselves, are dependent on proper
protein folding and degradation.
Keywords: ALS, SOD1, mitochondria, proteome, transgenic mice, inclusion.
Table of contents
Abbreviations ....................................................................................... 1
Preface .................................................................................................. 2
Introduction .......................................................................................... 3
Amyotrophic lateral sclerosis ........................................................... 3
Epidemiology ............................................................................... 3
Pathology...................................................................................... 4
Etiology ............................................................................................ 6
Genetics of ALS ........................................................................... 6
Genetic risk factors..................................................................... 10
Other risk factors ........................................................................ 13
Excitotoxicity ............................................................................. 13
Superoxide dismutase-1 ................................................................. 15
The protein ................................................................................. 15
SOD1 in ALS ............................................................................. 17
How do mutant SOD1s cause disease? ...................................... 19
The “–omics” in ALS/SOD1 models ......................................... 28
Aims ................................................................................................... 31
Methods .............................................................................................. 32
SOD1 transgenic mice ................................................................ 32
G127X patient ............................................................................ 32
Homogenization of tissue ........................................................... 32
Density gradient separation ........................................................ 33
Marker enzyme assays ............................................................... 34
Dot-blot assay............................................................................. 35
Antibody-based assays ............................................................... 35
Size-exclusion chromatography ................................................. 37
Proteomic techniques ................................................................. 37
Network analysis ........................................................................ 39
Statistics ..................................................................................... 40
Results and Discussion ....................................................................... 41
Paper I ............................................................................................ 41
Paper II ........................................................................................... 44
Paper III .......................................................................................... 45
Paper IV.......................................................................................... 47
Conclusions ........................................................................................ 51
Acknowledgements ............................................................................ 53
References .......................................................................................... 55
Abbreviations
ALS
CCS
CHAPS
CNS
DIGE
DTT
EDTA
EGTA
ER
FALS
GWAS
HEPES
hSOD1
HSP
ir
IMS
IPKB
LBHI
LC-MS
LMN
MALDI-TOF
MND
MOWSE
mRNA
mSOD1
NRF2
NP-40
OPLS-DA
PAGE
PLS
PMA
ROS
SALS
SDH
SDS
SLI
SMA
SOD1
SOD2
SOD3
UI
UMN
UPR
UPS
wt
Amyotrophic lateral sclerosis
Copper chaperone for SOD1
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate
hydrate (1)
Central nervous system
Differential in-gel electrophoresis
Dithiothreitol
Ethylenediaminetetraacetic acid
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′ tetraacetic acid
Endoplasmic reticulum
Familial amyotrophic lateral sclerosis
Genome-wide association study
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid
Human superoxide dismutase-1
Heat shock protein
immunoreactive
Intermembrane space (mitochondria)
Ingenuity pathway knowledge base
Lewy body-like hyaline inclusion
Liquid chromatography mass spectrometry
Lower motor neuron
Matrix-assisted laser desorption ionization time of flight
Motor neuron disease
Molecular weight search
Messenger RNA
Mouse superoxide dismutase-1
Nuclear factor erythroid 2-related factor 2
Nonidet P-40 (octyl phenoxylpolyethoxylethanol)
Orthogonal projections to latent structures discriminant analysis
Polyacrylamide gel electrophoresis
Primary lateral sclerosis
Progressive muscular atrophy
Reactive oxygen species
Sporadic amyotrophic lateral sclerosis
Succinate dehydrogenase
Sodium dodecylsulfate
Skein-like inclusion
Spinal muscular atrophy
CuZn superoxide dismutase
Mn superoxide dismutase
Extracellular superoxide dismutase
Ubiquitinated inclusion
Upper motor neuron
Unfolded protein response
Ubiquitin proteasome system
Wild type
1
Preface
In this thesis, I have tried to target the pathogenic events taking place when
mutant superoxide dismutase-1 (SOD1) causes amyotrophic lateral sclerosis
(ALS). Although more than 17 years have passed since the original report on
this subject, no consensus on the disease mechanism has been reached. In the
Introduction I will review the main characteristics of the disease and the
most common theories of pathogenesis. The focus is, however, on previous
and current work relevant to the experiments described here, including
mitochondrial toxicity, SOD1 aggregates, and the use of proteomic methods
in ALS. The four papers that constitute this thesis are listed below.
I.
Daniel Bergemalm, P. Andreas Jonsson, Karin S. Graffmo, Peter M.
Andersen, Thomas Brännström, Anna Rehnmark, Stefan L.
Marklund. Overloading of stable and exclusion of unstable human
superoxide dismutase-1 variants in mitochondria of murine
amyotrophic lateral sclerosis models. Journal of Neuroscience.
2006 Apr 19;26:4147-54.
II.
P. Andreas Jonsson, Daniel Bergemalm, Peter M. Andersen, Ole
Gredal, Thomas Brännström, Stefan L. Marklund. Inclusions of
amyotrophic lateral sclerosis-linked superoxide dismutase in ventral
horns, liver, and kidney. Annals of Neurology. 2008 May;63:671-5.
III.
Daniel Bergemalm, Karin Forsberg, Vaibhav Srivastava, Karin S
Graffmo, Peter M. Andersen, Thomas Brännström, Gunnar Wingsle
and Stefan L. Marklund. Superoxide dismutase-1 and other proteins
in inclusions of transgenic amyotrophic lateral sclerosis model mice.
(Submitted)
IV.
Daniel Bergemalm, Karin Forsberg, P. Andreas Jonsson, Karin S.
Graffmo, Thomas Brännström, Peter M. Andersen, Henrik Antti,
Stefan L. Marklund. Changes in the spinal cord proteome of an
amyotrophic lateral sclerosis murine model determined by
differential in-gel electrophoresis. Molecular and Cellular
Proteomics. 2009 Jun;8:1306-17
2
Introduction
Amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is
invariably fatal. The progressive loss of motor neurons in the spinal cord, the
brain stem, and the motor cortex leads to muscle wasting, paralysis, and
eventually respiratory failure. As originally described in the 19th century by
Charcot and others, the symptoms are derived from loss of upper and lower
motor neurons (UMNs and LMNs) (1;2). Modern criteria are still based on
signs of degeneration from both of these compartments in combination with
progression and spread of symptoms (the so-called El Escorial criteria) (3;4).
The control of muscle movement involves both UMNs and LMNs, and
symptoms reflect their functional characteristics. UMNs are involved in
voluntary movements and regulation of reflexes. Degeneration of UMNs and
the corticospinal tract (descending nerve fibers from UMNs in the spinal
cord) therefore leads to weakness in groups of coordinated muscles,
symptoms of increased muscular tonus (spasticity), hyperreflexia and
repetitive reflexes (clonus). LMNs are responsible for the direct innervations
of individual muscle fibers. Depending on the requirements of fine motor
ability, one LMN can innervate from a few up to hundreds of muscle fibers.
Degeneration of LMNs therefore leads to loss of reflexes, muscle paralysis,
spontaneous muscle contractions (fasciculations), and muscle atrophy due to
loss of signaling.
Despite the fact that there are clearly defined diagnostic criteria, the
clinical situation is often more complex and establishing an ALS diagnosis
takes a lot of time and resources; it is sometimes never established (5). This
complexity is partly due to motor neuronal diseases that affect only UMNs
(primary lateral sclerosis, PLS) or LMNs (progressive muscular atrophy,
PMA) but which sometimes eventually develop the criteria of ALS (6;7). As
the long-term prognosis is different, for example, for PLS, PMA, and
classical ALS, clinical studies have recently been carried out to establish the
criteria for and prognosis of these diseases and similar intermediate
syndromes with accentuation of UMN or LMN symptoms (6;8-10).
Epidemiology
The incidence, prevalence, and mortality of ALS have been shown to have
increased slightly over the past 50 years (11). Most studies have been
performed in Europe and the United States, where the incidences have been
quite similar with a rough mean of about 2 per 100,000 person-years
(variation depending on study ~0.6–3.2 per 100,000) (12-16). In Sweden, the
incidence has been shown to have increased from 2.3 per 100,000 personyears in 1991 to 3 per 100,000 person-years in 2005 (17). This corresponds
to an approximate lifetime risk of developing ALS of 1 in 400 for the
3
population studied. Cronin and colleagues performed a review of global
epidemiology and concluded that few studies have been performed outwith
Caucasian populations but that the incidence is most probably lower in other
ethnic groups (18). The mortality rates follow the incidence with marginally
lower counts, probably due to under-reporting of ALS on death certificates
(19). Historically, it has been shown that males have an increased risk of
developing ALS. In the 1960s, a male-to-female risk ratio of about 2:1 was
reported whereas this ratio was reported to average 1.3 in the 1990s (11).
The decrease in male preponderance (or increase in incidence in women)
now appears to have stabilized and recent reports have shown similar
numbers (13;15;17). However, a small study recently reported a higher
incidence in women than in men in an Italian population (14). It has been
speculated that the gender difference may be attributed to protective female
hormones and increased exposure to potential risk factors in males, such as
smoking (20;21).
Age of onset and survival
Most ALS patients are diagnosed at ages ranging between 55 and 85, with a
peak incidence at 68 years of age (17;19;20). The variation is, however,
great and in a recent report the patients included were between 20 and 90
years of age—which is typical of studies in large populations (15). Though
juvenile forms exist, with very early onset of disease, it is nevertheless
unusual to be diagnosed before the age of 30 (22). The mean survival time of
ALS patients has been estimated to be 32.6 months (range 26.3–43) (11) and
this is partly dependent on the site of onset, with the bulbar form being more
aggressive (23). Between 5% and 10% of patients have a disease duration of
more than 10 years (24;25). Familial ALS (FALS, see below) shows a
significantly lower age of onset and higher variability of disease duration
(26;27).
Pathology
The main characteristics of ALS pathology are due to selective loss of motor
neurons in affected areas of the motor cortex, the brain stem, and the anterior
horns of the spinal cord (28;29). This affects the fibers that are involved in
connecting these different cellular populations, the corticospinal tract, and
efferent axons in the anterior roots. Macroscopically, atrophy of the spinal
cord and anterior roots is often quite obvious, while atrophy of the precentral
gyrus (motor cortex) can only be seen in patients with a very long disease
duration (28). Microscopically, the loss of motor neurons is followed by
astrocytic gliosis and shrinkage of the few remaining motor neurons. Few of
these findings are, however, specific for ALS even though their
simultaneous presence clearly strengthens the diagnosis. As a further aid to
diagnosis, some characteristic cellular inclusions are visible with routine
staining, and these are more or less specific for ALS.
4
Inclusions
Neurodegenerative diseases are often characterized by the presence of
intracellular and extracellular inclusions in the CNS. In Alzheimer’s disease
and Parkinson’s disease, inclusions such as senile plaques, neurofibrillary
tangles, and Lewy bodies are fundamental for the pathological diagnosis and
they have been studied extensively. The mechanisms that give rise to the
different types of inclusions are as yet unknown. Studies in cell lines have
involved modeling of different entities that arise due to, for example, high
expression of mutant proteins or inhibition of protein degradation (e.g. the
ubiquitin-proteasome pathway). Examples of such entities are aggresomes,
JUNQ/IPOD, and ERAC (30-32). A series of different inclusions are known
in ALS; some are more common in SALS or FALS, but there is often
overlap (29).
Bunina bodies. Bunina bodies are almost pathognomonic for ALS and are
generally found in the cytoplasm of surviving motor neurons. They are
round or irregular in shape, with a diameter ranging from 1 to 6 µm. When
stained with hematoxylin and eosin (H&E), they are eosinophilic hyaline
inclusions occasionally surrounded by a thin halo. Bunina bodies stain with
antibodies to cystatin C and transferrin, and they have been suggested to be
derived from the endoplasmic reticulum or Golgi apparatus (28;29;33).
Ubiquitinated inclusions (UIs). UIs are very common in ALS patients
(close to 100%) and can be further subdivided according to morphology into
Skein-like inclusions (SLIs) and Lewy-body like hyaline inclusions (LBHIs).
It has recently been shown that one of the major proteins in UIs from SALS
and non-SOD1 FALS patients is TAR DNA binding protein 43 (TDP-43)
(34) (discussed further below). SLIs range from 5 to 25 µm and have a
filamentous morphology of aggregated fibrils. Similar to SLIs, LBHIs are
composed of filaments and are coated with small granules. LBHIs are seen
with routine H&E staining and are rounder and denser than SLIs. LBHIs
have been found in both astrocytes and neurons from SALS and mutant
SOD1-associated FALS, and are the most frequently found inclusions in
these patients as well as in SOD1 transgenic mouse models. In both SOD1
FALS and SOD1 transgenic mice, the LBHIs stain intensively for the SOD1
protein (35;36). A range of other proteins have been reported to be present in
LBHIs such as neurofilaments, peripherin, and ER chaperones (28;29;37).
Spheroids. Spheroids are mainly composed of accumulated
phosphorylated neurofilaments, found in the proximal part of motor neuronal
axons. They are variable in size, eosinophilic on H&E staining, and are
clearly seen in silver impregnations. Spheroids are non-specific and can be
found in tissue from both controls and patients with other neurological
diseases, but the numbers are far greater in ALS patients (28;29).
5
Other inclusions. Several other inclusions have been described in ALS
patients. One is hyaline conglomerate (neurofilament) inclusions (HCIs),
which appear to be rather specific for FALS rather than SALS but have been
found in other neurodegenerative diseases (20). Another is basophilic
inclusions, which are found in juvenile ALS (28).
Etiology
Genetics of ALS
Most cases of ALS develop without any familial history whereas about 5–
10% are regarded as inherited (FALS) (38). The majority of these families
have an autosomal dominant disease with variable penetrance, but recessive
inheritance also exists (39;40). To be regarded as “familial”, the proband
must have at least one or two first- or second-degree relatives that are also
affected (41). As the clinical and pathological presentations between SALS
and FALS are often similar (42), it is obviously of great importance to
characterize familial forms and identify the gene or genes that are involved.
So far, mutations in ten genes have been coupled to typical or atypical ALS,
and there are six more loci whose genes are as yet unidentified (Table 1).
Table 1. Loci and genes identified in familial ALS
Disease Type
ALS1
ALS2
ALS4
ALS6
ALS8
LMND
ALS
ALS
ALS-FTD
ALS-FTD
Gene
SOD1
ALSIN
SETX
FUS
VAPB
DCTN1
Angiogenin
TARDP
CHMP2B
PRGN
Locus
21q22.11
2q33
9q34
16q21
20q13.3
2p13
14q11.2
1p36
3p11.2
17q21.32
Inheritance
AD (AR)
AR
AD
AD
AD
AD
AD
AD
AD
AD
Clinical Pattern
Classical
Young onset
Young onset
Classical
Varied
LMND
Classical
Classical
FTD and ALS
FTD and ALS
ALS3
ALS5
ALS7
ALS-X
ALS-FTD
ALS-FTD
?
?
?
?
?
?
18q21
15q15
20p13
Xp11-q12
9q21-q22
9p21.3
AD
AR
AD
XD
AD
AD
Classical
Young onset
Classical
Classical
ALS and FTD
ALS and FTD
AD, autosomal dominant; AR, autosomal recessive; XD, X-linked dominant;
FTD, frontotemporal dementia; LMND, lower motor neuron disease; SETX,
senataxin; DCTN1, dynactin.
