Mutant SOD1 and familial ALS:
from the molecule to man
Milan (Italy) September 13-16, 2007
MARIO NEGRI Institute
Organizers
Caterina Bendotti; bendotti@marionegri.it
Maria Teresa Carrì; carri@Bio.uniroma2.it
Alberto Ferri; a.ferri@hsantalucia.it
Lavinia Cantoni; cantoni@marionegri.it
Scientific Committee
Caterina Bendotti
Maria Teresa Carrì
Stefan L. Marklund
Giuseppe Rotilio
Pamela Shaw
Joan S. Valentine
Secretariat
Annalisa Volpe; a.volpe@hsantalucia.it
Dear Colleagues and Friends,
It is a great pleasure for us to welcome you to this first meeting
on mutant SOD1 and familial ALS in Milano, Italy.
The conference is focussed on the selective neurotoxic properties
of mutant SOD1, from biochemical and biophysical data in vitro to
results in vivo from studies in animal models and patients.
Hopefully, this meeting will offer the opportunity to review and
discuss existing evidence and new hypotheses on the mechanisms of
mutant SOD1-linked ALS, and encourage scientific collaboration. We
are certain that this will be of help both to all the neuroscientists
already working in the field and to those that are now joining in the
attempt to understand the cause of this rather obscure disease.
Since the conference is running in Milano, we are also pleased to
be able to offer you the exclusive opportunity to visit the famous “Last
Supper” painted by Leonardo da Vinci from 1495 to 1498.
We wish to thank all participants, all the people who have
assisted us in the organization of the meeting and our sponsors that
made it possible.
Last, but not least, many thanks are due to Prof. Silvio Garattini,
director of the Mario Negri Institute, for kindly hosting the meeting.
Caterina Bendotti
Maria Teresa Carrì
2
Scientific Program
Thursday 13 September
Registration 3.30 pm
Welcome Ceremony 5.30 pm
Caterina Bendotti, Maria Teresa Carrì
Silvio Garattini ( Director of Mario Negri Institute)
Mario Melazzini ( President of AISLA)
Opening lecture 6.00 pm
S1 - Giuseppe Rotilio (Italy)
From erythrocuprein to SOD1: memories of a Biochemist.
Welcome party 7.00 pm
Friday 14 September
Session I 8.30 am – 10.45 am
Biochemistry of Mutant SOD1s
Chairpersons: J.S. Valentine – G. Rotilio
S2 - Joan S. Valentine (USA)
How do ALS-associated mutations in superoxide dismutase 1 promote aggregation
of the protein?
S3 - Mikael Oliveberg (Sweden)
ALS-associated SOD1 mutations preferentially reduce the proteins repulsive charge
S4 - Lucia Banci (Italy)
Interplay between structural and dynamical properties and protein aggregation in
SOD1
S5 - Joseph S. Beckman (USA)
Direct measurement of zinc-deficient SOD localized the ventral gray matter in G93A
SOD rats
Selected oral communication
SO1 - Albrecht Clement (Germany)
Dismutase-inactive mutant SOD1 has different functional properties when
heterodimerized with wild type SOD1
Coffee break (10.45 am -11.15 am)
3
Session II 11.15 am – 1.00 pm
Mutant SOD1s toxicity in cell models
Chairpersons: S.L. Marklund – M.T. Carrì
S6 - Pamela Shaw (UK)
Mutant SOD1 in motoneuronal cells: mechanisms from the genomic and proteomic
profile
S7 - Gen Sobue (Japan)
Mutant SOD1s and protein aggregation/degradation in motoneuronal cells
S8 - Maria Teresa Carrì (Italy)
Mutant SOD1s and mitochondria in motoneuronal cells
Selected oral communication
SO2 - Mauro Cozzolino (Italy)
Cysteine 111 affects aggregation of mutant SOD1 in NSC34 cells
Lunch (1.00 pm – 2.00 pm)
Session III 2.00 pm – 4.15 pm
Mutant SOD1s toxicity in animal models
Chairpersons: Z.S. Xu – W. Robberecht
S9 - Stefan L. Marklund (Sweden)
Soluble misfolded SOD1 species in ALS
S10 - Jean-Pierre Julien (Canada)
From secretion of SOD1 mutants to immunotherapeutic approaches
S11 - Jasna Kriz (Canada)
Live imaging of disease progression in SOD1 mutant mice
S12 - Valentina Bonetto (Italy)
Proteomics of spinal cord aggregates in G93A SOD1 transgenic mouse
Selected oral communication
SO3 - Avi Chakrabartty (Canada)
Detection of misfolded SOD1 in sporadic and familial ALS
Coffee break (4.15 pm – 4.45 pm)
4
Session IV 4.45 pm – 6.45 pm
Genetics of Familial ALS
Chairpersons: C.E. Shaw – A. Chiò
S13 - Peter M. Andersen (Sweden)
Genetics of SOD1-linked fALS
S14 - Christopher E. Shaw (UK)
Genetics of non-SOD1-linked fALS
S15 - Stefania Battistini (Italy)
SOD1 in Italian ALS patients
S16 - Wim Robberecht
The pathogenesis of sporadic amyotrophic lateral sclerosis
Selected oral communication
SO4 - Richard Wroe (UK)
The Evolution of the Amyotrophic Lateral Sclerosis Database (ALSOD)
Social Event - Visit to Leonardo’s “Last Supper” 7.30 pm
Social Dinner – 8.30 pm
Saturday 15 September
Session V 8.30 am – 10.15 am
Motor neuron death in SOD1-linked fALS: intra-cellular mechanisms
Chairpersons: L. Cantoni – P. Pasinelli
S17 - Piera Pasinelli (USA)
The double life of two pro-survival proteins: when SOD1 and Bcl-2 get mutated, wild
and pro-death
S18 - Claudia Crosio (Italy)
Role of Bcl2a1 in SOD1-linked fALS
S19 - Angelo Poletti (Italy)
The nucleus as target for mSOD1 toxicity
S20 - Massimo Tortarolo (Italy)
Role of MAP kinases cascade in SOD1-linked fALS
Selected oral communication
SO5 - Haining Zhu (USA)
Interaction between familial ALS-linked SOD1 mutants and the dynein complex
5
Coffee break (10.15 am – 10.45 am)
Session VI 10.45 am – 12.30 noon
Motor neuron death in SOD1-linked fALS: inter-cellular mechanisms
Chairpersons: M. Bentivoglio – L. Barbeito
S21 - Luis Barbeito (Uruguay)
SOD1 mutations and astrocyte dysfunction in ALS
S22 - Marina Bentivoglio (Italy)
Neuroinflammatory signalling in the motor circuit in SOD1-linked fALS
S23 - Davide Trotti (USA)
Post-translational processing of the glial glutamate transporter EAAT2 in ALS
Selected oral communication
SO7 - Giambattista Bonanno (Italy)
Excessive exocytotic release of glutamate in the spinal cord of SOD1 G93A mutant
mice
Lunch (12.30 noon – 1:30 pm)
Session VII 1:30 pm – 3.15 pm
Motor neuron death in SOD1-linked fALS: remote contributions
Chairpersons: J.P.Loeffler – A.Musarò
S24 - Antonio Musarò (Italy)
Muscle control of motor neuron survival and activity
S25 - Linda Greensmith (UK)
Axonal Transport deficits in models of motor neuron disease
S26 - Jean-Philippe Loeffler (France)
Energy homeostasis and denervation processes at the neuro-muscular junction in
ALS
Selected oral communication
SO8 - Valeria Padovano (Italy)
Effects of physical exercise and the steroid nandrolone on neuromuscular junctions
in G93A -SOD1 transgenic mice
Coffee break (3.15 pm – 3.45 pm)
Session VIII 3.45 pm – 5.45 pm
Poster Session
6
Sunday 16 September
Session IX 8.30 am – 12.30 noon
Therapeutics in SOD1 mutant mice
Chairpersons: V. Silani – C. Raoul
S27 - Cedric Raoul (Switzerland)
Lentiviral and adeno-associated viral vectors for motoneuron gene therapy
S28 - Zuoshang Xu (USA)
An RNAi approach to understanding the mechanism and therapy of ALS caused by
mutant SOD1
S29 - Vincenzo Silani (Italy)
Translational research: possible explanations for the failure in ALS
Coffee break (10.00 am -10.30 am)
S30 - Jonathan Glass (USA)
SOD1-People: It's time for a trial
S31 - Lucie Bruijn (USA)
Drug discovery and development for ALS
Selected oral communication
SO9 - Rita Perlingeiro (USA)
The potential of endothelial progenitors to recover the stem cell niche in ALS
Closing remarks
Caterina Bendotti (Italy)
________________________________________________________________
Individual departure (Sandwiches available)
7
Invited Lectures
8
S1
From erythrocuprein to SOD-1: memories of a Biochemist
Giuseppe Rotilio
Department of Biology, “Tor Vergata” University of Rome. Italy
Recollecting steps of his lifelong experience over decades of biochemical research on Cu,Zn SOD,
the author will touch upon some findings obtained in the Rome laboratory, which scan the evergreen
primacy of this green protein on different stages of biochemistry, even with changing pen – names.
Born as a humble vehicle for copper in blood and tissues (erythrocuprein), grown up unexpectedly
to an unrivalled leading role as superfast enzyme, top model for metal–binding protein centres and
primary defence against ROS (superoxide dismutase), then rejuvenated in the play of metal traffic in
the cell (cytocuprein), it is presently recognized as an invaluable key to understand biochemical
mechanisms of life, death and disease (SOD 1).
9
S2
How do ALS-associated mutations in superoxide dismutase 1 promote aggregation
of the protein?
Joan Selverstone Valentine
Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095-1569, USA.
More than 100 different mutations in the gene encoding copper-zinc superoxide dismutase (SOD1)
cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease in
which aggregation of the SOD1 protein is widely believed to be the primary mode of pathogenesis.
We have studied the mutant SOD1 proteins using two different approaches: characterization of
expressed and purified protein and analysis of SOD1 in transgenic mouse spinal cords. In vitro,
mutant SOD1 proteins show remarkably diverse structures, activities and native state stabilities.
Some of the mutations cause enormous changes while others seem to have no measurable effect on
its biophysical and biochemical properties. To address the question of how such widely differing
proteins can all cause the same disease, we have turned to analysis of the SOD1 aggregates
isolated from the spinal cords of transgenic ALS mice. Analysis of spinal cords from three different
strains of ALS-SOD1 mutant mice by mass spectrometric and shotgun-proteomic methods revealed
that the SOD1 proteins that are recovered from the isolated aggregates are predominantly the fulllength SOD1 polypeptide. The recovered SOD1 proteins did not bear post-translational modifications
such as ubiquitination, carboxymethylation, or extensive oxidation. The aggregates were composed
almost entirely of SOD1, although various proteins that are highly abundant in spinal tissue were
found in very low amounts, along with SOD1. The significance of these findings for ALS will be
discussed.
10
S3
ALS-associated SOD1 mutations preferentially reduce the proteins repulsive charge
Mikael Oliveberg
Department of Biochemistry and Biophysics, Stockholm University, Sweden.
We provide bioinformatical evidence that protein charge plays a key role in the disease mechanism of
amyotrophic lateral sclerosis (ALS). Analysis of 100 ALS-associated mutations in Cu/Zn superoxide
dismutase (SOD1) shows that these are site-selective with a preference to decrease the proteins net
repulsive charge. For each SOD1 monomer this charge is normally –6. As biomolecules as a rule
maintain net negative charge to assure solubility in the cellular interior, the result lends support to the
hypothesis of protein aggregation as an initiating event in the ALS pathogenesis. The strength of this
preferential reduction of repulsive charge is higher in SOD1-associated ALS than in other inherited
protein disorders.
11
S4
Interplay between structural and dynamic properties and aggregation in SOD1
Lucia Banci
CERM & Department of Chemistry, University of Florence, Italy
SOD1, in its mature form, is characterized by several post-translational modifications, such as metal
binding, disulfide bond formation, and quaternary structure, i.e. a dimeric state, all of which are
essential for a fully active enzyme. These features are interlinked and affect the conformational and
dynamic properties of SOD1 as well as dramatically affect SOD1 tendency to aggregate.
SOD1 has been suggested to be related to the onset of Familial Amyotrophic Lateral Sclerosis
(FALS) as a very large number of single point mutations have been found in FALS patients. It has
been also suggested that the pathogenic action of SOD1 mutants resides in their gain of a toxic
function, the latter being related to their tendency to form protein aggregates. One of the striking
features of these mutants is that they are spread all over the protein sequence without any apparent
general correlation between their nature and location and the potential effects on SOD1 properties.
We have characterized the structural and dynamic features of various SOD1 states, both for WT and
mutants, in terms of metallation and oxidation, and we have investigated the interlinks/correlations
among these properties(1, 2). We have identified some of the key factors which induce protein
aggregation in SOD1. They are independent of the mutation and operative also in WT SOD1. This
knowledge allowed us to suggest a general mechanism for SOD1 aggregation, which can reconcile
the common behavior of such a diverse set of mutations (1).
1
Banci, L.; Bertini, I.; Girotto, S.; Martinelli, M.; Vieru, M.; Whitelegge, J.P.; Durazo, A.; Valentine,
J.S., submitted.
2
Banci, L.; Bertini, I.; Boca, M., Girotto, S.; Martinelli, M.; Vieru, M.; unpublished data.
12
S5
Direct measurement of zinc-deficient SOD1 localized in the ventral gray matter in
G93A SOD rats
Beckman JS, Nylin K, Morré J and Arbogast B
Linus Pauling Institute, Environmental Health Sciences Center, Oregon State University
The loss of zinc from both wild type and mutant SOD offers a compelling hypothesis to explain how
SOD causes the selective degeneration of motor neurons in ALS. Using 0.5 mm punches of frozen
spinal cord and carefully optimized methods using electrospray-ToF mass spectrometry, we can
quantify SOD with its metals fully retained by a rapid procedure. We found that zinc-deficient SOD
accumulates to be nearly equamolar with Cu,Zn SOD (each was approximately 15 µM) specifically in
ventral gray matter of G93A SOD transgenic rats and mice at the time when disease symptoms first
develop (around 60 days). There was remarkably little evidence of protein aggregation until the
animals are terminal. Zinc-deficient SOD is not present in dorsal spinal cord or in the brain of the
same animals. X-ray structures show the loss of zinc causes both the electrostatic loop and the zinc
binding loop become disordered, causing the copper to become more exposed as well as affecting
the dimer interface. The increased redox activity of copper in zinc-deficient SOD depletes motor
neurons of ascorbate and glutathione and increases formation of peroxynitrite and tyrosine nitration.
The increased oxidative and nitrative stress catalyzed by zinc-deficient SOD is exceeding toxic to
motor neurons. In addition, zinc-deficient SOD makes astrocytes reactive, which would contribute to
the progressive death of motor neurons.
13
S6
Mutant SOD1 in motoneuronal cells: gene expression profiling to unravel
mechanisms of cellular injury.
Janine Kirby, Laura Ferraiuolo, Paul Heath, Hazel Holden, Pamela Shaw.
Sheffield Care and Research Centre for Motor Neuron disorders, Academic Neurology Unit, Section
of Neuroscience, University of Sheffield, UK
Despite intensive research effort, the causes of motor neuron injury in the presence of mutant SOD1
remain incompletely understood. Microarray analysis has been applied to the problem of motor
neuron degeneration. Early studies employed tissue analysis of whole spinal cord, in which changes
in the motor neuron transcriptome are masked by changes in other cell types. Recently, laser
capture microdissection has allowed the identification of gene expression changes in specific cell
types.
The initial aims of the functional genomics programme at the University of Sheffield are to: 1. Identify
the cell specific properties of motor neurons and differences in gene expression between groups of
MN vulnerable and spared in the disease process in ALS; 2. To understand the cellular pathways of
motor neuron injury in the presence of mutant SOD1 at different time points; 3. To identify targets for
drug therapies.
A cellular model (NSC34 cell line stably transfected to express mutant or normal human SOD1) and
the G93A mSOD1 mouse model have been employed. Gene expression changes are verified in
human ALS using optimally processed CNS tissue from patients. Motor neurons are isolated from
spinal cord sections using the Arcturus Pixcell laser capture microdissector. RNA is extracted using
Picopure kit (Arcturus), amplified (where necessary) using the RiboAmp Amplification kit (Arcturus)
and labelled using the BioArray High Yield RNA g cRNA is applied to the r Transcript Labelling Kit
(Enzo). 10elevant Affymetrix GeneChip, and data analysis is performed using ArrayAssist System
(Iobion); Pathway Architect and GenMAPP1.1.
New insights arising from 2 key studies will be discussed:
1. Transcriptome changes in the NSC34 cell line in the presence of mutant SOD1. 268 genes were
differentially expressed and key insights were the overall transcriptional repression and the downregulation of the Nrf2 anti-oxidant response element genes. The development of this pathway as a
target for neuroprotective therapy with Nrf2 inducing drugs will be illustrated.
2. Gene expression changes in motor neurons at 3 disease time points in the G93A mouse model
have been investigated. Key changes at the pre-symptomatic stage (252 genes differentially
expressed) include a marked upregulation of lipid and carbohydrate metabolism and mitochondrial
function, as well as genes involved in transcription and translational functions. At the late disease
stage (120days) 167 genes are significantly altered, with the development of marked transcriptional
repression, but up-regulation of complement system components and key cyclins involved in cell
cycle regulation.
Gene expression profiling of motor neurons in health and disease generates greater understanding of
the cell specific features of motor neurons and the cellular pathways which become dysfunctional
during motor neuron injury. These pathways represent rational targets for neuroprotective therapy
development.
14
S7
Mutant SOD1 and protein aggregation/ degradation in motorneuronal cells
Niwa J, Yamada S, Ishigaki S, Sone J, Takahashi M, Katsuno M, Tanaka F, Doyu M, Sobue G
Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan
Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene cause familial amyotrophic lateral
sclerosis (ALS) through the gain of a toxic function. Ubiquitylated aggregates of mutant SOD1
proteins in affected lesions are pathological hallmarks of the disease and are involved in motor
neuron death. Ubiquityl ligases, Dorfin has been reported to ubiquitylate mutant SOD1. Dorfin is a
RING-finger/IBR (in-between ring-finger) domain-containing ubiquityl ligase, which we previously
identified from human spinal cord. Dorfin physically binds and ubiquitylates various familial ALSlinked SOD1 mutants and subsequently targets them for proteasomal degradation. We have
investigated the effect of overexpression of Dorfin and found that it protects neuronal cells against the
toxic effects of mutant SOD1 and reduces the number of aggregates composed of mutant SOD1 in
cell culture model system. Furthermore, we crossed Dorfin- overexpressing mice onto G93A SOD1
mice and found that double transgenic mice showed longer survival and reduced number of
aggregates formation in spinal cord as compared to controls, although motor performance was not
affected. In order to get more direct and powerful effect of proteasomal degradation of mutant SOD1,
we next used 20 S proteasomes of archaebacteria (archaea). Archaeal 20 S proteasomes are
structurally simple and proteolytically powerful and thought to be an evolutionary precursor to
eukaryotic proteasomes. We successfully reproduced the archaeal proteasome in a functional state
in mammalian cells, and we show that the archaeal proteasome effectively accelerated degradation
of mutant SOD1 and reduced the cellular toxicities. Further, we demonstrated that archaeal
proteasome can also degrade other neurodegenerative disease- associated proteins such as mutant
polyglutamine tract extended androgen receptor, α-synuclein and tau. Meanwhile, recent studies
suggest that mutant SOD1 readily forms an incorrect disulfide bond upon mild oxidative stress in vitro
and the insoluble SOD1 aggregates in spinal cord of ALS model mice contain multimers cross-linked
via intermolecular disulfide bonds. Here we show that a non-physiological intermolecular disulfide
bond between cysteines at positions 6 and 111 of mutant SOD1 is important for high molecular
weight aggregate formation, ubiquitylation, and neurotoxicity; all of which were dramatically reduced
when the pertinent cysteines were replaced in mutant SOD1 expressed in Neuro-2a cells. Also we
found that Dorfin ubiquitylated mutant SOD1 by recognizing the Cys6, 111- disulfide cross-linked
form and targeted it for proteasomal degradation. From a perspective of protein aggregation/
degradation in motorneuronal cells, our results presented here open an avenue to develop a new
therapeutic approach for ALS.
(1) Yamada S, Niwa J, Ishigaki S, Takahashi M, Ito T, Sone J, Doyu M, and Sobue G. (2006)
Archaeal proteasomes effectively degrade aggregation-prone proteins and reduce cellular toxicities in
mammalian cells. J Biol Chem. 281, 23842-23851.
(2) Niwa J, Ishigaki S, Hishikawa N, Yamamoto M, Doyu M, Murata S, Tanaka K, Taniguchi N, and
Sobue G. (2002) Dorfin ubiquitylates mutant SOD1 and prevents mutant SOD1-mediated
neurotoxicity. J Biol Chem. 277, 36793-36798.
15
S8
Mutant SOD1s and mitochondria in motoneuronal cells
Ferri A (1,2), Cozzolino M (2), Crosio C (2,3), Nencini M (2), Casciati A (2), Gralla EB (4), Rotilio G
(5), Valentine JS (4) and Carrì MT (2,5).
(1) Inst. of Neuroscience CNR, Rome, Italy. (2) Lab. of Neurochemistry, Fondazione S.Lucia, Rome,
Italy. (3) Department of Physiological, Biochemical and Cell Sciences, University of Sassari, Italy. (4)
Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095-1569, USA. (5)
Department of Biology, University of Rome “Tor Vergata”, Rome, Italy
Recent studies suggest that the toxicity of familial amyotrophic lateral sclerosis (fALS) mutant Cu,Zn
superoxide dismutase (SOD1) arises also from its selective recruitment to mitochondria (1). We have
demonstrated that each of twelve different fALS-mutant SOD1s with widely differing biophysical
properties are associated with mitochondria of motoneuronal cells to a much greater extent than wild
type SOD1 and that this effect may depend on the oxidation of Cys residues. We demonstrated
further that mutant SOD1 proteins associated with the mitochondria tend to form cross-linked
oligomers and that their presence causes a shift in the redox state of these organelles and results in
impairment of respiratory complexes. The observation that such a diverse set of mutant SOD1
proteins behave so similarly in mitochondria of motoneuronal cells and so differently from wild type
SOD1 suggests that this behavior may explain the toxicity of ALS-mutant SOD1 proteins that causes
motor neurons to die (2). Considering that ALS is not only a multifactorial, but also a multi-system
disease, and that signals from non-neuronal cells contribute in determining the progression of the
disease, we have extended our observations by studying the effect of inflammatory cytokines on the
localization of mutant SOD1 proteins. Our data indicate that the amount of SOD1 associated with
mitochondria and the impairment of mitochondria functionality are increased in motoneuronal cells
treated with cytokines, thus supporting the view that death of motor neurons is a non-cell
autonomous event (3).
(1) Manfredi G. and Xu Z. (2005) Mitochondrial dysfunction and its role in motor neuron degeneration
in ALS. Mitochondrion 5, 77-87.
(2) Ferri A., Cozzolino M., Crosio C., Nencini M., Casciati A., Gralla E.B., Rotilio G., Valentine J.S.
and Carri M.T. (2006) Familial ALS-superoxide dismutases associate with mitochondria and shift
their redox potentials. Proc Natl Acad Sci U S A. 103, 13860-13865.
(3) Boillee S., Vande Velde C. and Cleveland D.W. (2006) ALS: a disease of motor neurons and
their nonneuronal neighbors. Neuron 52, 39-59.
16
S9
Soluble misfolded SOD1 species in ALS
Marklund SL (1), Zetterström P (1), Bergemalm D (1), Stewart H (2,3), Graffmo KS (1), Andersen P
(2), Brännström T (1), and Oliveberg M (4).
(1) Department of Medical Biosciences, and (2) Pharmacology and Clinical Neuroscience, Umeå
University, Sweden. (3) Brain Research Center, University of British Columbia, Canada. (4)
Department of Biochemistry and Biophysics, Stockholm University, Sweden.
Mutants of superoxide dismutase-1 (SOD1) cause amyotrophic lateral sclerosis (ALS) by an
unidentified cytotoxic mechanism. We have previously shown that the stable human (h) SOD1
mutants D90A and G93A are abundant and show highest levels in liver and kidney in transgenic
murine ALS models, while the unstable G85R and G127insTGGG (G127X) mutants are scarce but
enriched in the CNS. These data indicated that minute amounts of misfolded SOD1 enriched in the
motor areas might exert the ALS-causing cytotoxicity.
The objective of this study was to determine amounts and analyse the structure of soluble
misfolded hSOD1 species in tissues of murine transgenic ALS models. For the analyses we used
antibodies specific for misfolded hSOD1 generated with peptides in the sequence of the enzyme.
These were used in an immunocapture protocol for analysis of misfolded hSOD1 in 20.000 g
supernatants of murine tissue homogenates. A hydrophobic interaction chromatography protocol was
also developed and used for that purpose.
All G127X and the major part of the G85R hSOD1s bound in the assays, but only minute
subfractions of the G93A and D90A mutants. Wild-type hSOD1 bound even less. The absolute levels
of misfolded hSOD1 were, however, similar in the murine ALS models and they were enriched in the
susceptible spinal cord. This enrichment was seen from birth until death, and the levels of soluble
misfolded hSOD1 were comparable to the amounts of hSOD1 that become sequestered in
aggregates in the terminal stage. The misfolded hSOD1 was composed of disulfide-reduced subunits
lacking metal ions, and also subunits that apparently carried non-native intrasubunit disulfide bonds.
Misfolded hSOD1, released from the antibodies by the peptides used for immunization, was shown
by gel chromatography to be composed of monomers, trimers and oligomers.
The soluble misfolded hSOD1 species expose sticky hydrophobic internal structures which might
interact with essential cellular factors in ways that cause cytotoxicity. They form a least common
denominator amongst hSOD1 mutants with widely different molecular characteristics, and are thus
potential culprits for the cytotoxicity that causes ALS. The susceptibility of the motor areas of the CNS
may be caused by an inadequate ability to recognize and degrade misfolded SOD1 species.
17
S10
From secretion of SOD1 mutants to immunotherapeutic approaches
Urushitani M (1,2), Abou Ezzi (3), Gros-Louis F and Julien JP (1-3)
Research Centre of CHUL, Laval University, Quebec, Canada
Despite a decade of investigation on familial ALS caused by missense mutations in the superoxide
dismutase (SOD1) gene, the mechanism of toxicity to motor neurons has remained elusive. Although
it is well known that SOD1 is a cytosolic protein without specific translocation sequence, a yeast twohybrid screen led us to discover that chromogranins, components of neurosecretory vesicles, are
interacting partners of SOD1 mutants linked to ALS, but not of wild-type SOD1. The existence of
such interactions was confirmed by co-immunoprecipitation assays using either lysates from Neuro2a
cells co-transfected with chromogranins and SOD1 mutants or from spinal cord of ALS mice. Cell
culture studies showed that chromogranins may act as chaperones to promote the secretion of
mutant SOD1. Cell-free translocation assay using recombinant SOD1 and microsomes showed that
apo-form SOD1 of both wild and mutant types translocated into microsomes in ATP-dependent
fashion. Furthermore, we discovered that extracellular SOD1 mutant can trigger microgliosis and
death of motor neurons in culture suggesting a pathogenic mechanism based on toxicity of secreted
SOD1 mutant proteins. This led us to test immunization protocols aiming to reduce the burden of
extracellular SOD1 mutant in nervous tissue of mice models of ALS, using recombinant SOD1 mutant
protein as an immunogen. At first, a vaccination protocol was tested on a G37R SOD1 mouse strain
with late-onset disease exhibiting moderate levels of mutant SOD1. The repeated injections of
adjuvant-SOD1 mutant with a last boost injection before symptoms at 6 month-old was effective in
delaying disease onset and extending life span of G37R SOD1 mice by over 4 weeks. Western blot
analysis using a monoclonal antibody specific to mutant SOD1 forms provided evidence of clearance
of SOD1 species in the spinal cord of vaccinated G37R SOD1 mice. In addition, a passive
immunization through intraventricular infusion of purified anti-human SOD1 antibody with osmotic
minipump succeeded in alleviating disease symptoms and prolonging the lifespan of G93A SOD1
mice. From these results, we propose that immunization should be considered as potential
therapeutic strategy for familial ALS caused by SOD1 mutations.
(1) Urushitani, M, Sik A, Sakurai T, Nukina N, Takahashi, R. and Julien, J.-P. (2006) Chromograninmediated secretion of mutant superoxide dismutase proteins linked to ALS. Nature Neuroscience 9,
108-118.
(2) Urushitani, M., Abou Ezzi S. and Julien, J.-P. (2007) Therapeutic effects of immunization with
mutant superoxide dismutase in mice models of amyotrophic lateral sclerosis. Proc.Natl. Acad. Sci,
104, 2495-2500.
(3) Abou Ezzi, S., Urushitani, M. and Julien, J.-P. (2007) Wild-type superoxide dismutase acquires
binding and toxic properties of ALS-linked mutant forms through oxidation. J. Neurochem., on line.
18
S11
Live imaging of disease onset and progression in SOD1 mutant mice
Jasna Kriz
Dept. Anatomy and Physiology, Laval University, Centre de Recherche CHUL (CHUQ), Québec, QC,
Canada
Transgenic mice expressing a mutant sodium dismutase 1 (SOD1) develop phenotype with many
pathological features resembling human disease. Although major clinical symptoms in amyotrophic
lateral sclerosis (ALS) arise from neurodegeneration and death of motoneurons, recent studies
suggest that non neuronal cells, such as astrocytes and microglia, play a role in the toxicity to motor
neurons. Their precise role in onset and progression of the disease remain however unknown. To
further investigate the role of non-neuronal cells in the disease onset and progression, we developed
a mouse model for live imaging of astrocyte activation in ALS.