ALS1/SOD1. The identification of mutations in the gene for superoxide
dismutase-1 (SOD1) in 1993 was a real breakthrough in our understanding
6
of ALS pathogenesis (43). It had been preceded by a publication of a
disease-linked locus on chromosome 21, found in families with autosomal
dominant ALS (44). The numbers of SOD1 mutations detected in ALS are
still increasing, and currently more than 150 different mutations are known
(38) (alsod.iop.kcl.ac.uk/als/summary /summary.aspx). As SOD1 toxicity is
the main subject of this thesis, a more detailed description follows.
ALS2/Alsin. In 2001, it was shown that mutations in the gene encoding a
184-kDa protein called alsin could cause a slowly progressing atypical ALS
mainly with UMN involvement (45;46). Mutations were also found in cases
of recessively inherited juvenile ALS. Alsin contains three guanine
nucleotide exchange factor (GEF) domains and is involved in the cycling of
GDP/GTP in GTPases (46). GTPases are involved in important intracellular
signaling such as the RAS pathway, and alsin has been shown to interact
with Rab5, a small GTPase involved in endosomal dynamics (47).
There are long and short splice products of the alsin gene transcript.
Somewhat surprisingly, Kanekura and colleagues showed that
overexpression of the long form of alsin was able to rescue cells from the
toxicity of mutant SOD1. The long form of alsin could interact physically
with mutant SOD1 but not wild-type (wt) SOD1 (48). Subsequently, alsin
has been coupled to glutamate signaling and excitotoxicity (49), and shown
to be involved in the survival of motorneurons through its interaction with
Rac1 (50;51). As most ALS patients with alsin mutations are homozygous
and most mutations are predicted to truncate the protein, it has been
suggested that ALS is caused by a loss of function (42). There are, however,
no clear signs of motor neuron disease in the four different alsin knock-out
models that have been presented (reviewed in (52)).
ALS4/Senataxin. In 2004, mutations in the senataxin gene were shown to
be involved in juvenile-onset, autosomal dominant ALS (53). In the same
year, it was shown that the autosomal recessive disease ataxia-ocular apraxia
2 was due to mutations in the same gene (54). The phenotype of senataxincaused ALS is quite atypical, with juvenile onset, distal weakness, and
muscle atrophy but normal lifespan (53). From sequence homology, the
protein has been suggested to function as a DNA/RNA helicase, and it has
been shown to be distributed in both the cytosol and the nucleus in multiple
cell types of the CNS (with highest density in the cerebellum and the
hippocampus) (55).
ALS6/FUS. Recently, mutations in the gene encoding the protein fused in
sarcoma (FUS) were shown to be responsible for the familial ALS variant
previously called ALS6 (56;57). A total of 16 mutations were described. FUS
is mainly a nuclear protein involved in transcriptional regulation and
splicing, with functions that partly overlap with those of TDP-43 (see below)
(58). In patients with FUS mutations, similar to TDP-43, the mutant protein
was shown to aberrantly accumulate and aggregate in the cytoplasm.
7
ALS8/VAPB. In 2004, Nishimura et al. published the identification of a
locus (20q13.3, called ALS8) associated with an atypical form of autosomal
dominant ALS with slow progression (59). Later that same year, they
reported that a mutation in a gene called VAPB is responsible for ALS8 (60).
They found the same mutation in several families with different phenotypes
including classical rapid-progression ALS, atypical ALS, and late-onset
spinal muscular atrophy (SMA)—another motor neuron disease. Since then,
screens for mutations other than the P56S mutation originally described have
been fruitless (61;62). These studies found polymorphisms present in both
ALS patients (e.g. the deletion mutant Delta S160) and healthy controls.
From this, it was speculated that the P56S mutation confers a specific
toxicity that is not conferred by other mutations (61;63). The normal
function of VAPB was initially suggested to involve regulation of the
unfolded protein response (UPR) in the endoplasmic reticulum, a function
that is lost with the P56S mutation (64). Furthermore, it has been shown that
the mutant protein interacts with wt subunits and causes them to coaggregate (64-66). This results in a dominant negative effect with decrease
in UPR even in the presence of wt protein. A reduction in the level of VAPB
protein has also been found in both ALS patients and SOD1 transgenic mice
without VAPB mutations, indicating that there may be a functional link
between general ALS pathogenesis, SOD1 toxicity, and VAPB protein
(66;67). Apart from actions involving the UPR, Tsuda et al. showed that a
domain of VAPB, including the P56S mutation, can be secreted and can act
as a trophic factor at Eph receptors. The aggregation of VAPB abolished
secretion and resulted in depletion of this non-cell autonomous function (68).
Dynactin. Dynactin is a multiprotein complex, required for functioning of
the microtubule motor protein dynein. In 2003, a G59S mutation in the
largest subunit, p150(glued), was found to be associated with familial ALS
(69). Mutational screening of p150(glued) has later revealed four other
mutations in SALS, FALS, and ALS that are coupled to frontotemporal
dementia (ALS-FTD) (70;71). In 2008, Laird and colleagues showed that
expression of mutant p150(glued) protein, but not the wt protein, resulted in
a motor neuron disease phenotype in transgenic mouse models (72). The
model showed defects in vesicular transport and motor neuron pathology
with axonal swelling. Another mouse model, aimed at impairing
dynactin/dynein function (through means other than point mutation), was
published the same year (73). Although showing neuronal pathology, the
mice never developed symptoms of motor neuron disease. Interestingly, the
authors showed that inhibition of dynactin/dynein function in mutant SOD1
transgenic mice resulted in extended survival, indicating that there was a
functional link. Earlier, two mouse strains, loa and cra1, with chemicallyinduced mutations shown to be located in the dynein gene, had been found to
show motor neuron disease (74). Co-expression of different mutant SOD1
proteins in these model mice has recently been shown to be beneficial (75-
8
77). The result was, however, not uniform for all the SOD1 mutant models
tested (77). Furthermore, mutant SOD1 has been shown to interact with the
dynactin/dynein complex, which is important for inclusion formation by
mutant SOD1 proteins (78). These authors also showed that overexpression
of a dynactin subunit can prevent the deposition of large SOD1 inclusions.
Angiogenin. Since the original finding of ALS-associated mutations in the
angiogenin gene in 2004 (79), several more have been identified in SALS,
FALS, and in ALS-FTD (80-86). Angiogenin belongs to the ribonuclease A
superfamily and has been shown to induce angiogenesis through four major
pathways: degradation of RNA, basement membrane degradation, signal
transduction, and nuclear translocation (87). Recent studies have indicated
that angiogenin mutations lead to loss of function, affecting several
pathways mentioned above, with consequences for neurons dealing with
hypoxia (82;88-92).
TARDP. Since the discovery of ubiquitinated TDP-43 protein in
inclusions from both FTD and ALS patients in 2006 (34), there has been an
explosion of reports regarding this protein and the relationship between the
two diseases. The gene TARDP, encoding the TDP-43 protein, is located on
chromosome 1 and is ubiquitously expressed in multiple splice variants
(reviewed in (93)). TDP-43 binds RNA/DNA and is involved in diverse
functions such as gene transcription, splicing, and mRNA stability. The
initial histopathological studies indicated that anti-TDP-43 staining of
ubiquitinated inclusions (UIs) was located in both the nucleus and the
cytoplasm of neuronal and glial cells, but that TDP-43 was excluded from
the nuclei and accumulated in the cytoplasm of affected cells (34;94). TDP43 was also shown to be cleaved, producing a 25-kDa C-terminal fragment,
and to be phosphorylated in extracts from patients but not from controls (34).
TDP-43 positive staining of neuronal and glial cell UIs was later shown to be
present in SALS patients, FALS patients without SOD1-mutations, and
ALS-FTD patients but not in ALS patients with SOD1 mutations (95;96) or
in SOD1 transgenic mouse models (97). This has, however, been challenged
by a more recent report based on transgenic mice (98).
During the spring of 2008, multiple—almost simultaneous—reports
appeared on mutations in the TARDB gene in SALS and in autosomal
dominant FALS (99-103). Since then, reports on findings of new ALSassociated mutations have cointinued to appear; today, 30 mutations in 22
unrelated families and 29 sporadic cases have been found (reviewed in (58)).
Although there are similarities in pathology between FTD and ALS
regarding TDP-43 positive ubiquitinated inclusions, only one ALS patient
with a TDP-43 mutation has so far been reported to develop cognitive
impairment (of Alzheimer type) (104). As to the mechanism(s) of toxicity of
mutant TDP-43 in ALS, there is still considerable confusion. Some recent
reports have come with possible explanations. Mutated TDP-43 and also its
9
truncated C-terminal fragment have been shown to sequester the wt protein
into cytoplasmic inclusions and thereby deprive the cell of TDP-43 activity
(105;106). In contrast, expression of different TDP-43 fragments in a cell
line showed that the C-terminal fragment alone was responsible for toxicity
without affecting the activity of full-length TDP-43 (107). An antibody
specific for the cleaved C-terminal fragment was also shown to selectively
stain neuronal cytoplasmic inclusions in tissues from ALS patients (107).
Furthermore, TDP-43 has been shown to stabilize neurofilament-light (NFL) mRNA through direct interaction, and it might therefore be involved in
the formation of neurofilament inclusions in ALS (108). Iguchi and
colleagues studied the effect of siRNA knock-down of TDP43 in Neuro-2a
cells (109). This resulted in reduced viability, which was at least partly due
to loss of regulation of Rho-family GTPases. As judged by the enormous
amount of work invested in such a short time, we are only at the beginning
of understanding these mechanisms, and the field will probably become
clearer in the near future.
CHMP2B. In 2005, mutations in charged multivesicular body protein 2b
(CHMP2B) were reported in families with FTD (110). Later, mutations were
found in one patient with PMA and in another with ALS-FTD (111).
CHMP2B is involved in the endosomal sorting complex (ESCRT) and
mutations were suggested to disturb endosome dynamics, as with alsin and
dynactin (111). Recently, it has been suggested that mutations in CHMP2B
inhibit autophagy, which is an important function for clearance of
aggregated proteins (112;113).
Progranulin. Mutations in progranulin (PRGN) were initially found in
cases of FTD and linked to ALS through one family with a history of both
diseases (114). Progranulin is a secreted growth factor with multiple
functions and has recently been shown to be involved in caspase-dependent
cleavage of TDP-43 (115). In a mutational screen of a spectrum of ALS
variants, PRGN mutations were found in one SALS patient and one patient
with ALS-FTD (116). In another study in Belgian and Dutch ALS patients,
mutations were found but sequence variants were also present in control
subjects (117). The authors instead suggested that allelic PRGN variants
modify the disease progression and might therefore be more correctly
regarded as a genetic risk factor.
Genetic risk factors
The distinction between a genetic risk factor and a causative genetic
alteration is not always simple, as many findings have been in single SALS
patients and not all inheritable mutations have complete penetrance. The
number of genes that have been associated with ALS and suggested to
modulate the risk in one way or another are numerous, and beyond the scope
10
of this review (recently reviewed in (118). I will briefly mention a few of the
more interesting findings.
Neurofilament-heavy subunit and peripherin. Neurofilaments (NFs) have
been implicated in ALS from pathological findings of accumulated NFs in
inclusion bodies (discussed above) and from NF overexpression and knockout models in mice that in some cases develop motor neuron deficits (119).
Different genetic alterations in the C-terminal domain of the gene encoding
the heavy variant of neurofilaments (NF-H) have been found in ALS patients
(120-122). Another intermediate filament, peripherin, generates a motor
neuron disease phenotype when overexpressed in mice (123). In mutational
screening of the peripherin (PRPH) gene in SALS and FALS patients, 20
polymorphisms were found. One gave a truncated protein and was found
only in one SALS patient (124). Another homozygous mutation has been
found in one patient (125). Although there is not a strong correlation
between ALS and neurofilament genes, these proteins appear to be important
in the pathogenesis in several ways, including aggregation and axonal
transport for example.
VEGF. Oosthuyse et al. demonstrated that neuronal knock-down of
expression of the gene for vascular endothelial growth factor (VEGF)
through deletion of the hypoxia-response element resulted in an adult-onset
motor neuronal disease in mice (126). This was followed by the finding that
polymorphisms in the promoter sequence of VEGF, causing lower levels of
circulating protein, increased the risk of ALS by 80% (127). Later studies
have not been able to demonstrate this strong correlation in other
populations, but they have found differences in risk attributed to gender
(128;129). Like angiogenin, VEGF is involved in response to hypoxia and
neovascularisation. The association of both of these molecules with ALS,
although not clearly ruled out, indicates that these mechanisms are
fundamental for motor neuron survival. Administration of VEGF through
injection of viral vectors or as a purified protein has also shown to modify
disease in a positive way in ALS transgenic mice (130;131).
SMN1/2. The protein survival of motor neuron (SMN) is found in the
cytoplasm and nucleus, and is involved in RNA processing through
assembly of small ribonucleoproteins into functional complexes (132).
Homozygous mutations in SMN1 are responsible for most cases of spinal
muscular atrophy (SMA), a motor neuronal disease with juvenile onset with
variable survival (but adult forms exist). Another copy of the gene, SMN2,
produces a homologous protein that can modulate disease depending on
copy number, with more copies being favorable for survival and resulting in
less aggressive disease. As SMA is caused by the selective death of motor
neurons, similar to ALS, the role of SMN in ALS has been addressed by
several studies. Although the results are divergent, there are indications that
SMN genetic variants that produce less SMN protein are a risk factor for
11
ALS (133-135). In co-transfection models with simultaneous overexpression
of SMN and mutant SOD1, SMN has been shown to upregulate chaperone
activity and to rescue cells from SOD1 toxicity (136). Recently, transgenic
mice expressing mutant SOD1 (G93A) were produced in an SMN
heterozygote deletion background (SMN +/-) (137). SMN -/- is
embryonically lethal but SMN +/- mice show no sign of motor neuron
disease. Co-expression of G93A/SMN +/- modulated disease, resulting in a
more aggressive ALS phenotype.
APOE. Patients with late-onset Alzheimer’s disease have an overrepresentation of the epsilon 4 allele of the apoliporotein E (APOE4) gene
(138). Several correlation studies in various populations of ALS patients
regarding APOE genotype have been conducted (139-145). All such studies
have shown that there is no increase in risk of ALS associated with APOE
genotype. However, APOE4 carriers have been suggested to have an
younger age of onset and a worse prognosis (139;142) while the opposite
appears to be true of APOE2/3 (140;145).
Promoter deletions in SOD1. In sporadic ALS cases, the homozygous
deletion of a 50-bp region of the SOD1 promoter has been shown to be
correlated with increased age of onset in cases from British and Irish
populations (146). The deletion resulted in a 50% lower promoter activity
when expressed in various cell lines, but a reduction in SOD1 protein levels
could not be confirmed in brain extracts of SALS patients.
Genome-wide studies
The recent advances in genetics have revealed new methods (e.g. microchip
technology) for screening of genetic material, which makes it possible to
find DNA sequence polymorphisms that are associated with disease in large
patient material. Genome-wide association studies (GWAS) use dense maps
of single nucleotide polymorphisms (SNPs) to scan the human genome for
disease-common alleles (147). This approach has been successful in
common diseases such as diabetes and breast cancer where the patient
material is huge, and during the last few years they have been performed on
different ALS materials (reviewed in (118;148)). The first GWAS performed
on ALS in 2007 was conducted in a material of 276 patients. Thirty-four
SNPs were found that were potentially associated with disease, but none
survived correction for multiple testing (Bonferroni correction) (149). Later
that year, a GWAS in a total of 1,337 European patients showed association
with a genetic variant in the inositol 1,4,5-triphosphate receptor 2 gene
(ITPR2), a protein involved in regulation of intracellular calcium
concentration (150). These and other studies in different populations has
been conflicting and unable to corroborate each other (151-156). Single
studies have reported an association with dipeptidyl-peptidase 6 (DPP6)
(154;155) and FGGY (153). These inconclusive results have been suggested
12
to be due to the problem of sample size in ALS, which will need to be
overcome by the use of global multicenter studies in the future (118;148).
In a recent, comparatively large study involving 1,821 SALS patients,
Landers and colleagues found an association between survival and alleles in
the KIFAP3 gene (157). Homozygosity for the allele was associated with a
long survival time but not with risk, site of onset, or age of onset. With a
slightly different genetic approach using microsatellite markers, another
recent study presented an association between a component of RNA
polymerase II (ELP3) and SALS (158). This is interesting in the context of
other RNA processing enzymes that have been implicated in ALS
pathogenesis, but the association must be confirmed in other studies.
Other risk factors
Besides genetic factors, age, and gender, the only risk factor that has been
found to be somewhat associated with ALS is smoking (21). This association
has been strengthened by a recent prospective study (159). As smoking
among women has increased during the last fifty years, this has also been
suggested to partly explain the shift in gender ratio towards equality. During
the last decade, an unforeseen clustering of ALS cases in professional soccer
players has drawn new attention to a possible link between extensive
physical activity and motor neuron disease (reviewed in (160)). The studies
performed do, however, suffer from small sample sizes and the possible
confounding effects of other common exogenous or lifestyle risk factors. In
a recent report comparing cohorts of professional soccer players, basketball
players, and road cyclists, a higher than expected incidence of ALS was
found only in soccer players, indicating that high physical activity per se
may not be a risk factor (161).
Excitotoxicity
Glutamate is the dominant excitatory neurotransmitter in synapses of the
brain and spinal cord. Upon release, glutamate interacts with various
presynaptic and postsynaptic receptors (NMDA, AMPA, and KA receptors)
and is finally taken up by astrocytes where it is turned into inactive
glutamine. This uptake is mediated through glutamate transporters known as
EAAT1 to -5, where glial EAAT2 is the predominant species in the CNS and
is responsible for removal of glutamate from the synapses. Excessive
glutamate results in toxic effects that are mediated by, e.g. Ca2+ influx and
increased oxidative stress (162). This pathway has been implicated in ALS
for a number of reasons. (I) Motor neurons of the ventral horn have been
shown to be especially vulnerable to excessive glutamate levels and
signaling through AMPA receptors (163-166). It has also been shown that a
subset of AMPA receptors, lacking a GluR2 subunit that makes them
permeable to Ca2+, might be specially toxic for motor neurons (167;168).
Activation of AMPA receptors by administration of agonists in vivo has also
13
resulted in motor deficits in mice (165). (II) An early finding was that
deficits in glutamate transport were specific to ALS patients in affected
regions of the spinal cord and motor cortex (169). This was shown to be due
to a loss of EAAT2 protein in ALS patients (170). Although controversial,
this has been suggested to be caused by aberrant EAAT2 mRNAs (171).
These species have also been found in controls, however (172). More
recently, high levels of glutamate have been found in the plasma of ALS
patients (173), as well as reduced glutamate uptake in platelets (174),
suggesting that alterations in glutamate levels are not confined to the CNS.
(III) In experiments on cultured cells from the cerebral cortex and spinal
cord, cerebrospinal fluid (CSF) from ALS patients has been shown to
mediate toxic effects (175-177). One measurable effect was an increase in
cellular Ca2+ levels (175) and these studies also showed that administration
of glutamate receptor inhibitors was protective. (IV) The finding that
riluzole, an antiglutamate agent, had an effect on disease progression was a
great breakthrough since no approved drugs existed (178). This effect was
also confirmed in later studies (179;180). How this antiglutamate effect is
produced is not fully known, but it has been suggested that levels of
glutamate in serum decrease after intake of riluzole (181), a finding that
could not be reproduced in a recent study (182). In cell culture experiments,
it has been shown that riluzole significantly increases the activity of
glutamate transporters such as EAAT2, with increased clearance of
extracellular glutamate (183). It has also been suggested that riluzole
regulates glutamate release and postsynaptic receptor activation, and inhibits
voltage-sensitive channels (162). The riluzole effect appears to be the
strongest evidence for the involvement of glutamate metabolism in ALS
pathogenesis. Whether excitotoxicity is a primary toxic event or just
secondary due to the neurodegenerative disease process requires further
study. How this correlates to other etiological pathways such as mutant
SOD1 toxicity also requires investigation.
14
Superoxide dismutase-1
The protein
Activity and subcellular distribution. Seventy years ago, Mann and Keilin
described a protein that was responsible for binding most of the Cu2+ in
bovine erythrocytes, which they called haemocuprein (184). It took another
thirty years before McCord and Fridovich discovered and reported the
function of this copper protein and renamed it superoxide dismutase (SOD,
later SOD1) (185). The function described, dismutation of superoxide, is a
copper-dependent reaction that dismutes two molecules of superoxide (O2·-)
into hydrogen peroxide (H2O2) in a two-step reaction:
O2·- + SOD-Cu2+
+
-
2H + O2· + SOD-Cu
O2 + SOD-Cu+
+
H2O2 + SOD-Cu
(Step 1)
2+
(Step 2)
Oxygen, a molecule crucial for all human cells, is used as substrate for
energy production mainly in the mitochondrial respiratory chain (MRC).
During these reactions, O2 is reduced to water in multistep reactions (186).
The superoxide anion is a free radical that can be formed when oxygen picks
up one electron in reactions involving the complexes of the MRC. Another
major source of superoxide is the group of membrane-bound enzymes
known as the nicotinamide-adenine dinucleotide phosphate (NADPH)
oxidases (several other cellular oxidases that produce superoxide are
known). These enzymes are highly expressed by cells of the immune system,
such as neutrophils, microglia, and macrophages, and are activated during
inflammation for example. Depending on the type of NADPH oxidase
(several Nox genes are known), superoxide can be formed both
extracellularly and intracellulary. It is of particular interest that recent
experiments have shown that massive activation of these enzymes takes
place both in SALS and in murine models of ALS (187), and Nox2 knockout (the predominant species in microglia) has been shown to slow disease
progression in ALS transgenic mice (187;188). The activation of NOX2 and
increased amounts of superoxide have also been shown to be positively
regulated by different mutant SOD1s when expressed in cellular models
(189). Superoxide is considered to be a moderately reactive molecule but it
reacts with FeS clusters, which occur in several enzymes. It can also be
highly toxic through its very rapid reaction with NO to form peroxynitrite
(ONOO-). Peroxynitrite reacts quickly with CO2 to form
nitrosoperoxycarbonate (NOOCO2-) a highly toxic species that can react
with virtually all macromolecules in the cell (e.g. through tyrosine nitration).
NO is produced by three different nitric oxide synthetases (NOS):
endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS
(iNOS) (190). Endothelial NOS produces the NO that is essential for
15
vascular homeostasis whereas nNOS is expressed in neurons (as well as in
other cells), where NO is involved in diverse processes, e.g. neurogenesis
and memory. Superoxide can also cause adverse effects by interfering with
these normal physiological functions of NO. The activities of eNOS and
nNOS are regarded as essentially constitutive whereas the level of iNOS
depends on different cellular stimuli such as infection, inflammation, and
ischemia. A number of pathological conditions have been shown to cause a
significant increase in iNOS, one of them being ALS (191;192), where high
NO levels have been speculated to contribute to pathogenesis.
OH·, by far the most reactive free radical, can be formed from O2·- and
H2O2 through reactions involving transition metals such as Fe2+ (or Cu2+);
this is known as the Haber-Weiss reaction.
O2·- + Fe3+
O2 + Fe2+
H2O2 + Fe2+
OH· + OH- + Fe3+
(Fenton)
The second reaction is also called the Fenton reaction. Fe3+ and Cu2+ can also
be reduced by other reductants such as ascorbate. As O2·- and H2O2 are
involved in reactions resulting in the formation of OH·, the cell needs
efficient systems for handling of these molecules. The oxidation of H2O2 into
water is catalyzed by several cellular enzymes (e.g. peroxyredoxins,
glutathione peroxidases, thioredoxin, and catalase) and is extremely rapid.
Superoxide can be scavenged by ascorbate but is mainly taken care of
through dismutation via SOD. The importance of this activity is highlighted
by the existence of two other SOD enzymes besides SOD1, manganese-SOD
(SOD2) (193;194) and EC-SOD (SOD3) (195). These three enzymes are
located in different compartments, with SOD1 being responsible for the
cytosol (194), the nucleus (193;194;196;197) and the intermembrane space
(IMS) of mitochondria (193;198), whereas SOD2 is located in the
mitochondrial matrix (194) and SOD3 is secreted into the extracellular space
(195;199). The subcellular distribution of SOD1 is, however, controversial
and the subject of discussions involving other compartments such as
peroxisomes (196;200;201) and the endoplasmic reticulum (ER)
(198;202;203). As the mitochondrion is one of the main sites for superoxide
production (204) it is not surprising that the matrix has its own enzyme in
combination with protection from SOD1 in the IMS. Deletion of SOD2 in
mice has also been shown to be lethal during the first days of life (205;206).
SOD1 knock-out mice, although not completely healthy (e.g. reduced
fertility, axonal repair deficiencies, increased oxidative stress, cataract), do
not display any severe phenotype (207-212). Similar to knock-out of SOD1,
knock-out of SOD3 has no obvious spontaneous phenotype (212;213). This
is apparently caused by adaptions in the germ-line SOD3 knock-outs. Partial
conditional knock-out of SOD3 in the lungs of adult mice leads to death
from lung failure in just a few days (214). The relatively mild phenotype of
16
germ-line SOD1 knock-out mice might also be explained by compensatory
adaptions.
Structure and properties. SOD1 is composed of two equivalent 153-aa
long subunits, each containing one Cu2+ ion and one Zn2+ ion, with a
molecular weight of roughly 16 kDa (whereby the enzyme is also known as
Cu, Zn-SOD). The gene is located on chromosome 21q22.11. SOD1 is
widely expressed, with the highest amounts in the liver and kidneys (215).
The core of the SOD1 structure is eight β-strands connected through seven
loops and arranged as a β-barrel (216;217). As mentioned, the Cu2+ ion is
involved in the dismutase reaction but is also important for structure/folding,
whereas the Zn2+ ion functions as a stabilizer (218). Four histidines (His46,
His48, His63, and His120) are the ligands for the binding of copper and
His63, His71, His80, and Asp83 are the ligands for Zn (219). Many ligands
are present in the Zn-binding loop IV (His48-Asp83). Apart from the
binding of the metal ions, the tertiary structure of the monomer is stabilized
through an intrasubunit disulfide bond between Cys57 and Cys146. The
presence of a structural disulfide bond is an unusual property of a protein
located in the reducing environment of the cytosol, and it may be an Achilles
heel of SOD1.
There is a specific copper chaperone (copper chaperone for SOD1, CCS),
responsible for Cu loading of SOD1 in eukaryotes (220). CCS knock-out
mice were shown to display reduced SOD1 activity and a phenotype similar
to that seen in SOD1 deficiency (e.g. reduced fertility) (221), but other
experiments have shown that human SOD1 can be Cu-loaded to some extent
by other means (222). SOD1 is one of the most stable proteins known, and it
retains most of its activity in 10 M urea, 4% SDS, and heating to 80°C
(218;223). This stability is totally lost in the apo state, when the protein is
deprived of metals and upon reduction of the intrasubunit disulfide bond
between cysteines 57 and 146. After loss of metals and reduction of the
disulfide bond, SOD1 is unable to dimerize and the monomeric form is
favored (224-226).
SOD1 in ALS
As discussed above, the number of SOD1 mutations found in ALS cases is
steadily increasing and today mutations are known in approximately half of
the codons (74 out of 153). Most cases are missense mutations, but insertion
and deletion mutations exist and for the purposes of this thesis, the
truncation mutation G127insTGGG (G127X) serves as an example
(discussed below). The mutations confer widely different effects on the
SOD1 molecule, with some—such as G127X—causing a severely truncated
protein without SOD activity and loss of Cu/Zn binding and normal folding,
whereas the common D90A mutant is fully active and properly folded.
Despite such diversity, these mutations result in a similar disease phenotype,
indicating the existence of an ALS-causing common toxic SOD1 species.
17
When caused by mutated SOD1, the disease is generally dominantly
inherited with varying penetrance. However, the most common of the
mutations, D90A, causes mostly recessively inherited ALS (39).
Interestingly, this mutation has been found to be inherited as a dominant trait
among FALS and SALS cases in populations where the mutation is rare
(227). About 20% of FALS has been reported to be caused by mutations in
SOD1, which represents about 2% of all cases (223;228). In a study of a
mixed population of ALS patients, the frequency of SOD1 mutations was
found to be 7.2% (38). This is in accordance with other findings of rather
frequent SOD1 mutations in apparently sporadic ALS (229-231).
The phenotype of mutant SOD1 ALS is generally that of typical ALS,
although some mutations are known to be associated with atypical features
such as long-term survival and bladder control disturbances (e.g. D90A)
(231). The similarities between mutant SOD1 ALS and typical ALS have
drawn attention to the wild-type SOD1 protein, and recent experiments
suggest that SOD1 may be involved in the majority of cases (232-238).
Transgenic murine SOD1 models
Since taking CNS tissue samples from living ALS patients is associated with
great risks, the need for model systems has been obvious. Different cell lines
expressing various mutant SOD1s are routinely used, but since the spinal
cord is a very complex tissue with multiple cell types, animal models are
often needed. Mice have a highly developed CNS that is in many ways
similar to ours, and genetic techniques have been used successfully for many
years in this species. Since the discovery of mutant SOD1-induced ALS,
various transgenic murine models have emerged. By far the most frequently
used one is the G93A mouse, which was first described in 1994 (239). The
widespread use of this model is mainly due to the conveniently short
survival time (about 125 days, depending on the copy number) and the
predictable course of the disease. These characteristics have made it the
model of choice for pharmacological trials and virtually all potential
therapies have been tested in G93A. The poor reproducibility between
preclinical G93A trials and human drug trials, including riluzole, has
recently pulled into question the suitability of the model and the relevance of
positive drug reports to date (240). From a pathological point of view, there
are similarities with human disease, e.g. ubiquitin-staining inclusion bodies,
motor neuron loss, and reactive gliosis. During disease progression, the
G93A mouse develops overt vacuolar pathology of the CNS (241). These are
speculated to be remnants of damaged mitochondria and are common to
other “high-level” models (discussed below), but are rarely found in human
ALS subjects. The G93A mutation has also been transgenically expressed in
rats (Rattus norvegicus), a model similar in most ways to the mouse strains
used (242). The next mutant mouse model to be published was that with the
G85R mutation in 1997 (243). This model develops an ALS-like disease
18
within approximately one year, despite having a mutant SOD1 level no
greater than the endogenous mSOD1 (i.e. low-level, as discussed below).