Glial fibrilary acidic protein (GFAP) is strongly up-regulated in ALS. It is a known marker of astrocyte
activation, and GFAP up-regulation has been associated with the disease progression. To generate
a mouse model for live imaging of astrogliosis, we took advantage of reporter mice carrying the firefly
luciferase gene under the transcriptional control of GFAP promoter (GFAP-luc, Xenogen, CA) and
crossed them with SOD1G93A mutant mice. Live imaging of astrocyte activation was performed weekly
starting as early as 3-4 weeks of age till the end stage of the disease using a high resolution CCD
camera for small animal optical imaging. Data collected by in vivo imaging showed that photon
emission/GFAP signal was first detected at the lumbar spinal cord area. The signal first arose from
small multiple areas of astrocytes activation which then converged into a larger signal around 80100days of age. Moreover the peak signals arising from the spinal cord at approx. 100 days
correlated with the abrupt onset of sensori-motor deficits and paralysis. GFAP-luc/SOD1G93A mice will
provide unique tools for understanding disease pathology and longitudinal responses to drug testing.
19
S12
Proteomics of spinal cord aggregates in G93A SOD1 transgenic mouse
Valentina Bonetto
Dulbecco Telethon Institute and Mario Negri Institute for Pharmacological Research.
Proteinaceous inclusions rich in mutant Cu, Zn superoxide dismutase (SOD1) and ubiquitin have
been found in tissues of amyotrophic lateral sclerosis (ALS) patients and mutant SOD1 animals, even
before disease onset. However, very little is known on the aggregate protein constituents and
therefore on the actual role and mechanism of aggregation in ALS pathogenesis. We have recently
shown that mutant SOD1 progressively accumulates in a Triton X-100-insoluble fraction from the
spinal cord of the G93A SOD1 mouse model of ALS, and that part of insoluble SOD1 is
oligoubiquitinated, therefore not targeted to the proteasome. To investigate the impact and role of
aggregation in disease pathogenesis we attempted a comprehensive characterization of the proteins
and their post-translational modifications of the spinal cord Triton-insoluble fraction from G93A SOD1
mice at different stages of the disease. Proteins were separated by two-dimensional gel
electrophoresis and identified by MALDI-TOF mass spectrometry. The identified proteins are
cytoskeletal proteins, mainly intermediate filaments, several mitochondrial proteins, chaperones,
proteins of the endoplasmic reticulum, proteins involved in metabolic pathways and signaling. Now
we are performing the characterization of the post-translational modifications, including nitration,
carbonylation and phosphorylation. Interestingly, we could see that the majority of the aggregated
proteins are oxidized suggesting a possible link between oxidative stress and aggregation pathways
in ALS pathogenesis.
20
S13
Genetics of SOD1-linked ALS
Peter M. Andersen
Pharmacology and Clinical Neuroscience, Umeå University, Sweden.
In 1993 a consortium reported the finding of 11 missense mutations in the gene encoding the enzyme
CuZn-superoxide dismutase (EC 1.15.1.1, superoxide:superoxide oxidoreductase, SOD1) in 13 of 18
FALS pedigrees (Rosen et al., 1993). The human SOD1 gene spans 11 kb of genomic DNA on
21q21-22 with 5 exons separated by 4 introns. The 5 exons codes for 153 highly-conserved
aminoacids which together with a catalytic Cu ion and a stabilizing Zn ion forms a subunit. Each
subunit is also stabilized by an disulfid bridge spanning C57 and C146. Two identical subunits
combine through non-covalent binding to form the homodimeric SOD1enzyme. The only known
function of SOD1 is to catalyze the reduction of superoxide anion O2.- to molecular oxygen O2 and
hydrogenperoxide H2O2, which in turn is reduced to H2O by gluthation peroxidases and catalase.
SOD1 is ubiquitously expressed in all cells in all organisms above the bacteria, and constitutes about
0.5-0.8% of the soluble protein in the human brain. At the cellular level, it is found in the cytosol,
nucleus, and between the two mitochondrial membranes. There are some evidence that it may be
secreted through a chromogranin-mediated mechanism. In the mitochondrial matrix is the isoenzyme
manganese containing SOD (Mn-SOD, SOD2), and extracellularly body fluids the tetrameric CuZnSOD (SOD3).
The discovery of mutations in SOD1 in FALS sparked world-wide search for mutations in the gene.
Since 1993 , a total of 153 mutations have been found:137 exonic mutations alters the amino acid
sequence of the CuZn-SOD and are assumed to be disease causative. Also, eight silent mutations
and six intronic variants have been reported and are presumably non-causative. Two more intronic
variants close to the border between intron 4 and exon 5 have been predicted to either introduce 3
novel amino acids FLQ between exon 4 and exon 5 (creating a CuZn-SOD monomer of 156 amino
acids) or introduce five novel amino acids FFTGP after exon 4 truncating the mutated polypeptide
after amino acid number 123 (Andersen et al., 2003). Of the 139 disease-associated mutations, 123
are missense mutations causing a change of one amino acid to another but keeping the polypeptide
lenght of 153 aminoacids. The 123 missense mutations are in 72 different codons throughout the 5
exons, including eight in exon 3 which codes for the catalytic site. The remaining 16 mutations are
non-sense and deletion mutations that either introduces new nucleotides or removes nucleotides in
the DNA sequence in exons 2, 4 or 5, or intron 4 as described. The result is a change in the lenght of
the final polypeptide. The shortest of reported mutant polypeptides is 121 and the longest 156 amino
acids long (Andersen et al., 2003) Though most missense mutations are in exon 4 and the non-sense
mostly in the beginning of exon 5, there is no obvious correlation of the mutated sites and conserved
residues through evolution, enzymic stability or enzymatic function. Little clinical data have been
published for patients with SOD1 mutations. A 1996-review concluded that with few exceptions there
are no clinical correlate between a specific mutation and phenotype. However, there are no blindedstudy comparing patients with with patients without a SOD1 gene mutation to support this statement.
Now more data are available and some of the mutants can be grouped according to survival time
(Table 1), variability in site of onset (Table 2), complete or incomplete disease penetrance (Table 3)
with reservations for small numbers for some mutants. Inexplainable some mutants (i.e. A4V, G41S,
D83G, G93A) are consistently associated with a very rapid disease independently of site of onset,
while other mutants (G41D, H46R, A89V, homozygosity for D90A, E100K) are always associated
with onset in the lower limbs and very slowly ascending paresis. It is the authors clinical experience
that patients carrying any of the five latter mutations are remarkably similar in every aspect. In a third
group of mutants, some individuals have very short survival while others in the same family survive
for a decade or more. This is best illustrated by the G37R (survival range 2 to 36 years), I104F (3 to
38 years) and I113T (2-20 years). Without any obvious correlation to survival time, some mutants
consistently have onset in the lower limbs (G37R, H46R, D76V, L84F, D90A homozygous, E100K) or
upper limbs (L84V). Variable site of onset is the rule for many mutations. For most mutants, there
appears to be both intrafamilial and interfamilial variation in sites of onset with somewhat more
frequent onset in the lower limbs than in ALS not associated with SOD1 mutations. Bulbar onset has
been claimed to be rare among patients with SOD1 mutations but has been reported (Table 2).
The pedigrees used to find linkage to the SOD1 gene were pedigrees with high penetrance.
Unfortunately, only few detailed pedigrees have been published and at present it is only safe to state
21
that 24 mutations are associated with high if not complete penetrance (Table 3). Likewise, some
mutations appears to regularly be inherited with reduced penetrance sometimes obscuring the
heredity of the disease and making genetic counseling difficult. This is particular the case for the
widespread I113T, which is associated with a higher mean age of onset (58 years) than is most
commonly reported for SOD1 mutations (47 years) (Jones et al., 1994). It is documented that I113T
can pass asymptomatic from a patient down through to the grand-children or even great
grandchildren before becoming manifest again (Suthers et al., 1994). The grouping listed in Tables 1- 5 are based on available published data and may change as data are published.
For many SOD1 mutants no clinical data are available or the mutation has only been found in
singular patients making characterisation impossible at present.
The mean age at onset of first symptom is 47 years for ALS patients with a SOD1 mutation, 50.5
years for FALS and 56-58 years for SALS both without SOD mutation (Cudkowicz et al., 1997). The
youngest onset of ALS in a patient with a SOD1 mutation was 6 years (I104F) and 18 years (G16S),
and the oldest 84 years (A4V; I113T) and 94 years (D90A homozygous), respectively. The shortest
documented survival time was 14 weeks (N86S homozygous) and the longest 36 years (G37R), 38
years (I104F) and 44 years (L144F), respectively. There is surprisingly little variability in the mean
age of onset for nearly all mutants, with the exception of L37R, V38L and G114A with somewhat
lower mean age of onset (perhaps biased by small numbers) and I113T with somewhat higher as
mentioned earlier. That the mutants have the same mean age of onset but very different disease
progression rates, uniformity in site of onset or disease penetrance implies, that onset of the disease
and expression may be different processes (Andersen et al., 1997; Cudkowicz et al., 1997).
While many patients with SOD1 mutations reportedly are clinically identical to patients without SOD1
mutations, a predominantly lower motor neuron pattern is the rule for patients with a SOD1 mutation.
No case with predominantly upper motor neuron (UMN) features have been reported. A feature that
may be unique to ALS cases with SOD1 mutations is very prolonged central motor conduction times
upon transcranial magnetic stimulation of the motor cortex (MEP). Delayed central latency has been
found in many patients with slowly progressing ALS heterozygous for D76Y or homozygous for D90A
as well as in fast progressing cases heterozygous for G12R, G41S and N139H (Weber et al., 2000).
Some mutants may show features of involvement of other parts of the nervous system. Autonomic
failure has been reported in cases with G93S and V118L. Sensory symptoms, paresthesies,
lancinating pain in the back, localized neuralgic pain in the buttocks, hips or knees, ataxia, or bladder
disturbance (in some cases progressing to incontinence) have been reported for some mutants
(Table 4). These atypical features may precede the onset of paresis and may cause difficulty in
setting the ALS diagnosis. In H46R and in D90A homozygous patients, this preparetic disease phase
may last for a few months to several years (Andersen et al., 1996). Patients with SOD1 gene
mutations have frequently been claimed to be cognitively intact but no formal studies of this have
been published. On the contrary, Masé et al. (2001) reported a patient heterozygous for the L144F
SOD1 with ALS with severe frontallobe-type dementia and Battistini et al. (2005) reported two
patients heterozygous for the G41S SOD1 gene mutation with aggressive ALS and frontallobe-type
dementia.
The most common SOD1 mutation globally is the D90A followed by the A4V (accounting for about
half of all cases in the U.S.A.), and I113T. Patients with the D90A SOD1 mutation has been found in
almost every country where patients have been screened for SOD1 mutation. The D90A is also the
only one of the 139 mutations to show recessive inheritance and has as such been found in many
apparently SALS cases. Homozygosity for the D90A gives rise to an easily identifiable phenotype
with initially a preparetic phase followed months to years later by slowly ascending creeping paresis
and wasting always beginning asymmetrically in the lower limbs. After a mean of some 4 years from
onset in the legs, paresis appears in the upper limbs and a year and a half later bulbar symptoms
appears. With time bulbar symptoms have progressed to anarthria and aphagia, and some patients
have shown pseudo-bulbar palsy. A few patients have shown ataxia in the earlier stages of the
diseases but it later disappears. At end stage, the patient is completely tetraparalytic with generalized
wasting, cachectic and in some cases with bladder incontinence. Dementia has not been observed in
D90A-homozygous ALS patients surviving for more than two decades. The mean survival time is 14
years (Andersen et al., 1996). Presently, only the D90A has been proven to show recessive
inheritance but homozygosity for three other mutations (L84F, N86S, L126S) have been found in
single individuals in heavily inbred families (Boukaftane et al., 1998). In these families heterozygous
individuals also develops ALS and the inheritance is therefore not recessive. Interestingly, the
phenotype in two of these homozygous cases appears to be far more aggressive than in the
heterozygous cases suggesting a dose-effect. Recently, two siblings with ALS were found to be
carriers of both a D90A and a D96N SOD1 alleles and the phenotype was rather similar to the D90A
homozygous cases. This is the only instance of compound heterozygosity in ALS, but the published
pedigree is also consistend with dominant inheritance of D96N with incomplete penetrance.
22
The prevalence of patients with SOD1 mutations varies greatly from country to country: No patient
with a SOD1 mutation has been found in Poland, and only 9 mutations in a few patients in Germany
contrasting with four mutations in Scotland, seven in Sweden and 26 in Japan. In Scotland, the I113T
has been found in several FALS and apparently SALS cases (all shown to have the same common
ancestor), while in northern Scandinavia the D90A is very common in particular in SALS cases. Other
mutations reported in apparently SALS are listed in table 5. For only one of these, the H80R, has it
been possible through paternity studies to shown that it is a de novo mutation in one of the parents.
The others are likely to be FALS with low penetrance though this has only been proven for N19S,
D76Y, L84F, D90A and I113T. The high prevalence of the A4V in the caucasian population in North
America has in a haplotype study been shown to be unrelated to the three A4V families found in Italy
and Sweden and is probably of Asian-Indian origin. I113T is most commonly found in the United
Kingdom and its former colonies, L144F is widespread on the Balkan and in northern Italy, while
L84F is the most frequent mutation in central Italy. The most common mutation in Germany appears
to be R115G and in Japan H46R though large epidmiological studies have not been performed.
Some mutations have been found in very different ethnic groups (H46R in caucasians in eastern
Norway, Germany and the U.S.A., as well as in Pakistan and Japan; E100K in Afro-Americans as
well as in caucasians in eastern Germany) and are probably separate mutational events in the past.
While 26 mutations have been reported in the 128-million Japanese population, only recently were
the first four mutations reported from Korea and China. The global distribution of different mutations
may change radically when mass screening is performed in all of Asia, Africa and South America.
The present knowledge is based mainly on screening a small proportion of the ALS patients in the
western countries. The national differences in the reported prevalences of SOD1 mutations is not
only explained by the different ethnic backgrounds but also whether only FALS cases were studied,
the number of studied cases (in some countries as few as 30-40 cases have been studied) and the
laboratory technique used to analyze for mutation.
Screening for SOD1 mutations has revealed that SOD1 mutations are not found in other diseases
than ALS, that the only SOD1 polymorphism is the D90A and that about a fifth-a sixth of all
diagnosed FALS cases and a few percentage of apparently SALS cases carries a SOD1 mutation
(Table 6). The 139 mutations that causes a change in the CuZn-SOD polypeptide are probably all
disease causative though for only a small part statistical analysis have shown linkage to ALS or have
been shown to cause a motor neuron disease when expressed in transgenic mice (G37R, G85R,
G93A, G93D, D90A, G127X) or transgenic rats (H46R, G93A). The possibility that some mutants
found in single patients (i.e. V14G, G16S, S134N) are co-incidental findings can not be excluded
untill further studies have been done.
TABLES
Table 1: Disease survival time in ALS associated with SOD1 mutations
(without artificial ventilation. Het = heterozygous, hom = homozygous)
Fast (< 3 years)
A4T
A4V
C6F
C6G
V7E
L8Q
G10V
G41S
H43R
H48Q
L84V
D90V
G93A
D101H
L106V
I112M
I112T
R115G
G127X
A145T
V148G
V148I
Medium (3-10 years)
Slow (>10 years) Variable
G85R
G41D
E21G
G93R
H46R
G37R
G93V
D76V
L38V
E100G
A89V
D76Y
D101G
D90A hom
L84F
D101N
G93D
N86S
G108V
G93S
D90A het
L126X
E100K
G93C
G141E
G93R
I104F
I113T
L144F
L144S
23
Table 2: Site of onset in ALS associated with a SOD1 mutation
Uniform
Variable
Bulbar onset
G37R
H46R
D76V
L84F
L84V
D90A hom
E100K
E100G
A4V
C6G
G41S
N86S
D90A het
I113T
L144F
V148I
A4V
C6G
L8Q
D76Y
D90A het
V148I
I151T
Table 3: Disease penetrance in SOD1 gene mutations associated with ALS:
Complete (>90% by age 70)
Incomplete
A4V
A4T
G37R
L8Q
L38V
N19S
G41S
E21G
H43R
N65S
H46R
G72S
D76V
D76Y
L84F
N86S
L84V
A89V
N86K
D90A hom
D90A het
E100G
A95T
D101H
G93S
I104F
I113T
G108V
L126S
C111Y
S105L
I112M
N139H
G114A
L126GQRWKX
G127GGQRWKX
G141E
L144F
V148G
V148I
Table 4: SOD1 mutationes associated with atypical features:
A4V (neuralgic pain), V14G, E21G, H46R, H48Q, L84F, D90A, G93S, E100G, I104F, V118L (only
after having been placed on IV), L144F, I151T.
Sexual disinhibition: G41S
Cognitive disturbance: L144F, G41S
Table 5: SOD1 mutations reported in cases with apparently sporadic ALS (SALS):
A4V, L8V, G12R, V14G, G16S, N19S, E21K, V29A, H48Q, C57R, N65S, G72S, D76Y, H80R (only
de novo mutation confirmed), L84F (of unknown father), N86I, N86S, N86D, A89V, D90A,G93S,
A95T, V97L, D101N, S105delSL, L106P, D109Y, I113T, T116R, V118L, V118KTGPX, D124G,
E133E, K136X, L144F
Table 6: Frequency of SOD1 mutations
In SALS:
:
7.3% (3/41) in Italy (Corrado L et al., Neuromuscul Disord 2006;16:800-804)
7% (4/56) in Scotland (Jones CT et al., J Med Genet 1995;32:290-292)
6% (3/48) in Italy (Gellera C et al., ALS & other MND’s 2001;2(suppl 1):543-546)
4% (14/355) in Scandinavia (Andersen PM et al., Brain 1997;10:1723-1737)
3% (5/175) in the UK (Shaw CE et al., Ann Neurol 1998;43:390-394)
3% (5/155) in England (Jackson M et al., Ann Neurol 1997;42:803-807)
24
1.2% (1/87) in Spain (García-Redondo A et al., Muscle & Nerve 2002;26:274-278)
0% (0/225) in Italy (Battistini S et al., J Neurol 2005;252:782-788)
In FALS:
23.5% (12/51) in Scandinavia (Andersen PM et al., Brain 1997;10:1723-1737)
23.5% (68/290) in the USA (Cudkowicz ME et al., Ann Neurol 1997;2:210-221)
21% (8/38) in the UK (Shaw CE et al., Ann Neurol 1998;43:390-394)
19.7% (14/71) in the UK (Orrell R et al., Neurology 1997;48:746-751)
18% (2/11) in Spain (García-Redondo A et al., Muscle & Nerve 2002;26:274-278)
18% (7/39) in Italy (Battistini S et al., J Neurol 2005;252:782-788)
14.3% (10/70) in France (Boukaftane Y et al., Can J Neurol Sci 1998;25:192-196)
12% (9/75) in Germany (Niemann S et al., JNNP 2004;75:1186-1188)
Without classification to hereditary disposition:
7.2% (148/2045) in North America (Andersen PM et al., ALS & Other MND’s 2003;4:62-73).
25
S14
Genetics of non-SOD1-linked fALS
Christopher E. Shaw
Department of Clinical Neuroscience, King’s College London, Box 43 Institute of Psychiatry, De
Crespigny Park, London SE5 8AF, UK. chris.shaw@iop.kcl.ac.uk
Traditional gene hunting strategies in autosomal dominant (AD) conditions rely on ascertaining DNA
and detailed clinical information from multiple affected individuals in large kindreds. Because classical
amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal illness the identification of a
robust locus has only been achieved in a handful of kindreds. The identification of SOD1 on
Chromosome 21q21 was followed by linkage to 18q21 (ALS3), 16q21 (ALS6) and 20p13 (ALS7).
Disappointingly the genetic mutation responsible for ALS in these kindreds has been elusive despite
exhaustive gene sequencing. Confirmation of linkage in multiple kindreds to these loci has only been
achieved for ALS 6 and genome-wide scans of the linked kindreds using microarrays has challenged
our confidence that these are the only potentially linked regions.
Relatively recently it has been recognised that ALS and fronto-temporal dementia exist in a
phenotypic spectrum and can occur within the same AD kindred as a single gene disorder. Five
families with AD ALS-FTD were previously linked to 9q21. More recently we have identified a locus
on chromosome 9p13-21, (maximal multipoint LOD score of 3.02 (=0) at D9S1878). Four other
kindreds have now been linked to the same overlapping locus. Recombination has narrowed the
conserved haplotype in our kindred to 12cM (11Mb) at 9p13.2-21.3 (flanking markers D9S2154 and
D9S1874). Bioinformatic analysis of the region has identified 103 known genes but no pathogenic
mutation has been identified to date.
New genomic research platforms are being developed which we anticipate will advance our ability to
identify new loci and gene mutations.
26
S15
SOD1 in Italian ALS patients
Stefania Battistini
Department of Neuroscience, Neurology Section, University of Siena, Siena, Italy
Presently, more than 100 different mutations located in the five exons of the SOD1 gene have been
identified worldwide in ALS patients with an autosomal dominant transmission or rarely, recessive
transmission. In surveys of different populations in Europe and North America, the frequency of
SOD1 mutations in familial ALS (FALS) was reported to be 12% to 23.5% and in sporadic ALS
(SALS) 1.2% to 7% (1). We and others have shown that, in Italy, the frequency of SOD1 gene
mutations in FALS cases is 18% to 22% and in SALS cases 1% to 4.5% (2-4), a figure consistent
with those of other surveys. Among the more than 100 SOD1 mutations associated with ALS, some
occur as recurrent mutations while others as founder mutations and a geographic distribution of
SOD1 mutations is beginning to emerge. The A4V mutation results the most prevalent mutation in the
United States while the D90A mutation the most prevalent in Europe and a founder effect has been
demonstrated for both. In Italy, at the present time, 18 different SOD1 gene mutations have been
reported in ALS patients (2-5). Overall, the D90A results the most common mutation among SOD1
mutated Italian ALS cases. Recently, we first reported two Italian ALS patients heterozygous for the
D90A mutation, providing evidence of the coexistence in Italy, like in other countries, of ALS cases
with heterozygous and homozygous D90A mutation. The G41S, together with the L84F, results the
most prevalent SOD1 gene mutation found in SOD1 mutated Italian FALS patients (3). These
mutations seem to be responsible for ALS clustering in Central Italy. Indeed, three of the L84F
families originated from the same mountainous area of the Marche region, while all the seven ALS
patients carrying the G41S mutation identified by us, belonged to five unrelated FALS pedigrees
originating from the same restricted area of northwest of Tuscany. Interestingly, the G41S mutation
has only been found so far in Italian families. This mutation is associated with a quite uniform clinical
phenotype with early UMN and LMN involvement in one or both lower limbs and a rapidly progressive
disease course with appearance of bulbar signs within 1 year and death few months later.
Genotyping with markers from the SOD1 locus showed that the seven Italian G41S FALS patients
share a common haplotype (6). Our findings strongly suggest that the G41S mutation in the Italian
population originates from a common ancient founder.
(1) Andersen PM. (2006) Amyotrophic lateral sclerosis associated with mutations in the CuZn
superoxide dismutase gene. Current Neurology and Neuroscience Reports 6, 37-46.
(2)Gellera C, Testa D, Passariello P, et al. (2003) SOD1 gene mutations in amyotrophic lateral
sclerosis Italian patients. Amyotroph Lateral Scler Other Motor Neuron Disord. 4 (Suppl 1), 98.
(3) Battistini S, Giannini F, Greco G, et al. (2005) SOD1 mutations in amyotrophic lateral sclerosis.
Results from a multicenter Italian study. J Neurol. 252, 782-8.
(4) Corrado L, D’Alfonso S, Bergamaschi L, et al. (2006) SOD1 mutations in Italian patients with
sporadic amyotrophic lateral sclerosis (ALS). Neuromuscul Disord. 16,800-4.
(5) ValentinoP, Conforti FL, Pirritano D, et al. (2005) Brachial amyotrophic diplegia associated with a
novel SOD1 mutation (L106P). Neurology. 64, 1477-1478.
(6) F. Giannini, S. Battistini, C. Ricci, et al. (2005) Phenotypic-genotypic study of amyotrophic lateral
sclerosis Italian families with the G41S SOD1 gene mutation. Amyotroph Lateral Scler Other Motor
Neuron Disord. 6 (Suppl 1),79.
27
S16
The pathogenesis of sporadic amyotrophic lateral sclerosis
Wim Robberecht
Dept. of Neurology and Section of Experimental Neurology,University Hospital Leuven, Leuven
Belgium.
The pathogenesis of sporadic amyotrophic lateral sclerosis (ALS) remains unknown. It is generally
accepted that a genetic component and/or an environmental factor on the background of an aging
nervous system are involved. Several attempts to identify an environmental factor have been
unsuccessful. Genetic susceptibility factors or modifying genes can be identified in a candidate gene
approach based on molecular studies of models of motor neuron degeneration in vivo or in vitro,
based upon genome wide association studies in ALS patients, or through the genetic screening of
small animal models for the disease. These different approaches are likely to be complementary as it
is hypothesized that many factors are likely to be involved.
Excitotoxicity, the vascular endothelial growth factor (VEGF) system and abnormalities of RNA
metabolism have been identified as pathogenic contributors to the mechanism of motor neuron
degeneration. In the present presentation, data supporting these hypotheses will be highlightened,
and new supportive evidence will be presented. In addition, a zebrafish model generated to identify
new modifying genes will be presented.
28
S17
The double life of two pro-survival proteins: when SOD1 and Bcl-2 get mutated, wild
and pro-death
Piera Pasinelli
Farber Institute for Neurosciences, Thomas Jefferson University
900 Walnut Street, Philedelphia, PA 19107 USA
Mitochondrial abnormalities and activation of the mitochondrial apoptotic pathway are both
characteristic features of mutant SOD1-mediated motor neuron death.
Contrary to WT SOD1 (a well known anti-apoptotic protein), mutant SOD1 is pro-apoptotic. The
mechanisms by which WT SOD1 protects and mutant SOD1 kills the cells are unknown.
Because SOD1 represents one of the major anti-oxidant defenses in the cells, and an increased
production of reactive oxygen species (ROS) and oxidative stress have been implicated in many
paradigms of cell death, the anti-apoptotic and pro-survival properties of SOD1 have mainly been
attributed to its anti-oxidant activity. We found that Sod1 interacts with Bcl-2 raising the intriguing
question of whether the anti-apoptotic properties of SOD1 are a consequence of both its
dismutase/anti-oxidant function and, in parallel but perhaps independently, its interactions with Bcl-2
and the anti-apoptotic machinery.
The molecular switches that confer toxic properties to mutated SOD1 are unknown. Similarly to WT
SOD1, mutant SOD1 binds Bcl-2. However, the nature of mutant SOD1 binding to Bcl-2 differs from
that of WT SOD1. Contrary to WT SOD1, mutant SOD1 specifically localizes to spinal cord
mitochondria were it forms SDS-resistant high molecular weight aggregates that bind and entrap Bcl2 Thus, opposed to the WT SOD1/Bcl-2 complex, mutSOD1/Bcl-2 aggregates could be a molecular
determinant of cell death in ALS.
In the present study, we began a series of in vitro and cell-based studies to determine whether
indeed both WT and mutant SOD1, at least partially, depend on Bcl-2. We will show our unpublished,
preliminary evidence indicating that both WT and mutant SOD1 may require Bcl-2 to exert their
protective and toxic function respectively. Consequences for development of therapies against
mutant SOD1-mediated cell death will be discussed.
29
S18
Role of Bcl2a1 in SOD1-linked fALS
Crosio C (1,2), Casciati A (2), Iaccarino C (1,2), Rotilio G (3) and Carrì MT (2,3)
(1) Department of Physiological, Biochemical and Cell Sciences, University of Sassari, Italy.
(2) Lab. of Neurochemistry, Fondazione S.Lucia, Rome, Italy.
(3) Dept. of Biology, University of Rome “Tor Vergata”
Motor neurons are the only cells in both animal models and ALS patients that undergo degeneration
upon mutant SOD1 over-expression. Up to date, the molecular bases of mutant SOD1-induced cell
death are still debated. A crucial step in filling this gap is the identification of genes whose expression
is altered by mutant SOD1 in motor neurons. We provide evidence of the selective induction of
Bcl2a1 in motor neurons of a mouse model of ALS and its functional switch upon Tumor Necrosis
Factor alpha (TNFalpha) treatment in neuronal cell lines. Bcl2a1 up-regulation may represent an
early event aimed to protect cells from oxidative stress induced by mutant SOD1, but at later stages,
when neuroinflammation occurring in ALS is maximal, Bcl2a1 expressed early in motor neurons, may
switch from anti-apoptotic to pro-apoptotic. In these conditions increase in Bcl2a1 constitutes a
reinforcing event in the progressive loss of motor neurons occurring in ALS.
30
S19
The nucleus as target for mSOD1 toxicity
Sau D (1,4), Crippa C (1,4), De Biasi S (2), Vitellaro-Zuccarello L (2), Simonini F (1,4), Onesto E
(1,4), Rusmini P (1,4), Bolzoni E (1,4), Bendotti C (3), Poletti A (1,4).
(1) Institute of Endocrinology, Center of Excellence on Neurodegenerative Diseases, University of
Milan (Italy). (2) Department of Biomolecular Sciences and Biotechnologies, University of Milan
(Italy).
(3) Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri, Milan (Italy). (4)
InterUniversity Center on Neurodegenerative Diseases, Universities of Florence, Rome and Milan
(Italy).
Cu/Zn Superoxide-Dismutase type-1 (SOD1) is one of the major intracellular antioxidant enzyme
responsible for the removal of toxic free radical species. The enzyme is a homodimer of about 32
kDa, which is mainly localized in the cytoplasm, but smaller amounts are present in the nucleus, in
the peroxisomes and lysosomes, as well as in mitochondria (1, 2). It is well know that point mutations
in the gene coding for SOD1 are linked to almost 15% of cases of familial amyotrophic lateral
sclerosis (fASL), but in the majority of the mutations, the SOD1 enzymatic activity is preserved,
suggesting that motor neuron death is due to a gain of neurotoxic function(s) of the aberrant SOD1.