Histopathologically these mice differ from the G93A strains, as they lack
vacuoles and show SOD1 inclusions in astrocytes as well as in neurons. The
G127X mouse was generated in our laboratory and the mice are now bred as
homozygotes (35). This mutation was found in a Danish family and is an
insertion of four novel nucleotides after the glycine 127 codon
(Gly127insTGGG) (231). The insertion leads to five novel amino acids
followed by a premature stop codon. This protein is present at very low
levels, about half that in the wt mSOD1 situation. G127X mice live
approximately 216 days and succumb to motor neuron disease after an
extremely rapid disease progression (of less than one week). D90A mice
were also generated in our laboratory and, similarly to G93A, produce high
levels of mutant protein (234). Multiple mutant SOD1 transgenic mouse
models exist, but they will not be discussed further here. Several lines of
mice expressing wt hSOD1 have been generated (239;244;245). None of
them have displayed an ALS phenotype, but pathologically some similar
characteristics are apparent such as loss of neurons in the ventral horn,
vacuolization of mitochondria, and late accumulation of aggregated SOD1
(234;246;247).
There are several other animal models that express mutant SOD1s, e.g.
Drosophila melanogaster (248), Caenorhabditis elegans (249), zebrafish
(250), and pigs (unpublished). Although not extensively studied, these might
prove to be important in new ways. Recently, canine degenerative
myelopathy, a disease in dogs similar to human ALS, was found to be
caused by a point mutation in the SOD1 gene (251). Pathological
examination also revealed neuronal SOD1 inclusions similar to those seen in
human mutant SOD1 ALS cases.
How do mutant SOD1s cause disease?
Loss of function
It soon became obvious from multiple sources that loss of SOD1 function
does not cause ALS.
(i)
SOD1 knock-out mice do not present an ALS phenotype.
(ii)
Transgenic overexpression of mutant SOD1 leads to ALS
despite there being normal levels of endogenous wild-type
enzyme.
(iii)
Transgenic overexpression of most mutations results in several
times more functional SOD1 enzyme.
(iv)
Disease is almost always dominantly inherited, and some
mutations do not reduce the total SOD activity (e.g. D90A).
19
Thus, mutations in SOD1 confer a toxic property to the protein, which is
most likely of the same basic character for all mutants.
A non cell-autonomous toxicity
Several reports have suggested that the toxic effect of mutant SOD1 is noncell autonomous. This conclusion is based on the following findings:
(I)
Neuron-specific expression of mutant SOD1 does not generate
an MND phenotype (252;253). (This was challenged recently;
see below).
(II)
Astrocyte-specific expression of mutant SOD1 does not generate
an MND phenotype (254).
(III)
Chimeric mice that have been generated from mice expressing
wt and mutant SOD1 (mixed cell-types), develop motor neuron
pathology but do not develop a symptomatic MND. In these
mice, motor neuron pathology is dependent on a mutant SOD1
context (255). Another recent report has shown that selective
expression of mutant SOD1 in motor neurons and
oligodendrocytes surrounded by wild-type neurons results in a
delayed onset of disease (256).
(IV)
Transplantation of wild-type myeloid cells into G93A mice was
shown to slow down disease progression (257) and microglia
expressing mutant SOD1 were shown to have higher production
of ROS and nitric oxide than those expressing WTSOD1 (258).
(V)
During disease progression, an increasing amount of
lymphocytes migrating into the CNS has been reported (259)
and the embryonic knock-out of T-lymphocytes in G93A mice
has shown an accelerated disease progression (259;260).
(VI)
Extracellular mutant SOD1 has been shown to be toxic to motor
neuronal cells only when co-cultured with microglia (192)
(VII)
Removal of mutant SOD1 expression in neurons through the
Cre/lox system resulted in delayed onset and extended survival
but did not abolish MND (261). Using the same technique,
mutant SOD1 expression was removed from microglia (261) and
astrocytes (262), which slowed disease progression but did not
alter the time of onset.
(VIII) Somewhat contradictory, knock-down of mutant SOD1
expression in Schwann cells has been shown to enhance disease
progression (263).
20
(IX)
Deletion of mutant SOD1 expression in muscle does not alter
the phenotype, suggesting that muscle cells is not involved in
the disease (264).
(X)
The expression of mutant SOD1 in glial-type cells has been
shown to induce apoptotic mechansims in co-cultured
neuroblastoma cells and embryonal spinal motor neurons from
mutant SOD1 transgenic mice (265). Co-culture of wt SOD1
motor neurons and G93A glial cells from differentiated
embryonic stem cells has resulted in motor neuron damage,
indicating a soluble factor (266-268). It has also been shown that
mutant SOD1 can interact with chromogranins and thereby be
secreted to the extracellular compartment (269).
Recently, it has been shown that neuron-specific expression of high levels
of SOD1 is indeed able to produce an MND phenotype in transgenic mice
(270). Similar findings have been reported elsewhere (271). These results
argue against the initial findings (I) and they probably depend on the level of
expression.
In combination, these results argue that multiple cell types present in
motor areas modulate disease through different parameters such as disease
onset and progression. Although neuron-specific expression of mutant SOD1
might be sufficient, the natural form of the disease is probably dependent in
different ways on interactions between most cells of the CNS.
Aberrant redox chemistry and peroxidase activity
During the first decade of mutant SOD1 studies, it was speculated whether
mutant SOD1 could cause ALS through altered redox chemistry. It was
suggested that the active site copper became increasingly exposed and could
therefore participate in reactions with peroxynitrite and induce damage to
proteins by tyrosine nitration (272-274). Another similar theory suggested
that mutant SOD1 could catalyze copper-mediated conversion of hydrogen
peroxide to hydroxyl radicals, which—like peroxynitrite—is one of the most
reactive ROS (275). Other data contradict these theories. Several SOD1
mutations have either been shown or predicted to be deficient in copper
binding and to lack activity (35;243;276;277). An artificial mutant lacking
all four copper-binding histidines can still cause disease in transgenic mice
(278). In line with this, the knock-out of CCS did not ameliorate the MND
phenotype in mutant SOD1 mice despite substantial loss of SOD1 copper
binding (279). This theory has not been completely discarded, however, and
recently suggestions have been made that metal-deficient SOD1 can bind
copper or other transition metals in the zinc site, and thereby take part in
aberrant redox reactions (223;280). Recent studies have also suggested that
the excessive nitration of proteins, found in the G93A mouse model, may
contribute to disease (281).
21
SOD1 and mitochondria
As already mentioned, SOD1 was shown in the early 1970s to be localized
not only in the cytosol, but also in the intermembrane space (IMS) of
mitochondria (193). This partioning was due to an unknown mechanism, as
SOD1 does not contain a mitochondrial localization sequence. It was later
shown that SOD1 is translated in the cytosol and imported in a disulfidereduced state, without any metals bound (the apo state) (282;283). It was
also shown that CCS is present in the IMS to aid apo-SOD1 turn into mature
holo-SOD1, and thereby to get trapped in this compartment (282;284). The
mitochondrion is the major cellular site for energy production through
oxidative phosphorylation, and therefore also a potential site of generation of
oxidative free radicals (204). As mentioned, Mn-SOD is present in the
mitochondrial matrix (193) for scavenging of free radicals and it was
therefore not surprising that SOD1 was found to be localized in the IMS,
probably for the same purpose. It has been shown that knock-out of SOD1
leads to increased carbonyl levels in mitochondria, which can be used as a
marker of increased oxidative stress (284).
The first reports on mitochondrial pathology in ALS came from studies on
autopsy specimens and from functional measurements on peripheral tissues
of patients (reviewed in (285)). Later, similar studies carried out on CNS
tissue have revealed aggregated, swollen mitochondria and functional
deficits (286-289). This has, however, not been a uniform feature of several
other reports (290-292). Autopsy studies are also subject to several
drawbacks, such as the issue of post-mortem time, tissue preservation, and
the fact that only end-stage disease can be studied (285).
The real take-off in the subject of mitochondrial pathogenesis came with
the first reports of severe vacuolization of mitochondria present in the early
lines of mutant SOD1 transgenic mice (241;244). These vacuoles were later
shown by electron microscopy to be derived from expansion of the IMS
evident before the onset of symptoms (293-296). The process of
vacuolization was described by Higgins et al. to be distinct from earlier
reported vacuole processes (mitochondrial permeability transition (MPT) or
autophagy-derived), and it was referred to as Mitochondrial Vacuolation by
Intermembrane Space Expansion (293). It was also found that mutant SOD1
accumulates in vacuolated mitochondria and co-localizes with cytochrome
C, suggesting a direct toxicity through interaction (293;294). This idea is,
however, challenged by the findings that mice overexpressing wt hSOD1
also show vacuolar pathology and accumulation of wt hSOD1 protein
without any signs of ALS disease (234;241;244;246;247;294). Other work
has shown that not all SOD1 mouse models, e.g. the G85R and H46R
(243;297), display this pathology and that it is rarely found in autopsy
samples (298). In line with the mitochondrial pathology, several reports have
indicated that expression of mutant SOD1 in both cells and transgenic mice
22
results in alterations in mitochondrial function (299-302) similar to what was
originally found in human subjects.
After these initial findings of mitochondrial pathology, dysfunction, and
direct association with SOD1, the obvious question was how mutant SOD1
might be toxic to the organelle. Several somewhat divergent mechanisms of
toxicity have been suggested. These are not mutually exclusive but are more
or less linked.
(I) Mitochondrial accumulation of SOD1 protein aggregates. Several
studies have shown that mutant SOD1, and under certain circumstances wt
SOD1, can oligomerize and form protein aggregates on the outer surface of
the outer mitochondrial membrane or in the IMS (293;303-308). Another
report suggested that mutant SOD1 could aggregate in the mitochondrial
matrix (309). Although not a well-defined mechanism, the idea of SOD1 that
accumulates in mitochondria being toxic was supported by the report that
overexpression of CCS along with mutant SOD1 in transgenic mice induced
an even heavier mitochondrial load of SOD1 followed by a remarkable
decrease in survival time (310). These findings are the subject of some
debate due to findings by us (extensively discussed in paper 1) and others
showing that mutant SOD1s appear to accumulate (to a relative level higher
than mSOD1) in mitochondria in vivo only if the total cellular level of
mutant protein is extremely high (e.g. the G93Amouse model) (297).
(II) Mitochondrial release of apoptotic molecules. As mitochondria are
important in the regulation of apoptotic pathways, these have been studied in
several reports. Cytochrome C is a mitochondrial IMS protein that, upon
mitochondrial release, induces apoptosis through activation of cellular
caspases. The discovery of mutant SOD1 and cytochrome C co-localization,
(293;294) was followed by reports on its cytosolic release (311-313).
Cozzolino et al. showed that mutant SOD1 expression caused apoptosis
through a pathway involving Apaf1, a scaffold protein of the apoptosome (a
protein complex involved in activation of caspases) (311). When Apaf1 was
knocked out, apoptosis and mitochondrial pathology was abolished despite
similar SOD1 mitochondrial accumulation. Mutant SOD1 has also been
shown to interact directly with the anti-apoptotic protein Bcl-2 and, through
co-aggregation, to deplete cells of this important function (304). This finding
has not, however, been reproduced by another group (314). These and other
findings of increase in apoptotic mediators have been partly challenged by
experiments in transgenic mice expressing mutant SOD1 in which different
important apoptotic mediators have been knocked out: caspase-11 (315) and
BAX (314). Both of these mouse models develop an ALS phenotype despite
the inhibition of apoptotic pathways. Remarkably, BAX knock-out mice
were found to develop motor neuronal disease without loss of motor
neuronal cell bodies in the spinal cord. If apoptosis is involved in motor
neuronal death in ALS, clearly apoptotic routes other than these must be
involved.
23
(III) Increased production of reactive oxygen species (ROS). There have
been several reports on increase in oxidative stress and ROS produced from
mitochondria following expression of mutant SOD1s (300-302;306;316318). The precise source of ROS has not been established, but mutant SOD1
expression appears to inhibit the normal function of the electron transport
chain, either through direct interaction/aggregation or through secondary
effects, generating a higher leakage of free electrons and thereby more ROS
(discussed above). Increase in ROS triggers several pathways such as
cytochrome C release/apoptosis, induces damage to mitochondrial DNA,
causes protein modifications/alterations, and stimulates nuclear transcription
of genes involved in protection and inflammation (285;287;318-323).
(IV) Mitochondrial damage through cytosolic/non-cell autonomous
events. It has been suggested that binding of mutant SOD1 to proteins in the
cytosol, such as heat shock proteins (HSPs), causes depletion of and toxicity
to mitochondria (283;285;324). SOD1 has been shown to interact with a
lysyl-tRNA synthetase (mitoKARS) in the cytosol and to co-aggregate at the
cytoplasmic face of the mitochondrial outer membrane, thus contributing to
mitochondrial dysfunction (325). Recently, it has been shown that selective
expression of mutant SOD1 in astrocytes leads to mitochondrial pathology
and dysfunction in motor neurons, indicating that the toxicity can be
transferred from one cell to another (non-cell autonomous), possibly through
oxidative stress (326;327). Cassina et al. also showed that inhibition of
mitochondrial function in astrocytes caused by means other than mutant
SOD1 resulted in similar toxicity to motor neurons.
(V) Excitotoxicity. Excitotoxicity through extracellular glutamate
signaling (discussed above) is a well-studied pathogenic mechanism in both
in vivo and in vitro models (285). Mitochondrial dysfunction may enhance
the sensitivity to this assault even more in motor neurons than in other cells
(285;328). Mitochondria are the main storage site of intracellular Ca2+, and
dysfunction of Ca2+ regulation seems to be important for excitotoxicity
(329). Defects in intracellular Ca2+ regulation have been found in SOD1
mutant mice (330) and cell models (299;331).
(VI) Impairment of axonal transport. In motor neurons, adequate
anterograde and retrograde transport of mitochondria is necessary for
cellular function and energy metabolism all through their extremely long
axons. This is achieved through interaction with motor proteins such as
kinesin/dynein and movement along microtubules (332). There is recent
evidence to suggest that these movements are inhibited either by
mitochondrial dysfunction or through dysfunction of the transport systems
(reviewed in (332)). Disturbance of mitochondrial transport has been
reported in SOD1 transgenic mice (333;334) as well as in SALS patients
(289). This also suggests functional links between SOD1 toxicity and
mutations in dynactin (as discussed above). Furthermore, mutant SOD1 has
24
recently been shown to co-aggregate with and possibly cause depletion of
components of the anterograde transport system (335).
SOD1 aggregation and inclusions
As mentioned above, CNS tissues from FALS patients with mutant SOD1
and transgenic murine models show inclusions with SOD1 immunoreactivity
(36;336). The formation of cellular inclusions could be toxic as they may,
for example, trap essential proteins and block or interfere with cellular
functions (283;335). There is no consensus on how mutant SOD1 ends up in
inclusions, but most of the evidence points to impaired degradation,
misfolding, and aggregation (32;337;338).
SOD1 degradation. Although the routes of protein degradation in cells of
the CNS have not been fully worked out, proteasomal degradation and
autophagic/lysosomal degradation appear to be the two main pathways
(339). These systems are partly complementary, but the ubiquitinproteasome system (UPS) is mainly responsible for degradation of ubiquitintagged soluble cytosolic proteins and the lysosome for endocytosed or
autophagocytosed proteins (339).
Wild-type and mutant SOD1 has been shown to be degraded by the UPS
(337;340-343). Important mediators in this degradation have been found to
be the chaperones HSP70/HSC70 and the ubiquitin E3-ligases CHIP, dorfin,
and NEDL1 (342;344-347). Inhibition of the UPS accelerates aggregation of
mutant SOD1 and formation of cellular SOD1 immunoreactive (ir)
inclusions in cell models and tissue cultures with similarities to those found
in transgenic mice and FALS patients (337;343;348-350). Further on,
SOD1ir inclusions co-stain for ubiquitin, chaperones, and proteasomal
components (336;342), and SOD1 has been shown to be oligo-ubiquitinated
in cell models (351) and in vivo (35;352). Apart from the constitutive
proteasome, there is an inducible form called the immunoproteasome (353).
The activities of these two entities have been measured in tissues from
mutant SOD1 transgenic mice, and it appears that at least the constitutive
proteasome is inhibited (354-356). Reduced proteasomal activity has also
been found in cells expressing mutant SOD1 (351) as well as in mice
overexpressing wild-type protein (357).
Although the proteasomal route to SOD1 degradation seems important, its
involvement in ALS pathogenesis is not clear. Transgenic mice expressing
G93A SOD1 have been crossed with mice that are deficient in the
immunoproteasome (LMP2 -/-) (358). This model did not show a significant
alteration in phenotype. Furthermore, inhibition of proteasomal function in
organo-typical spinal cord cultures showed no difference in survival of
motor neurons between those expressing mutant SOD1 and those from
normal tissue (359).
25
Our knowledge of the role of autophagy in degradation of proteins of the
CNS was given a remarkable turnabout when mice deficient in this pathway
(atg7, atg5 -/-) were found to show severe neurodegeneration (360;361). The
disease was associated with neuronal pathology at several levels, with
protein aggregates and inclusion bodies. It was later shown that SOD1 is
partly degraded by macroautophagy (362) and that this route is altered in
G93A mice (363;364). Ligation of ubiquitin monomers to proteins targeted
for autophagic degradation is mediated by lysine 63 (K63) on ubiquitin.
When a ubiquitin K63 mutant was co-expressed with mutant SOD1, it
resulted in generation of more cellular inclusions (365).
Misfolding/aggregation. The formation of cellular inclusions and SOD1
aggregates could thus be caused by deficiencies in degradation and cellular
overloading. Other theories and complementary ones suggest that SOD1
aggregation is caused by misfolding of mutant SOD1 monomers either in
combination with deficient degradation, loss of folding of chaperones, or by
intrinsic aggregation—through aberrant disulfide coupling, for example. In
vitro studies of wt apo-SOD1 have shown that it can aggregate even under
physiological conditions (366;367), although the wt holo protein is regarded
as an extremely stable protein. These propensities to misfold may be
aggravated in mutant SOD1 (368-370). For nucleation of aggregation, the
least common denominator is disulfide-reduced, non-metallated SOD1
(367;367;367;371;372). Similar species have been identified in vivo, in a
series of transgenic mouse models (247;373). Although intermolecular
disulfide bonding exists in SOD1 aggregates both in vitro and in vivo
(247;366;374;375), resulting in SOD1 oligomers, mutation of all four
cysteines does not prevent SOD1 aggregation (371;376). C57-C146
disulfide-reduced SOD1 has also recently been shown to be the predominant
species in detergent-resistant aggregates (375). This highlights the
importance of this in vivo phenomenon and indicates that nucleation and
building of SOD1 aggregates derives from the constant pool of soluble,
disulfide-reduced apo-SOD1 (discussed in paper III).
The composition of SOD1ir inclusions has mainly been explored by
histochemical approaches in transgenic mice and FALS patients. Co-staining
of inclusions has been found for a number of proteins such as CCS, heat
shock proteins, ER chaperones, neurofilaments, kinesin-associated protein 3,
p38MAPK (202;298;335;336;377;378), and parts of the UPS mentioned
above (discussed in paper III). Analysis of detergent-resistant aggregates has
revealed similar proteins such as different heat shock proteins
(278;281;324;379). Proteomic approaches have recently emerged as a
promising tool for identification of aggregate composition, and they will be
discussed below.
Although it is clear that SOD1 aggregates/inclusions are present in most
murine models and ALS cases carrying SOD1 mutations, it is not clear
whether they are a toxic prerequisite. In G93A mice, overexpression of CCS
26
has resulted in a more aggressive phenotype without any signs of SOD1ir
inclusions (310). Other experiments, on inhibition of proteasomal function in
mutant SOD1 cell models for example, have induced SOD1 aggregation but
have not resulted in more pronounced cell death than in controls (380).
SOD1-interacting heat shock proteins. Apart from the proteins found to be
co-aggregated with SOD1 in inclusions, a number of proteins have been
found to interact with SOD1, through co-immunoprecipitation experiments
for example. These are mainly proteins from the family of heat shock
proteins (HSPs), a group of proteins involved in stress responses and protein
folding. The most frequently reported SOD1 interaction partner has been
HSP70/HSC70 (283;324;344;345). As mentioned above, HSP70 has been
proposed to bind misfolded SOD1 and mediate degradation or possibly
refolding. The initial reports stated that this interaction was indeed confined
to mutant SOD1, but later experiments have shown that oxidized wild-type
enzyme acquires mutant-like properties and binds HSP70 (232). Other HSPs
found to interact with SOD1 are HSP105, HSP90, HSP40, HSP25, and αβcrystallin (283;324;344;381). The heat shock response is crucial for the
cellular response to misfolded proteins, and it has been reported that these
pathways may be deficient in SOD1 transgenic mice (382). Furthermore, the
interaction of HSPs and SOD1 has been suggested to be the reason for
cellular depletion of chaperones, as they may be trapped in inclusions (283).
In this way, the initially beneficial SOD1/HSP interaction eventually leads to
loss of important functions and a vicious circle arises through generation of
more misfolded proteins. From the above-mentioned experiments, it was
suggested that induction, overexpression, or exogenous delivery of HSPs
could be beneficial in ALS. Overexpression of HSP70 in cultured cells has
been shown to reduce the amount of insoluble SOD1 aggregates (349) but
not to ameliorate lifespan in SOD1 transgenic mice (383). Administration of
recombinant HSP70 intraperitoneally in SOD1 transgenic mice has been
shown to be beneficial when treatment is started before the onset of disease
(384). In 2004, Kieran and colleagues reported that treatment with
arimoclomol, a co-inducer of heat shock proteins, significantly ameliorated
the disease in SOD1 transgenic mice (385). Although arimoclomol induces
expression of several HSPs, increases in HSP70 and 90 were the most
pronounced. This treatment has also been shown to reduce the number of
ubiquitin-positive inclusions (386). To be effective in survival, treatment has
to be started before or very early after onset of disease, which of course is
difficult to achieve in human ALS subjects (385;386). Still, this is one of the
most promising therapies and human trials are ongoing (387).
27
The “–omics” in ALS/SOD1 models
The expanding field of proteomic and genomic experimental methods has
introduced new ways of exploring pathogenic events without input of prior
knowledge. In genomics, microarrays confer the possibility of exploring
gene expression patterns in practically the whole genome—during
progression of a disease, for example. Proteomic techniques are still more
limited, but they may be more reliable since mRNA levels do not always
reflect the amount of protein finally produced. Genomics also misses the
whole area of post-translational modification of proteins. This and other
technical discrepancies have led to partly conflicting results between
genomic and proteomic findings. These issues will probably be solved some
time in the future and the different approaches shown to complement each
other. Here, will follow a brief review of “global” genomic and proteomic
experiments performed in the context of ALS, i.e. those involving total
changes in a tissue or cell.
Genomics
In recent years, several gene-expression studies using the microarray
technique have been performed both in transgenic mice (321;388-392) and
on autopsy material from human ALS cases (393-397). One serious
drawback of autopsy material is the difficulties associated with post-mortem
time and major cellular/mRNA alterations that take place after death. While
the early studies were performed on whole spinal cord tissue or parts thereof
(motor cortex in (396;397)) with a wide range of cell types, more recent
genomic studies have been specifically performed on motor neurons
collected by laser-capture techniques. As all of these studies have been
highly individual in terms of the techniques and models used, the findings
have been quite diverse regarding alterations in individual genes. The focus
here must be to find similarities in expressional regulation of pathways and
functional classes of genes. Most studies have found alterations in
mitochondria, in oxidative stress, in the citric acid cycle, and in
neurotransmission (GABA and glutamate metabolism). The effect on energy
metabolism appears to be a common denominator.
Proteomics
A series of global proteomic studies have been performed in slightly
different murine models of ALS. In a study on pooled homogenates from
terminal G93A mice, Strey et al. found seven proteins to be differentially
regulated compared to non-transgenic mice and mice expressing wt hSOD1
(398). These protein alterations were not present in asymptomatic mice.
Atkin et al. performed a study at disease onset in the G93A rat model (202).
Out of 15 proteins that were altered, 12 were identified of which some were
ER chaperones involved in the unfolded protein response (UPR). In a study
28
comparing G93A mice to transgenic mice expressing wt hSOD1, Massignan
et al. reported a mutant-specific change in 15 proteins in presymptomatic
mice (399). Similarly to the study by Atkin et al., they found upregulation of
the ER chaperone protein disulfide isomerase (PDI). However, most
alterations were in proteins that are involved in metabolism, particularly
from mitochondria. Lukas et al. performed a similar study on
presymptomatic and terminal G93A mice but also included non-transgenic
mice as controls (400). Using fractionation of spinal cord homogenates and
LC-MS/MS, they compared the proteome from different fractions on the
basis of functional categories (gene ontology analysis). This was preferred,
since LC-MS generates semi-quantitative data and the alterations in
individual proteins become less reliable. Interestingly, they performed a set
of microarray experiments for comparison of proteome and gene expression
data in the same model. As in other reports, the most obvious alterations
were found in mitochondrial pathways, results that were not, however,
corroborated by gene expression analysis. A study looking for differentially
expressed proteins in the hippocampus of mice overexpressing wt hSOD1
found changes in 41 out of 157 proteins identified (401). This model has a
high steady-state level of SOD1, much like the G93A model, and does
develop loss of motor neurons but not overt symptoms. Several of the
differentially expressed proteins involved mitochondria and the
cytoskeleton.
Using a cell model expressing different mutant and wt hSOD1s, with
proteomic techniques Di Poto et al. found 17 proteins to be differentially
regulated (402). Most proteins were involved in cellular metabolism and
antioxidant defense. The most serious drawback of studies with cell lines
may be the lack of a diverse cellular context, which in recent years has been
shown to be important for motor neuronal disease (403) (discussed above).
Proteomic methods have also been used for screening of potential
biomarkers in cerebrospinal fluid and plasma (404-409). Although no
consensus biomarker has been found, cystatin C may be a candidate as its
potential as a biomarker has been able to replicate in the CSF of ALS
patients.
Proteomics on SOD1-interacting proteins
Different methods have been used to try to identify SOD1 interaction
partners. Proteomic methods have recently given new insights in this area.
With the addition of a chemical cross-linker in lysates from mutant SOD1
expressing cells, Zhang et al. were able to immunoprecipitate and—by
proteomic methods (LC-MS/MS)—identify SOD1 interaction partners (410).
Interestingly, apart from known partners such as CCS and HSP70, the heavy
chain of dynein was implicated, thus linking mutant SOD1 with dysfunction
of motor proteins as suggested previously. Using a similar strategy,
Watanabe et al. used transgenic mice expressing FLAG-tagged truncated
29
SOD1 for immunoprecipitation and identification of SOD1 interaction
partners by LC-MS/MS (411). Thirty proteins were found to co-precipitate
with SOD1 in mutant mice, whereas only a few were present in homogenates
from wild-type expressing or non-transgenic mice. Wang and colleagues
immunoprecipitated soluble and insoluble fractions containing mutant SOD1
(G85R coupled to yellow fluorescent protein) from murine spinal cord (379).
The soluble fractions contained CCS and various HSPs, whereas the
insoluble fractions consisted mainly of cytoskeletal components. In a similar
study on spinal cords from terminal G93A mice, proteins in detergentresistant (insoluble) pellets were analyzed with 2-dimensional gels and
MALDI-TOF (281). They found 42 protein spots selectively accumulated in
insoluble pellets from G93A mice, as compared to a strain expressing wt
hSOD1. Shaw et al. performed a study on detergent-insoluble aggregates and
immunoprecipitated SOD1 aggregates for identification of co-aggregation
proteins in mutant SOD1 transgenic mice with different characteristics
(412). The only other protein found with reasonable consistency was
vimentin, suggesting that SOD1 was the main constituent of these species.
Metabolomics
In the search for possible biomarkers of ALS disease, the expanding field of
metabolomics may be more promising than genomic and proteomic
methods. These new methods deal with quantification and analysis of small
molecules (of 60–1,000 Da), such as peptides and degradation products,
derived from accessible extracellular fluids. Work in identifying biomarkers
in ALS is in progress (413).
30
Aims
•
To determine whether ALS can be ascribed to toxic effects
of mutant SOD1 accumulating in mitochondria. Are
different mutant SOD1 species, like the truncated G127X
protein, uniformly present in mitochondria? (paper I)
•
To relate tissue vulnerability to distribution of the
defective, truncated G127X mutant SOD1. Are the toxic
properties of mutant SOD1 expression confined to the CNS
in human ALS? (paper II)
•
To determine the composition of SOD1 inclusions in the
spinal cord of mutant SOD1 transgenic mice and the
properties of SOD1 protein trapped in them (paper III)
•
To gain insight into ALS pathogenesis from analysis of
changes in individual protein levels in the spinal cord of
SOD1 transgenic mice at onset of disease. (paper IV).
31
Methods
SOD1 transgenic mice
A variety of transgenic mouse models of ALS were used in the different
experiments discussed here. The high-expressing G93A mutant line
(G93AGur) was obtained from the Jackson laboratory, as were the mice
expressing wt hSOD1. The mouse strain expressing the G85R mutant was
obtained from Dr D.W. Cleveland. G127X and D90A mice were generated
in our laboratory (discussed above). Mice were deemed terminally ill when,
owing to limb weakness, they could no longer reach for food in the cages.