However, we recently found that the two mutant SOD1s (G93A and A4V) misfold generating
insoluble species and aggregates, that alter the nuclear diffusion of the enzyme. The lack of the
antioxidant defense mechanisms in the nucleus correlates with an increased sensitivity to DNA
damage, induced by the free radical species formed after treatment with high doses of hydrogen
peroxide. Thus, although, we confirmed that the formation of mutant misfolded SOD1 aggregates is
associated to the impairment of the ubiquitin-proteasome pathway (UPP), and possibly the trigger of
the disease, the alteration of the genomic DNA in postmitotic motor neuronal cells may be a
contributing factor in the pathogenesis and progression of fALS.
In order to counteract the primary cause of mutant SOD1 toxicity linked to protein misfolding, we
have then analysed the role of a small chaperone protein on the solubility and degradation of this
antioxidant enzyme, and shown that the clearance of the mutant SOD1 is greatly enhanced by the
HspB8 protein, without affecting the UPP. This suggests that the modulation of the levels of this small
Hsp protein may provide novel potential therapeutical approaches for fALS.
1) Fridovich I., (1975) Superoxide dismutases. Annu. Rev. Biochem. 1975, 44, 147-159.
2) Valentine, JS, and Hart PJ (2003) Misfolded CuZnSOD and amyotrophic lateral sclerosis. PNAS.
100, 3617-3122.
GRANTS: Telethon - Italy (#GGP06063), MIUR-FIRB (#RBAU01NXFP), MIUR-Cofin
(2005057598_002),
University of Milan-FIRST,
FONDAZIONE CARIPLO
31
S20
Role of MAP kinases cascade in SOD1-linked fALS
M. Tortarolo, G. Spaltro, D. Lidonnici, P.Veglianese, D. Lococo and C. Bendotti
Dept. Neuroscience, Institute for Pharmacological Research” Mario Negri”, Milano, Italy
Activation of mitogen activated protein kinases (MAPKs), which consists of three main kinase
subfamilies, JNK, p38MAPK and ERK, represents an important step in the signaling transduction
cascades leading to neuronal death in response to a variety of extracellular stimuli including
excitotoxicity, oxidative stress and inflammatory processes. These pathways are involved in several
neurodegenerative diseases and have been implicated in the mechanisms underlying of motor
neuron degeneration in amyotrophic lateral sclerosis (ALS). In particular we have found that
p38MAPK cascade, including MKK3-6, MKK4 and ASK1, is selectively activated in the motor neurons
of transgenic mice overexpressing SOD1 mutants before the symptoms onset in concomitance with
the accumulation of phosphorylated neurofilaments in the perikarya, an early sign of axonal
dysfunction and neuronal degeneration. Increased activated p38MAPK and ASK1 immunoreactivity
was also found in the spinal motor neurons from sporadic ALS patients indicating that this
phenomenon is likely to play a relevant role in the pathogenesis of ALS. No changes of JNK
activation was found in the motor neurons of mouse models and ALS patients. We have also found
that activation of p38MAPK pathway in the motor neurons is associated with a concomitant
upregulation of the TNFalpha but not IL1 beta, receptors. With the progression of the disease
p38MAPK activation becomes prominent also in the reactive astrocytes and microglia of SOD1
mutant mice although this effect was not accompanied by the activation of the upstream cascade,
suggesting different regulatory mechanisms of this kinase in motor neurons and glial cells. JNK is
also activated in the glial cells of mouse models and patients with ALS. Overactivation of these
kinases in the glial cells may induce the over production of inflammatory cytokines and nitric oxide
and this may strongly contribute to propagate the damage to adjacent healthy cells. Studies using
spinal neurons-astrocytes co-culture system expressing or not the mutant SOD1,are underway to
verify this hypothesis. p38MAPK has been also implicated in a specific FAS-linked death pathway
occurring in motor neurons. Recently, a specific inhibition of FAS ligand has demonstrated a delay in
the onset and increased survival in SOD1 mutant mice mediated by the inhibition of p38MAPK.
However a direct inhibitor of p38MAPK function was shown to protects motor neurons but did not
prevent disease progression in SOD1 mutant mice. Thus, the direct involvement of p38MAPK as
primary event or epiphenomenon in the degeneration of motor neurons is still controversial and will
be discussed.
Supported by Telethon.
32
S21
SOD1 mutations and astrocyte dysfuction in ALS
Luis Barbeito
Institut Pasteur de Montevideo, Uruguay.
Glial cells surrounding motor neurons can greatly influence neurodegeneration in mice expressing
the mutations of the superoxide dismutase-1 (SOD1) linked to familiar ALS. We analyzed
mitochondrial activity in astrocytes expressing the SOD1G93A mutation and determined whether such
astrocytes influenced survival of neighboring motor neuron. Mitochondria from astrocyte cultures
obtained from transgenic rats expressing the SOD1G93A displayed a defective respiration,
decreased oxygen consumption, lack of ADP-dependent respiratory control and reduced membrane
potential. Similar defects were found in mitochondria isolated from the spinal cord of SOD1 G93A rats.
Furthermore, protein 3-nitrotyrosine was detected immunochemically in mitochondrial proteins from
SOD1G93A astrocytes revealing that mitochondrial defects were associated to nitro-oxidative damage.
Pre-treatment with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) restored mitochondrial
function, forming adducts with mitochondrial proteins in vivo. Remarkably, SOD1G93A astrocytes
induced death of motor neurons cultured on the top of the astrocyte monolayers, as compared
to non-transgenic ones. Motor neuron loss was prevented by pre-incubation of SOD1G93A astrocytes
with antioxidants and NOS inhibitors. We also tested recently designed mitochondrially-targeted
antioxidants, ubiquinone or carboxy proxyl nitroxide covalently coupled to a triphenylphosphonium
cation (MitoQ and Mito-CP, respectively), since they could act as multimodal drugs, having multiple
cellular synergistic effects. In our hands, both MitoQ and Mito-CP completely restored mitochondrial
respiration in SOD1G93A-expressing astrocytes and stimulated the ability of astrocytes to support
motor neuron survival. Remarkably, neuroprotection was accomplished at low nanomolar
concentrations of the oxidants, which make them much more effective than therapies with
conventional antioxidants. We hypothesize that the systemic administration of these drugs to
SOD1G93A mice will down-regulate glial-mediated neurotoxicity. Taken together, our results indicate
that mitochondrial dysfunction in astrocytes critically influence motor neuron survival, which may
explain key features of ALS progression.
33
S22
Neuroinflammatory signalling in the motor circuit in SOD1-linked fALS
Bentivoglio M (1), Kassa R.M. (1), Cupidi C. (1,2), Mariotti R. (1)
(1) Department of Morphological and Biomedical Sciences, University of Verona, Verona, Italy, (2)
Department of Experimental Medicine, University of Palermo, Italy.
A wealth of data obtained in the murine model of SOD1-linked familial amyotrophic lateral sclerosis
(fALS), as well as experimental findings on motoneuron degeneration after peripheral nerve damage,
recall attention on the role of neuroinflammatory signalling in motoneuron death, but this role is still
unclear. In our laboratory, we investigated alterations of central motor circuits in SOD1-mutant mice,
with special reference to inflammatory parameters and to the response to peripheral challenges. The
role of retrograde inflammatory signals in motoneuron degeneration was studied in presymptomatic
SOD1(G93A) mice, applying a “double hit” paradigm. This sequential insult consisted of inflammation
elicited in the target muscles by injections of the endotoxin lipopolysaccharide (LPS), followed by
facial axotomy. The data obtained with these manipulations revealed an enhancement of motoneuron
loss and microglial activation in the facial nucleus of SOD1-mutant mice after nerve transection with
respect to wild-type (wt) mice, and inflammatory priming further enhanced the degeneration of
axotomized SOD1-mutant facial motoneurons. In addition, recruitment of T lymphocytes in the injured
facial nucleus was affected by inflammatory priming in a different manner in SOD1-mutant and wt
mice, since the transgenic animals, at variance with wt ones, showed a low degree or no T cell
infiltration in the axotomized facial nucleus after LPS pretreatment. This abnormal lymphocytic
response was not due to impairment of cellular adhesion mechanism or to alternative recruitment of
neutrophils. The data point out the interesting findings of i) an effect of retrograde inflammatory
signalling on the enhanced susceptibility of SOD1-mutant motoneurons to death-inducing axonal
injury, and ii) an effect of mutant SOD1 on immune-related responses to peripheral inflammatory
challenges. Furthermore, investigation of the motor cortex of SOD1(G93A) mice revealed alterations
of corticospinal neurons and surrounding glia. Shrinkage of the cell bodies of corticospinal neurons,
characterized with retrograde tract tracing from the spinal cord, as well as marked activation of the
surrounding astrocytes and microglia were found in the presymptomatic stage of disease and
worsened in the terminal stage. Upregulation of mRNAs of pro-inflammatory mediators (interleukin,
IL,-1α and IL-1β, inducible nitric oxide synthase and NF-κB) was documented in the deep layers of
the motor cortex, where corticospinal neurons reside, of SOD1-mutant mice. Altogether the findings
point to damage of the entire central motor circuit in murine SOD1-linked fALS, and highlight that
such alterations are accompanied by inflammatory events and implicate alterations of immunerelated responses. (Supported by FIRB RBNE01B5WW and MIUR PRIN 2005057598)
34
S23
Post-translational processing of the glial glutamate transporter EAAT2 in ALS
Davide Trotti
Farber Institute for Neurosciences, Thomas Jefferson University
900 Walnut Street, Philedelphia, PA 19107 USA
EAAT2, also known as GLT-1 in rodents, is a high affinity, Na+-dependent glutamate transporter of
glial origin that is essential for the clearance of synaptically released glutamate and prevention of
excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in transgenic
mutant SOD1 mice models of the disease, expression and activity of EAAT2 is remarkably reduced.
We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit
EAAT2 and the impairment was largely brought about by caspase-3 cleavage at a single defined
locus, giving rise to two fragments that we termed truncated EAAT2 (Tr-EAAT2) and C-terminus of
EAAT2 (CTE). In this study we report that analysis of spinal cord homogenates prepared from
mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected due to
conjugation with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal
cords of these mice as early as pre-symptomatic stage (70 days of age) and not in other central
nervous system areas which are unaffected by the disease. CTE-SUMO-1 appearance is specific to
the ALS mice, as it does not occur in the R6/2 mouse model for Huntington’s disease. Using an
astroglial cell line, primary culture of astrocytes and tissue samples from G93A-SOD1 mice, we show
that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies (PML-NBs). As one of the
proposed functions of PML-NBs is regulation of gene transcription we suggest a possible novel
mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS.
35
S24
Muscle control of motor neuron survival and activity
Dobrowolny G (1), Aucello M (1), Fanò G (2), Protasi F (2), Molinaro M(1), Rosenthal N (3), Sandri M
(4), and Musarò A (1)
(1) Department of Histology and Medical Embryology, Sapienza University of Rome, Italy. (2) CeSI,
Centro Scienze dell'Invecchiamento, Universita G. d'Annunzio, Chieti, Italy. (3) EMBL Mouse Biology
Program, Monterotondo, Italy; (4) Venetian Institute of Molecular Medicine, Univ Padova, Italy.
One of the crucial systems severely affected in several neuromuscular diseases is the loss of
effective connection between muscle and nerve, leading to a pathological non-communication
between the two tissues.
The best examples of impaired interplay between the two tissues is the disease Amyotrophic Lateral
Sclerosis (ALS), due to mutation in the SOD1 gene.
Motor neurons degeneration and muscle atrophy are the major pathological processes associated
with ALS suggesting that nerve activity plays an important role in muscle homeostasis and
remodeling. However, other cells may be involved in the pathogenesis of ALS and open the
possibility that alteration in skeletal muscle homeostasis represents one of the principal mediators of
motor neurons degeneration.
In this context, skeletal muscle can play a direct role in the pathogenesis of ALS or, alternatively,
provide the appropriate factors to sustain neuron survival.
In this presentation I will discuss the contribution of skeletal muscle to motor neuron survival and
activity and the potential muscle signals, including IGF-1, that influence neuron survival, axonal
growth and maintenance of synaptic connections.
36
S25
Axonal transport deficits in models of motor neuron disease
Linda Greensmith
Institute of Neurology, University College London, Queen Square, London, UK
Increasing evidence suggests that deficits in axon transport pathways play an important role in MND
pathogenesis. In SOD1 mice, defects in both retrograde and anterograde transport have been
observed prior to disease onset. Indeed, we previously observed deficits in retrograde axonal
transport in SOD1 motoneurons as early in development as embryonic day 13, which represents one
of the earliest pathological changes reported in this model1. Results from other mouse models of
MND have also shown a link between axonal transport deficits and motoneuron degeneration. For
example, Loa mice homozygous for mutations in the molecular motor dynein, show an impairment of
axonal retrograde transport in addition to significant motoneuron degeneration and postnatal overexpression of the dynein subunit dynamitin, also induces motoneuron degeneration in mice.
Furthermore, human disorders involving degeneration of motoneurons have been linked to mutations
in motor proteins e.g. the anterograde motor Kinesin 1 and dynactin which is an essential component
of the dynein motor complex. Surprising results from our laboratory provided further evidence linking
transport deficits to motoneuron degeneration, when we found that the deficit in retrograde transport
in SOD1 motoneurons is completely rescued when SOD1 mice are crossed with Loa mice. Moreover,
Loa/SOD1 mice show amelioration in disease symptoms and a significant extension in lifespan. It has
been recently been shown that mutant SOD1 interacts and co-localizes with dynein, suggesting a
direct gain of function of mutant SOD1 which may result in impaired axonal transport. However, the
mechanism by which a mutation in a motor protein ameliorates symptoms and axon transport defects
in SOD1 motoneurons remains unclear. Nevertheless, our unexpected findings not only indicate that
defective transport may play a significant role in MND pathogenesis but also suggest that
manipulation of axonal transport pathways may have therapeutic value. We are currently
investigating the mechanism by which axonal transport deficits may cause motoneuron degeneration
for example by investigating mitochondrial transport in models of MND. We are also studying axonal
transport in a number of alternative mouse models of MND, for example a new mouse strain called
Abnormal rear legs (Arl), which has a different point mutation in the cytoplasmic dynein heavy chain
to that described for the Loa mouse. Finally, we are also studying axonal transport in motoneurons of
a mouse model of Spinal Bulbar Muscular Atrophy (SBMA), in which motoneurons die as a
consequence of an expansion of CAG trinucleotide repeats in the androgen receptor gene.
37
S26
Energy homeostasis and denervation processes at the neuro-muscular junction in
ALS
Loeffler JP, Dupuis L, Gonzalez de Aguilar JL, René F and Meininger V.
Laboratoire INSERM U692 “Signalisations Moléculaires et Neurodégénérescence”, Université Louis
Pasteur, Strasbourg France and (MV) Centre SLA Salpêtrière Fédération de Neurologie, Paris
France.
Despite the traditional view of ALS as a pure motor neuron (MN) disease, growing evidence suggests
that the disease is, in fact, a multisystem disorder with additional extramotor neurological
manifestations. Beyond the nervous system, intriguing metabolic alterations have also been observed
in asssociation with the course of the disease. In particular, recent studies revealed that two thirds of
ALS patients present with a stable hypermetabolism that correlates with survival (Desport et al.,
2005).
The cause of the hypermetabolism in ALS is still unknown but our research on genetic animal models
has shed some light into this question. Very early, transgenic mice with mutated SOD1 genes present
with reduced adiposity and increased rates of energy expenditure, which reveals the presence of a
metabolic deficit before any apparent sign of motor impairment. The burden of these metabolic
alterations exerts some influence on the neurodegenerative process, since increasing the lipid
content of the diet offers neuroprotection and extends survival (Dupuis et al., 2004). We have also
shown that the gastroinstestinal absorption of lipids as well as the peripheral clearance of triglyceriderich lipoproteins are markedly increased in the mutant SOD1 mice, which strongly suggests an
increase in the consumption of lipids by muscles, a situation that could account for the protective
effect of the high fat regimen in these animals (Fergani et al., 2007). Our recent studies demonstrates
that hyperlipidemia is a typical feature of ALS patients and, most importantly, that bearing an
abnormally elevated LDL/HDL ratio significantly increased survival by more than 12 months (Dupuis
et al., 2007).
An early hallmark of ALS is the destruction of the neuromuscular junction (NMJ), an event that might
suggest that the anatomical target of MNs is somehow involved in the disease initiation. To test
whether increased energy expenditure in skeletal muscle is sufficient to initiate denervation, we
analyzed transgenic mice overexpressing uncoupling protein 1 (UCP1) under the control of the
muscle specific creatine kinase (MCK) promoter. We show that these mice present progressive
denervation (as shown by electromyographic recording), loss of functional NMJ, loss of MN and
muscle atrophy.
These results show that an increased energy demand in the muscle fibre is sufficient to initiate major
disorders in the motor unit. The metabolic disorder associated with ALS may represent a physiopathological mechanism whose importance in disease initiation and progression has been largely
underestimated.
References
Desport et al., 2005 Hypermetabolism in ALS: correlations with clinical and paraclinical parameters.
Neurodegener Dis 2:202-207.
Dupuis et al., 2004 Evidence for defective energy homeostasis in amyotrophic lateral sclerosis:
benefit of a high-energy diet in a transgenic mouse model. Proc Natl Acad Sci USA 101:1115911164.
Dupuis et al., 2007 Dyslipidemia is a protective factor in amyotrophic lateral sclerosis. Neurology (in
press).
Fergani et al., 2007 Increased peripheral lipid clearance in an animal model of amyotrophic lateral
sclerosis. J Lipid Res 48:1571-1580.
38
S27
Lentiviral Vectors and Adeno-associated Vectors for Motoneuron Gene Therapy
Raoul C 1,2, Towne C 1 and Aebischer P1
1
Integrative Biosciences Institute, SV LEN, Ecole Polytechnique Fédérale de Lausanne (EPFL),
Lausanne, Switzerland.
2 Present address: Institut National de la Santé et de la Recherche Médicale (INSERM) Avenir team,
INMED, campus de Luminy, Marseille, France.
Approximately 20% of familial ALS cases are caused by mutations in the Cu/Zn superoxide
dismutase (SOD1) gene that result in a deleterious gain of function for this enzyme. The posttranscriptional gene silencing mechanism of RNA interference (RNAi) thus represents a promising
therapeutic strategy for this disease. We have previously described the proof of principle for RNAibased knockdown of SOD1 mutant in a fALS mouse model. Lentiviral vectors expressing short
hairpin RNA (shRNA) targeting mutated SOD1 were injected into the lumbar spinal cord of
presymptomatic SOD1 mice retarding disease onset and progression in the hindlimbs. The next
challenge is therefore to enhance the delivery of these silencing instructions to reach more cells
involved in disease pathogenesis. Towards this goal, we are evaluating the use of systemic injections
of adeno-associated viral vectors (AAV), which have been shown to infect large quantities of skeletal
muscle following intravenous delivery. Since AAV has been shown to transduce motoneurons
through retrograde transport following local intramuscular delivery, we hypothesised that this
systemic approach would facilitate widespread transduction of motoneurons. Accordingly, we have
generated an AAV serotype 6 vector driving expression of shRNA against SOD1 (AAV6-shSOD1)
with an EGFP reporter to trace transduced cells. We first observed efficient retrograde transport of
AAV6-shSOD1 to the motoneuron soma following intramuscular injection and have also
demonstrated that this vector was capable of silencing SOD1 in muscle at the mRNA and protein
level. We have thus performed systemic administration of the vector by tail vein injection. Intravenous
AAV6-shSOD1 injections resulted in high levels of transduction in skeletal muscles, including
gastrocnemius, biceps, tongue, masseter, intercostals and diaphragm. Strikingly, anaylsis of the
transduction pattern of AAV6 in the central nervous system revealed transduced motoneurons at
lumbar, thoracic and cervical levels of the spinal cord and also within the motoneuron nuclei of the
brain stem. We are currently examining the effect of this delivery technique on disease onset,
progression and ultimately, survival, by injecting presymptomatic adult mice with the AAV6-shSOD1
vector.
39
S28
An RNAi approach to understanding the mechanism and therapy of ALS caused by
mutant SOD1
Zuoshang Xu
Department of Biochemistry and Molecular Pharmacology,University of Massachusetts Medical
School, Worcester, Massachusetts, USA.
RNAi is a conserved cellular mechanism in metazoan. Triggered by double stranded RNA, RNAi
causes degradation of RNA molecules that share homology to the double stranded RNA. Therefore,
RNAi can be used to target specific mRNA for degradation, leading to gene silencing. My lab tries to
harness this mechanism to accomplish two goals: establishing transgenic RNAi as a reverse genetics
tool for silencing specific genes in mice and developing RNAi as a therapeutic means for treating
neurodegenerative diseases. Currently, the main method for reverse genetics in mammals is gene
knockout by homologous recombination. Although highly effective, this method is complex,
expensive, time-consuming and limited to mouse. These limitations hinder rapid and wide application
of reverse genetics in mammalian species. We have developed a transgenic RNAi method to
silencing specific genes in transgenic mice and demonstrated that this method can reproduce
phenotypes of gene knockout with lower cost and shorter time. Recently we have developed an
inducible silencing method with which one can induce gene silencing in specific cell types. We are
applying this technology to study genes that are relevant to neurodegenerative diseases including
ALS.
Because of its specificity, RNAi has a great potential as a therapeutic method for a variety of
diseases. We proposed to develop RNAi as a therapy for ALS caused by mutations in SOD1 and
other neurodegenerative diseases that are caused by dominant, gain-of-function type of genetic
mutations. We demonstrated that RNAi is effective in knock down the expression levels of mutant
SOD1 in vitro, in cultured cells and in vivo. We are testing delivery of RNAi in animals by several
different ways, including gene therapy using recombinant adenovirus and adeno-associated virus,
and direct infusion of chemically stabilized siRNA. Our preliminary results suggest that both delivery
methods can be effective in extending the survival of mice expressing mutant SOD1G93A. These
results hold promise that RNAi can be applied to treat ALS caused by mutations in SOD1 gene as
well as other neurodegenerative diseases caused by gain-of-function type of genetic mutations.
40
S29
Translational research: possible explanations for the failure in ALS
Vincenzo Silani
Department of Neurology and Laboratory of Neuroscience,
University of Milan Medical School - IRCCS Istituto Auxologico Italiano, Milano, Italy
The most popular animal model in ALS is the SOD1 G93A mutant mouse. The use of this transgenic
in understanding the pathogenesis of the disease has become invaluable since 1994 (Gurney et al,
1994). As natural extension of neurobiological studies, any treatment to ALS patients has been
pretested in the SOD1 mouse. Today obligate informations in pre-clinical settings indicate drug trials
in this animal model as a prerequirement for any therapeutical approach in humans. The number of
treatments tested in the SOD1 mutant mouse has been endless but the applicability to ALS patients
on the basis of the efficacy in the model really poor. Success in human clinical trials have been
extremely limited, calling into question the utility of preclinical data for identifying therapeutical
agents that are worthy of further studies in humans. There are several possible explanations for the
failure of the successful animal studies to translate into effective therapies in humans. The main
possibility is that the SOD1 mouse really represents an animal model for familial rather than sporadic
ALS. A second possible explanation is that therapies in the mouse are most commonly initiated prior
to the clinical onset of the disease. These considerations and many others have been recently
reviewed (Benatar, 2007). The scientific community has reached a large awareness of the scarce
therapeutical translation from mice to humans in a desperate disease as ALS. A preliminary
document has been produced and guidelines for the preclinical in vivo evaluation of pharmacological
active drugs assembled (Ludolph et al., 2007). We need animal models mimicking motoneuronal
selective vulnerability such as SOD1 transgenic mouse that, compared to transgenics for other
neurodegenerative disease, represents an elegant study model to define the pathobiology of ALS.
However, we need to further set accepted standards for drug testing. Translation of the SOD1
transgenic data to humans needs to be perfected but the ALS scientific community has the skill and
commitment to achieve the goal.
References

Benatar. Neurobiol Dis. 2007 Apr;26(1):1-13. Epub 2007 Jan 3;

Gurney et al. Science 1994, 264, 1772-1775;

Ludolph et al. Amyotrophic Lateral Sclerosis First published on 05 April 2007; DOI:
10.1080/17482960701292837.
41
S30
SOD1-People: It's time for a trial
Jonathan Glass
Emory Center for Neurodegenerative Disease
Woodruff Memoriall Research Building, Atlanta GA 30322, USA
A small minority of people with ALS carry pathogenic mutations in the SOD1 protein. This discovery
led to the development of transgenic models of SOD1-related ALS in mice and rats, as well as in
non-mammals such as fish and worms. The biological consequences of SOD1 mutations are many,
and there are a number of hypotheses regarding how SOD1 mutant proteins lead to motor neuron
dysfunction and death. Even so, it is still unclear how mutant SOD1 leads to clinical disease in
animals, and there is precious little data on the mechanism(s) of disease in SOD1 people.
There have been more than 150 reports of therapeutic interventions in animal models of SOD1
related ALS (Benatar, 2007), many of which demonstrate the "effectiveness" of treatments directed at
one or several of the hypothetical mechanisms of disease. Several of these promising animal studies
have led to human clinical trials. Unfortunately, none of these trials has shown beneficial effects in
humans with ALS. These failures in human clinical trials must force us to re-evaluate how we
evaluate preclinical data from SOD1 models as tools for translation to humans. There are several
potential problems with this approach:
1.
The overwhelming majority of participants in ALS clinical trials have sporadic ALS, and not
familial ALS. Very few would be expected to have SOD1-related ALS.
2.
There are no data supporting the idea that the mechanism(s) of disease in sporadic and
familial ALS or SOD1-related ALS are the same.
3.
Many of the therapeutic "successes" in animal models were demonstrated in experimental
paradigms requiring presymptomatic treatment of animals.
4.
The animal models of SOD1-related ALS require that the mutant protein be massively
overexpressed in order to generate a clinical or pathological phenotype. This is not the case in
humans with SOD1-related ALS.
A logical and reasonable approach to better understanding the pathogenesis of ALS and developing
new treatments is to focus more attention on people and families with SOD1-related ALS. These
people represent the true human counterpart to the mutant SOD1 that have been studied so
intensively. We have found this to be highly motivated to participate in epidemiologic and genetic
surveys, and many have expressed their willingness to participate in a preventive clinical trial. Much
can be learned from this population: What is the true risk of carrying an SOD1 mutation? What
factors other than genetics influence the onset of disease? Are there proteomic, physiologic, or other
biomarkers that can be used to predict disease onset? Are there similarities or differences in disease
mechanisms in SOD1-related ALS and sporadic ALS? Will a therapeutic trial in people carrying
SOD1 mutations show positive results that correspond to similar trials in animals?
SOD1 people are an experimental resource that are waiting to be studied. Its time.
This work is presented as a collaborative effort with Michael Benatar, MD, and Meraida Polak, RN,
along with Samantha Kaplan and Crystal Richards, MPH. Funding for this project is from Emory
University's Woodruff Health Science Center and a fellowship grant to MB from the American
Academy of Neurology and the ALS Association.
42
S31
Drug discovery and development for ALS
Lucie Bruijn
The ALS Association, 4338 Lavender Drive, Palm Harbor FL34685; email: lucie@alsa-national.org
Currently there is only one FDA approved drug for ALS, Riluzole, which has a modest effect on
patient survival. There is a real need for the development of effective drugs for the disease. There is
no single cell based assay that best represents the disease mechanism in ALS (it is likely that
multiple mechanisms are involved in disease progression). In partnership with The National Institute
of Neurological Disorders (NINDS), The ALS Association tested 1000 FDA approved compounds in
10 different assays modeling aspects of the disease. The most promising lead from this screen has
moved forward into clinical trials for ALS. Expanding on these efforts and taking a more directed and
focused approach to ensure that projects once initiated and promising will be taken through the
necessary steps to move better chemical entities into model systems and finally the clinic, The ALS
Association launched its new program Translational Research Advancing Therapies for ALS (TREAT
ALS) in 2005. The intent of this new program is to develop novel compounds and take advantage of
drugs in development that have potential for ALS from the academic and biotech sector. This
presentation will provide an overview of ongoing efforts and future directions for Drug Discovery and
Development in ALS.
43
Selected oral communications
44
SO1
Dismutase-inactive mutant SOD1 has different functional properties when
heterodimerized with wild type SOD1
Heidrun Witan (1), Ingrid Koziollek-Drechsler (1), Rebecca Wade (2), Christian Behl (1) and Albrecht
M. Clement (1)
Institute for Physiological Chemistry and Pathobiochemistry, University of Mainz, Medical School,
55099 Mainz; (2) EML Research, Heidelberg.
The toxic properties of amyotrophic lateral sclerosis (ALS)-causing mutant Cu/Zn superoxide
dismutase (SOD1) proteins are still elusive. Among the proposed, but recently challenged, toxic
qualities are the increased aggregation capacity and an aberrant enzymatic activity. Furthermore,
contradictory reports exist to which extend the presence of wild type SOD1 ( WTSOD1) contributes to
disease onset and progression.
In order to investigate the role of hWTSOD1 in ALS, we initially tested if mutant monomers can form
functional dimers with hWTSOD1 in HEK-293 cells. The wild type-like mutants A4VSOD1, G37RSOD1
and G93ASOD1 but also copper-free G85RSOD1 form dismutase active SOD1 dimers after transient
transfection. In order to study the biochemical and functional properties of heterodimers, we
generated SOD1 dimer constructs allowing the expression of a fusion protein of two covalently linked
SOD1 monomers with an EGFP tag. Although the two SOD1 monomers are in close proximity, they
do not form a static functional dimer as others have reported by introducing intermolecular disulfide
bonds. We demonstrated that hWTSOD1, A4VSOD1, G37RSOD1 and G93ASOD1 homodimers as well as
mutant-WT heterodimers display dismutase activity, suggesting that the dimer formation and 3Dstructure remain unaffected by the linker and the EGFP-tag. Even G85RSOD1 heterodimers showed
activity, whereas G85RSOD1 homodimers are inactive as previously published. Homo- and
heterodimers show different aggregation behaviour. When expressed transiently, mutant homodimers
form visible aggregates, whereas heterodimers have a largely reduced aggregation potential. These
results have been confirmed by centrifugation assays. Mutant homodimers are enriched in the pellet
fractions, whereas heterodimers remain largely in the cytosolic fractions.