This occurred for the D90A, G85R, G127X, and G93A mice at an average
age of 407, 345, 216, and 124 days, respectively. The presymptomatic
D90A, G85R, and G127X mice used were around 100 days old, whereas the
G93A mice were around 50 days old. For the experiments in paper IV, mice
were collected at their peak body weight. This occurred at day 195 ± 14 for
the G127X mice and at day 325 ± 22 for G85R mice. As controls, we used
non-transgenic littermates from the G93A and D90A breeding program, or
occasionally pure C57BL/6JBomTac (B6) mice that were used for all
breeding.
G127X patient
With a post-mortem delay of only 10 h, CNS and peripheral tissue from a
Danish patient carrying the G127X mutation were quick-frozen at –80°C or
stored in formalin after the post-mortem examination. This patient had
showed rapidly progressing disease with truncal-thoracic onset and a
survival time of 6 months.
Homogenization of tissue
Generally, tissues were homogenized in 25 volumes of appropriate buffer. In
all experiments on organelles and inclusions with density gradient
separations, an iso-osmotic buffer was used (mitochondrial buffer, consisting
of 0.21 M mannitol, 0.11 M sucrose, 10 mM K HEPES, pH 7.2, 1 mM
EGTA, and the Complete antiproteolytic cocktail without EDTA (Roche
Diagnostics, Basel, Switzerland)). Tissues for immunoblot were supplied
with 25 volumes of PBS (10 mM K phosphate, pH 7.0, 0.15 M NaCl and
EDTA-free Complete antiproteolytic cocktail). For discrimination between
disulfide-reduced and oxidized SOD1, the homogenization buffer was
32
supplied with 40 mM iodoacetamide. For 2-D gels, the spinal cords were
homogenized in 10 volumes of lysis buffer (1 M Tris-HCl, pH 8.5, 2 M
thiourea, 7 M urea, and 4% CHAPS). For 2-D gels and immunoblot, tissue
was homogenized using an Ultraturrax (IKA, Stufen, Germany) followed by
sonication using a Sonifier Cell Disruptor (Branson, Danbury, CT) for 30 s.
For the preservation of organelles and separation of inclusions, a more gentle
approach using dounce glass homogenizers was used.
Density gradient separation
Separation of organelles from tissue extracts can be performed in different
ways, although density gradient centrifugation is the most commonly used
method. Other methods involve gel filtration and cell sorting/flow
cytometry. For density gradient separations, sucrose solutions have been the
medium that is traditionally used. The advantage has mainly been low cost
and availability, but for the separation of organelles the hypertonic
characteristics of sucrose gradients have been a great disadvantage. This
causes organelles to shrink due to osmotic water loss and affects their
density (414;415). As organelles such as lysosomes, mitochondria, and
peroxisomes exhibit very similar densities, hypertonic gradients make them
inseparable. Furthermore, the high viscosity of sucrose solutions requires
long centrifugation times at high speed. The introduction of non-ionic,
iodinated, high-density substances developed as contrast media for X-ray
purposes, such as metrizoic acid (415;416), brought a great improvement to
organelle separation. The latest addition, iodixanol (5,5’-[(2-hydroxy-1-3propanediyl)-bis (acetylamino)] bis [N,N’bis (2,3-dihydroxypropyl)-2,4,6triiodo-1,3-benzenecarboxamide]) has a molecular weight of 1,550 Da and a
density of 2.08 g/ml combined with a relatively low viscosity and low
osmolarity (417). These characteristics preserve organelle morphology and
increase the resolution of particles of similar density. Also, iodixanol is
compatible with antibody-based and spectrophotometric assays. Density
gradients are generally used as discontinuous gradients or continuous
gradients. Discontinuous gradients are made with a series of different
concentrations (at least two) of the medium that are put on top of each other.
In this way, particles of similar density are trapped at the interfaces between
gradient steps and can be easily identified. The major drawback in organelle
separation is the overlap in density distribution between different organelles
or other subcellular particles. A well-known example is the similarity in
buoyant density between mitochondria and lysosomes from liver tissue. The
problem can be partially avoided by the use of continuous gradients. In this
case, the gradients are premade, more or less linearly, depending on the
method used. We used the “two-chamber” technique, which is simple and
highly reproducible (418). The two chambers are loaded with iodixanol
diluted to the two appropriate densities representing the higher and lower
33
ends of the density range; here we used 12% and 38%. The chambers are
connected at the bottom, which allows passage of the denser medium into
the chamber of the lighter. A peristaltic pump draws the mixed medium from
the chamber of the lighter medium and delivers it to the bottom of a
centrifuge tube. During this process, the denser medium is mixed
consecutively with the lighter medium, bringing a denser mix to underlay the
medium already present (Figure 1).
Figure 1. Two-chamber set-up used for making linear gradients.
The preformed gradient is close to linear and the centrifugation will not
affect the slope in any significant way, at least during the short
centrifugation times used here. Although vertical and fixed-angle rotors are
efficient for gradient separation, swinging-bucket rotors are traditionally
used as the effect on the preformed gradient is minimal. These also minimize
the risk of material adhering to the walls of the tube, and they were therefore
used for all gradient separations.
For our separations, a 9-ml gradient was produced with a linear density
covering 1.07–1.21 g/ml using 12–38% iodixanol in mitochondrial buffer.
Homogenates were passed though a nylon filter of 5-µm pore size by
centrifugation at 20 g for 5–10 min. Filtered homogenates were supplied
with iodixanol to a final concentration of 8% to minimize the formation of a
thick lipid layer at the homogenate/gradient interface, and loaded on top of
the gradient. Centrifugation was carried out at 100,000 g for 90 min at 4°C
and fractions were collected from the bottom of the tube.
To separate mitochondria from peroxisomes, the homogenate was supplied
with 6 mM CaCl2, 5 mM succinate, and 5 mM K2HPO4 prior to separation in
order to cause permeability transition pore opening and swelling of the
mitochondria.
Marker enzyme assays
For determination of organelle separation in density gradients, enzymatic
activities or protein content (by immunoblot) of certain compartmentspecific markers were measured. For mitochondria, the activity of succinate
34
dehydrogenase (SDH) was determined. Samples were incubated with 100
mM Na HEPES, pH 7.4, 1 mM EDTA, 10 mM succinic acid, and 0.08% 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for
90 min. To stop the reaction, three volumes of 2-propanol were added,
followed by centrifugation at 14,000 g for 10 min. The absorbance of the
supernatants at 550 nm was then measured. For detection of lysosomes,
alkaline phosphatase activity was determined with p-nitrophenyl phosphate
as substrate. The activity of lactate dehydrogenase (LD) was measured using
the CytoTox96 kit (Promega, Madison, WI) and used as a cytosolic marker.
The distribution of other organelles was determined by immunoblotting.
Dot-blot assay
A filter-trap assay for aggregated proteins was used in papers I and III,
somewhat modified from that originally described by Wang et al. (419).
Fractions from separations were filtered through 0.15- or 0.2-µm cellulose
acetate membranes (Schleicher and Schuell, Dassel, Germany) in a dot-blot
apparatus. In paper I, the homogenates and separated fractions were supplied
with 0.1% SDS and gently sonicated before centrifugation and filtration.
This increased the sensitivity of the assay 25-fold. After filtration, the
membranes were subjected to immunoblotting. In paper III, the fractions
from density separations were subjected to filtration without any prior
treatment. Individual wells with trapped aggregates were then punched out
and incubated in 1 × SDS-PAGE sample buffer for immunoblotting or
DeStreak rehydration buffer (GE Healthcare) for 2-dimensional (2-D)
electrophoresis.
Antibody-based assays
Immunoblotting
Immunoblotting (western blots) were performed according to standard
procedures. Samples were diluted with the same amount of 2 × SDS-PAGE
sample buffer (100 mM Tris-HCl, pH 6.8, 10% β-mercaptoethanol, 20%
glycerol, 4% SDS, and bromophenol blue) and separated on SDS-PAGE
(Criterion™ Cell, Bio-Rad). When discrimination between C57-C146
disulfide-reduced and oxidized SOD1 was required (paper III), βmercaptoethanol was omitted from the sample buffer. Gels were blotted onto
polyvinylidine difluoride (PVDF) membranes and probed with primary
antibody. All secondary antibodies were conjugated with horseradish
peroxidase and chemiluminescence was generated with the addition of ECL
Advance substrate (GE Healthcare). Separated bands were visualized using
film and digital imaging (Chemidoc XRS, Bio-Rad). For quantification of
35
hSOD1, wt hSOD1 with its concentration determined by quantitative amino
acid analysis was used as original standard (420).
Histopathology
Tissues were fixed with 4% paraformaldehyde in phosphate-buffered salt
solution (10 mM K phosphate, pH 7.4, 0.15 M NaCl), dehydrated and
embedded in paraffin. Transverse paraffin sections were prepared for
immunohistochemistry or stained with hematoxylin/eosin. In paper II, liver
and kidney sections were stained with periodic acid-Schiff, van Gieson, and
reticulin stains and liver also with periodic acid-Schiff diastase, Fe, and
Faucet’s stain. For immunohistochemistry, enhancement using microwaveheating for 20 min in citrate buffer, pH 6.0, was performed and the sections
were blocked for 30 min in goat sera prior to the staining procedures. Single
immunohistochemistry was performed on the sections using the Ventana
AEC (Ventana Medical Systems, Tucson, AZ) and alkaline phosphatase red
immunohistochemistry system, according to the protocol of the
manufacturer.
Antibodies
SOD1 antibodies. The primary antibodies used for detection of human and
murine SOD1 were mainly polyclonal rabbit antibodies raised against
keyhole limpet hemocyanin-coupled peptides corresponding to amino acids
(aa) 24–39 (hSOD1-specific) or 24–36 (mSOD1-specific) (35). For analysis
of the G127X protein in human tissue, an antibody raised against the novel
C-terminus of the truncated protein was used (ADDLGGGQRWK, with
new amino acids highlighted) (35).
Commercial antibodies. The antibodies to lamin A/C, Tim23, and β-COP
(Golgi β-coatomer protein) were obtained from BD Biosciences (Franklin
Lakes, NJ), anti-PMP70 (70-kDa peroxisomal membrane protein) was from
Affinity BioReagents (Golden, CO) or Zymed (San Francisco, CA), and the
anti-synaptotagmin antibody was from Stressgen (Victoria, British
Columbia, Canada). Antibodies to HSC70, isocitrate dehydrogenase, UCHL1, isopeptide, and CNP were from Abcam (Cambridge, UK), antibodies to
Cop9s8 were from PTGlab (Chicago, IL), antibodies to FABP were from
R&D Systems (Minneapolis, MN), and antibodies to FIS1 were from Biosite
(Täby, Sweden). Anti-rabbit or anti-mouse IgG secondary antibodies were
from GE Healthcare (Uppsala, Sweden), anti-goat IgG was from Santa Cruz
(Santa Cruz, CA), anti-sheep IgG was from DakoCytomation (Glostrup,
Denmark), and anti-rat IgG secondary antibodies were from Abcam
(Cambridge, UK).
36
Size-exclusion chromatography
Tissues were homogenized after the addition of 20 mM iodoacetamide using
an Ultraturrax and sonication as described above. The iodoacetamide was
added to alkylate free thiol groups in proteins and low-molecular-weight
compounds. After centrifugation at 20,000 g for 30 min, the supernatants
were subjected to chromatography in 1 cm × 30 cm Superdex 75 columns
(GE Healthcare, Uppsala, Sweden) using PBS with 5 mM iodoacetamide.
The elution rate was 45 ml/h, and the eluate was collected in 0.3-ml
fractions. The SOD activities (421) and hSOD1 protein content of the
fractions were determined.
Proteomic techniques
Differential in-gel electrophoresis (DIGE)
DIGE uses the properties of different protein-binding fluorophores with
similar mobilities on SDS-PAGE. At present, three different fluors are
available, which makes it possible to separate two different samples along
with an internal standard on every gel in a 2-D gel experiment. This
minimizes the number of gels needed and the internal standard makes it
possible to compare a great number of different samples from different gels.
The procedure was performed according to personal communications and the
handbook of the manufacturer (GE Healthcare).
For each new DIGE experiment, the internal standard was generated by
mixing the same amount of protein from all the animals included. Quantified
homogenates were mixed with Cy2 (internal standard), Cy3, or Cy5 (0.25
mM in dimethyl formamide) and incubated on ice for 30 min in the dark.
Samples from transgenic or control mice were labeled with either Cy3 or
Cy5 in a random way, as suggested by the manufacturer. Reactions were
quenched by addition of 1 µl of 10 mM lysine (Riedel-De Haën AG, Seelze,
Germany) and incubated on ice. Cy2-, Cy3-, and Cy5-labeled samples were
mixed, to be run on a single gel, and incubated with the same amount of 2 ×
sample buffer for DIGE (1 M Tris-HCl, pH 8.5, 2 M thiourea, 7 M urea, 4%
CHAPS, and 40 mM DTT).
2-D electrophoresis
DIGE samples were run on 24-cm pI 3-11NL or pI 4-7 strips (GE
Healthcare, Uppsala, Sweden). The pooled samples were supplied with IPG
buffer (GE Healthcare) and DeStreak rehydration buffer (GE Healthcare),
and rehydrated into IPG strips for 12–16 h. Isoelectric focusing (IEF) was
performed on a Protean IEF cell (Bio-Rad) at 20°C and maximum current
was set to 50 µA/strip, with increase of the voltage stepwise until 35,000 or
45,000 Vh was reached (3-11NL and 4-7, respectively). Following IEF, the
37
strips were equilibrated for 15 min in equilibration buffer (6 M urea, 50 mM
Tris-HCl, pH 8.8, 30% (v/v) glycerol, 2% SDS, and 0.002% bromophenol
blue) containing 1% DTT and then for 15 min with 2.5% iodoacetamide in
the same buffer. The second dimension was run using the Ettan DALTsix
electrophoresis system (GE Healthcare) with low-fluorescence glass plates.
In paper III, unlabeled samples were run on 11-cm pI 3-11NL strips and
the Criterion™ Cell electrophoresis system (Bio-Rad) was used for the
second dimension. For analysis, gels were post-stained with a silver stain
(422). Briefly, the gels were fixed for 30 min in 40% methanol/10% acetic
acid, sensitized (with 30% methanol, 0.1% sodium thiosulfate, and 6.8%
sodium acetate), stained with silver nitrate (0.25% silver nitrate and 0.015%
formaldehyde), developed (2.5% sodium carbonate and 0.03%
formaldehyde), and the reaction was stopped in 1.46% disodium EDTA. For
Coomassie staining, the gels were fixed in 10% methanol/7% acetic acid for
1 h. Staining was performed overnight with G-250 staining solution (0.8%
phosphoric acid, 8% ammonium sulfate, 20% methanol, and 0.08% CBBG250), briefly washed with 25% methanol, and stored in deionised water.
Computer analysis
CyDye-labeled DIGE gels were scanned in a Typhoon 9410 imager (GE
Healthcare) using excitation and emission filters specified by the
manufacturer (Cy2, Cy3, and Cy5; excitation 480 nm, 540 nm, and 620 nm,
and emission 530 nm, 590 nm, and 680 nm, respectively). Further analysis
was done using DeCyder 6.5 software. Spot detection was done using the
differential in-gel analysis (DIA) module and data from this application were
then entered into the biological variation analysis (BVA) software for spot
matching between gels and variation analysis. Statistical significance was
calculated using Student’s t-test.
MALDI-TOF
Selected spots were picked manually and dehydrated with acetonitrile, dried
in a Speed-vac (Savant; GMI, Ramsey, MN) and finally incubated with
trypsin (Promega, Madison, WI). The protein digest was applied to a
MALDI-TOF target plate (Applied Biosystems) with α-cyana-4 hydroxycinnamic acid solution (Agilent Technologies, Santa Clara, CA). Calibrant
was spotted onto the plate and used for external calibration. The plate was
put in a Voyager DE-STR mass spectrometer (Applied Biosystems) and the
peptide spectra recorded. Peptide peak lists were generated using Data
Explorer software version 4 (Applied Biosystems). Proteins were identified
by searching the Swissprot and IPI databases using an in-house Mascot
server (Matrix Science Inc., Boston, MA). To obtain a Mowse score for the
G127X dimer, this sequence was added to the Swissprot database searched.
Search parameters were set to allow mass deviations of ± 50 ppm and one or
two missed cleavage sites, as well as fixed modifications such as
38
carbamidomethylation of cysteine and oxidation of methionine.
Identifications that were statistically significant (with a Mowse score in the
range of at least 60–66, p-value < 0.05), and that showed a gel position close
to the theoretical mass and isoelectric point, were regarded as positive.
LC-ESI MS/MS and data analysis
Samples were incubated in 10 mM dithiothreitol (DTT) and 6 M guanidine
hydrochloride with Tris, pH 9.0. Iodoacetamide was added and the samples
were kept at 37°C in the dark. The reaction mixture was filtered and washed
twice with 0.2 M ammonium bicarbonate and trypsin added. The resulting
peptides were collected by centrifugation, lyophilized, and resuspended in
0.1% formic acid.
Peptide analysis was done by reverse-phase liquid chromatography
electrospray ionization-tandem mass spectrometry as described by
Srivastava et al. (423) using a capillary HPLC system coupled to a
quadrupole time-of-flight mass spectrometer (CapLC Q-TOF UltimaTM,
Waters Corporation). ProteinLynx Global Server software version 2.2.5 was
used to convert raw data to peak lists for database searching. Proteins were
identified by a local version of the MASCOT search program and the
Mascot Daemon application (version 2.1.6) using the Swissprot protein
sequence database with mouse taxonomy. Peptides with Mascot ion scores
exceeding the threshold for statistical significance (p < 0.05) were selected.
Proteins were classified as having been identified either if at least two
peptides with a Mascot score above the statistically relevant threshold (p <
0.05) were found or if only one peptide achieved the required Mascot score
with at least four consecutive y- or b-ions and with a significant signal-tobackground ratio.
Network analysis
Proteomic studies generate huge amounts of data. To find common themes
among altered proteins, network analysis can be used. Ingenuity Pathways
Knowledge Base (IPKB) uses published information on interactions between
proteins and clusters the data into networks, and identifies cellular pathways
that may be significantly altered (www.ingenuity.com). The data set
containing protein identifiers (Swissprot) and corresponding expression
values was uploaded to the IPKB. Networks of the identified proteins were
then generated algorithmically along with identification of functional and
canonical pathways, and diseases in which the uploaded proteins are known
to be involved. Fisher’s exact test was used by the software to calculate a pvalue for biological functions and diseases. The significance of canonical
pathways was measured in 2 ways: (1) a ratio of the number of genes from
the data set that map to the pathway divided by the total number of genes
that map to the canonical pathway, and (2) Fisher’s exact test.
39
Statistics
Means and standard deviations were calculated using Microsoft Excel (paper
I). In paper IV, DeCyder 6.5 used Student’s t-test for calculation of p-values.
To minimize the risk of false positives due to multiple comparisons in paper
IV, orthogonal projections to latent structures discriminant analysis (OPLSDA) was used for multivariate statistical evaluation in the SIMCA-P_
software version 11.5 (Umetrics AB, Umeå, Sweden).
40
Results and Discussion
Paper I
Experiments in our laboratory had shown that the extremely high level of
SOD1 protein present in G93A, D90A, and wthSOD1 transgenic mouse
models of ALS was associated with incomplete Cu2+-charging of 30–50% of
the hSOD1. This was probably caused by CCS insufficiency (247). In
contrast to these models (called high-level models), other models expressed
SOD1 at similar rates (in terms of mRNA levels) but accumulated mutant
protein at levels that were similar to the normal level of mSOD1 or even less
(G85R, G127X) (low-level models). This is probably due to rapid
recognition for degradation, as these two mutant SOD1s are highly
destabilized by their mutations. At that point in time, one line of evidence
had suggested that mutant SOD1 preferentially localizes to mitochondria,
where it interferes with normal function and may be responsible for vacuole
pathology (293;294;303;304;309) (discussed above). It had also been shown
that SOD1 is imported to the mitochondrial IMS in the apo state, where it is
loaded with metals and disulfide-oxidized—at least partly due to activities of
CCS (282;283). As high-level models accumulate comparable high levels of
metal-deficient, C57-C146 disulfide-reduced subunits (247), we
hypothesized that this might lead to a propensity for excessive mitochondrial
accumulation. Furthermore, we wanted to explore whether destabilized
mutants like G85R and G127X would be retained inside the mitochondrial
IMS as they, at least in the G127X case, cannot be disulfide-oxidized and are
unlikely to bind the prosthetic metals. Ultracentrifugation in density
gradients is the method of choice for separation of organelles (as discussed
in Methods). We chose to work with continuous iodixanol gradients, as this
system offers the advantages of high resolution and iso-osmotic properties.
From these gradients, we were able to look at patterns of segregation rather
than accumulation of similar-density particles at interfaces.
As also shown by others, we found an extremely high association of
mutant SOD1s with mitochondria in the high-level models, and they reached
levels 100-fold higher than that of endogenous mSOD1. The quantity of this
association was similar for wild-type enzyme and mutant enzymes, with only
slightly higher levels for mutants, and there was no difference between
spinal cord and brain—or between time points (presymptomatic stage and
terminal stage). This showed that the mitochondrial association is due to an
intrinsic property of SOD1 that is not qualitively different between the wt
protein and mutant proteins. In the low-level G85R model, the relative levels
that appeared to co-fractionate with mitochondria were similar to what was
41
found for the wild-type murine enzyme in control mice (~2%). This
contrasted with the relative levels found in high-level models (> 9% of all
mutant SOD1) and suggested that the level of SOD1 protein and also the
disulfide-reduced/oxidized state are major determinants of mitochondrial
association. In presymptomatic G127X mice, very low levels were found at
the position of mitochondria (0.6%) and the pattern of gradient distribution
suggested that there was no co-fractionation. When the cytosolic marker
lactate dehydrogenase (LD) was measured, the contamination from this
compartment was at a similar level. In terminal G127X mice, the levels of
aggregated SOD1 distributed all over the gradient (showed by a filter-trap
assay) made it impossible to quantify any mitochondrial association but
there were no signs of apparent co-fractionation. These results did indeed
indicate that the truncated G127X protein does not accumulate in
mitochondria, probably due to its inability to achieve a holo state (i.e. to bind
the metals and form the disulfide bridge). We also explored the association
between murine wt SOD1 and mitochondria. In the controls and low-level
models, this was quantified to be approximately 2%, with no difference
between tissue (brain and spinal cord) and time point (presymptomatic or
terminal). In the high-level models, however, the relative associations were
similar to that of the mutant human enzyme: ~8–9%. In the study by Jonsson
et al., it was shown that the Cu2+ and CCS deficiency caused by
overexpression of mutant enzyme cause a similar amount of murine SOD1 to
be trapped in the apo state (at least disulfide-reduced) (247). These results
suggested that the accumulation of disulfide-reduced protein in these models
is responsible for the high association between SOD1s and mitochondria.
These findings were somewhat different to those in the report by Liu and
colleagues, who studied a similar set of transgenic animals (303). They
found preferential association of mutant SOD1 (but not wt hSOD1) with
mitochondria and an age-dependent accumulation and aggregation at the
cytoplasmic surface of the mitochondrial outer membrane. The discrepancies
are mainly due to different methods of separation. Liu et al. used
discontinuous gradients, which collect particles of similar density at
interfaces, in combination with differential centrifugations that are likely to
co-pellet heavy aggregates and mitochondria, resulting in artificial
association. As shown by us in this work, and as explored further in paper
III, all of these models accumulate high levels of aggregated SOD1 material
with age. These aggregates are trapped in inclusions that show overlapping
densities with all organelles when subjected to gradient separation in the
absence of detergent (paper III). In a recent study by the same group, these
authors tried to overcome the issue of aggregate co-fractionation by flotation
of mitochondria through loading of the sample at the bottom of the tube
(305). This is unlikely to overcome the problem, however, since such SOD1
inclusions behave like organelles, in terms of density, when not exposed to
detergents. A weakness of our study is that, despite gentle treatment, a
42
significant amount of mitochondria are trapped at the top of the gradient,
probably due to the sticky lipid materials released from CNS tissue. This loss
of separation might, however, be due to swollen or broken mitochondria
with low density. The accumulation of mutant SOD1 in such organelles
cannot be ruled out.
A recent study has shown that physiological association of wt SOD1 with
mitochondria is dependent on intramolecular disulfide bonding and
interaction with CCS, but that this regulation is absent in mutant SOD1s—
causing aggregation and intramitochondrial accumulation (308). Another
similar study has indicated the importance of misfolded/aggregated SOD1 in
mitochondrial toxicity and that the aggregation is partly dependent on SOD1
cysteines (424). As the association between mitochondria and aggregated
SOD1 is impossible to explore in terminal mice by density separation
methods, we cannot rule out the possibility that such interaction takes place
even in the context of G127X. In recent unpublished experiments, we have
been able to concentrate mitochondria by a filter-trap assay but we are still
unable to find any mitochondrial association in presymptomatic G127X.
Is the mitochondrial pathogenic hypothesis, then, a simple artifact in
transgenic mouse models? This is unlikely, since there is overwhelming
evidence from patients and transgenic mouse models that there are signs of
dysfunction and pathology in mitochondria (discussed above). In paper IV,
we also found evidence of mitochondrial dysfunction in the G127X mice.
Similarly to our own findings, in a recent immunohistochemical study on
H46R mice—another model with low-levels of SOD1 devoid of
mitochondrial vacuolization (but with other mitochondrial pathology)—the
authors were unable to find any association between SOD1 and
mitochondria (297). This strengthens the conclusion that models with
extreme levels of mutant protein are those that display mitochondrial
vacuoles. From my point of view, mitochondria are, according to these
findings, most probably involved in the pathogenic process of ALS but
might also suffer from unspecific damage due to high accumulation of
mutant SOD1. The main damage is not related to direct
interaction/accumulation of mutant SOD1 and mitochondria, but is due to
other mechanisms (as discussed above). In the G93A model where extreme
amounts of mutant protein accumulate in the IMS, this probably causes overt
pathology (e.g. vacuoles) and modulates disease with an effect on the
survival of motor neurons. Depletion of mitochondrial S-nitrosothiol, caused
exclusively by mutant SOD1s with intact copper binding, has been suggested
to be responsible for vacuole pathology in these kinds of models (425). The
toxic effect of G93A SOD1 overloading was recently shown to be a disease
modulator by simultaneous overexpression of CCS and G93A in transgenic
mice, leading to even higher loads of mitochondrial SOD1 and a marked
shortening of survival (310). This could be the explanation for why certain
43
therapeutic interventions in this model have had an effect on the course of
disease in mice but lack any effect in human trials.
Paper II
As discussed above, the G127X mutation was first described in a Danish
pedigree in 1997 (231). The insertion of TGGG after codon 127 leads to a
terminal stop codon preceded by five novel amino acids. This allows
generation of mutant-specific antibodies and analysis of mutant SOD1 in the
presence of wild-type SOD1 from the non-mutated allele. In control humans,
SOD1 levels are by far the highest in the liver and the kidneys (215). When
mutant SOD1 levels are measured from different tissues in low-level
transgenic mice such as G127X, the highest protein levels accumulate in the
spinal cord (247). As the spinal cord is the main tissue affected by ALS, the
vulnerability may be caused by inadequate recognition and degradation of
misfolded SOD1. From studies of CNS tissues from a G127X patient, it had
previously been shown that mutant protein accumulates at very low levels
and is mainly seen in inclusions (35). In paper II, we studied both CNS
tissue and peripheral tissues from a second G127X patient who succumbed
to ALS.
Similar to what has been found in G127X mice, we found the highest
levels of mutant protein to be in the CNS. By far the highest levels were
found in the vulnerable ventral horns. The amount was estimated to be about
8% of the total amount of SOD1 protein. The wild-type SOD1 levels were
highest in liver and kidney, as expected. To explore the properties of mutant
and wild-type protein further, tissue homogenates were subjected to density
gradient ultracentrifugation as described in paper I. As in brain tissue of
terminal G127X mice, we did not find detectable levels of dense SOD1
material in tissue from the frontal lobe. From ventral horns, about 50% of the
mutant protein entered the gradient with a pattern identical to that seen in
spinal cord of terminal G127X mice. Interestingly, similar to spinal cord, a
substantial amount of the G127X mutant protein from homogenates of liver
and kidney entered the gradient. Thus, aggregated mutant SOD1 can also
appear in peripheral tissues. From histopathological examinations using the
G127X-specific antibody, we found SOD1 inclusions in motor areas of the
ventral horn as well as in hepatocytes and kidney tubular epithelium.
These results suggest that degradation of G127X protein is subject to
difficulties, not only in the CNS but in peripheral tissues. In quantitative
terms, this accumulation is by far the most abundant in the ventral horns of
the spinal cord. Whether aggregated/inclusion SOD1 reflects the disease
process or actually causes it is, however, unclear (discussed above). If
defined as a marker of disease, the ALS pathogenesis might also involve
peripheral tissues but at a much slower rate and obvious organ dysfunction
might not be prominent during the lifespan of a human. This particular
44
subject had signs of chronic liver disease, for which we found no definite
explanation. Future studies in peripheral tissues will be able to tell us
whether ALS is a disease that is restricted to the CNS alone or if some signs
of damage appear elsewhere.
Paper III
In the density gradient ultracentrifugation studies of spinal cord
homogenates from terminal transgenic mice, we had found that dense SOD1
material was distributed all over the gradient (paper I). This material was not
present in presymptomatic mice and was aggregated, as it could be captured
in a filter-trap assay. Aggregated SOD1 is known to accumulate in large
inclusions in both ALS transgenic mice and human ALS patients carrying
SOD1 mutations and it has been suggested that this process could be
deleterious, e.g. through depletion of essential proteins. Pure protein
aggregates have a very high density and had been shown to accumulate in
the bottom of the tubes when the homogenate was subjected to detergent
(paper I). The composition and properties of such detergent-resistant
aggregates have been extensively analyzed before (discussed in introduction)
and the difference between such entities and what we refer to as “inclusions”
are described in figure 2.