Based on their differential activity and aggregation behaviour, we propose that studying the functional
properties of homo- and heterodimeric SOD1 proteins may give new insights into the role of hWTSOD1
in ALS pathogenesis and possibly will shed more light on the toxic properties of mutant SOD1
This work was supported by the MAIFOR Program and the IFZN of the University of Mainz. H.W. was
supported by the “Graduiertenkolleg Neuroscience” of the University of Mainz.
45
SO2
Cysteine 111 affects aggregation of mutant SOD1 in NSC34 cells
Cozzolino M. (1), Amori I. (1), Pesaresi M.G. (1), Ferri A. (1,3), Nencini M. (1), Carrì M.T. (1,2).
(1) Lab. of Neurochemistry, Fondazione S. Lucia IRCCS, Rome, Italy; (2) Department of Biology,
University of Rome “Tor Vergata”, Rome, Italy; (3) Inst. of Neuroscience CNR, Dept. of
Psychobiology and Psycopharmacology, Rome, Italy
Converging evidence indicates that aberrant aggregation of mutant SOD1 is strongly implicated in
FALS. Mutant SOD1 forms high molecular weight oligomers which disappear under reducing
condition, both in neural tissues of FALS transgenic mice and in cells overexpressing mutSOD1,
indicating a role of aberrant intermolecular disulfide crosslinking in the oligomerization and
aggregation process 1, 2. To study the specific contribution of cysteines in the mechanism of
aggregation, we mutated human SOD1 in each of its cysteine residues in position 6, 57, 111 and
146. Using a cell transfection assay for SOD1 solubility and aggregation, we analysed the behaviour
of those SOD1s. Preliminary results showed that cysteines affected hSOD1 solubility to different
extents, with mutation of Cys6 the more effective in promoting SOD1 aggregation and mutation of
Cys111 having no effect. A mutated cysteine background was also used to test the solubility of G93A
mutSOD1. Interestingly, the removal of Cys111 strongly reduced the ability of G93A SOD1 to form
aggregates in cultured NSC34 cells. Accordingly, insoluble G93A mutSOD1 showed an altered
reactivity of Cys11 to modification by maleimide, suggesting the occurrence of cysteine oxidation.
Moreover, treatments that depleted the cellular pool of reduced glutathione (GSH) exacerbated
mutSOD1 insolubility, while an overload of intracellular GSH significantly rescued mutSOD1
solubility. These data are consistent with the view that the redox environment influences the
oligomerization/aggregation pathway of mutant SOD1 and point to Cys111 as a key mediator of this
process.
1.
Ferri, A. et al. Familial ALS-superoxide dismutases associate with mitochondria and shift their
redox potentials. Proc Natl Acad Sci U S A 103, 13860-5 (2006).
2.
Deng, H. X. et al. Conversion to the amyotrophic lateral sclerosis phenotype is associated
with intermolecular linked insoluble aggregates of SOD1 in mitochondria. Proc Natl Acad Sci U S A
103, 7142-7 (2006).
46
SO3
Detection of misfolded SOD1 in sporadic and familial ALS
Rakhit R (1), Robertson J (1), Vande Velde C (2), Horne P (1), Ruth DM (1), Griffin J (1), Cleveland
DW (2), Cashman NR (3), Chakrabartty A (1)
(1) Dept Med Biophysics/CRND, University of Toronto, Toronto, Canada. (2) Ludwig Inst., UCSD, La
Jolla, USA. (3) Dept Medicine, UBC, Vancouver, Canada.
Protein misfolding diseases result from the toxicity associated with conversion of the native state of a
protein into a pathologically misfolded conformation. In 20% of familial ALS, mutations in SOD1
cause the protein to misfold and form intracellular inclusions. Toxicity of these cytoplasmic
aggregates is thought to arise from aberrant interactions with the protein-folding chaperones or from
inhibition of proteasomes. Toxicity has also been proposed to result from aberrant interactions with
mitochondrial proteins (Tom20/Bcl-2) because SOD1 has been detected in mitochondria from spinal
cord, and mitochondrial vacuolization is an early event in ALS models.
The ability to detect misfolded proteins in vivo would be very beneficial for diagnosis and for
furthering research into the molecular basis of the disease. Our approach to developing in vivo
conformational probes involves generation of antibodies that detect non-native states of a protein, yet
do not bind natively folded molecules.
We have developed an antibody that specifically recognizes monomer/misfolded forms of SOD1. This
antibody was raised to an epitope within the SOD1 dimer interface, which is normally buried within
the native obligate homodimer. This SOD1 exposed dimer interface (SEDI) antibody, only recognizes
SOD1 conformations where the native dimer is disrupted/misfolded, exposing the hydrophobic dimer
interface. We have used this antibody to detect misfolded SOD1 in transgenic rodent ALS models
and in human tissue.
Using the SEDI antibody we establish the presence of monomer/misfolded SOD1 in three ALS
mouse models, with G37R, G85R or G93A -SOD1 mutations, and in a human individual with an A4V
SOD1 mutation. Despite ubiquitous expression, misfolded SOD1 is found primarily within
degenerating motor neurons and is preferentially enriched in mitochondrial and membrane fractions
from affected spinal cords. Misfolded SOD1 appears before symptom onset and decreases at
disease end-stage, concomitant with motor neuron loss. Extracellular misfolded SOD1 was also
observed by exclusion of double staining with markers of CNS cell types. Misfolded SOD1 was
present in brain and spinal cord, but absent in tissues that are unaffected by disease: liver, heart,
muscle, and kidney. Spinal cord sections from human individuals with sporadic ALS and familial ALS
with and without SOD1 mutations are being examined.
47
SO4
The Evolution of the Amyotrophic Lateral Sclerosis Database (ALSOD)
Wroe RJ, Butler AW, Powell JF and Al-Chalabi A
MRC Centre for Neurodegeneration Research, Department of Neuroscience, Institute of Psychiatry,
King’s College London, UK.
To date, more than 100 different point mutations spanning across the 153 amino acid SOD1
sequence have been identified causing ALS. In 1999 the ALSOD database was generated to store
these point mutations along with ALS patient information, in an attempt to correlate ALS genotype
with ALS phenotype (1). The aim of the database was to further understand the disease producing
allelic variations and genetic diversity associated with ALS. Here we report our ongoing development
and redesign of the ALSOD database and its automated procedures. The significant new features
have improved ALSOD helping to link the genetic mutations of the SOD1 protein to the hypothetical
3D structural rearrangements caused by the mutation and the resulting patient phenotype. In addition
to the ALS genotype to phenotype relationship, the new version of ALSOD now provides a more
comprehensive knowledgebase for ALS, detailing genetic, proteomic, and bioinformatics information
associated with the disease. The redeveloped site also enables ALSOD to be searched for a specific
ALS subject based on their demographic and disease information. We believe that ALSOD provides
an ideal vehicle so that ALS data can not only be recorded but also searched.
(1) Radunovic A, Leigh PN. ALSODatabase: database of SOD1 (and other) gene mutations in ALS
on the Internet. European FALS Group and ALSOD Consortium. Amyotroph Lateral Scler Other
Motor Neuron Disord. 1999 Dec;1(1):45-9.
48
SO5
Interaction between familial ALS-linked SOD1 mutants and the dynein complex
Fujian Zhang (1), Anna-Lena Ström (2), Kei Fukada (2), Sangmook Lee (3), Lawrence J. Hayward (3)
and Haining Zhu (1,2)
(1) Graduate Center for Nutritional Sciences, (2) Department of Molecular and Cellular Biochemistry,
College of Medicine, Lexington, KY 40536, (3) Department of Neurology, University of Massachusetts
Medical School, Worcester, MA 01655
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive
motor neuron death. More than 90 mutations in the copper-zinc superoxide dismutase (SOD1) gene
cause a subset of familial ALS. Toxic properties have been proposed for the ALS-linked SOD1
mutants, but the nature of the toxicity has not been clearly specified. Cytoplasmic inclusion bodies
containing mutant SOD1 and a number of other proteins are a pathological hallmark of mutant SOD1
mediated familial ALS, but whether such aggregates are toxic to motor neurons remains unclear. In
this study, we identified a dynein subunit as a component of the mutant SOD1-containing highmolecular-weight complexes using proteomic techniques. We further demonstrated interaction and
colocalization between dynein and mutant SOD1, but not normal SOD1, in cultured cells and also in
G93A and G85R transgenic rodent tissues. Moreover, the interaction occurred early, prior to the
onset of symptoms in the ALS animal models and increased over the disease progression. Motor
neurons with long axons are particularly susceptible to defects in axonal transport. Our results
demonstrate a direct “gain-of-interaction” between mutant SOD1 and dynein, which may provide
insights into the mechanism by which mutant SOD1 could contribute to a defect in retrograde axonal
transport or other dynein functions. The aberrant interaction is potentially critical to the formation of
mutant SOD1 aggregates as well as the toxic cascades leading to motor neuron degeneration in
ALS.
49
SO7
Excessive exocytotic release of glutamate in the spinal cord of SOD1 G93A mutant
mice
Milanese M. (1), Zappettini S. (1), Raiteri L. (1), Popoli M (2), Barbiero V.S. (2), Bonanno G. (1)
(1) Department of Experimental Medicine, Pharmacology and Toxicology Section, and Centre of
Excellence for Biomedical Research, University of Genoa, Italy
(2) Department of Pharmacological Sciences, Center of Neuropharmacology, and Center of
Excellence on Neurodegenerative Diseases, University of Milan, Italy
Among the different hypotheses to explain motor neurons death in amyotrophic lateral sclerosis
(ALS), glutamate-mediated excitotoxicity may play a mayor role. Abnormalities in glutamate transport,
mainly a reduced expression and function of GLT-1, were observed in synaptic preparations of motor
cortex and spinal cord in ALS. It has been suggested that this reduction in GLT-1 activity could
explain the higher levels of glutamate in ALS patients and in animal models of the disease Since
GLT1 is mainly localized in astroglial processes, it was hypothesized that glial cells play a role in the
development of the disease. Alternatively, elevated extra-cellular concentrations of glutamate may
well be due to augmentation of neuronal glutamate release rather than to the astrocyte-localized
inhibition of reuptake.
We have previously shown that the heterotransporter-mediated glycine- ad GABA-induced release of
[3H]D-aspartate ([3H]D-Asp) from mouse spinal cord synaptosomes was more elevated in mice
expressing human SOD1 with the Gly93A substitution [SOD1(+)/G93A(+)], a transgenic model of
ALS, respect to mice expressing the unmodified human SOD1 [SOD1(+)] and in non transgenic
littermates [SOD1(-)/G93A(-)] (1, 2). We have here studied the release of 3HD-Asp and of
endogenous glutamate induced by depolarizing and non depolarizing stimuli known to induce
exocytotic neurotransmitter release.
The spontanoeus outflow of the excitatory amino acid was higher in SOD1/G93A(+) than in SOD1(+)
or SOD1(-)/G93A(-) mice. Exposure to 15 or 25 mM KCl or to 0.3 µM ionomycin provoked an almost
complete Ca2+-dependent release of [3H]D-Asp from spinal cord synaptosomes. The exocytotic
release of 3HD-Asp and glutamate induced by KCl or ionomycin was dramatically increased in
symptomatic SOD1(+)/G93A(+) mice than in controls. The higher glutamate release in mutant
animals was already present in pre-symptomatic 70-90 and 30-40 day-old mice. Noticeably, both the
stimulus-evoked release of GABA in spinal cord and of [3H]D-Asp in motor cortex of
SOD1(+)/G93A(+) mice did not differ from controls
As to the molecular determinants of this increased glutamate exocytosis, we have studied the
SNARE-complex proteins expression, the SNARE-complex formation, CaM kinasi II
phosphorylation, its interaction with syntaxin-1. The results indicate that spinal cord glutamatergic
nerve terminals undergoes to some presynaptic modifications which may sustain the increased
glutamate exocytosis.
Our in vitro results show that the exocytotic release of glutamate is enhanced mutant SOD1 mice. If it
occurs in vivo, the different modulation of GLU and GABA release here reported could induce an
unbalance between spinal inhibitory and excitatory transmission in ALS.
(1) Raiteri L., Paolucci E., Prisco S., Raiteri M. and Bonanno G. (2003) Activation of a glycine
transporter on spinal cord neurons causes enhanced glutamate release in a mouse model of
amyotrophic lateral sclerosis
Br. J. Pharmacol. 138, 1021-1025.
(2) Raiteri L., Stigliani S., Zappettini S., Mercuri N.B., Raiteri M. and Bonanno G. (2004)
Excessive and precocious glutamate release in a mouse model of amyotrophic lateral sclerosis
Neuropharmacology 46, 782-792.
50
SO8
Effects of physical exercise and the steroid nandrolone on neuromuscular junctions
in G93A -SOD1 transgenic mice
Francolini M.(1), Cappello V.(1), Matteo Fossati (1), Mariotti R.(2), Kassa R. M.(2), Padovano V. (1),
Bentivoglio M (2), and Pietrini G. (1)
(1) Department of Pharmacology, School of Medicine, Università degli Studi di Milano; CEND- Center
of neurodegenerative diseases; CNR-Institute of Neuroscience , Milan, Italy. (2) Department of
Morphological and Biomedical Sciences, University of Verona, Verona, Italy.
Evidence in mice and man indicates that ALS is a distal axonopathy, with pathological changes of
motoneurons beginning at the distal axons (with early mitochondrial damages and selective loss of
neuromuscolar synapses) and proceeding in a "dying back" pattern (1). Quantitative analysis
demonstrated denervation at the neuromuscular junction (NMJ) by day 47 in SOD1(G93A) mice in
which signs of clinical disease appeared at about 80 days. A confirmation of ALS as a distal
axonopathy also in the human pathology derived from autoptic findings in a patient, demonstrating
denervation and reinnervation changes in muscle but normal appearing motoneurons (2). The
molecular mechanisms underlying these defects are not known, but they suggest an altered plasticity
of NMJs. SOD1(G93A) transgenic mice have been used as a model to study the effects of strenuous
exercise and/or the anabolic steroid nandrolone on NMJs by immunofluorescence and electron
microscopy. Transgenic SOD1-G93A mice and their wild type littermates were sacrificed at disease
onset (P90) and diaphragm and triceps has been dissected, fixed by perfusion and subsequent
immersion in 4% paraformaldehyde for 2 hours and extensively washed with phosphate buffer.
Teased muscles bundles composed of 20-30 fibers were excised from diaphragms and stained with
TRITC-conjugated alpha-bungarotoxin (Molecular Probes) to label the nAchR in the postsynaptic
membrane. These bundles were then immunolabeled with anti-synapsin in order to evaluate the
percentage on innervated/denervated fibres in the different experimental models to evaluate the
protective or detrimental effect of the treatments in this muscle. For the ultrastructural analyses
bundles of fibers were dissected, fixed with 2% glutaraldehyde and further processed for
transmission electron microscopy.
Our preliminary results indicate that strenuous exercise
decreases the number of innervated fibres in both transgenic and wild type littermates, with a
remarkable effect in transgenic mice. On the contrary, the chronic steroid treatment appears
beneficial, although insufficient to prevent the negative effect of physical exercise in G93A transgenic
mice.
It has been recently proposed that mutant SOD1 accumulated in the presynaptic terminal may
contribute directly to denervation or to failure of successful reinnervation of neuromuscular junctions
(3), we are therefore looking for the presence and the localization of mutant SOD1 in the NMJs of
transgenic mice diaphragms.
(1) Frey D, Schneider C, Xu L, Borg J, Spooren W, Caroni P. (2000). Early and selective loss of
neuromuscular synapse subtypes with low sprouting competence in motoneuron diseases.J
Neurosci.20, 2534-2542.
(2) Fischer LR, Culver DG, Tennant P, Davis AA, Wang M, Castellano-Sanchez A, Khan J, Polak
MA, Glass JD (2004). Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and
man.Exp Neurol. 185, 232-240.
(3) Gould TW, Buss RR, Vinsant S, Prevette D, Sun W, Knudson CM, Milligan CE, Oppenheim RW.
(2006) Complete dissociation of motor neuron death from motor dysfunction by Bax deletion in a
mouse model of ALS. J Neurosci. 26, 8774-8786.
51
SO9
The potential of endothelial progenitors to recover the stem cell niche in ALS
Akbarloo N., Darabi R. and Perlingeiro R.C.R.
Department of Developmental Biology, UTSouthwestern Medical Center, Dallas-TX, USA
Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder in which motor
neurons die beginning in mid-adult life. The loss of large projection neurons results in severe
movement dysfunction. Transgenic mice that express mutant SOD1 develop a progressive motor
neuron disease that resembles human ALS, representing a valuable animal model to study novel
therapies for this disease. To date, ALS is still incurable and current cellular approaches to treat ALS
have attempted to replace damaged or deteriorating neurons by stem cell transplantation. Although
some improvement has been observed, regeneration of large projection neurons capable of
establishing appropriate connections to their targets is still out of reach. An alternate approach to
treat ALS would be to improve the microenvironment, which in turn would stimulate cell regeneration.
Endothelial progenitors mediate revascularization, which promotes improved blood flow in ischemic
tissues, a fundamental requirement for tissue regeneration. In this regard, human umbilical cord
blood (UCB) has been pointed out as a rich potential supply of endothelial progenitors, which are
known to express the surface marker CD133. To test the hypothesis that endothelial progenitors
isolated from human cord blood are able to repair the neuronal microenvironment in ALS, we have
generated an immunodeficient SOD1 mouse model, SOD1;Rag2-/-;gammaC-/-. These mice are
devoid of B, T, and NK cells and make excellent xeno-transplant recipients, allowing us to perform
human to mouse transplantations without immunosuppression. This immunodeficient mouse model
shows similar disease progression to conventional SOD1 mice. By week 18, untreated
immunodeficient SOD1 mice as well as the ones that had been transplanted with unfractionated
mononuclear cells (MNCs) presented the usual symptoms of disease, including significant weight
loss, marked kyphosis, and hind limb muscle wasting, while mice that had received transplantation of
UCB-derived CD133+ cells presented no signs of disease by this time. Overall, an extension of 4
weeks was observed in the lifespan of SOD1;Rag2-/-;gammaC-/- infused with endothelial progenitors.
Rotarod analyses further demonstrated a delay in the impairment of motor function in mice that
received CD133+ cell transplantation when compared to untreated mice (an approximate 4 week
latency in motor performance deficits: week 20 vs week 16, respectively). Analyses of spinal cord
cryosections showed that transplanted mice had twice as many surviving motoneurons as did nontransplanted mice. These findings corroborate our hypothesis that early endothelial progenitors can
preserve or repair the neurogenic niche and provide scientific rational for the therapeutic application
of UCB transplantation in ALS.
52
Posters
Posters
Posters
53
P1
The Molecular Mechanism of Superoxide Dismutase Aggregation in Lou Gehrig's
Disease
Sagar D. Khare, Michael Caplow, Kyle Wilcox and Nikolay V. Dokholyan
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill,
School of Medicine, Campus Box 7260 Chapel Hill, NC 27599 USA
Mutation-induced aggregation of the dimeric enzyme Cu, Zn superoxide dismutase (SOD1) has been
implicated in the familial form of the amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease, but
the mechanism of aggregation is not known. We studied the folding thermodynamics and kinetics of
SOD1 using a hybrid molecular dynamics approach. We found that the residues which contribute the
most to SOD1 thermal stability are also crucial for apparent two-state folding kinetics. Surprisingly,
we found that these residues are located on the surface of the protein and not in the hydrophobic
core. Mutations in some of the identified residues are found in patients with the disease. We argue
that the identified residues may play an important role in aggregation (1,2).
We further investigated in silico the sequence and structural determinants of SOD1 aggregation: (a)
we identified sequence fragments in SOD1 that have a high aggregation propensity, using only the
sequence of SOD1, and (b) we performed molecular dynamics simulations of the SOD1 dimer folding
and misfolding. In both cases, we identified identical regions of the protein as having high propensity
to form intermolecular interactions. These regions correspond to the N- and C-termini, and two crossover loops and two -strands in the Greek-key native fold of SOD1. Our results suggest that the high
aggregation propensity of mutant SOD1 may result from a synergy of two factors: the presence of
highly amyloidogenic sequence fragments ("hot-spots"), and the presence of these fragments in
regions of the protein that are structurally most likely to form inter-molecular contacts under
destabilizing conditions.
Therefore, we postulated that the balance between the self-association of aggregation-prone
sequences and the specific structural context of these sequences in the native state determines the
aggregation propensity of proteins (3,4).
We further experimentally measured dissociation rate and equilibrium constants for human SOD1
dimer dissociation. We have also determined that loss of Zn is required for SOD1 aggregation. Using
computation and experiments, we determined the rates of Zn loss and further SOD1 aggregation.
Thus, we have identified a minimal aggregation sequence of SOD1 and measured the rate and
equilibrium constants for each step of this sequence. Our findings suggest the role of mutations that
are associated with familial ALS (5).
(1) S. D. Khare, F. Ding, and N. V. Dokholyan, "Folding of Cu, Zn superoxide dismutase and Familial
Amyotrophic Lateral Sclerosis" Journal of Molecular Biology, 334: 515-525 (2003)
(2) S. D. Khare and N. V. Dokholyan, "Common dynamical signatures of FALS-associated
structurallydiverse
Cu, Zn superoxide dismutase mutants" Proceedings of the National Academy of Sciences
USA, 103: 3147-3152 (2006)
(3) S. D. Khare, K. C. Wilcox, P. Gong, and N. V. Dokholyan, "Sequence and structural determinants
of
Cu, Zn superoxide dismutase aggregation" Proteins: Structure, Function, and Bioinformatics, 61: 617632 (2005)
(4) S. D. Khare, M. Caplow, and N. V. Dokholyan, "FALS mutations in Cu, Zn superoxide dismutase
destabilize the dimer and increase dimer dissociation propensity: a large-scale thermodynamic
analysis" Amyloid: the Journal of Protein Folding Disorders, 13: 226 - 235 (2006)
(5) S. D. Khare, M. Caplow, and N. V. Dokholyan, "The rate and equilibrium constants for a multi-step
reaction sequence for the aggregation of superoxide dismutase in ALS" Proceedings of the National
Academy of Sciences USA, 101: 15094-15099 (2004)
54
P2
Early misfolding events in human Cu-Zn superoxide dismutase apo-enzyme are
revealed by molecular dynamics
Richard W. Strange*, Chin W. Yong*, William Smith & S. Samar Hasnain
CCLRC Daresbury Laboratory, Warrington, Cheshire WA4 4AD
Mutations of the gene encoding Cu-Zn superoxide dismutase (SOD1), a vital antioxidant enzyme,
cause 20 % of the familial cases of the progressive neurodegenerative disease amyotrophic lateral
sclerosis (FALS). A growing body of evidence suggests that it is the molecular behaviour of the
dimeric molecule in the absence of metals that leads to gain of toxic properties via misfolding,
unfolding and aggregation. Structural studies have provided static snapshots on the behaviour of the
molecule in the absence of the metals. Using atomic resolution structure of fully metallated human
SOD1 and highly parallelised MD code on a high performance capability computer, we have
employed molecular dynamics to reveal the first stages of misfolding due to metal-deletion. These
calculations reveal the first steps in the formation of experimentally observed aggregations of FALS
mutant SOD1. This result could have implications for the role of de-metallated wild-type SOD1 in
sporadic cases of ALS, for which the molecular cause still remains undiscovered.
55
P3
Oxidized wild type SOD1 acquires toxic properties reminiscent of mutant SOD1
species
Abou Ezzi S. (1), Urushitani M. (2), Julien J.-P. (1)
(1) CHUL research center, Laval University, Quebec, QC, Canada; (2) Molecular Neuroscience
research center, Shiga university, Shiga, Japan
Sporadic and familial ALS cases are clinically indistinguishable, suggesting that common pathogenic
mechanisms are involved. Oxidative stress is implicated in the pathogenesis of neurodegenerative
diseases including Parkinson’s, Alzheimer’s and ALS (1). Recent studies suggest that superoxide
dismutase (SOD1) may represent a major target of oxidative damage in neurodegenerative diseases.
To test the possibility that oxidized species of wild type (WT) SOD1 might be involved in pathogenic
processes, we analyzed the properties of the WT human SOD1 protein after its oxidation in vivo or in
vitro by hydrogen peroxide (H2O2) treatment. Using transfected Neuro2a cells expressing WT or ALSlinked SOD1 species, we show that exposure to H2O2 modifies the properties of WT SOD1. Oxidized
WT SOD1 can be conjugated with poly-ubiquitin and can interact with Hsp70. Chromogranin B, a
neurosecretory protein that interacts with mutant SOD1 but not with WT SOD1(, was coimmunoprecipitated with oxidized WT SOD1 from lysates of Neuro2a cells treated with H 2O2.
Treatment of microglial cells (line BV2) with either oxidized WT SOD1 or mutant SOD1 recombinant
proteins induced tumor necrosis factor-α and inducible nitric oxide synthase. Furthermore, exposure
of cultured motor neurons to oxidized WT SOD1 caused cell death like mutant SOD1 proteins. These
results suggest that WT SOD1 may acquire binding and toxic properties of mutant forms of SOD1
through oxidative damage. Hence, the possibility that sporadic and familial ALS may share a
common pathogenic pathway involving abnormal SOD1 species must be considered.
(1) Shaw P. J. (2005) Molecular and cellular pathways of neurodegeneration in motor neurone
disease. J Neurol Neurosurg Psychiatry 76, 1046-1057.
(2) Urushitani M., Sik A., Sakurai T., Nukina N., Takahashi R. and Julien J. P. (2006) Chromograninmediated secretion of mutant superoxide dismutase proteins linked to amyotrophic lateral sclerosis.
Nat Neurosci 9, 108-118.
56
P4
SOD1 intracellular localization in copper-depleted NSC-34 motoneuronal cells
Arciello M (1), Capo CR (1), Ferri A (2), Cozzolino M (2), Carrì MT (1,2), Rossi L (1)
(1) Department of Biology, “Tor Vergata” University of Rome. Italy; (2) Laboratory of Neurochemistry,
Fondazione S.Lucia IRCCS, Rome, Italy.
A small fraction of SOD1 locates in the intermembrane space of mitochondria, where it enters as
apo-protein (1); it has been postulated that the accumulation of mutant SOD1 in mitochondria may be
a possible cause of motoneuron death in familial amyotrophic lateral sclerosis (fALS) (2). We have
previously demonstrated that the treatment of neuroblastoma cells with the copper chelator Trientine
depletes SOD1 activity (3); this might increase the amount of copper-devoid SOD1 entering
mitochondria. Contradictory evidence has been reported on the correlation between copper-depletion
and ALS: treatment of fALS model mice with Trientine prolongs survival (4), while copper-deficient
patients show evidence of lower motor neuron disease, similar to ALS patients (5).
In order to clarify the role of copper deficiency in SOD1 sub-cellular localization and motor neuron
degeneration, we treated murine motoneuronal NSC-34 cells, transfected with human wild-type
SOD1, with Trientine for 72 hours. Intracellular copper level was measured by atomic absorption
spectrometry, showing a decrease of about 50%. The activity of transfected SOD1 (assessed by
specific staining of gels, after electrophoresis under non-denaturing conditions) decreased, as well as
its protein content, evaluated by Western blotting. Mitochondria were isolated from transfected NSC34 cells following copper depletion and showed accumulation of wild-type SOD1. Cell viability as
well as cell cycle, analysed by flow cytometry, were not affected, indicating that accumulation in
mitochondria of wild-type SOD1 is not detrimental to cells. The same experimental design, but carried
out on NSC-34 expressing mutants SOD1, will elucidate whether these proteins behave differently
than the wild-type form under conditions of copper-depletion, thus affecting motorneuron viability.
(1)
Sturz L.A., Diekert K., Jensen L.T., Lill R. and Culotta V.C. (2001). A fraction of Cu,Znsuperoxide dismutase and its metallochaperone , CCS, localize to the intermembrane space of
mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage. J.
Biol. Chem. 276, 38084-38089.
(2)
Ferri A., Cozzolino M., Crosio C., Nencini M., Casciati A., Gralla E.B., Rotilio G., Valentine
J.S. and Carrì M.T. (2006). Familial ALS-superoxide dismutases associate with mitochondria and
shift their redox potentials. Proc. Natl. Acad. Sci. 103, 13860-13865.
(3)
Rossi L., Marchese E., Lombardo M.F., Rotilio G. and Ciriolo M.R. (2001). Increased
susceptibility of copper-deficient neuroblastoma cells to oxidative stress-mediated apoptosis.
Free Radic. Biol. Med. 30,1177-1187.
(4)
Nagano S., Fujii Y., Yamamoto T., Taniyama M., Fukada K., Yanagihara T. and Sakoda S.
(2003)The efficacy of trientine or ascorbate alone compared to that of the combined treatment with
these two agents in familial amyotrophic lateral sclerosis model mice. Exp. Neurol. 179, 176-180.
(5)
Weihl C.C. and Lopate G. (2006). Motor neuron disease associated with copper deficiency.
Muscle nerve. 34, 789-793.
57
P5
Doubling expression of SOD1 in SH-SY5Y cells leads to altered SOD1 forms that
are increased on G93A mutant expression
Cerqueira FM (1), Medinas DB (1), Demasi M (2), Carrí MT (3), Augusto O (1).
(1) Departmento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
(2) Departmento de Biologia, Instituto Butantan, São Paulo, Brazil. (3) University of Roma “Tor
Vergata”, Rome, Italy.