Firstly, we wanted to address the physical character of SOD1 inclusions
from murine models expressing mutant SOD1 of widely different character
(G93A, D90A, G85R, and G127X). The inclusions were shown to be
distributed all over the gradient at densities lower than expected from pure
protein aggregates, and to be disrupted upon addition of weak detergent.
SOD1 protein in inclusions was not accessible to antibodies without the
addition of detergent, but it was sensitive to digestion by a proteinase. This
suggested that the SOD1 protein is not present on the surface of inclusions
and that it is not completely covered by a continuous lipid membrane, for
example.
In presymptomatic high-level models G93A and D90A, large amounts of
SOD1 protein enter the gradient. This material was shown in paper I to
accumulate mainly in mitochondria. To characterize the properties of SOD1
in the gradient of these high-level models in greater detail, non-reducing
immunoblotting was used to discriminate between SOD1 species with the
C57-C146 disulfide bond oxidized and reduced. It was shown that SOD1
with the disulfide bond intact closely followed the distribution of
mitochondria. In presymptomatic G85R and G127X models, only minute
amounts entered the gradient.
In gradients from terminally ill mice of all models, large amounts of
mutant protein entered the gradient, some at higher densities than in
gradients from presymptomatic animals. When subjected to weak detergent
for disruption of organelles and filter-trapping followed by non-reducing
45
immunoblotting, only disulfide-reduced SOD1 and, in some cases, another
SOD1-species with a non-native disulfide bond, were trapped, showing that
these are the predominant species in aggregates. A minute portion of the
aggregated SOD1 was found to be ubiquitinated and about 30% was shown
to oligomerize or bind to other proteins through disulfide coupling.
Figure 2. Biochemical isolation of inclusions. There are different routes for
isolation of aggregated materials. The standard procedure involves different
detergents and sequential centrifugations producing a detergent-resistant
pellet (middle). In contrast to simple centrifugations (left), more or less
stringent protocols produce a reproducible pellet containing detergentresistant material free from detergent-soluble cellular components. In
contrast, density gradient centrifugations on homogenates free from
detergents (right), enables the separation of in vivo particles containing both
soluble and detergent-resistant materials with much less contamination than
though a pure centrifugation protocol.
46
In G85R and G127X mice, a proportion of the inclusions were denser than
most organelles. These dense SOD1 inclusions gave us the opportunity to
study their composition without any major contamination from organelles
and cytosolic proteins. When separated on 2-D gels, apart from SOD1, 10
proteins not present in the controls could subsequently be identified using
MALDI-TOF MS. The most abundant was mutant SOD1, representing at
least 50% of the total protein. Two were cytoplasmic chaperones, 4 were
cytoskeletal proteins, and 4 were proteins that normally reside in the
endoplasmic reticulum. Most of these proteins had been found to interact or
co-aggregate with SOD1 before, but the ER chaperone calreticulin had not
previously been found to associate with SOD1.
When tissue sections are stained for mutant SOD1s, inclusions are clearly
visible in motor neurons, astrocytes, and in the neuropil. The entities isolated
here most probably represent a mixture of such inclusions. The set of
proteins found to co-fractionate with SOD1 also shows that the inclusions
are not only derived from motor neurons, but also from glial cells. The fact
that the pathology induced by mutant SOD1s involves not only motor
neurons but also glial cells is well established (non-cell autonomous), and
the presence of inclusions in other cell types has been reported before
(35;243;336). It is possible that there may be different inclusions, with high
densities that overlap, formed in the terminal phase of the disease—some
containing SOD1 and some not. Although they might possibly be different
cellular entities, they might all include aggregated material that could deplete
the cell of important functions.
Several immunohistochemical studies have explored the composition of
ALS inclusions. The most frequently described inclusions from SOD1
transgenic mice have been ubiquitinated inclusions (UIs, described in
introduction) in motor neurons and astrocytic hyaline inclusions (AST-His).
These are also present in both sporadic and familial ALS patients. In mice,
UIs are frequently SOD1-positive but they also stain for proteins such as
CCS, heat shock proteins (HSPs), GRP78, neurofilaments, NEDL1, dorfin,
proteasome 20s, EAAC1, and p38MAPK with varying frequency
(336;342;347;377;378). AST-HIs contain similar proteins, but with the
astrocytic markers αβ-crystallin and GFAP at high frequency (243;426). The
relationship between such histological entities and the biochemical isolates
from these experiments is not easy to determine, however.
Paper IV
Proteomic techniques allow screening for pathogenic alterations with
minimal input of prior knowledge. Use of the latest techniques makes it
possible to look at thousands of proteins at the same time. Differential in-gel
electrophoresis (DIGE) enables the separation of two different samples and
an internal standard on the same 2-D gel using pre-labeling with
47
fluorophores of similar molecular weight (discussed in more detail in
Methods). We hypothesized that expression of mutant SOD1 in transgenic
mice affects several different pathways in affected cells. One way of
exploring these events simultaneously is to subject tissue homogenates from
suitable time points to DIGE. We chose to work with G127X mice, as their
mutant protein level is only half the level of the endogenous mSOD1, thus
reducing the risk of overexpression artifacts (discussed in paper I). As time
point, we choose their peak body weight, as this has been shown to reflect
the onset of symptoms where the systemic effect of motor neuron disease is
still small (383).
The DIGE gels revealed about 1,800 protein spots that could be processed
further. From multivariate analysis, we found that about 420 protein spots
contributed significantly to the difference between controls and transgenics.
This is more than expected and suggests that 20–25% of proteins are altered
by pathogenesis in affected tissue. We can only speculate that the majority of
these alterations probably reflect compensatory actions taken for the survival
of the cell. From another angle, this suggests that protein alterations are
taking place in most cells of the spinal cord and not only in motor neurons.
This is in line with recent reports of SOD1 pathogenesis being non-cell
autonomous.
Owing to limitations of 2-D gels and MALDI-TOF, 53 significantly
altered protein spots could be reliably identified. As it is inevitable to
encounter false-positive hits in multiple comparisons such as this, we used
stringent exclusion criteria. The DIGE handbook suggests that differences
down to 10% can be viewed as reliable, but we chose 25%. The use of
stringent statistics with a univariate p-value of 0.01 and a multivariate
correlation of p(corr)p > 0.07 reduced the risk of false positives significantly.
In fact, when the different samples were assigned randomly (controls and
trangenics mixed), virtually no significant spots appeared using these
criteria.
For interpretation of the 53 protein alterations identified, the material was
subjected to analysis using Ingenuity pathway knowledge base (IPKB,
described in Methods). One of the main findings was the representation of
altered mitochondrial proteins. We had shown in paper I that the G127X
protein does not associate with mitochondria (a finding strengthened in
paper III), but still mitochondrial functions are clearly altered in this model.
This suggests that the mitochondrial involvement may be secondary to
primary noxious action of mutant SOD1s elsewhere. Furthermore, we found
alterations in the NRF2-mediated stress response, a pathway involved in
protection from oxidative stress. Regulation of proteins involved in oxidative
stress has been reported previously (427;428). It is particularly interesting
that all proteins found in this pathway were upregulated. A previous geneexpression study on NSC34 cells expressing mutant G93A found NRF2 and
some downstream targets to be downregulated (429). This was also reported
48
in a study on isolated motor neurons of G93A rats (430). As our study only
detected downstream targets of this oxidative response, we can only
speculate that (I) this response might be heavily upregulated in other cells of
the spinal cord but still be downregulated in motor neurons, or that (II) the
isolated system of motor neuronal/NSC34 cells does not adequately reflect
the in vivo situation, or that (III) the upregulation of the proteins in this study
is due to stimulation of another pathway similar to NRF2. In a study by Kraft
et al. (431), double transgenic mice expressing mutant SOD1 with a reporter
for NRF2 activation were generated. When these authors followed NRF2
activation in different cell types during the course of the disease, they found
a clear activation of this antioxidant response in several cell types of the
spinal cord. This correlates well with our finding, and might indicate that the
NRF2-mediated response activation in transgenic mice is dependent on an
accurate cellular context. One consequence of increased oxidative stress is
protein oxidation, which has been shown to be elevated in G93A mice (432).
Protein degradation was another pathway that appeared to be affected by
mutant SOD1 expression. Proper degradation of protein is essential to cells,
particularly for neurons as they lack the ability to regenerate. The UPS is one
of the major degradation pathways (discussed in introduction), and here it
was found to be differentially regulated. Oxidation and altered expression of
UCH-L1 has been found in brains from both Alzheimer’s and Parkinson’s
disease cases by proteomic methods (433). The UCH-L1 upregulation found
here is supported by an earlier report from our group showing intense
immunostaining in motor neurons and astrocytes of a G127X patient (35).
The COP9 signalosome complex is a multifunctional complex involved in
apoptosis, protein degradation, and hypoxia (reviewed in(434)). An
important function of the signalosome is to inhibit ubiquitination through
deneddylation of cullin-type E3 ubiquitin ligases. As subunits 4 and 8, which
are scaffold proteins in this complex, were found to be upregulated, this
could indicate inhibition of ubiquitin ligase activity and of proper protein
degradation.
In immunoblots of samples from G127X patients and transgenic mice, a
SOD1 band is present at double the molecular weight of the monomeric
species (35). A similar band has been seen in other SOD1 transgenic mouse
models, and also in ventral horn extracts from SALS cases (233;247). This
species was identified in the DIGE gels, and MALDI-TOF analysis revealed
a spectrum including only G127X SOD1 peptides. This indicates that the 32kDa band seen in immunoblots is a covalently-coupled SOD1 dimer,
previously uncharacterized.
In conclusion, we found alterations in cellular pathways such as oxidative
stress, mitochondrial function, and protein degradation. This is in agreement
with earlier proteomic studies on the G93A model, although few specific
proteins overlap (399;400). We also found evidence for a novel dimeric
49
SOD1-species that might be common in ALS regardless of genetic
background.
50
Conclusions
Paper I
•
High-level mutant SOD1 models accumulate more than
100 times more SOD1 protein in mitochondria than nontransgenic mice, probably due to high levels of metaldeficient, disulfide-reduced SOD1 species. There is no
obvious quantitative difference between brain and spinal
cord or between presymptomatic stage and terminal stage.
•
The truncated G127X protein is not retained in the
mitochondria of presymptomatic animals.
•
The difference in mitochondrial accumulation of SOD1
between different mouse models correlates with differences
in mitochondrial pathology (such as vacuoles) and
indicates that these may represent overexpression artifacts.
•
If mitochondrial deficits are universally present in mutant
SOD1 ALS, this is apparently not caused by mitochondrial
accumulation of SOD1 protein.
Paper II
•
Aggregation of mutant SOD1 is not confined to the CNS of
a human ALS patient, but is also evident in peripheral
tissues such as the liver and kidney.
•
Defects in recognition and degradation of misfolded SOD1
might explain the vulnerability of motor areas.
Paper III
•
SOD1 inclusions are complex entities containing both
detergent-resistant and detergent-susceptible protein
aggregates, and they are not enclosed by a continuous
membrane.
•
Disulfide-reduced SOD1 protein is the major species in
SOD1 inclusions from the spinal cord of transgenic mice.
•
Besides cytoskeletal proteins, the main constituents of
SOD1 inclusions are protein chaperones, normally residing
in the ER. This is in line with recent reports on the
51
involvement of the ER-controlled
response in the pathogenesis of ALS.
unfolded
protein
Paper IV
•
Expression of the G127X mutant of SOD1 in transgenic
mice affects the expression of up to 25% of other proteins
in the spinal cord. Alterations in important mitochondrial
pathways, response to oxidative stress, and protein
degradation are most prominent.
•
The G127X protein gives rise to a previously
uncharacterized, covalently-coupled dimer. This species
may be formed by other SOD1 mutants and wild-type
SOD1 and might be a common modification in both ALS
patients and transgenic mouse models.
52
Acknowledgements
To my supervisor, Stefan Marklund. I feel favored to have been
guided by someone with such genuine knowledge and devotion. Your
leadership is a true model for how to handle confused students and
make them feel as if they have actually achieved something.
To Peter Andersen and Thomas Brännström, my co-supervisors.
Thank you for ideas, fruitful discussions, and inspiration.
Per Zetterström, my room-mate and travel companion for many
years. Thank you for the discussions about work and other important
areas of life. Looking forward to the next big party you need to
accomplish in a short time. Andreas Jonsson, my unofficial
supervisor. You are a brilliant scientist and your extensive teaching in
practical and theoretical science has made a great difference. Karin
Graffmo, for handling all the mouse issues and for being an excellent
travel companion across the world. Karin Forsberg, for all your help
with histochemistry and for just being such a great fellow Ph.D.
student. I know that all your work will soon pay off and that you will
finally get your reward.
Eva Bern and Agneta Öberg, the two number-one western-blotters of
the world. You have been so important for this work that to try to
quantitate your efforts would look silly. Thanks also for the very nice
atmosphere you have created in the SOD lab.
Micael Granström, for being an excellent room-mate – where are
you? Former lab companions Anna Rehnmark andLena Larsson,
for productive collaboration. Heather Stewart and Monica Vestling,
for interesting discussions.
All former and present collaborators in the SOD lab. The atmosphere
mentioned above can also be attributed to you. Jörgen Andersson,
Anna Birve, Caroline Dahlberg, Sabine Eriksson, Ingalis
Fransson, Johan Bergh, Karin Hjertqvist, Ann-Charlotte Nilsson,
Eva Olofsson and Ulla-Stina Spetz.
53
The Mikael Oliveberg group in Stockholm and Robert Byström in
Umeå: thank you for making protein folding an issue for those who
don’t understand it.
To all the wonderful administrators at the department of Medical
Biosciences whom I have worked with during these years. Birgitta
Berglund, Karin Bodén, Åsa Lundstén, Terry Persson, Iris
Svanholm, and Monica Sörman. Your helpful attitude and
knowledge are some of this department’s most valuable treasures.
To all the technical stuff who handle our numerous mice. Elisabeth
Nilsson, Karin Wallgren, and all the others at UTCF and GDL.
Kjell Granqvist, Parviz Behnam-Motlagh, Johan Hultdin, and the
others at 6M, 2 tr.
My collaborators: Henrik Antti – for brilliant statistics. Thomas
Kieselbach, Gunnar Wingsle, and Vaibhav Srivastava for
proteomic expertise and helpful discussions.
All my friends at Umeå Vineyard, especially families Skog, Rydin,
and Petersson. You underestimate your own importance.
To my family in Örebro: Pappa, Mamma, Jacob, Anna, Johannes,
and Ellinor. To Runar, Christina, Ann-Christine, Bosse, AnnaCarin, and Klas. And to all my friends back home in Örebro. For
more than ten years I have longed to say: “we are coming home”.
To my heavenly Father, without whom the quest would never end.
Most of all to my wife, Jenny, and my two wonderful children,
Hanna and Ludvig. You know that this would never have been
possible without your loving support.
54
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