About 20% of familial ALS (fALS) cases are due to mutations in the Cu,Zn-superoxide dismutase
(SOD1) gene. It was thought that the mutated enzymes would have impaired SOD activity, but this
has not been corroborated so far. Presently, it is more accepted that the mutated enzymes acquire a
new toxic function. What this new toxic function is and how it relates to the degeneration of motor
neurons remains debatable. Here, we compared human neuroblastoma cells transfected with fALS
mutant G93A (SH-SY5YG93A) or wild-type SOD1 (SH-SY5YWT) with parent cells (SH-SY5Y) (1) in
regard to growth, viability, basal oxidant production, SOD activity and SOD forms. Transfected cells
presented increased growth rate and higher basal oxidant production. SH-SY5YWT and SH-SY5YG93A
cells in early culture stages showed the expected 2-fold increase in SOD expression and activity.
After 4 weeks of culture, SH-SY5YG93A maintained SOD1 expression levels but dismutase activity
decreased to parent cell levels. Cells transfected with SOD1 presented increased levels of altered
SOD1 forms such as the reduced enzyme, disulfide multimers and anionic detergent-insoluble forms,
particularly in SH-SY5YG93A cells. Among the insoluble forms, a thiol-resistant SOD dimer was
detected by SDS-PAGE. A similar dimer has been previously detected during the bicarbonatedependent peroxidase activity of human SOD1 (2). Altered SOD forms are probably responsible for
the increase in the chymotrypsin-like activity of the proteasome, verified in transfected cells.
In conclusion, doubling SOD1 expression was sufficient to increase altered SOD1 forms and the
G93A mutation enhanced their yields. To establish SOD1 dimer structure appears to be particularly
relevant because dimeric SOD forms have been detected in ALS animal models and patients, and
shown to shadow the onset and progression of motor neuron degeneration (3). Supported by:
FAPESP and CNPq.
(1) Carri, M. T.; Ferri, A.; Battistoni, A.; Famhy, L.; Gabbianelli, R.; Poccia, F.; Rotilio, G. (1997)
Expression of a Cu,Zn-superoxide dismutase typical of familial sclerosis induces mitochondrial
alteration and increased of cytosolic Ca2+ concentration in transfected neuroblastoma SH-SY5Y
cells. FEBS Lett., 414, 365-368.
(2) Zhang H, Andrekopoulos C, Joseph J, Crow J, Kalyanaraman B. (2004) The carbonate radical
anion-induced covalent aggregation of human copper, zinc superoxide dismutase, and alphasynuclein: intermediacy of tryptophan- and tyrosine-derived oxidation products. Free Radic Biol Med.
36, 1355-1365.
(3) Shaw BF, Valentine JS. (2007) How do ALS-associated mutations in superoxide dismutase 1
promote aggregation of the protein? Trends Biochem. Sci. 32, 78-85.
58
P6
Effect of the of small heat shock protein HspB8 in a cellular model of familial
Amyotrophic Lateral Sclerosis
Crippa V (1), Simonini F (1), Bolzoni E (1), Onesto E (1), Rusmini P (1), Sau D (1), Carra S (2),
Landry J (2), Poletti A (1)
(1) Institute of Endocrinology, Centre of Excellence on Neurodegenerative Diseases of the University
of Milan and InterUniversity Centre on Neurodegenerative Diseases (University of Florence, Rome
and Milan)
Via Balzaretti 9, 20133 Milan. tel 02-5031.8215; fax 02-5031.8204; E-mail: angelo.poletti@unimi.it
(2) Centre de recherche en cancerologie de l'Universite Laval. L'Hotel-Dieu de Quebec,
9, rue McMahon, Quebec, QC, CANADA G1R 2J6. tel: (418) 691-5281/fax: (418) 691-5439
About 10% of cases of amyotrophic lateral sclerosis (ALS) are familial (fALS) and 20% of them are
linked to mutations in Cu/Zn Superoxide Dismutase 1 (SOD1), a soluble enzyme present in cytosol,
nucleus, peroxisomes, lysosomes, and mitochondria (1,2). Compelling data support the idea that
fALS should be considered a protein misfolding disorder, in which a non native, toxic, oligomeric
conformation is generated in the mutant protein.
To study fALS we have produced a model based on immortalized motoneuron transfected with
plasmids coding for wild type (wt) or mutant SOD1 (wtSOD1/G93A-SOD1). While, as expected,
wtSOD1 was widely diffused in the cell, G93A-SOD1 was mainly present in the perinuclear region
forming citoplasmic and nuclear aggregates. High molecular weight (MW) species of G93A-SOD1
oligomers and SDS-resistant forms were detectable only in G93A-SOD1 samples. Filter retardation
assay revealed that wt protein was not retained by the cellulose acetate membranes, instead
significant amounts of insoluble G93A-SOD1 were detectable, suggesting that G93A-SOD1
generates misfolded species capable to produce aggregates. Using the YFPu, a reporter protein
targeted to degradation by the proteasome, we have shown a proteasome impairment when G93ASOD1 was expressed.
We have studied the effects of overexpression of HspB8 protein, a small heat shock protein, that has
already shown its ability on reducing aggregate formation of mutant polyglutamine proteins.
Overexpression of HspB8 reduced the levels of monomeric form of either wt and G93A-SOD1 and
increased the clearence of G93A-SOD1 oligomeric high MW species. Similar results were obtained in
presence of the proteasome inhibitor MG132. In fact, robust decrease of G93A-SOD1 insolubile
species was observed in presence of HspB8 both in basal condition and in presence of MG132.
Moreover, HspB8 reduced YFPu levels, suggesting a desaturation of the proteasome system.
By immunoprecipitation analysis we found no interaction between HspB8 and SOD1 proteins,
indicating that, probably, HspB8 does not need a direct interaction to exert its chaperone function.
The ability of HspB8 to reduce G93A-SOD1 levels even in presence of MG132 and to desaturate the
proteasome suggests that this chaperone might be involved in an alternative degradative pathway.
(1) Fridovich, I. Annu. Rev. Biochem. 1975, 44, 147.
(2) Valentine, JS, and Hart PJ. PNAS 2003. 100, 3617
Grants Telethon - Italy (#GGP06063),
MIUR-FIRB (#RBAU01NXFP);
(2005057598_002), University of Milan-FIRST, FONDAZIONE CARIPLO.
MIUR-Cofin
59
P7
Altered redox environment in a motor neuron-like cell model for SOD1-related forms
of amyotrophic lateral sclerosis
D’Alessandro G (1), Tartari S (1), Babetto E (2), Rizzardini M (1), Conforti L (2) and Cantoni L (1).
(1) Laboratory of Molecular Pathology, Dept. of Molecular Biochemistry and Pharmacology, Istituto di
Ricerche Farmacologiche “Mario Negri”, Milan, Italy.(2) Babraham Institute, Cambridge, United
Kingdom.
About 20% of familial ALS cases are associated to mutations in the Cu/Zn superoxide dismutase
gene (SOD1). The toxic functions acquired by these mutant SOD1 forms are still unclear. The
presence of oxidative damage was documented in ALS patients and in experimental models.
Glutathione is the main low-molecular weight thiol in mammalian cells. The GSSG/2GSH couple
provides a very large pool of reducing equivalents and is considered the most important cellular
redox buffer. However, data on the levels of GSH or GSSG in tissues of ALS patients and in SOD1
transgenic mice are scanty. Even in the SOD1-mouse model it has not been studied whether a
different disease stage might differently affect this parameter. Furthermore, when analyzing a whole
tissue, these investigations are made difficult by the heterogeneous nature of cell populations in the
sample. This study investigated how a prolonged exposure to the mutant G93ASOD1 affects the pool
of reduced glutathione (GSH) and glutathione disulfide (GSSG) in a cell model. Motor neuron-like cell
lines expressing a high level of wtSOD1 and a high or low level of G93ASOD1 were cultured for four,
seven and ten weekly passages. GSH and GSSG were determined spectrophotometrically in the
total cell lysates. In parallel, an untransfected control cell line was studied, that showed constant
levels of GSH and GSSG in a ratio of 100:1. At the fourth passage, GSH and GSSG were
significantly higher than in controls in all the transfected cells; however, the largest increase (about
three-fold) in GSH was in the high-G93ASOD1 cells; while GSSG increased more (about 2.5-fold) in
the wtSOD1 cells. At the tenth passage, the GSH increases were no longer evident, while GSSG
remained higher than in control cells. These changes increased the GSH/GSSG ratios in the
G93ASOD1 cells expressing a high level of the mutant protein at the early passages. They also
shifted the half-cell reduction potential values (Ehc) (calculated by the Nernst equation) towards a
more negative value only in the G93ASOD1 cells. In conclusion, G93ASOD1 altered not only the
reducing capacity but also the redox environment differently from the wtSOD1; exposure time
influenced this effect. This might modify key cellular processes such as signal transduction, DNA and
RNA synthesis, protein synthesis, enzyme activation and even regulation of the cell cycle and be
relevant to G93ASOD1 toxicity to motor neurons.
Financial support was provided by MIUR, FIRB, Protocol RBIN04J58W_000
---
60
P8
Induction of oxidative DNA damage in cells expressing mutant Superoxide
Dismutase 1 (SOD1)
Sau D(1), De Biasi S (4), Vitellaro L (4), Crippa V (1), Bolzoni E (1), Onesto E (1), Simonini F (1),
Riso P (2), Bendotti C (3), Poletti A (1)
(1) Inst. of Endocrinology, Centre of Excellence on Neurodegenerative Diseases, University of Milan,
Via Balzaretti 9, 20133 Milan, Italy. Tel/Fax 02-5031.8215/04 - angelo.poletti@unimi.it
Inter-University Research Centre on the molecular basis of neurodegenerative diseases
(2) Department of Food Science and Microbiology (DiSTAM), Human Nutrition Unit, University of
Milan;
(3) Institute of Pharmacological Research Mario Negri, Via Eritrea 62- 20157 Milan, Italy.
(4) Department of Biomolecular Sciences and Biotechnology, University of Milan,ViaCeloria 2620133 Milan, Italy.
Amyotrophic Lateral Sclerosis (ALS) is characterized by a selective loss of both upper and lower
motorneurons. About 10% are inherited cases (familial ALS or fALS) and, among them, about 20%
are linked to Superoxide Dismutase 1 (SOD1) mutations. SOD1, one of the major cellular antioxidant
enzyme is localized in the cytosol, nucleus, peroxisomes, lysosomes, and mitochondrial
intermembrane space (1, 2); the active form is composed of a soluble homodimer of ~32 kDa., whose
enzymatic activity is preserved in most mutant forms. Immunocytochemical analysis, performed on
spinal cord sections from transgenic mice, expressing either wild type (wt), or mutant G93A-SOD1
(Gly to Ala in 93), showed that human wtSOD1 was detectable both in cytoplasm and, at lower levels,
in the nuclei of motorneurons, while nuclear levels of mutant SOD1 were reduced. We have
confirmed this peculiar distribution of the mutant SOD1 in immortalized motor neurons (NSC34),
transfected with wt and mutant SOD1. In these cells, we also observed the formation of cytoplasmic
and nuclear inclusions. Moreover, western blot and filter retardation assay showed that G93A-SOD1
aggregates formed insoluble species. We have analyzed the Ubiquitin-Proteasome-Pathway (UPP),
using reporter plasmids expressing Yellow Fluorescent Protein (YFP), fused with a short degron
which direct s YFP to proteasome degradation, and also carrying either a Nuclear Localization Signal
(YFP-NLS) or a Nuclear Exporting Signal (YFP-NES); we found proteasome impairment only in the
cytoplasm. The effect of mutant SOD1 exclusion from nuclei on genomic DNA integrity was analyzed
using a COMET assay (Single Cell Gel Electrophoresis), on NSC34 expressing SOD1s, after
induction of oxidative stress with H2O2. Cells expressing G93A-SOD1 showed a higher DNA damage
compared to those expressing wtSOD1, possibly because of a loss of nuclear protection. Therefore,
an increased nuclear concentration of free radical species may results from their lower clearance due
to the sequestration of the SOD1 enzyme into insoluble inactive forms. The data obtained suggest
that the nucleus may be a target of G93A-SOD1 neurotoxicity in fALS which might arise from an
initial misfolding (gain-of-function), generating nuclear deprivation of the active enzyme (loss-offunction in the nuclei), a process that may be involved in ALS pathogenesis.
1) Fridovich, I. Annu. Rev. Biochem. 1975, 44, 147.
2) Valentine, JS, and Hart PJ PNAS 2003. 100, 3617
Grants
Telethon
Italy
(#GGP06063),
MIUR-FIRB
(#RBAU01NXFP);
(2005057598_002), University of Milan-FIRST, FONDAZIONE CARIPLO.
MIUR-Cofin
61
P9
Glutathione and protein-glutathione mixed disulfides compartmentalization in intact
motor neuron-like cells
Tartari S (1), D’Alessandro G (1), Veglianese P (1), Rizzardini M (1), Ottersen OP (2) and Cantoni L
(1).
(1) Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy. (2) Institute of Basic Medical
Sciences, University of Oslo, Oslo, Norway
Mutations in the Cu/Zn superoxide dismutase gene (SOD1) are associated with 20% of familial
amyotrophic lateral sclerosis (fALS) cases, oxidative stress being one of the mechanisms possibly
involved in the toxicity of the mutant protein(s). Glutathione is an ubiquitous tripeptide. Its reduced
form (GSH) has important antioxidant properties and is in equilibrium with oxidized species, including
protein-GSH mixed disulfides (Pr-SSG). This equilibrium is altered in the presence of
oxidative/nitrosative stress. Usually, levels of intracellular GSH and PrSSG are measured using total
cell lysates or subcellular fractions obtained by differential centrifugation, but very poor information
and methodology are available to determine the compartmentalization of GSH and Pr-SSG in the
intact cell. This could be relevant to understand the pathogenic mechanisms of mutant forms of
SOD1, since it was suggested that toxicity could be associated with depletion of the mitochondrial
pool of GSH in motor neurons, the cell population preferentially affected in ALS. The aim of this study
was to assess the localization and the relative distribution of GSH and PrSSG in a motor neuron-like
cell line (NSC-34-tTA40) established in our laboratory. Cells were fixed and stained with a polyclonal
GSH-specific antibody coupled to an ALEXA-Fluor 594 secondary antibody and analyzed by laser
confocal microscopy. GSH immunostaining was clearly evident in the cell body and in the neurites
while the central area of the cell, likely corresponding to the nucleus, was almost completely devoid
of staining. Treatment with buthionine sulfoximine (20 M for 24 hr), an inhibitor of GSH synthesis,
drastically diminished the intensity of the labelling confirming the antibody selectivity. In another
experiment, NSC-34-tTA40 cells were exposed to the thiol oxidant diamide (1mM for 5 minutes),
fixed and treated with a specific washing procedure to remove unbound GSH. Marked
immunoreactivity for Pr-SSG (virtually absent in the controls) became evident and it was localized
mainly on the cellular surface and within plasma membrane blebs. To investigate the presence of
GSH in mitochondria, cells were double stained with the anti-GSH and an anti-HSP60 (as a marker
for mitochondria). Immunoreactivity for HSP60 showed only partial overlap with that for GSH,
suggesting that mitochondria might be heterogeneous with respect to GSH content. In conclusion,
this confocal microscopy analysis appears suitable to study the compartmentalization of GSH and
PrSSG. Experiments are in progress to apply this technique to study the effect(s) of the mutant
G93ASOD1 in a conditional cell model that utilizes the NSC-34-tTA40 cell line developed in our
laboratory.
Financial support was provided by MIUR, FIRB, Protocol RBIN04J58W_000
62
P10
Interaction between SOD1 and cystatin b in “in vitro” models of ALS
Ulbrich L (1), Cozzolino M (2), Melli M (3), Carrì MT (2,4), Augusti-Tocco G (1)
(1) Dept. of Cell and Developmental Biology, University of Rome “La Sapienza”, Italy
(2) Fondazione Santa Lucia IRCCS, Rome, Italy
(3) Department of Biology, Bologna University, Italy.
(4) Dept. of Biology, University of Rome “Tor Vergata”, Italy
Cystatin B (CSTB) is an inhibitor of the cysteine proteases of the cathepsin family (1). Furthermore,
CSTB, invitro, is used as model to study the formation of amyloid fibrils (4-6). The cstb gene is
frequently mutated in a rare neurodegenerative disease called Progressive Myoclonus Epilepsy (2,
3). The interaction of CSTB with a number of proteins involved in the cytoskeletal function, is also
reported (7, 9). Recently, Wootz et al. (8) have shown increased levels of CSTB and C in the spinal
cord of amyotrophic lateral sclerosis (ALS) transgenic mice. This may correlate with the influence of
the redox environment on aggregate formation of cystatin B and its mutants (9) Western blot analysis
of neuroblastoma SH-SY5Y cells transfected with wild type or mutant copper/zinc superoxide
dismutase (SOD1) shows that overexpression of SOD1 correlates with incresed levels of CSTB, as
compared to non transfected cells. Using anti-CSTB abs, co-immuneprecipitation of SOD1 and CSTB
from WT SH-SY5Y protein extract was also observed. Similar results were obtained by the
immuneprecipitation of CSTB from the NSC34 hybrid cell line described as a model for motor
neurons, transfected with both Cystatin b and SOD1. Furthermore, in agreement with Cipollini et al.
(9) overexpression of WT CSTB in WT and parental SH-SY5Y cells triggers aggregation of the
protein in the cytoplasm. Altogether, these results suggest interaction between SOD1 and Cystatin b
and their possible cooperation in the functional alteration of motoneurons in ALS.
1 Turk V. and Bode W. (1991) The cystatins: Proteins inhibitors of cysteine proteinases. FEBS Lett.
285, 213-219
2 Lalioti M., Mirotsou M, et al. (1997) Identification of mutation in cystatin b, the gene responsabile for
the Unverricht-Lundeborg type of progressive myoclonus epilepsy (EPM1).Am.J.Hum.Genet. 60,
342-351
3 Pennacchio L.A., Lehesjoki A.E. et al (1996) Mutations in the gene encoding cystatin b in
progressive myoclonus epilepsy (EPM1). Science 271, 1731-1733.
4 Staniforth RA, Giannini S, Higgins LD, Conroy MJ, Hounslow AM, Jerala R, Craven
CJ, Waltho JP (2001) Three-dimensional domain swapping in the folded and molten-globule states of
cystatins, an amyloid-forming structural superfamily. EMBO J 20: 4774-4781
5 Zerovnik E., Pompe-Novak M., et al. (2002) Human stefin B readily forms amyloid fibrils in vitro,
Biochim. Biophys 1594,1-5.
6 Zerovnik E., Zavasnik-Bergant V., et al (2002) Amyloid fibril formation by human stefin B in vitro:
immunogold labelling and comparison to stefin A- Biol. Chem 383, 859-863.
7 Di Giaimo R., Riccio M., et al. (2002) New insights into the molecular basis of progressive
myoclonus epilepsy: a multiprotein complex with cystatin B. Human Molecular Genetics 11, 29412950.
8 Wootz H., Weber E., Korhonen L, and Lindholm D., (2006) Altered distribution and levels of
cathepsin D and cystatins in amyotrophic lateral sclerosis transgenic mice: possible roles in motor
neuron survival. Neuroscience 143, 419-130.
9 Cipollini E., Riccio M., et al Wild type cystatin B and its EPM1 mutants are polymeric and generate
amyloid-like aggregates in vivo (submitted).
63
P11
Studies on zinc and metallothioneins in the G93A SOD1 rat mutant
Averill SA, Yee J, Ismajli M., Malaspina A. and Michael-Titus AT.
Neuroscience Centre, ICMS, Bart’s and the London School of Medicine, Queen Mary, University of
London, 4 Newark Street, London E1 2AT, UK.
Zinc is a divalent cation with essential structural and functional roles in the central nervous system
(1). In the context of acute neurological injury, it has been suggested that excess zinc is associated
with neurotoxicity. Metallothioneins are small proteins which bind zinc and have been shown to be
associated with neuronal injury (2). An increase in the expression of these proteins has been
reported in the mouse G93A SOD1 model of amyotrophic lateral sclerosis (ALS) (3) and in ALS
patients. This study explored the distribution of zinc and of metallothioneins in the G93A SOD1 rat
model of ALS. Adult transgenic G93A SOD1 rats and matched wild-type animals were used
throughout. Zinc was detected using an autometallographic technique, following the injection of
sodium selenite (10 mg/kg, i.p.) at various times before sacrifice, and metallothionein I/II (MT)
expression was detected by immunocytochemistry. In wild-type rats zinc was present both at spinal
and supraspinal level and also in the dorsal root ganglia (DRG). In the spinal cord, the staining
appeared in the neuropil at 2.5 h after the injection of sodium selenite, and became more intense at 6
h after injection. At 24 h, zinc was also detected in neuronal cell bodies. In the DRG, the zinc-labelled
cells were mainly small and medium sized IB4-positive cells. In ALS rats at late stage disease (20-25
weeks), there was a marked decrease in zinc in the spinal cord. In contrast, in the same animals
there was a marked increase in MT immunoreactivity, in particular in the ventral horn and white
matter tracts. However, motorneurones did not appear to express MT. Most of the MT
immunoreactivity was present in astrocytes, confirmed by double labeling with GFAP. The
observations on MT confirm previous observations on MT up-regulation in a murine ALS model and
in ALS patients, and suggest that pathogenetic events associated with the disease (e.g. an increased
oxidative stress) may trigger an increased MT expression, which may have a neuroprotective role.
This is the first report of changes in zinc in the spinal cord of an ALS animal model at an advanced
stage of the disease. It remains to be seen if these changes are linked with the pathogenesis of the
disease.
(1) Frederickson C.J., Suh S.W. Silva D., Frederickson C.J. and Thompson R.B. (2000) Importance
of zinc in the central nervous system: the zinc-containing neuron. J. Nutr. 130, 1471S-1483S
(2) Stankovic R.K., Chung R.S. and Penkova (2007) Metallothioneins I and II; neuroprotective
significance during CNS pathology. Int. J. Biochem. Cell Biol. 39, 484-489.
(3) Gong Y.H. and Elliott J.L. (2000) Metallothionein expression is altered in a transgenic murine
model of familial amyotrophic lateral sclerosis. Exp. Neurol., 162, 27-36.
64
P12
Force plate for measuring G93A SOD1 mice forces by digital speckle pattern
interferometry
Calvo AC (1), Arroyo MP (3), Bea JA (2), Manzano R (1), Andres N (3), Grasa J (2), Zaragoza P (1),
Doblare M (2) and Osta R (1).
(1) LAGENBIO-INGEN.I3A, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain. (2)
GEMM.I3A, Centro Politécnico Superior, Universidad de Zaragoza, Zaragoza, Spain. (3) Gnomo,
Instituto de Investigación en Ingeniería de Aragón (I3A), Universidad de Zaragoza, Zaragoza, Spain.
Digital Speckle Pattern Interferometry (DSPI) is a well established technique for measuring small
displacements of scattering surfaces (1). This technique can be very useful when an objective test for
measuring the muscle strength is necessary. In particular, we studied the state of development of the
amyotrophic lateral sclerosis (ALS) in G93A transgenic mice, in terms of muscle strength as a
parameter to measure the forces exerted by the animal during locomotion over a sensing plate. The
experimental apparatus for obtaining surface displacements consists of two acrylic (PMMA) walls
inserted in a box and attached to an aluminium plate which has a central hole where the sensing
force plate is placed. The walls define a central corridor where the animal is allowed to walk freely. A
mirror placed at 45º under the box and a camera above it are used to take photos of the animal while
it walks on the sensing plate. A diode pumped solid state (DPSS) laser was used as a source of
illumination. The scattered light from the plate was collected onto a charge-couple device (CCD)
sensor where a focused plate image was formed. The calculation of applied forces magnitudes and
position involves a first step that includes the calculation of the phase for each specklegram, using a
Fourier transform method (2), and its difference with the reference specklegram phase. Then, the
maps are filtered, unwrapped and transformed into displacement fields. At first stage, we carried out
calibration experiments for estimating the sensing plate elastic parameters. We observed that 3 m is
the maximum displacement produced by the 10 grams static weight and occurred when the weight is
placed on the plate centre. We then challenged experiments from wild type and G93A SOD1 mice
varying in age and weight. Manual triggering is used to record time sequences of 500 specklegrams
every time the mouse walks on the sensing plate. One of the specklegrams, corresponding to no
force on the plate, is taken as the reference specklegram. We have obtained force traces for singlelimb contacts in mice with only 20 grams weight body. The maximum force magnitude is around 50 %
of the animal weight, which is within the range of values previously reported form mice (3). We report
that DSPI can be used as a tool to objective measure the force plate displacement fields produced by
G93A SOD1 mice, shedding light on the knowledge of ALS.
(1) Rastogi P.K. (2001) Measurement of static surface displacements, derivatives of displacements
and three-dimensional surface shapes-examples of applications to non-destructive testing. Digital
speckle pattern interferometry and related techniques, John Wiley & Sons, Chichester.
(2) Lobera J., Andrés N. and Arroyo M.P. (2004) Digital speckle pattern interferometry as a
holographic velocimetry technique. Meas. Sci. Technol. 15, 718-724.
(3) Zumwalt A.C., Hamrick M. and Schmitt D. (2006) Force plate for measuring the ground reaction
forces in small animal locomotion. J. Biomechanics 3, 2877-2881.
65
P13
Characterization of SOD1G93A mouse muscle by a combined genomic and proteomic
approach
D. Capitanio (1), C. Fallini (2), M. Vasso (1,3), A. Ratti (2), G. Grignaschi (4), M. Volta (2), C. Daleno
(4), S. Calza (5), C. Bendotti (4), C. Gelfi (1,3), V. Silani (2)
(1) Department of Biomedical Sciences and Technologies, University of Milan, Segrate, Italy. (2)
Dept. Neuroscience, University of Milan Medical School – IRCCS Istituto Auxologico Italiano, Milano,
Italy. (3) Institute of Molecular Bioimaging and Physiology, CNR, Segrate, Milano. (4) Lab. Molecular
Neurobiology, Dept. Neuroscience, Mario Negri Institute for Pharmacological Research, Milano. (5)
Department of Biomedical Statistics, University of Brescia, Brescia, Italy.
ALS is a fatal, progressive paralysis arising from the premature death of motoneurons. Alterations in
the morphology and metabolism of skeletal muscle have also been identified both in animal models
and in patients affected by the disease. Furthermore, muscular modulation of IGF-1 and GDNF have
been proved to delay the onset and prolong survival of mutant SOD1 mice. On the contrary, recent
evidence suggests that SOD1mut-mediated damage within muscle is not a significant contributor to
non-cell-autonomous pathogenesis of ALS. Despite these contrasting results, muscle tissue may
represent a valuable source for identification of disease biomarkers and for the understanding of the
pathogenetic mechanisms of ALS.
We have characterized the gene expression profile of triceps and gastrocnemius muscles from
transgenic SOD1G93A and control mice at presymptomatic (7 weeks) and symptomatic (14 weeks)
stages of the disease by the use of Affymetrix GeneChip technology. This analysis was combined
with the proteomic profiling of gastrocnemius muscles using a two-dimensional difference in gel
electrophoresis (2D-DIGE) approach coupled to mass spectrometry. To evaluate and to exclude from
our data gene and protein expression changes associated to physiological denervation processes
and muscular atrophy, we have also analysed gastrocnemius muscle after crush of the sciatic nerve.
At presymptomatic stage of the disease, few changes in gene expression profile were observed in
gastrocnemius and triceps muscles. On the contrary, at a symptomatic stage, Gene Ontology
analysis of both muscles revealed their different involvement in the disease. In particular, triceps
seemed to be still engaged in regenerative processes, while gastrocnemius was already atrophic with
a predominant activation of the apoptotic pathway.
Proteomic profiling of SOD1G93A versus control mice revealed the differential expression of 82 spots
at presymptomatic stage and 153 spots at 14 weeks of age. Interestingly, 55 out of 82 spots (67%)
were common to the two disease stages, and 16 of them were identified. Moreover, we identified 8
additional proteins specifically changed only at 7 weeks of age. These proteins were all associated
to mitochondrial metabolism and architecture.
Our data support the importance of skeletal muscle in the study of ALS since it well recapitulates the
pathological processes that lead to the degeneration of motor neurons. The use of comparative
genomic and proteomic technologies may represent a valid approach for the identification of clinically
relevant biomarkers for neurological disorders.
66
P14
Effect of phrenic nerve degeneration on diaphragm contraction in a murine
transgenic model of ALS
Severini C. (1), Carunchio I. (2,3), Pieri M. (2,3), Curcio L. (2,3), Panico M.B. (2), Pisani V. (2) ,
Massa R. (2), Zona C. (2,3).
(1) Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy, (2) Department of
Neuroscience, University of Rome ‘‘Tor Vergata’’, Rome, Italy, and (3) IRCCS Fondazione S. Lucia,
Rome, Italy.
Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disease affecting
motor neurons. The etiology of most ALS cases remains unknown, but 2% of instances are due to
mutations in Cu/Zn superoxide dismutase enzyme (SOD1). Transgenic mice overexpressing human
mutant SOD1 genes (G93A) have been obtained and are the most widely used animal model for the
study of ALS. They develop the disease and die at 130-140 days.
In this work we studied electrophysiological and histopathological features of phrenic nerve and
diaphragm muscle obtained from control and G93A mice at the age of 90 and 130 days. We
examined the contractile activity by physiological assessment of muscle force of isolated phrenic
nerve-diaphragm in vitro preparations.
By 90 days of age the muscular tension, measured through direct and indirect electric stimulation,
was similar in control and G93A mice. By 130 days muscular contractile characteristics remained
unvaried in control and G93A mice following a direct stimulation, whereas the indirect stimulation of
the muscle through the phrenic nerve was significantly less efficient in G93A mice compared to
control mice. Samples of the phrenic nerve and diaphragm muscle were analysed from mice at the
same age for histopathological analysis. At 90 days, initial axonal degerneration was observed in the
phrenic nerve. At 120 days widespread axonal degeneration characterized the phrenic nerve
whereas there was a mild myofiber atrophy in diaphragm.
The present results show that the muscle contractility is not significantly impaired also at the endstage of the disease, whereas at this stage an axonal degeneration rapidly involves the majority of
motor nerve fibers, inducing diaphragm paralysis.
67
P15
Role of the ubiquitin proteasome pathway dysfunction in a mouse model of
amyotrophic lateral sclerosis
Cheroni C (1), Marino M. (1), Dantuma N (2), Bendotti C (1)
(1) Department of Neuroscience, M. Negri Institute for Pharmacological Research, Milan, Italy
(2) Department of Cell and Molecular Biology, Karolinske Institute, Stockholm, Sweden
Accumulation of aggregated and ubiquitinated proteins are pathological hallmarks of both familial and
sporadic Amyotrophic Lateral Sclerosis (ALS)and are found in the spinal cord of transgenic mice
carrying human mutant SOD1, a model of the disease. The ubiquitin-proteasome pathway (UPP) is
the main proteolytic system in eukaryotic cells; increasing evidence suggest a link between UPP
impairment and the aggregate formation in neurodegenerative disease; however, it is still unclear
whether an impairment of the UPP occurs in the motor neurons and/or in glial cells of ALS mouse
models and if it is related with accumulation of protein aggregates.
Real time PCR experiments on lumbar spinal cord of SOD1G93A mice revealed no changes in the
mRNA levels of the catalytic constitutive 20S proteasome subunits prior to symptom onset while both
19S and 11S resulted significantly decreased. At the symptomatic stage, the mRNA levels of 20S
subunit still remained unchanged between G93A and NTg littermates. On the contrary, LMP7,
MECL1 were significantly increased in G93A lumbar spinal cord, while the nonresulted decreased. At the end stage of disease progression a decrease of the mRNA levels for all
the constitutive subunits of proteasome was found in the lumbar spinal cord of SOD1G93A mice
The phenomena resulted restricted in the pathological area, since no changes were detected in the
hippocampus (Beta5, LMP7, alpha and 19S tested).
In order to evaluate the UPP function at the cellular levels, SOD1G93A mice were cross-bred with the
UbG76V-GFP transgenic model, that expresses a reporter protein for UPP functionality.
Immunohistochemical analyses of symptomatic double transgenic mice revealed the presence of rare
GFP-positive cells in the spinal cord; at the end stage the accumulation of the reporter protein was
more extended in the spinal cord and involved also the motor nuclei of the brainstem. Co-localization
experiments in the spinal cord of end stage double transgenic mice revealed that Ub-GFP
accumulation was rarely observed in the glial population. In fact, the majority of GFP-positive cells
showed a neuronal morphology. At the symptomatic stage, about one third of GFP positive neurons
showed perikaryal staining of phosphorylated neurofilaments, while two third of all GFP positive cells
were also ubiquitin positive.These data suggest that early changes of transcript levels for 19S and
11S in ventral spinal cord may be the first sign of UPP change in G93A mice. Progressive decrease
of constitutive proteasome subunits observed at the later stages may contribute to accumulation of
protein aggregates found in neurons and neurites. Supported by Telethon
68
P16
Spinal cord degeneration in amyotrophic lateral sclerosis: the role of retinoid
signaling
Natasa Jokic, Yong Yong Ling, Rachael E. Ward, Adina T. Michael-Titus, John V. Priestley, Andrea
Malaspina
Neuroscience Centre, Institute of Cell and Molecular Science, Barts and the London School of
Medicine and Dentistry, Queen Mary University of London, London E1 2AT, United Kingdom
Retinoid signaling (ReS), a complex of molecular pathways that mediate the effects of vitamin A and
its derivatives, is increasingly recognized as a component of the repair capacity that could be
activated to induce protection and regeneration in the mature nervous tissue. Changes in distribution
and expression of retinoid receptors have been described as part of a spinal cord protective response
to acute injury and to chronic degeneration. We have recently reviewed the expression of ReS gene
candidates and functionally synergic nuclear receptors in human spinal cord from healthy adults and
from patients with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder causing
extensive motor neurons loss and death from respiratory failure in 3 to 5 years from disease onset
(1). This tentative ReS expression profile included data generated from in-house gene expression
analysis and information retrieved from independent datasets, obtained through serial analysis of
gene expression and array investigations. In the present study (2), we have combined RNA and
protein expression analysis to characterize the expression profile of retinoid receptor isotypes in the
lumbar spinal cord of the superoxide dismutase 1 G93A transgenic rat model of ALS throughout the
disease development and in wild type littermates. We show that key pathogenic features associated
to disease progression, including gliosis and motor neuron loss appear to develop in parallel with a
specific pattern of retinoid receptors expression change, involving RXRbeta up-regulation in
astrocytes and a selective gamma motor neuron RARalpha staining at end-stage disease. Our work
provides experimental evidence supporting the role of retinoid signaling in neurodegeneration of the
spinal cord and suggests that future studies could explore new treatment strategies based on
retinoid-modulating agents.
(1)
Malaspina A, Turkheimer F. A review of the functional role and of the
expression profile of retinoid signaling and of nuclear receptors in human spinal cord.
Brain Res Bull. 2007; 71: 437-46.
(2)
Jokic N., Ling Y., Ward R., Michael-Titus A.T., Priestley J.V., Malaspina A. Retinoid receptors
in chronic degeneration of the spinal cord: observations in a rat model of amyotrophic lateral
sclerosis. Submitted to J. Neurochem.
69
P17
ATF-3 Expression in the SOD1 Rat Model of Amyotrophic Lateral Sclerosis
Ngoh SFA, Ward RE, Hall JCE, Jones C, Jokic N, Averill SA, Michael-Titus AT, Malaspina A,
Priestley JV.
Neuroscience Centre, Bart’s and the London School of Medicine and Dentistry, Queen Mary
University of London, Institute of Cell & Molecular Sciences, 4 Newark Street, London E1 2AT
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that leads to progressive
paralysis. The molecular pathogenesis of this ultimately fatal disease remains poorly understood. It
has previously been reported that expression of the stress-activated transcription factor ATF-3
precedes the death of motoneurones in mice that express G93A mutant superoxide dismutase SOD1
(1). We carried out this study to characterize the expression of ATF-3 in the SOD1 rat model of ALS.
Transgenic Sprague Dawley rats expressing the G93A SOD1 gene mutation, along with age and sexmatched wild-type littermates, were subject to behavioural analysis to monitor disease progression.
The rats were anaesthetized and perfusion fixed for histological analysis at either a presymptomatic
(10 week) or end-stage (20 - 25 weeks) of the disease. Tissue obtained from the spinal cord and
dorsal root ganglia (DRG) of these animals was processed for ATF-3 immunoreactivity and double
labeled with a range of antisera used to identify motoneurones, glia, and sub-populations of DRG
neurones.
Motoneurones in the spinal cord of the end-stage SOD1 transgenic rats exhibited ATF-3
immunoreactivity, in contrast with motoneurones in wild-type controls, which displayed no ATF-3.
Most of the ATF-3 positive cells were also immunoreactive for choline acetyl transferase (ChAT), and
some of the ATF-3 positive cells had low ChAT expression. Consistent with degeneration of
motoneurones, strong ATF-3 immunoreactivity was also observed in Schwann cells in the ventral
roots and spinal nerves. However, ATF-3 immunoreactivity was also present in a few Schwann cells
in the dorsal roots and in a sub-population of DRG neurones. ATF-3 immunoreactive DRG neurones
were predominantly large diameter, and immunoreactive for heavy-chain neurofilament. ATF-3
immunoreactivity was rarely found in small diameter calcitonin gene related peptide (CGRP)-positive
neurones, or in the DRG of wild-type animals.
Our results in SOD1 mutant rats mirror earlier results obtained in SOD1 mice (1) and in studies of
peripheral nerve injury (2). ATF-3 is a marker of cellular injury and its upregulation in motoneurones
and their associated Schwann cells may play a role in the pathogenesis of ALS. An upregulation of
ATF3 in sensory systems in ALS has not been reported previously, and ongoing studies are
investigating whether this is a secondary response to motoneuron loss or a parallel primary event.
We gratefully acknowledge support from St. Bartholomew and the London Charitable Foundation.
(1) Vlug, A.S., Teuling, E., Haasdijk, E.D., French, P., Hoogenraad, C.C., and Jaarsma, D. (2005)
ATF3 expression precedes death of spinal motoneurons in amyotrophic lateral sclerosis- SOD1
transgenic mice and correlates with c-Jun phosphorylation, CHOP expression, somato- dendritic
ubiquitination and Golgi fragmentation. European Journal of Neuroscience. 22: p1881- 1894
(2) Hunt, D., Hossain-Ibrahim, K., Mason, M.R., Coffin, R.S., Lieberman, A.R., Winterbottom, J.,
Anderson, P.N. (2004) ATF3 upregulation in glia during Wallerian degeneration: differential
expression in peripheral nerves and CNS white matter. BMC Neuroscience 5: 9
70
P18
Inclusions containing superoxide dismutase-1 are regularly present in amyotrophic
lateral sclerosis patients lacking mutations in the enzyme
Nilsson K (1), Jonsson, P.A.(2), Graffmo K(1), Andersen P.M.(3), Marklund S (2) and Brännström T
(1)
(1) Dep. of Medical Bioscience, Umeå University, Sweden. (2) Dep. of Clinical Chemistry, Umeå
University, Sweden. (3) Dep. of Pharmacology and Clinical Neuroscience, Umeå University, Sweden
Six percent of amyotrophic lateral sclerosis (ALS) is linked to mutations in the ubiquitously expressed
enzyme superoxide dismutase-1 (SOD1), making it the most common cause of the disease. The
cause of the remaining cases is largely unknown. Histopathologycally, three types of inclusions are
seen in neurons of the brain and spinal cord – Bunina bodies, Lewybody-like-hyaline inclusions and
skein inclusions. In addition, in familial ALS (FALS) caused by mutated SOD1 another type of
inclusions are observed, which contain aggregated units of the misfolded enzyme.
Misfolded and aggregation-prone forms of mutant SOD1s are thought to trigger the disease.
The aim of this study was to investigate if SOD1 is involved in ALS patients lacking mutations in the
enzyme and to find the subcellular localization of the SOD1 inclusions.
CNS tissue was collected from 30 patients with sporadic ALS (SALS), 7 patients with FALS, and from
46 control patients with and without other neurodegenerative diseases. Paraffin embedded spinal
cord sections were immunostained using immunohistochemistry and immunofluorescence for
detection and co-localization of SOD1 with lysosomes, endoplasmatic reticulum (ER) and
mitochondria. An antibody recognizing the human TAR DNA-binding protein (TDP-43) was used to
study a possible co-lozalisation with SOD1 inclusions. Confocal microscopy was used for analysis of
the samples.
Numerous small somal round inclusions in motoneurons, immunoreactive for SOD1, were a feature
of both SALS and FALS. They were relatively homogenous in size and measured 0.5-3µm. All
investigated SALS and FALS patients had SOD1 containing inclusions. In contrast none or very few
controls had inclusions. Double immunofluorescence staining showed a partial co-localization with
the lysosomal marker. No co-localization was found between SOD1 inclusions and ER, mitochondria
or TDP-43.
Our results suggest that inclusions of misfolded, aggregated SOD1 are a general histopathological
feature, regularly present in the motoneurons of ALS patients lacking mutations in the enzyme.The
inclusions are situated in the cytoplasm of the neurons and do not co-localize with TDP-43,
mitochondria or ER and only partly with lysosomes.
Our results suggest that SOD1 is involved in the pathogenesis of all forms of ALS.
71
P19
Direct Detection and Quantitation of Metals Bound to Cu,Zn Superoxide Dismutase
from discrete regions of the central nervous system of ALS animals models
Keith E. Nylin (1,2), Brian Arbogast (3), Nathan Lopez (1), Mark Levy (2), Jeff Morre (3), Joseph S.
Beckman (1,2,3)
(1) Department of Biochemistry & Biophysics, Oregon State University, Oregon, USA, (2) Linus
Pauling Institute, Oregon State University, Oregon, USA, (3) Environmental Health Science Center,
Oregon State University, Oregon, USA
We have hypothesized the loss of zinc from Cu,Zn superoxide dismutase (SOD1) plays a direct role
in the disease mechanism for amyotrophic lateral sclerosis (ALS). Several lines of evidence support
this hypothesis, however, because the affected tissue regions of ALS are so minute, it has previously
been impossible to quantify metals bound to SOD from tissue, and therefore Zn-deficient SOD has
never been measured directly from ALS tissue. Consequently, we have developed a sensitive
method to measure the metal content of SOD directly from discrete regions of the spinal cord using
electrospray mass spectrometry. We collect small necropsy punches (250 g) from flash frozen
central nervous tissue then thaw in 5 L of buffer. The supernatant from the thawed tissue is
collected after centrifugation and loaded onto a C4 ZipTip from Millipore and eluted into the mass
spectrometer with 40% acetonitrile/water. In addition to SOD, we have identified several proteins
which elute from the ZipTip including ubiquitin, CCS, and -synuclein. Furthermore, by analyzing
blood with this new assay, we are able to genotype an animal in less than two minutes. By extensive
testing of controls we show the metal status of SOD is preserved throughout the assay and the
results obtained from the mass spectrometer accurately reflect the metal status in vivo. By using
Cu,Zn bovine SOD as an internal standard, we find nearly 15 M of SOD in the ventral grey matter of
a rat model of ALS is missing zinc, equal to the amount of Cu,Zn SOD, while over 20 M is apo.
Strikingly, we find the dorsal grey matter is almost void of Zn-deficient SOD, but still maintains 20 M
apo SOD. Furthermore, we do not see Zn-deficient SOD in 30 days old transgenic rats, long before
pathology of the disease begins. By using this new sensitive assay we developed we have found a
spatial and temporal correlation between the presence of Zn-deficient SOD and affected tissue in
ALS, further supporting the hypothesis that Zn-deficient SOD plays a direct role in the ALS disease
mechanism.
72
P20
Flavoproteins and ALS: expression of Fmo gene family in SOD1 mutated mice
Ogliari P. (1), Corato M (1), Cova E. (1), Cereda C. (1), Gagliardi S. (1), Daleno C. (2), Bendotti C.
(2) and Ceroni M (1,3,4).
(1) Lab. of Experimental Neurobiology, Neurological Institute IRCCS C. Mondino, Pavia, Italy, (2)
Dept. of Neuroscience, “Mario Negri” Institute for Pharmacological Research, Milano, Italy (3) Dept.
of Neurosciences, Policlinico di Monza, Monza, Italy (4) Dept. of Neurology, University of Pavia,
Pavia, Italy.
Amyotrophic Lateral Sclerosis (ALS) is an adult-onset, progressive and fatal neurodegenerative
disease, whose pathogenesis is unknown. Motor neuronal degeneration represents the main
pathological feature in ALS, for which two major pathogenetic hypotheses have been proposed:
oxidative stress and excitotoxicity. Flavin-containing monooxigenases (FMO) represent a gene family
coding for microsomal enzymes which are involved in the oxidative metabolism of a variety of
xenobiotics. This family contains five genes, FMO1, FMO2, FMO3, FMO4, FMO5, and at least six
pseudogenes in humans. Function of FMO genes is largely studied in liver, kidney and lung but not in
nervous system. Recently, the observation of a 80% reduction in FMO1 mRNA expression in spinal
cord of sporadic ALS patients pointed to a relation between ALS and FMO genes. In addition, in a
previous study, we found an association between two 3’-UTR single nucleotide polymorphisms of
FMO1 gene and the development of sporadic ALS. To investigate the mRNA levels of FMO1, FMO2,
FMO3, FMO4, FMO5 in murine nervous system in normal mice and in a transgenic murine model of
ALS.The C57BL/6J mouse strain dealing the human-SOD1 G93A mutation has been used as
disease model; C57BL/6J normal mice have been used as controls. Four brain areas have been
examined in all animals: cerebellum, cerebral hemispheres, brainstem and spinal cord. A Real-Time
PCR technique with TaqMan probes has been used for expression analysis. Normalization was
optimized using Hprt as housekeeping gene.Our study demonstrates that all FMO genes, except for
Fmo3, are expressed in the murine nervous system. The FMO expression is sex-dependent and
varies over different brain areas. Moreover, alterations in FMO expression have been found in G93A
mutated mice. Particularly, in G93A males, we observed in different cerebral areas a lower
expression of Fmo2 and Fmo4 gene compared to WT mice. Conversely, G93A female mice
expressed significantly greater amounts of Fmo2 and Fmo5 genes in cerebellum and cerebral
hemispheres.Our study represents the first attempt to analyse Fmo gene expression in different
areas of the nervous system and particularly in spinal cord. We confirmed the sex-dependent
expression of Fmo genes in nervous system, as already found in other tissues. The finding of altered
Fmo expression in a model of motor neuron degeneration might suggest a role for Fmo genes in the
development of ALS, even if the pathogenic link is presently unknown.
73
P21
Valosin-Containing Protein Mutation Accelerates Paralysis and Death in SOD1G93A
Transgenic Mice
Yan J , Fu R , Deng HX , Caliendo J , Zhai H , Chen W , Liu E , Siddique T,
Department Neurology, Northwestern Medical University, Chicago, IL USA
SOD1G93A transgenic mouse is an established animal model of human familial ALS. A subgroup of
ALS is associated with frontotemporal dementia (FTD). Mutations in the VCP gene cause autosomal
dominant FTD with inclusion body myopathy and Paget disease of bone (IBMPFD). VCP performs a
variety of essential cellular functions. Mutant SOD1 can be degradated via Dorfin, a ubiquitin ligase
(E3). VCP directly interacts with Dorfin and both colocalize in Lewy body-like inclusions in the spinal
cord motor neurons of ALS(1). We found a VCP R155P mutation in a IBMPFD family. To explore
the effect of mutant VCP on the onset and progression of SOD1 G93A transgenic mice, we established
VCPR155P mouse using a 17.9kb human genomic DNA fragment containing the entire VCP gene with
site-directed mutation. VCPR155P mouse were crossbred with SOD1G93A to generate SOD1G93A
/VCPR155P double transgenic mice. Transgenes were identified by PCR, DNA sequencing and
Southern blot analysis. Mice were observed weekly for changing weight and tested on the Rotarod
system. We generated four SOD1G93A /VCPR155P double transgenic mice, two littermates with
VCPR155P, two littermate with SOD1G93A and two nontransgenic littermates. SOD1G93A /VCPR155P
double transgenic mice stopped gaining weight after 61 days and the onset of hind legs paralysis
was at 77.7±3.9 days and end-stage was at 84.3±4.6 days. The SOD1G93A and VCPR155P littermates
were not affected by 112 days. The average onset of paralysis in SOD1G93A mice (n=273) was
120.4±8.2 days and end-stage time of SOD1G93A mice (n=397) was 128.5±10.7 days. The SOD1G93A
/VCPR155P vastus lateralis muscle showed remarkable muscle fiber atrophy and fiber size variation,
but without obvious rimmed vacuoles. Muscle morphology of SOD1G93A / VCPR155P littermates were
normal at the time SOD1G93A /VCPR155P reached end stage. It appears that VCP mutation significantly
accelerates the onset and progression of disease in SOD1G93A mice. VCP immunohistochemic
analysis and characterization of the muscles, brain and spinal cord is currently underway.
(1) Ishigaki S, Hishikawa N, Niwa J, et al. Physical and functional interaction between Dorfin and
Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative
disorders. J Biol Chem. 2004; 279(49):51376-85.
74
P22
p62 accumulates and enhances aggregate formation in model systems of familial
amyotrophic lateral sclerosis
Jozsef Gal, Anna-Lena Ström, Renee Kilty, Fujian Zhang and Haining Zhu
Department of Molecular and Cellular Biochemistry, University of Kentucky, 741 South Limestone
Street, Lexington, Kentucky 40536
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by
motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the
affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation,
accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel
to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies
showed that p62, ubiquitin and mutant SOD1 co-localized in the protein aggregates in affected cells
in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not
WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the
formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was
measured in the presence and absence of overexpressed p62 and the results suggest that the large
aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study.
Deletion of the ubiquitin-association (UBA) domain of p62 significantly decreased the p62-facilitated
aggregate formation, but did not completely inhibit it. Further protein interaction experiments also
showed that the truncated p62 with the UBA domain deletion remained capable of interacting with
mutant SOD1. The findings of this study show that p62 plays a critical role in forming protein
aggregates in familial ALS, likely by linking misfolded mutant SOD1 molecules and other cellular
proteins together.
75
P23
Identification of a novel splice-site mutation in the Cu/Zn superoxide dismutase
(SOD1) gene associated with amyotrophic lateral sclerosis
Corrado L (1), Negri G (1), Testa L (2), Oggioni GD (2), Momigliano-Richiardi P (1), Mazzini L (2),
D’Alfonso S (1)
(1) Human Genetics Laboratory, Dept. of Medical Sciences, Eastern Piedmont University , (2) Dept of
Neurology, Azienda Ospedaliera Maggiore della Carità, Novara, Italy
We report a novel mutation of the Cu/Zn superoxide dismutase (SOD1) gene localized in intron 4 and
found in an ALS patient with a family history for amyotrophic lateral sclerosis (FALS). The patient is a
56 years old woman, who presented at our outpatients’ department with a two-year history of speech
disturbances and progressive weakness in her left arm. At the time of our visit (June 2006)
neurological and neurophysiological examinations confirmed upper and lower motoneuron
involvement both at bulbar and spinal level. The patient was finally diagnosed as ALS with bulbar
onset. Her maternal grandmother died of motoneuron disease whereas her mother is 84 years old
and does not manifest any sign of motoneuron disease.
The mutation is a T nucleotide insertion at +3 downstream the exon4-intron4 junction Although the
invariant GT donor site was not apparently affected, bioinformatics analysis (http//:
l25.itba.mi.cnr.it/~webgene/wwwspliceview.html) predicted that a T at position +3 completely
abolished the exon 4 donor site suggesting a possible alternatively spliced mRNA. To determine
whether this mutation could affect splicing mechanisms, reverse trascription PCR (RT PCR) was
performed on polyA-RNA isolated from peripheral blood lymphocytes of the patient. The analysis
identified two different aberrant transcripts together with the normal isoform. One of these presented
the retention of intron 4 sequence. We predict that the addition of intronic extrabases would result in
an introduction of four aa (V-K-F-S- end) before a premature stop codon at aa 122. A second
aberrant transcript was generated using a different donor site localized at the 3’ end of exon 4 (three
nucleotides upstream the natural donor site) leading to an in frame 3bp deletion. We predict for this
transcript the production of a SOD1 protein lacking a single amino acid (val 118) in a conserved
region close to the active site loop (aa 121-144). We have no data about the quantitative ratio among
the three mRNA isoforms and about the pathogenic effect of the two aberrant transcripts. We can
hypothesize a main pathogenic role for the transcript with the intron 4 retention since at least two
similarly truncated proteins at residues 124 and 121 were reported in two patients with a familial and
a sporadic disease respectively.
In conclusion we report a likely pathogenic new SOD1 intronic mutation with reduced penetrance.
76
P24
Novel SOD1 Q23R mutation associated with muscle mitochondrial dysfunction in
familial ALS
Stefania Corti, Andreina Bordoni, Dario Ronchi, Domenico Santoro, Sabrina Salani, Chiara
Donadoni, Costanza Lamperti, Valeria Lucchini, Veronica Crugnola, Maurizio Moggio, Nereo
Bresolin, Giacomo P. Comi
Dino Ferrari Center, Department of Neurological Sciences. Fondazione I.R.C.C.S. Ospedale
Maggiore,
Policlinico, Mangiagalli and Regina Elena, University of Milan, Milan Italy
Superoxide dismutase 1 (SOD1) gene mutations are responsible for approximately 20% of all familial
amyotrophic lateral sclerosis (ALS) cases. Substantial evidence suggests that mitochondrial
dysfunction is involved in ALS pathogenesis. In fact, two mitochondrial DNA mutations have been
described in motor neuron disease patients, and recent studies suggest that the toxicity of mutant
SOD1 arises from its selective recruitment to motoneuron (MN) mitochondria. Here we describe the
clinical feature of a 40-year-old man from Bangladesh that presents progressive proximal lower limb
muscle weakness by the age of 37, involving after one year also the arms. At age 40 years,
neurological examination shows proximal and distal limb muscle weakness with distal atrophy,
characterized by lower rather than upper MN impairment. Diffuse fasciculations were present, also in
the tongue. The patient s mother died at 35 age for pneumonia, while a maternal uncle presented a
referred muscle impairment at the lower limbs. EMG showed acute and chronic denervation findings,
while brain and spinal MRI was normal. Direct sequencing revealed a heterozygous mutation CAG to
CGG in codon 23 substituting glutamine to arginine in the SOD1 gene (Q23R). A muscle biopsy
showed neurogenic pattern associated with cytochrome c oxidase (COX) deficieny in several muscle
fibers. No apoptotic nuclei were found with TUNEL reaction. The novel SOD1 Q23R mutation affects
a highly conserved aminoacidic position and results in a early-onset ALS phenotype. The mutation is
likely to confer to the mutant SOD1 protein a mitochondrial toxicity also in muscle tissue, as
demonstrated by partial cytochrome c oxidase dysfunction.
77
P25
Gender difference in levels of Cu, Zn-Superoxide Dismutase (SOD1) in
cerebrospinal fluid of patients with amyotrophic lateral sclerosis
Katrin Frutiger (1), Thomas J. Lukas (2), George Gorrie (3), Senda Ajroud-Driss (1), Teepu Siddique
(1)
(1) Davee Department of Neurology and Clinical Neurosciences, (2) Department of Molecular
Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Tarry
building, Room 13-715, 303 East Chicago Avenue, Chicago, IL 60611 USA, (3) Southern General
Hospital, Department of Neurology, Glasgow, UK
BACKGROUND: Currently the best studied mechanism for amyotrophic lateral sclerosis (ALS) is the
one caused by mutations in the gene for cytosolic Cu/Zn-binding superoxide dismutase (SOD1).
Mutant SOD1-protein causes motor neuron degeneration due to the gain of a novel toxic function.
OBJECTIVES AND METHODS: To evaluate the relevance of SOD1 levels in cerebrospinal fluid
(CSF) in ALS patients, the SOD1 concentration was immunoassayed in the CSF of 11 patients with
ALS and 19 neurological controls.
RESULTS: The mean level of SOD1 in CSF from all samples was 45.5 +/-11.3 ng/ml. There was no
statistically significant difference between the levels of SOD1 in CSF of ALS patients and
neurological control subjects. Here we show that the SOD1 concentration in the CSF is significantly
higher in male ALS patients (54.0 +/-9.0 ng/ml) compared to female ALS patients (38.1 +/-6.4 ng/ml)
(p=0.007). This gender difference is not observed in the CSF of neurological controls.
CONCLUSION: The observation of a gender difference in levels of SOD1 in CSF of ALS patients
needs further investigation to determine whether it is relevant to the observed gender related
difference in disease incidence.
78
P26
SOD1 Gene Mutations in Italian Amyotrophic Lateral Sclerosis Patients
Gellera C (1), Ticozzi N (2), Passariello P (1), Ratti A (2), Plumari M (1), Colombrita C (2), Castellotti
B (1), Lauria G (1), Fallini C (2), Testa D (1), Corbo M (2), Taroni F (1), Silani V (2).
(1) Fondazione IRCCS – Istituto Neurologico C. Besta – Milano – Italy. (2) Dipartimento
Neuroscienze, Università degli Studi di Milano - IRCCS Istituto Auxologico Italiano –– Milano – Italy.
Mutations in the SOD1 gene are present in 10-20% of ALS familial (FALS) and in 3-6% sporadic
(SALS) cases with more than one hundred different mutations described so far. Disease progression
and duration often correlate with specific SOD1 mutations, although a great variability has been
observed among and within families carrying the same mutation. A great variability in SOD1 gene
mutations has also been observed in populations with different ethnic backgrounds.
Our work has focused on the identification of mutations in SOD1 gene in a large cohort of Italian ALS
patients from two Neurological referral Centers.
We have screened 762 Italian patients including 149 FALS and 613 SALS cases. We have found
mutations in 18/149 (12%) unrelated FALS and in 7/613 (1%) SALS patients. 10/18 (55%) FALS
carried mutations frequently described in different ethnic backgrounds (A4V, L84F, G93D and
L144F). Interestingly, we identified 4 new SOD1 mutations.
In an attempt to speculate about genotype-phenotype correlations, we suggest that, excluding A4V
cases that associate with a severe and rapid course of the disease, FALS SOD1 cases show a
slower and less aggressive phenotype than idiopathic cases. It is possible that susceptibility genes
may be involved not only in ALS variants with unknown aetiology, but could also explain the
incomplete penetrance and variable expression observed in pedigrees with the same SOD1 gene
mutation.
Other candidate genes have also been screened in a subset of our SOD1-negative patients: alsin
gene in 75 patients with a juvenile onset and SETX gene in 44 patients with a dominant inheritance.
No mutations were identified in exons where mutations were previously described to occur.
We have analyzed Angiogenin gene as a risk factor for ALS pathogenesis in our cohort of patients.
We identified 7 different mutations in 13 patients and one mutation (I46V) in a control sample (1.9%
vs 0.2%, p 0,014). Six mutations were novel and were found both in familial (4.5%) and sporadic
(1.3%) patients (p 0.035).
A subset of sporadic patients (n=95) were also screened for deletions in SMN2 gene, another
candidate ALS susceptibility gene. We did not find a significant association of SMN2 deletions in ALS
patients respect to controls.
Our results indicate that SOD1 gene mutations still remain the main determinant in the pathogenesis
of hereditary ALS and ANG gene represents a strong susceptibility factor for both SALS and FALS
cases in the Italian population.
79
P27
S-adenosylmethionine-dependent methylation in amyotrophic lateral sclerosis:
preliminary results and pathogenetic hypotheses
Monsurrò MR (1), Trojsi F (1), Salvatore A (2), Piccirillo G (1), Capone F (1), Maiorano P (1), De
Bonis ML (2), D’Angelo S (2), Salemme S (2), Galletti P (2), Tedeschi G (1)
(1) Department of Neurological Sciences and (2) Department of Biochemistry and Biophysics
“Francesco Cedrangolo”, Second University of Naples, Naples, Italy
Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disorder of upper and lower motor
neurons. It is sporadic in 90% of cases, while familiar ALS (fALS) is mainly caused by missense
mutation of superoxide dismutase 1 (SOD1) gene, responsible of SOD activity decrease and reactive
oxygen species increase (1). The aim of this study was to evaluate levels of methylation in
erythrocyte membrane proteins of ALS patients, as a possible marker of oxidative stress.
Blood samples of 35 ALS patients (20 men, 15 women, 54±10 years) and 32 healthy age- and sexmatched controls were processed to assess methylation of intact erythrocytes membrane proteins by
electrophoretic analysis (as index of abnormal levels of L-isoaspartyl residues) and intracellular
concentrations of methyl donor S-adenosylmethionine (AdoMet) and AdoMet demethylated product
S-adenosylhomocysteine (AdoHcy) by high performance liquid chromatography.
Abnormal L-isoaspartyl residues were significantly higher in ALS patients erythrocyte membrane
proteins with an increased methyl accepting capability (P<0.05). AdoMet concentration in ALS
patients erythrocytes was about 50% lower, while AdoHcy levels were equivalent, measuring a low
AdoMet/AdoHcy ratio in ALS patients.
Several evidences underlined the role of reactive oxygen species and nitric oxide in
neurodegenerative mechanism (1). Furthermore, previous studies demonstrated that increased
formation of L-isoaspartyl residues is one of the major structural alterations occurring in erythrocyte
membrane proteins as a result of oxidative stress (2). These abnormal residues are physiologically
converted into normal L-aspartyl residues by protein-L-isoaspartyl methyltransferase (PIMT), which
methylates the alpha-carboxyl group of atypical L-isoaspartyl residues. PIMT is ubiquitous and its
repair function is particularly crucial in erythrocytes, as a consequence of molecular ageing and
oxidative damage (2). Our preliminary results suggest the presence of an injured action of PIMT in
ALS patients erythrocytes, with accumulation of L-isoaspartyl residues, notably responsible of
abnormal protein conformation. Thus, we hypothesize a concomitant injured mechanism of DNA
methylation, dependent on AdoMet/AdoHcy ratio, which probably causes a deregulated gene
expression. DNA hypomethylation in ALS could damage epigenetic control of long term silencing
genes expression, such as imprinted or X-linked genes, with possible involvement in ALS
pathogenesis, as described in some human genetic disorders. Further studies might be planned to
evaluate methylation and gene expression in motor neurons of ALS animal models.
(1) Rowland LP, Shneider NA (2001) Amyotrophic lateral sclerosis. N Engl J Med 344:1688-1700
(2) Ingrosso D, D'Angelo S, di Carlo E, Perna AF, Zappia V, Galletti P (2000) Increased methyl
esterification of altered aspartyl residues in erythrocyte membrane proteins in response to oxidative
stress. Eur J Biochem 267:4397-4405
80
P28
SOD1 mutations in ALS patients: an epidemiological study in Piemonte, Italy
Restagno G (1), Lombardo F (1), Sbaiz L (1), Fimognari M (1), Calvo A (2), Ghiglione P (2),
Cammarosano S (2), Cavallo E (2), Mutani R (2), Chiò A (2)
(1)
(2)
Laboratorio di Genetica Molecolare, ASO OIRM-Sant’Anna, Torino, Italy
Dipartimento di Neuroscienze, Università di Torino, Italy
According to series from ALS referral centres, superoxide dismutase 1 (SOD1) mutations may
account for 20% of FALS and 2-4% of apparently sporadic cases (1). However, no studies have
evaluated the frequency of SOD1 mutations in population-based series of cases. We assessed
SOD1 mutations in familial and sporadic ALS in an epidemiological setting. From 1995 all ALS cases
resident in Piemonte are actively enrolled in a population register. After 2000, most patients from
Torino province (population, 2,214,934) have been evaluated for SOD1 mutations. Genomic DNA
from patients and normal controls was isolated and purified from 150 µl of peripheral blood on ABI
PrismTM 6100 Nucleic Acid Prep Station. DHPLC was performed using the WAVE system
(Trasgenomic, Inc). Direct sequence of the entire coding region (five exons plus flanking introns) of
the SOD1 gene was performed using dideoxinucleotides method with the Big Dye kit (Applied
Biosystems) on ABIPrism 3100 automated sequencer. PCR products were band-purified on lowmelting-point agarose gel using QIAquick (QIAGEN) purification columns. Products were sequenced
directly from both ends using Big Dyes Terminator and then purified with Centrisep columns. The
products were run on an ABI PRISM 3100 DNA sequencer and analysed with softwares “Factura”
and “Sequence Navigator.” From 2000 to 2005 a total of 386 ALS patients (223 men and 163
women) were diagnosed in the Province of Torino (mean annual incidence rate, 2.91/100,000 [95%
CI, 2.63-3.21]). Twenty-two patients (5.7%) had a positive family history for ALS. FALS were younger
than SALS (FALS 57.4 [SD 12.2]; SALS 65,6 [SD 10.5]; p=0.00001) and had a lower gender ratio
(1:1 vs.1.4:1). SOD1 was analyzed in 325 patients (84.2%), including all FALS. Five patients had a
SOD1 exon mutation: 3 FALS (13.6% of FALS), and two apparently sporadic cases (0.7% of SALS).
The following mutations were found: G41S (2 FALS), G93D (1 FALS), N19S (1 SALS), and a deletion
of a glutamic acid at codon 133, E133ΔE (1 SALS). In our epidemiologic study, the frequency of
FALS was lower than that reported by clinical series, whereas the frequency of SOD1 mutations
among FALS was in the range of expected values. Only less than 1% of SALS cases had a SOD1
mutation. These discrepancies indicate that referral series overestimate both the frequency of FALS
and the frequency of SOD1 mutations in SALS patients, probably because they preferentially perform
SOD1 analysis in younger subjects, who have a higher risk of carrying SOD1 mutations.
(1) Andersen P. Amyotrophic lateral sclerosis associated with mutations in the CuZn superoxide
dismutase gene. Curr Neurol Neurosci Rep. 2006;6:37-46
81
P29
DNA damage, p53 activation and apoptosis in a cellular model of ALS
Barbosa LF (1), Cerqueira F (1), Macedo A (1), Sogayar MC (1), Augusto O (1), Carrì MT (2), Di
Mascio P (1) and Medeiros MHG (1).
(1)Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Sao Paulo, Brazil.
(2)Dipartimento di Biologia, Università di Roma “Tor Vergata”, Rome, Italy.
Mutations in Cu,Zn-Superoxide Dismutase (SOD) have been linked to familial amyotrophic lateral
sclerosis (FALS) (1) leading to a gain of a “toxic function”, not completely understood. It has been
suggested that FALS-mutant SOD induces motor neuron apoptosis (2). Oxidative damage in DNA (3)
and p53 upregulation (4) have been detected in ALS neuronal tissues. It is known that accumulation
of DNA damage triggers apoptosis by p53 activation (5). We examined DNA damage, p53 activity
and apoptosis in human neuroblastoma SH-SY5Y cells transfected with SOD wild-type (SOD-WT)
and a mutant SOD (SOD-G93A) typical of FALS. The levels of 8-oxo-7,8-dihydro-2’-deoxyguanosine
(8-oxodGuo) and 1,N2-etheno-2´-deoxyguanosine (1,N2-dGuo) were determined by HPLC coupled
with eletrochemical detection and mass espectrometry, respectively. DNA strand breaks were
analyzed by the comet assay. The activity of p53 was quantified by reporter gene assay and
apoptosis by DNA fragmentation followed by flow cytometry. Significantly increased levels of DNA
damage (3 fold), increased p53 activity (3 fold) and a higher level of apoptosis (4 fold) were observed
in SH-SY5Y cells transfected with SOD-G93A, compared to cells transfected with SOD-WT and
parental cells. Moreover, western blot analyses showed that SOD-G93A accumulates in the nucleus
and is more associated to DNA than SOD-WT. These results indicate that this mutant SOD has a
pro-oxidative and pro-apoptotic activity, and its accumulation in the nucleus and association to DNA
may induce DNA damage and trigger apoptosis by p53 activation.
Significantly increased levels of DNA damage (3 fold), increased p53 activity (3 fold) and a higher
level of apoptosis (4 fold) were observed in SH-SY5Y cells transfected with SOD-G93A, compared to
cells transfected with SOD-WT and parental cells. Moreover, western blot analyses showed that
SOD-G93A accumulates in the nucleus and is more associated to DNA than SOD-WT. These results
indicate that this mutant SOD has a pro-oxidative and pro-apoptotic activity, and its accumulation in
the nucleus and association to DNA may induce DNA damage and trigger apoptosis by p53
activation.
Acknowledgements: FAPESP, CNPq, FINEP, Instituto do Milênio Redoxoma, USP.
(1) Rosen D.R. et al (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with
familial amyotrophic lateral sclerosis. Nature, 362, 59-62.
(2) Cozzolino M. et al. (2006) Apaf1 mediates apoptosis and mitochondrial damage induced by
mutant human SOD1s typical of familial amyotrophic lateral sclerosis. Neurobiol Dis, 21(1), 69-79.
(3) Ferrante R.J. et al (1997) Evidence of increased oxidative damage in both sporadic and familial
amyotrophic lateral sclerosis. J. Neurochem. 69(5), 2064-2074.
(4) Martin L.J. (2000) p53 is abnormally elevated and active in the CNS of patients with Amyotrophic
Lateral Sclerosis. Neurobiol Dis 7, 613-622.
(5) Levine A.J. (1997) p53, the cellular gatekeeper for growth and division. Cell 88, 323-331.
82
P30
The slow Wallerian degeneration gene (WldS) blocks early formation of focal axonal
dystrophy in CNS axons undergoing degeneration
Bogdan Beirowski (1), Elisabetta Babetto (1), Guillermo Garcia Alias (2), Keith R.G. Martin (2),
Michael P. Coleman (2)
(1) The Babraham Institute and, (2) Centre for Brain Repair, Cambridge, UK
Degeneration of axons is a neuropathological hallmark in various neurodegenerative diseases such
as ALS. Generally, axons respond with diverse morphology, topology and speed to various lesion
paradigms and noxious effects and recent studies involving the slow Wallerian degeneration (Wld S)
phenotype suggest a common mechanism underlying these responses. Previously we have shown in
the PNS that individual wild-type and WldS axons following transection and crush injury degenerate
progressively displaying different morphological, topological and temporal degeneration patterns.
Here we extend these studies to the CNS and investigate the spatiotemporal patterns of axonal
degeneration in wild-type and WldS optic nerve and spinal cord following physical lesion.
Assessment of axonal survival on transverse optic nerve and spinal cord dorsal column sections
suggested a progressive nature of axon degeneration in the CNS following different lesion
paradigms. Surprisingly, in contrast to cut or crushed PNS axons, where no major morphological
changes are detectable within the ca. 36 h latent phase, optic nerve and dorsal column axons
showed focal hypertrophic swellings as early as 10 h, long before axonal fragmentation.
Ultrastructural analysis revealed that these swellings were either filled with diverse vesicular bodies,
disorganized neurofilaments and degenerating organelles or appeared empty. Interestingly, these
swellings showed a gradient along the lesioned nerve tracts with more swellings at proximal sites.
Lesioned optic nerves and dorsal columns of WldS rats contained significantly fewer hypertrophic
swellings, especially at sites far distal to the lesion. Observations of cultured YFP-H optic nerves
showed that hypertrophic axonal swellings arose on uninterrupted nerve fibres after a few hours. In
summary, we found that the morphology of axons undergoing Wallerian degeneration in the CNS
differs from that in the PNS and resembles focal axonal dystrophy that occurs in many
neurodegenerative disorders. We propose a novel working model for CNS axon degeneration where
lesioned optic nerve axons become dystrophic long before they fragment, possibly due to early focal
axonal transport defects. The WldS phenotype delays this focal axonal dystrophy.
83
P31
The Arl Mouse - A New Mouse Strain With A Mutation In the Cytoplasmic Dynein
Heavy Chain
Bros V (1), Golding M (2), Chia R (1), Schiavo G (2), Flenniken A (3), Adamson SL (3), Rossant J (3).
Fisher EMC (1), Greensmith L (1), Hafezparast M (4),
(1) Institute of Neurology, London, UK; (2) Cancer Research UK, London, UK
(3) Centre For Modeling Human Disease, Toronto, Canada;
(4) Department of Biochemistry, University of Sussex, Brighton, UK
Mutations in cytoplasmic dynein have been implicated in the pathogenesis of amyotrophic lateral
sclerosis (ALS), a fatal neurodegenerative disorder characterized by motor neuron degeneration and
muscle paralysis. For example, we have previously shown that Loa mice with a mutation in the
cytoplasmic dynein heavy chain show defects in retrograde axonal transport and reduced
motoneuron survival. Furthermore, postnatal over-expression of the dynein subunit dynamitin, also
induces motoneuron degeneration in mice. In addition, mutations in motor proteins such as the
anterograde motor Kinesin 1 or dynactin, an essential component of the dynein motor complex, have
been linked with motoneuron degeneration in human disorders.
Here we present a new mouse strain called the Abnormal rear legs (Arl), which has a different point
mutation in the cytoplasmic dynein heavy chain to that described for the Loa mouse. Our initial
examination suggests that the ‘Arl’ mouse may have a more severe phenotype than the Loa mouse.
For example, from a young age, the Arl mice show a distinctive “amphibian-like” gait. Their hind limbs
are splayed laterally so that they have difficulty in supporting their pelvis, which hinders forward
movement. We are therefore carrying out a full phenotypic characterisation of the Arl mice in terms of
axonal transport, physiological analysis of muscle force and motor unit survival as well as a
morphological assessment of motoneuron survival. Our initial results indicate that there is a clear
pathology in the sciatic nerves of these mice, resulting in a significant reduction in nerve diameter
compared to their wildtype (WT) littermates. The underlying pathology of these nerves is under
investigation.
84
P32
The nucleocytoplasmic transport system is altered in Motor Neurons of a mouse
model of familial ALS
Peviani M., Spano G., Bendotti C
.
Lab. Molecular Neurobiology, Istituto di Ricerche Farmacologiche “Mario Negri”, Milan (Italy).
Amyotrophic Lateral Sclerosis (ALS) is a rare neurodegenerative disease that affects upper and
lower motor neurons. It determines progressive muscle atrophy, leading to ultimate paralysis and
death. It has recently been suggested that alteration of distal axons may take part in the early steps
leading motor neurons to death in ALS (1). Moreover, evidence is emerging that a dysfunction of
nucleocytoplasmic transport system may also be involved in the mechanisms underlying motor
neuron degeneration in ALS (2). Importins are recruited for important steps of nucleocytoplasmic
transport system, and upregulation of importin beta has been found to be specifically triggered in
peripheral nerves to modulate regeneration of injured neurons after crush injury (3). These evidences
prompted us to investigate whether importin-mediated nucleocytoplasmic transport system is
modified in neuronal perikaria or at the periphery during progressive degeneration of neuronal cells,
in a mouse model of ALS. We observed a reduction of importin beta levels in homogenates from
ventral horn spinal cord concomitantly with the appearance of first symptoms of the pathology.
Moreover using confocal miscroscopy we detected an altered distribution of the importins in motor
neurons with impaired retrograde axonal transport, and in neurons showing accumulation of
phosphorylated neurofilaments, an hallmark of neural degeneration. We suggest that alterations of
nucleocytoplasmic transport-related proteins may represent an additional mechanism involved in the
degeneration of motor neurons in ALS pathology.
(1) Fischer LR, Culver DG, Tennant P, Davis AA, Wang M, Castellano-Sanchez A, Khan J, Polak
MA, Glass JD. (2004) Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and
man. Exp Neurol. 185(2):232-40.
(2) Zhang J, Ito H, Wate R, Ohnishi S, Nakano S, Kusaka H. (2006) Altered distributions of
nucleocytoplasmic transport-related proteins in the spinal cord of a mouse model of amyotrophic
lateral sclerosis. Acta Neuropathol (Berl). 112(6):673-80.
(3) Hanz S, Perlson E, Willis D, Zheng JQ, Massarwa R, Huerta JJ, Koltzenburg M, Kohler M, vanMinnen J, Twiss JL, Fainzilber M. (2003) Axoplasmic importins enable retrograde injury signaling in
lesioned nerve. Neuron. 18;40(6):1095-104.
85
P33
Proteomic analysis of astrocytic secretion in a mouse model of familial amyotrophic
lateral sclerosis
Basso M (1,2), Tortarolo M (2), Gabriella Spaltro (2), Dario Lidonnici (2), Salmona M (2), Bendotti C
(2), Bonetto V (1) (2)
(1) Dulbecco Telethon Institute, (2) Institute for Pharmacological Research “Mario Negri”, Milan, Italy
Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurological disease characterized by the
specific degeneration of motor neurons in the cortex, brainstem and spinal cord. The mechanisms
involved in this selective degeneration are still unidentified. In recent years, a clear interplay between
motor neurons and other cellular population have been proven (1-2). In particular, the role of
astrocytes seems to be extremely important for their contribution to spinal motor neuron degeneration
(3). It seems that the toxicity is caused by factors and proteins released into the extracellular space
by astrocytes and motor neurons (3).
The aim of this work is to investigate the role of astrocytes in the pathogenesis of familial ALS. For
this purpose we analyzed the secretome from primary culture of astrocytes expressing the
pathogenic mutated SOD1 or wild-type SOD1. Secreted proteins were separated by two-dimensional
electrophoresis, and the gel maps were compared by computerized image analysis. The most
abundant proteins secreted were identified by MALDI mass spectrometry. About forty proteins are
differentially secreted by astrocytes expressing the mutated SOD1 in comparison with controls. We
are currently identifying these proteins. These preliminary results suggest that the only expression of
mutated SOD1 has an impact on astrocyte secretion pathways. We are now comparing astrocyte
secretome with the secretome of motor neuron/astrocytes co-cultures carrying or not the mutation
with a special focus on SOD1. At the same time we are investigating the expression of astrocytic
proteins when the mutated SOD1 is present or not, in order to understand which are the intracellular
molecular pathways which determine alteration in the secretome and may be important in mediating
SOD1 toxicity.
(1) Clement, A. M., Nguyen, M. D., Roberts, E. A., Garcia, M. L., Boillee, S., Rule, M., McMahon, A.
P., Doucette, W., Siwek, D., Ferrante, R. J., Brown, R. H., Jr., Julien, J. P., Goldstein, L. S., and
Cleveland, D. W. (2003). Wild-type nonneuronal cells extend survival of SOD1 mutant motor neurons
in ALS mice. Science 302(5642), 113-7.
(2) Boillee, S., Yamanaka, K., Lobsiger, C. S., Copeland, N. G., Jenkins, N. A., Kassiotis, G., Kollias,
G., and Cleveland, D. W. (2006). Onset and progression in inherited ALS determined by motor
neurons and microglia. Science 312(5778), 1389-92.
(3) Nagai, M., Re, D. B., Nagata, T., Chalazonitis, A., Jessell, T. M., Wichterle, H., and Przedborski,
S. (2007). Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor
neurons. Nat Neurosci 10(5), 615-622.
86
P34
Astrocytes regulate GluR2 expression in motor neurons and their vulnerability to
excitotoxicity
Bogaert E. (1), Van Damme P. (1), Callewaert G. (2), Herijgers P. (3), Van Den Bosch L. (1),
Robberecht W. (1)
(1) Laboratory of Neurobiology, (2) Laboratory of Physiology, (3) Laboratory of Experimental Surgery
and Anaesthesiology, University of Leuven, Belgium.
Influx of Ca2+ through AMPA receptors contributes to the neuronal damage in stroke, epilepsy and
neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). The Ca 2+ permeability of
AMPA receptors is determined by the GluR2 subunit, receptors lacking GluR2 being permeable to
Ca2+. We identified a difference in GluR2 expression in motor neurons between two different rat
strains (Wistar and Holtzman), which correlated with a difference in vulnerability to AMPA receptormediated excitotoxicity. In an in vitro model Wistar and Holtzman motor neurons, were cultured on a
pre-established feeder layer of astrocytes. Compared to Holtzman motor neurons, Wistar motor
neurons had lower GluR2 expression levels, and were more vulnerable to AMPA receptor-mediated
excitotoxicity. Astrocytes were found to determine the neuronal GluR2 expression levels. In order to
demonstrate an increased vulnerability of Wistar motor neurons to AMPA receptor-mediated
excitotoxicity in vivo, transient spinal cord ischemia was studied. We found that the extent of paresis
and motor neuron degeneration induced by ischemia was more pronounced in Wistar compared to
Holtzman rats. We also studied survival of Wistar and Holtzman rats overexpressing mutant SOD1, a
reliable model for human ALS. However, no difference was found, in spite of the known contribution
of AMPA receptor-mediated excitotoxicity to mutant SOD1-induced motor neuron degeneration. The
lack of protection was found to be due to the presence of mutant SOD1 in Holtzman astrocytes.
Holtzman astrocytes overexpressing mutant SOD1 did no longer protect motor neurons against
excitotoxicity and abolished their ability to stimulate GluR2 expression.
All together, these results reveal a new mechanism through which astrocytes influence neuronal
functioning in health and disease.
87
P35
Glutamate release induced by glycine and GABA is enhanced in the spinal cord of
SOD1 G93A mutant mice.
Zappettini S. (1), Raiteri L. (1), Milanese M. (1), Raiteri M. (1), Bonanno G. (1)
(1) Department of Experimental Medicine, Pharmacology and Toxicology Section, and Centre of
Excellence for Biomedical Research, University of Genoa, Italy.
There is increasing evidence that the extra-cellular levels of the excitatory neurotransmitter glutamate
are enhanced in amyotrophic lateral sclerosis (ALS) and that glutamate-mediated excitotoxicity is
implicated in the disease. As a cause of excessive glutamate, abnormalities of the glutamate
transport were observed, which were subsequently ascribed to a selective loss of the GLT1
glutamate transporter. Since GLT1 is mainly localized at astroglial processes, the hypothesis that
glial cells play a role in the development of the disease appeared to be likely. An alternative way to
produce elevated extra-cellular concentrations of glutamate may well consists of the increase of
neuronal glutamate release rather than of astrocyte-localized inhibition of reuptake.
We have previously shown that glycine evokes glutamate and GABA release from mouse spinal cord
synaptosomes by penetrating in the glutamate- or GABA-releasing nerve terminals through
transporters (heterotransporters) of the GLYT1 and GLYT2 type (1, 2) and that GABA evokes
glutamate, but not glycine, release via GAT1 heterotransporters sited at glutamate-releasing nerve
terminals (3). We here compared the glycine-evoked release of glutamate or GABA and the GABAevoked release of glutamate in mice expressing a mutant form of human SOD1 with a Gly93Ala
substitution [SOD1(+)/G93A(+)], a transgenic model of familiar ALS; in mice, expressing the
unmodified human SOD1 [SOD1(+)] and in non-transgenic littermates [SOD1(-)/G93A(-)].
In [SOD1(+)] control animals, the basal efflux of 3HD-aspartate, a marker of the glutamate
intraterminal pools, was concentration-dependently increased by GLY (EC50=68.3 M; Emax=186% of
potentiation) and by GABA (EC50=3.6 M; Emax=113%) through a heterotransporter-mediated
process. Similar results were observed in SOD1(-)/G93A(-) mice. The effects of GLY and GABA
were significantly higher in symptomatic mutant SOD1(+)/G93A(+) mice than in controls (E max= 260%
vs 186% and 159% vs 113%, respectively, with no changes in EC 50 values). GLY and GABA
increased also the release of endogenous glutamate more efficiently in SOD1(+)/G93A(+) animals
respect to controls. On the contrary, the release of tritiated or endogenous GABA, induced by glycine,
was not modified in ALS mice. The higher release of 3HD-aspartate release in mutant animals was
already present in pre-symptomatic 70-90 and 30-40 day-old mice.
Our results show an excessive release of glutamate in an animal model of familiar ALS. The
understanding of the mechanisms of this excessive release requires detailed investigation. If it
occurs also in vivo, the different modulation of glutamate and GABA release here reported could
induce an unbalance between inhibitory and excitatory transmission in ALS.
(1) Raiteri L., Raiteri M. and Bonanno G. (2001) Coexistence and function of different
neurotransmitter transporters in the plasma membrane of CNS neurons. J. Neurochem. 76, 18231832,.
(2) Raiteri L., Stigliani S., Siri A., Passalacqua M., Melloni E., Raiteri M. and Bonanno G. (2005)
Glycine taken up through GLYT1 and GLYT2 heterotransporters into glutamatergic axon terminals of
mouse spinal cord elicits release of glutamate by homotransporter reversal and through anion
channels. Biochemical Pharmacology, 69, 159-168.
(3) Raiteri L., Stigliani S., Patti L., Usai C., Bucci G., Diaspro A., Raiteri M. and Bonanno G. (2005)
Activation of GABA GAT-1transporters on glutamatergic terminals of mouse spinal cord mediates glutamate
release through anion channels and by transporter reversal. J. Neurosci. Res. 80, 424-433.
88
P36
Glutamate uptake by presynaptic compartment is decreased in transgenic
SOD1G93A mice, an animal model of amyotrophic lateral sclerosis
Elena Fumagalli, Simona Colleoni, Caterina Bendotti and Tiziana Mennini
Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy
Excitatory amino acid transporters guarantee glutamate homeostasis in the CNS, and both glial and
neuronal transporter dysfunction could be of relevance in pathological conditions. While the role
played by glial cells in glutamate clearance in vivo is well documented, the involvement of the
presynaptic compartment is still unclear, also due to the difficulties in isolating pure functional cellular
fractions from the whole tissue.
A decreased glial GLT1 expression with reduced glutamate uptake has been reported in CNS tissues
of amyotrophic lateral sclerosis (ALS) patients and in transgenic SOD1G93A mice, one of the most
used animal model of familiar ALS. However, studies were done using crude synaptosomal fractions,
that are a mixed preparation containing both neuronal and glial elements.
We evaluated the glutamate uptake in purified gliosomes and synaptosomes obtained from spinal
cord of SOD1G93A mice and non transgenic C57 mice, and compared the results with those
obtained in the classical crude synaptosomal preparation. Western blot analysis showed 90%
enrichment of synaptophysin in the purified synaptosomes, with about 30% contamination from glial
cells (GFAP). Two time points were considered, the early symptomatic stage (16 weeks) and at the
late stage of the disease (24 weeks).
The different preparations showed similar Km and different capacities of glutamate uptake (Vmax),
with higher Vmax in the purified synaptosomes, both in transgenic and control mice. We found no
changes in Km and Vmax of glutamate uptake between transgenic and non transgenic mice at the
early stage of disease, while a significant reduction of Vmax (-30%) was evident at the late stage in
transgenic SODG93A mice, selectively in the purified synaptosomes. A slight reduction of the Vmax
was also found in crude synaptosomes (-12%) while value was unchanged in gliosomes.
Pharmacological properties of these preparations were also investigated, in order to try to identify the
glutamate transporter located at the nerve terminals. However, no differences were found in terms of
sensitivity to different glutamate transporters inhibitors (DHK, SOS, TBOA) between transgenic and
non transgenic mice and among the different purified preparations. We confirmed a decrease in the
expression of the glutamate transporter GLT1 and found a reduction of immunoreactivity for the
neuronal marker synaptophysin, likely a consequence of the degeneration of nerve terminals of the
corticospinal tract.
Thus we conclude that the loss of presynaptic glutamatergic neurons is responsible for reduced
glutamate uptake in the spinal cord of 24 week-old SOD1G93A mice.
89
P37
Longitudinal study of the onset and progression of Amyotrophic Lateral Sclerosis
disease in SOD1 mutant mice by non invasive imaging
KELLER A.Florence & KRIZ Jasna
Dept. Anatomy and Physiology, Laval University, Centre de Recherche CHUL(CHUQ), Québec, QC,
Canada
Amyotrophic lateral sclerosis (ALS) is a late onset neurological disease characterized by progressive
spinal motor neurons degeneration associated with paralysis and eventually death. Transgenic mice
expressing a mutant sodium dismutase 1 (SOD1) develop phenotype with many pathological features
resembling human familial and sporadic ALS. Although major clinical symptoms in ALS arise from
neurodegeneration and death of motoneurons, recent studies suggest that non neuronal cells could
play a role in the toxicity to motor neurons. Their precise role in onset and progression of the disease
remain however unknown.
To further investigate the role of non-neuronal cells in the disease onset and progression, we
developed mouse model for live imaging of astrogliosis in ALS.
As a glial fibrilary acidic protein (GFAP) is strongly up-regulated in ALS, we used it as a hallmark of
astrogliosis to create our model. We thus crossed mice carrying the firefly luciferase gene under the
transcriptional control of mouse GFAP promoter (GFAP-luc, Xenogen, CA) with mice carrying the
SOD1G93A mutation. The double transgenic GFAP-luc/SOD1G93A mice were used in the study as well
as GFAP (wt)-SOD1G93A and GFAP-luc as controls. Live imaging was performed weekly starting from
postnatal weeks 3-4 till the end stage of the disease. Loss of extension reflex, weight-loss and motor
deficits were used as indicators of clinical symptoms.
The results were obtained from 19 female/male mice matched-age littermates and were compared to
adequate controls (n=7). Data collected by in vivo imaging showed that photon emission/GFAP signal
was first detected at the lumbar spinal cord area. The signal first arose from small multiple areas of
astrocytes activation which then converged into a larger signal around 80-100days of age. The
correlation analysis between live imaging and behaviour data revealed that increase in GFAP signal
in the spinal cord at 70-80 days correlated with the initial disease onset (loss of extension reflex).
Moreover the peak signals arising from the spinal cord at approx. 100 days correlated with the abrupt
onset of sensorimotor deficit and paralysis. The end-stage of the disease (approx 133d) was
characterized by an increase of bioluminescent signals arising form the different brain structures that
coincided with the loss of body weight.
GFAP-luc//SOD1G93A mice will provide unique tools for understanding disease pathology and
longitudinal responses to drug testing. Acknowledgement: CIHR, FRSQ, RRTQ
90
P38
Effects of mutant FALS-SOD1 on microglia secretion
Padovano V., Massari S., Righi M. and Pietrini G.
Department of Pharmacology, School of Medicine, Università degli Studi di Milano; CEND- Center of
neurodegenerative diseases; CNR-Institute of Neuroscience , Milan, Italy.
Increasing evidence indicates a participation of the microglia to motor neuron degeneration in human
and murine ALS. Microglia represents the resident immune competent cells of the CNS, and noxious
stimulus may elicit microglia activation and its subsequent release of toxic factors that accelerate
neuronal degeneration and death. To unravel the contribution of microglia to the pathogenesis of
ALS, we are testing the hypothesis of altered pathways of secretion in microglia induced by the
expression of the G93A mutant of SOD1. To this purpose we have established microglia cell culture
models expressing constitutive or inducible human SOD1 (WT and G93A SOD1). Our data indicate
that the expression of mutant SOD1 alters the basal and LPS-stimulated release of potentially toxic
molecules including the G93A SOD1, whereas the expression of even higher amount of the WT
SOD1 decrease the release of these molecules. Moreover, our data suggest that these molecules
are released via unconventional pathways of secretion not involving the endoplasmic reticulum-Golgi
complex route. We are now performing experiments aimed at identifying the secretory pathways
followed by these molecules, which is a crucial step in order to develop appropriate therapeutic
strategies to prevent toxicity mediated by microglia.
91
P39
Microglial activation and MCP-1 staining pattern develop differently with disease
progression in the spinal cord of SOD1 mutant mouse and rat
Siklos L (1), Paizs M (1), Henkel J (2), Beers DR (2) and Appel SH (2).
(1) Institute of Biophysics, Biological Research Center, Szeged, Hungary
(2) Department of Neurology, Methodist Neurological Institute, Houston, USA
Neuropathological findings and experimental data support the decisive role of microglia in the
pathogenesis of amyotrophic lateral sclerosis (ALS) (1,2). Additionally, the involvement of monocyte
chemoattractant protein-1 (MCP-1) in neuron-microglia interaction has been suggested (3), which
chemokine is critical for the recruitment of inflammatory cells of monocytic lineage after injury in the
central nervous system. For modeling the disease, transgenic mice (4) as well as transgenic rats (5),
overexpressing mutant (G93A) Cu2+/Zn2+ superoxide dismutase (mSOD1) have been developed
which animals show signs of progressive motor neuron disease resembling clinical and pathological
features of human ALS.
In both transgenic animals, we characterized the change of microglial and MCP-1 immunostaining in
the lumbar segment of the spinal cords as the disease progressed. One set of animals was used
from each species: rats were sacrificed at age of 18, 40, 50, 70, 90, 102 days and at endstage (day
122); mice were killed at age of 16, 38, 80, 111 days and at endstage (day 141). After perfusion, the
spinal cords were dissected, frozen sections were cut (30 m) from the lumbar segment and
immunostaining was made on free-floating sections with the avidin-biotin technique.
In transgenic rats, MCP1-staining started to develop at age of 40 days, reached its maximum at day
70 and disappeared at day 102. Only the motor neurons were stained. According to the change in
their appearance, microglia activation commenced at days 50-70, then their proliferation progressed
until endstage, although activated microglia were mostly confined to the ventro-lateral region of the
spinal cord. In mSOD1 transgenic mice, MCP-1 staining could be seen at every time point, which
staining was not restricted to motor neurons. The staining intensity decreased from a high level (at
age of 16 day) to a low level (at age of 38-80 days), then started to increase again and reached a
high level for a second time at endstage. This unexpected U-shaped time dependence of the MCP-1
staining intensity was supported by data from RT-PCR measurements (6). Microglia activation in
mice was evident at age of 80 days then their proliferation continuously increased until endstage with
a homogeneous distribution in the whole grey matter. The contrasting time dependence of MCP-1
staining in mice and rats as well as the different distribution pattern of the activated microglia at close
to endstage suggest that the same mutant enzyme may lead to different pathological course even in
relatively close species.
(1) Kawamata T., Akiyama H., Yamada T. and McGeer P.L. (1992) Immunologic reactions in
amyotrophic lateral sclerosis, brain and spinal cord. Am.J.Pathol. 140, 691-707.
(2) Beers D.R., Henkel J.S., Xiao Q., Zhao W., Wang J., Yen A.A., Siklos L., McKercher S.R. and
Appel S.H. (2006) Wild-type microglia extend survival in Pu.1 knockout mice with familial amyotrophic
lateral sclerosis. Proc.Natl.Acad.Sci.USA. 103, 16021-16026.
(3) Flügel A., Hager G., Horvat A., Spitzer C., Singer G.M., Graeber M.B., Kreutzberg G.W. and
Schwaiger F.W. (2001) Neuronal MCP-1 expression in response to remote nerve injury.
J.Cereb.Blood Flow Metab. 21, 69-76.
(4) Gurney M.E., Pu H., Chiu A.Y., et al. (1994) Motor neuron degeneration in mice that express a
human Cu,Zn superoxide dismutase mutation. Science 264, 1772-1775.
(5) Howland D.S., Liu J., She Y., Goad B., Maragakis N.J., Kim B., Erickson J., Kulik J., DeVito L.,
Psaltis G., DeGennaro L.J., Cleveland D.W. and Rothstein J.D. (2002) Focal loss of the glutamate
transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis
(ALS). Proc.Natl.Acad.Sci.USA. 99, 1604-1609.
(6)Henkel J.S., Beers D.R., Siklos L. and Appel S.H. (2006) The chemokine MCP-1 and the dendritic
and myeloid cells are increased in the mSOD1 mouse model of ALS. Mol.Cell Neurosci. 31, 427-437.
92
P40
Ivermectin inhibits AMPA receptor-mediated excitotoxicity in vitro and extends the
life span of an ALS mouse model
Andries M (1), Van Damme P (2), Robberecht W (2), and Van Den Bosch L (2)
(1) Department of Molecular Cell Biology, Faculty of Medicine, K.U.Leuven, Campus Gasthuisberg,
Leuven, Belgium. (2) Laboratory of Neurobiology, Faculty of Medicine, K.U. Leuven, Campus
Gasthuisberg, Leuven, Belgium
We have shown before that α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptormediated excitotoxicity contributes to the selective motor neuron death in amyotrophic lateral
sclerosis (ALS) (1). In this study, we investigated the effect of P2 receptor-influencing substances on
kainate-induced motor neuron death in an in vitro model for AMPA receptor-mediated excitotoxicity.
Complete protection was found after preincubation of the motor neurons with ivermectin or Cibacron
Blue 3G-A. Preincubation with both P2X4 modulators did not influence the number or Ca2+
permeability of the AMPA receptors and addition during kainate stimulation alone had no effect.
Preincubation with a low concentration of ATP, the natural agonist of the P2X 4 receptor, also
protected the motor neurons against a subsequent excitotoxic stimulation, while high concentrations
of ATP were toxic. Moreover, ivermectin increased the toxicity of low ATP concentrations, indicating
that ivermectin can potentiate the effect of ATP on its receptor. Ivermectin and ATP also protected
against hypoxia/hypoglycemia. To further investigate the relevance of these findings for ALS, we
treated mutant SOD1G93A-mice with ivermectin. This resulted in an extension of the life span of these
mice with almost 10%. We conclude that ivermectin induces a mechanism in motor neurons, in vivo
and in vitro, that protects against subsequent excitotoxic insults. Our in vitro data indicate that this
protective mechanism is due to the potentiation by ivermectin of an effect of ATP mediated by the
P2X4 receptor.
(1) Van Damme P., Leyssen M., Callewaert G., Robberecht W. and Van Den Bosch L. (2003) The
AMPA receptor antagonist NBQX prolongs survival in a transgenic mouse model of amyotrophic
lateral sclerosis. Neurosci. Lett. 343, 81–84.
93
P41
Mesenchymal stem cell treatment induces survival prolonging and symptom
amelioration in mutant SOD1 G93A mice
Milanese M. (1), Zappettini S. (1), Casazza S. (2), Caponetto C. (2), Bonanno G. (1), Uccelli A. (2).
(1) Department of Experimental Medicine, Pharmacology and Toxicology Section, and Centre of
Excellence for Biomedical Research, University of Genoa, Italy
(2) Department of Neuroscience, Neurology Section, University of Genoa, Italy
Amyotrophic lateral sclerosis (ALS) is a chronic neuromuscular disorder clinically characterized by
muscle wasting, weakness and spasticity reflecting a progressive degeneration of upper and lower
motor neurons. To date hardly any treatments prolong survival in ALS patients of some extent.
Development of more effective neuroprotective therapies is impeded by lack of understanding of the
mechanisms of neuronal death and how the disease propagates. Mesenchymal stem cells (MSC), a
subset of adult stem cells derived from the bone marrow stroma, have generated much enthusiasm
as possible cell source for tissue repair including the nervous system. Recent studies have shown
that MSC can also modulate immune responses and exert an anti-apoptotic effect on different cells
including neurons. In addition, MSC can migrate into the central nervous system when i.v. injected.
We studied here the effects of MSC administration in mice expressing mutant human super oxide
dismutase (SOD1) with a G93A substitution [SOD1(+)/G93A(+)], a transgenic animal model of ALS.
Mesenchymal cells (106 cells/animal, i.v.) were injected at day 90, well after the onset of the first
disease symptoms, that can be recorded at about day 60. Saline injected SOD1(+)/G93A(+) were
used as controls. Control mice survived about 120 days while the MSC-treated mice exhibited a
statistically significant prolonged survival time compared to saline injected controls. Such effect was
associated with a significant amelioration in the performance of behavioral motor tests in the MSCtreated animals. Studying neurotransmitter release, we have found that glutamate exocytosis is
enhanced in the spinal cord of SOD1(+)-G93A(+) mice, respect to controls. Interestingly, MSC
treatment almost abolished this extra-release of the excitatory amino acid. Upon i.v injection, a few
luciferase-labeled MSCs were detected inside the mice spinal cord. Amelioration of some histological
parameters was observed irrespective neural trans-differentiation. We can conclude that the
treatment with MSC may be considered as an appealing therapeutic opportunity for ALS although the
effect does not seem to rely on tissue repair.
94
List of Participants
95
Chiara Abrescia
Bioindustry Park Canavese spa
Colleretto Giacosa (TO), Italy
abrescia@bipcamail.it
Ilaria Amori
Lab. Of Neurochemistry
Fondazione Santa Lucia IRCCS
Roma, Italy
ilariaamori@hotmail.com
Peter Andersen
Department of Neurology
Institute of Clinical Neuroscience
Umeå University
SE-901 85 Umeå, Sweden
peter.andersen@neuro.umu.se
Svetlana Antonyuk
Molecular Biophysics Group
Daresbury Laboratory
Warrington, U.K.
s.antonyuk@dl.ac.uk
Mario Arciello
Dept. of Biology
University of Rome "Tor Vergata"
Rome, Italy
arciello.mario@libero.it
Gabriella Augusti-Tocco
Dept. of Cell and Developmental Biology
University of Rome “La Sapienza”
Rome, Italy
gabriella.tocco@uniroma1.it
Elisabetta Babetto
The Babraham Institute
Cambridge, UK
elisabetta.babetto@bbsrc.ac.uk
Lucia Banci
Department of Chemistry
University of Florence
Sesto Fiorentino (FI), Italy
banci@cerm.unifi.it
Luis Barbeito
Istituto Clemente Estable
Montevideo, Uruguay
barbeito@pasteur.edu.uy
Livea Barbosa
Departamento de Bioquímica
Instituto de Química
Universidade de São Paulo
São Paulo, Brazil
livea@iq.usp.br
Manuela Basso
Department of Molecular Biochemistry and
Pharmacology
Istituto di Ricerche Farmacologiche “Mario
Negri”
Milano, Italy
basso@marionegri.it
Stefania Battistini
Azienda Osp. Univ. Senese
U.O.C. Neurologia
Policlinico “Le Scotte”
Siena, Italy
battistinis@unisi.it
Andrea Battistoni
Dept. of Biology
University of Rome "Tor Vergata"
Rome, Italy
andrea.battistoni@uniroma2.it
Joseph S. Beckman
Linus Pauling Institute
Department of Biochemistry and
Biophysics, Oregon State University
Corvallis OR 97331USA
joe.beckman@oregonstate.edu
Bogdan Beirowski
The Babraham Institute
Cambridge, UK
bogdan.beirowski@bbsrc.ac.uk
Caterina Bendotti
Dept. Neuroscience
Istituto di Ricerche Farmacologiche
"Mario Negri"
Milano, Italy
bendotti@marionegri.it
Marina Bentivoglio
Department of Morphological and
Biomedical Sciences
University of Verona
Verona, Italy
marina.bentivoglio@univr.it
96
Elke Bogaert
Laboratory of Neurobiology
University of Leuven
Leuven, Belgium
elke.bogaert@med.kuleuven.be
Irene Carunchio
Dept. of Neuroscience
University of Rome “Tor Vergata”
Rome, Italy
neurolab@virgilio
Giambattista Bonanno
Department of Experimental Medicine
University of Genoa
Genoa, Italy
bonanno@pharmatox.unige.it
Cristina Cereda
IRCCS Ist. Neurologico “Mondino”
Pavia, Italy
cristina.cereda@mondino.it
Valentina Bonetto
Istituto di Ricerche Farmacologiche "Mario
Negri"
Milano, Italy
bonetto@marionegri.it
Fernanda Cerqueira
Departmento de Bioquímica
Instituto de Química
Universidade de São Paulo
São Paulo, Brazil
fernandamcerqueira@yahoo.com.br
Virginie Bros-Facer
Institute of Neurology
University College
London, UK
v.bros@ion.ucl.ac.uk
Avi Chakrabartty
Dept Med Biophysics/CRND
University of Toronto
Toronto, Canada
chakrab@uhnres.utoronto.ca
Lucie Bruijn
The ALS Association
Palm Harbor, FL 34685, USA.
lucie@alsa-national.org
Albrecht Clement
Institute for Physiological Chemistry and
Pathobiochemistry
University of Mainz Medical school
Mainz, Germany
clement@uni-mainz.de
Lavinia Cantoni
Istituto di Ricerche Farmacologiche "Mario
Negri"
Milano, Italy
cantoni@marionegri.it
Daniele Capitanio
Dept. of Biomedical Sciences
and Technologies
University of Milan
Segrate (MI), Italy
daniele.capitanio@unimi.it
Concetta Capo
Dept. of Biology
University of Rome "Tor Vergata"
Rome, Italy
capo@uniroma2.it
Maria Teresa Carrì
Dept. of Biology
University of Rome "Tor Vergata"
Rome, Italy
carri@bio.uniroma2.it
Claudia Colombrita
Dip. Scienze Neurologiche
Università di Milano
Milano, Italy
claudia.colombrita@studenti.unimi.it
Mauro Cozzolino
Lab. of Neurochemistry
Fondazione S. Lucia IRCCS
Rome, Italy
m.cozzolino@hsantalucia.it
Valeria Crippa
Institute of Endocrinology
Centre of Excellence on
Neurodegenerative Diseases of the
University of Milan
Milan, Italy
valeria.crippa@unimi.it
97
Claudia Crosio
DiFBC
Università degli Studi di Sassari
Sassari, Italy
ccrosio@uniss.it
Elena Fumagalli
Istituto di Ricerche Farmacologiche "Mario
Negri"
Milano, Italy
fumagalli@marionegri.it
Giuseppina D’ Alessandro
Istituto di Ricerche Farmacologiche "Mario
Negri"
Milano, Italy
dalessandro@marionegri.it
Stella Gagliardi
Università degli studi di Pavia
Pavia, Italy
stellagag@libero.it
Ruth Danzeisen
International Copper Association
New York NY 10016 USA
rdanzaisen@copper.org
Paolo Di Mascio
Instituto de Química
Universidade de São Paulo
São Paulo, Brazil
pdmascio@iq.usp.br
Gabriella Dobrowolny
Dip. Istologia ed Embrionologia Medica
Università “La Sapienza”
Roma, Italy
gabriella.dobrowolny@uniroma1.it
Jacques Durand
Lab. de la Plasticitè et Physio-Pathologie
de la Motricité
UMR 6196 CNRS
Aix-Marseille Université
Marseille, France
durand@dpm.cnrs-mrs.fr
Samer Abou Ezzi
CHUL Research Center
Laval University
Quebec, Canada
samer_ezzi@yahoo.ca
Claudia Fallini
Dipartimento di Scienze Neurologiche
Università di Milano
Milano, Italy
claudia.fallini@unimi.it
Valentina Gallo
Imperial College of London
London, UK
v.gallo@imperial.ac.uk
Jonathan Glass
Emory Center for Neurodegenerative
Disease
Whitehead Biomedical Research Building
Atlanta, GA 30322 USA
jglas03@emory.edu
Genevieve Gowing
Laval University
Quebec, Canada
genevieve.gowing@crchul.ulaval.ca
Linda Greensmith
Sobell Department of Motor Neuroscience
and Movement Disorders
Institute of Neurology
London, UK
l.greensmith@ion.ucl.ac.uk
Francois Gros-Louis
Dept.of Oncology and Molecular
Endocrinology
CHUL Research Center
Laval University
Quebec, Canada
f_groslouis@hotmail.com
Jean-Pierre Julien
Dept.of Oncology and Molecular
Endocrinology
CHUL Research Center
Laval University
Quebec, Canada
jean-pierre.julien@crchul.ulaval.ca
Alberto Ferri
Lab. of Neurochemistry
Fondazione S. Lucia IRCCS
Rome, Italy
a.ferri@hsantalucia.it
98
Ali Murat Kaya
Institute for Physiological Chemistry and
Pathobiochemistry
University of Mainz Medical school
Mainz, Germany
kaya@uni-mainz.de
Antonio Musarò
Department of Histology and Medical
Embryology
University of Rome “La Sapienza”
Rome, Italy
antonio.musaro@uniroma1.it
Jasna Kriz
Dept.of Oncology and Molecular
Endocrinology
CHUL Research Center
Laval University
Quebec, Canada
jasna.kriz@crchul.ulaval.ca
Monica Nencini
Lab. of Neurochemistry
Fondazione S. Lucia IRCCS
Rome, Italy
monicanencini@hotmail.com
Michael Kyba
Dept.of Developmental Biology
University of Texas
Georgetown, Texas, USA
michael.kyba@utsouthwestern.edu
Jean-Philippe Loeffler
Lab. de Signalisations Mol. et Neurodeg,
Universite Louis Pasteur
Strasbourg, France
loeffler@neurochem.u-strasbg.fr
Andrea Malaspina
Institute of Cell and Molecular Science
Queen Mary University of London
London E1 2AT, UK
a.malaspina@qmul.ac.uk
Stefan Marklund
Department of Medical Biosciences
Umea University
SE-901 85 Umea, Sweden
stefan.marklund@medbio.umu.se
Marco Milanese
Department of Experimental Medicine,
Pharmacology and Toxicology Section
University of Genoa
Genoa, Italy
milanese@pharmatox.unige.it
Maria Rosaria Monsurrò
Department of Neurological Sciences
Second University of Naples
Naples, Italy
mrmonsurro@hotmail.com
Keith Nylin
Department of Biochemistry & Biophysics
Oregon State University
Oregon, USA
keith.nylin@oregonstate.edu
Paolo Ogliari
IRCCS Ist. Neurologico “Mondino”
Pavia, Italy
paolo_ogliari@yahoo.com
Mikael Oliveberg
Department of Biochemistry and Clinical
Neuroscience
Umea University
SE-901 85 Umea, Sweden
mikael.oliveberg@dbb.su.se
Valeria Padovano
Department of Pharmacology
School of Medicine
Università degli Studi di Milano
Milano, Italy
valeria.padovano@unimi.it
Piera Pasinelli
Farber Institute for Neurosciences
Thomas Jefferson University
Philadelphia, PA 19107 USA
piera.pasinelli@jefferson.edu
Rita Perlingeiro
Department of Developmental Biology
UTSouthwestern Medical Center
Dallas, TX USA
rita.perlingeiro@utsouthwestern.edu
Maria Grazia Pesaresi
Lab. of Neurochemistry
Fondazione S. Lucia IRCCS
Rome, Italy
lorpesaresi@libero.it
99
Grazia Pietrini
Department of Pharmacology
School of Medicine
Università degli Studi di Milano
Milan, Italy
grazia.pietrini@unimi.it
Daniela Sau
Inst. of Endocrinology
Centre of Excellence on
Neurodegenerative Diseases
University of Milan, Milan, Italy
daniela.sau@unimi.it
Angelo Poletti
Institute of Endocrinology
Center of Excellence on
Neurodegenerative Diseases
University of Milan
Milan, Italy
angelo.poletti@unimi.it
Christopher E. Shaw
Department of Neurology
King's College London School of Medicine
London, UK
chris.shaw@iop.kcl.ac.uk
Cédric Raoul
INSERM-AVENIR Team
Campus de Luminy
Parc Scientifique de Luminy
13200 Marseille cedex 09 France
cedric.raoul@ibdm.univ-mrs.fr
Antonia Ratti
Dipartimento di Scienze Neurologiche
Università di Milano
Milano, Italy
antonia.ratti@unimi.it
Claudia Ricci
Università di Siena
Siena, Italia
ricci6@unisi.it
Wim Robberecht
Department of Neurology and
Experimental Neurology
University Hospital Gasthuisberg
University of Leuven
Leuven, Belgium
wim.robberecht@uz.kuleuven.ac.be
Luisa Rossi
Dept. of Biology
University of Rome "Tor Vergata"
Rome, Italy
luisa.rossi@uniroma2.it
Giuseppe Rotilio
Dept. of Biology
University of Rome "Tor Vergata"
Rome, Italy
rotilo@bio.uniroma2.it
Pamela J. Shaw
Academic Neurology Unit
University of Sheffield
Sheffield, UK
pamela.shaw@shef.ac.uk
Laszlo Siklos
Institute of Biophysics
Biological Research Center
Szeged, Hungary
siklos@brc.hu
Vincenzo Silani
Dep.of Neurology and Lab.of
Neuroscience
IRCCS Istituto Auxologico Italiano
Milan, Italy
vincenzo@silani.com
Gen Sobue
Department of Neurology
Nagoya University Graduate School of
Medicine
Nagoya 466-8500, Japan
sobueg@med.nagoya-u.ac.ip
Richard Strange
CCLRC Daresbury Laboratory
Warrington
Cheshire WA4 4AD, UK
r.w.strange@dl.ac.uk
Michael Swash
Royal London Hospital
London, Uk
mswash@btinternet.com
Silvia Tartari
Istituto di Ricerche Farmacologiche "Mario
Negri"
Milano, Italy
tartari@marionegri.it
100
Nicola Ticozzi
IRCCS-Istituto Auxologico Italiano
Università di Milano
Scuola Specializzazione Neurologia
Milano, Italy
n.ticozzi@fastwebnet.it
Massimo Tortarolo
Dept.of Neuroscience
Istituto di Ricerche Farmacologiche "Mario
Negri", Milano, Italy
tortarolo@marionegri.it
Davide Trotti
Farber Institute for Neurosciences Thomas
Jefferson University Philadelphia
Philadelphia PA 19107, USA
davide.trotti@jefferson.edu
Joan S. Valentine
Department of Chemistry and Biochemistry
University of California
Los Angeles CA 90095, USA
jsv@chem.ucla.edu
Ludo Van den Bosch
Laboratory for Neurobiology
Experimental Neurology
University of Leuven
Leuven, Belgium
ludo.vandenbosch@med.kuleuven.be
Monika Vestling
Molecular Biology
Umezi University
Umea, Sweden
monika.vestling@molbiol.umu.se
Zuoshang Xu
Department of Biochemistry and Molecular
Pharmacology
University of Massachusetts Medical
School
Worcester, MA 01605 USA
Zuoshang.xu@umassmed.edu
Simona Zappettini
Farmacologia
Università degli studi di Genova
Genova, Italia
simonazappettini@libero.it
Haining Zhu
Department of Molecular and Cellular
Biochemistry
College of Medicine
University of Kentucky
Lexington, KY 40536 USA
haining@uky.edu
101
Alphabetical list of contributors
102
A
Adamson
Aebischer
Ajroud-Driss
Akbarloo
Al-Chalabi
Amori
Andersen
Andres
Andries
Appel
Arbogast
Arciello
Arroyo
Aucello
Augusti-Tocco
Augusto
Averill
P31
S27
P25
SO9
SO4
SO2
S9, S13, P18
P12
P40
P39
S5, P19
P4
P12
S24
P10
P5, P29
P11, P17
B
Babetto
Banci
Barbeito
Barbiero
Barbosa
Basso
Battistini
Bea
Beckman
Beers
Behl
Beirowski
Bendotti
Bentivoglio
Bergemalm
Bogaert
Bolzoni
Bonanno
Bonetto
Bordoni
Brännström
Bresolin
Bros
Bruijn
Butler
P7, P30
S4
S21
SO7
P29
P33
S15
P12
S5, P19
P39
SO1
P30
S19, S20, P8,
P13, P15, P20,
P32, P33, P36
S22, SO8
S9
P34
S19, P6, P8
SO7, P35, P41
S12, P33
P24
S9, P18
P24
P31
S31
SO4
C
Caliendo
Callewaert
Calvo
Calza
Cammarosano
Cantoni
Capitanio
P21
P34
P12, P28
P13
P28
P7, P9
P13
Caplow
Capo
Capone
Caponnetto
Cappello
Carra
Carrì
Carunchio
Casazza
Casciati
Cashman
Castellotti
Cavallo
Cereda
Cerqueira
Ceroni
Chakrabartty
Chen
Cheroni
Chia
Chiò
Clement
Cleveland
Coleman
Colleoni
Colombrita
Comi
Conforti
Corato
Corbo
Corrado
Corti
Cova
Cozzolino
Crippa C.
Crippa V.
Crosio
Crugnola
Cupidi
Curcio
P1
P4
P27
P41
SO8
P6
S8, S18, SO2,
P4, P5, P10, P29
P14
P41
S8, S18
SO3
P26
P28
P20
P5, P29
P20
SO3
P21
P15
P31
P28
SO1
SO3
P30
P36
P26
P24
P7
P20
P26
P23
P24
P20
S8, SO2, P4, P10
S19
P6, P8
S8, S18
P24
S22
P14
D
Daleno
D’Alessandro
D’Alfonso
D’Angelo
Dantuma
Darabi
De Biasi
De Bonis
Demasi
Deng
Di Mascio
Doblare
Dobrowolny
Dokholyan
Donadoni
P13, P20
P7, P9
P23
P27
P15
SO9
S19, P8
P27
P5
P21
P29
P12
S24
P1
P24
103
Doyu
Dupuis
S7
S26
E
Ezzi
P13, P26
S24
S8, SO2, P4
P31
P28
P31
SO8
SO8
P25
P21
SO5
P36
P20
P22
P27
P30
P13
P26
P28
S30
P31
S26
P25
S9, P18
S8
P12
S25, P31
SO3
P13
S10
H
Hafezparast
Hall
Hasnain
Hayward
Henkel
Herijgers
Horne
P31
P17
P2
SO5
P39
P34
SO3
I
Iaccarino
Ishigaki
Ismaili
P16, P17
P17
P18
S10, P3
K
G
Gagliardi
Gal
Galletti
Garcia Alias
Gelfi
Gellera
Ghiglione
Glass
Golding
Gonzales De Aguilar
Gorrie
Graffmo
Gralla
Grasa
Greensmith
Griffin
Grignaschi
Gros-Louis
Jokic
Jones
Jonsson
Julien
S10, P3
F
Fallini
Fanò
Ferri
Fisher
Fimognari
Flenniken
Fossati
Francolini
Frutiger
Fu
Fukada
Fumagalli
J
S18
S7
P11
Kassa
Katsuno
Keller
Khare
Kilty
Koziollek-Drechsler
Kriz
S22, SO8
S7
P37
P1
P22
SO1
S11, P37
L
Lamperti
Landry
Lauria
Lee
Levy
Lidonnici
Ling
Liu
Lococo
Loeffler
Lombardo
Lopez
Lucchini
Lukas
P24
P6
P26
SO5
P19
S20, P33
P16
P21
S20
S26
P28
P19
P24
P25
M
Macedo
Maiorano
Malaspina
Manzano
Marino
Mariotti
Marklund
Martin
Massa
Massari
Mazzini
Medeiros
Medinas
Melli
Meininger
Mennini
Michael-Titus
Milanese
Moggio
Molinaro
Momigliano-Richiardi
Morré
Monsurrò
Musarò
Mutani
P29
P27
P11, P16, P17
P12
P15
S22, SO8
S9, P18
P30
P14
P38
P23
P29
P5
P10
S26
P36
P11, P16, P17
SO7, P35, P41
P24
S24
P23
S5, P19
P27
S24
P28
104
N
Negri
Nencini
Ngoh
Nilsson
Niwa
Nylin
S
P23
S8, SO2
P17
P18
S7
S5, P19
O
Oggioni
Ogliari
Oliveberg
Onesto
Osta Pinzalos
Ottersen
P23
P20
S3, S9
S19, P6, P8
P12
P9
P
Padovano
Paizs
Panico
Pasinelli
Passariello
Pesaresi
Perlingeiro
Peviani
Piccirillo
Pieri
Pietrini
Pisani
Plumari
Poletti
Popoli
Powell
Priestley
Protasi
SO8, P38
P39
P14
S17
P26
SO2
SO9
P32
P27
P14
SO8, P38
P14
P26
S19, P6, P8
SO7
SO4
P16, P17
S24
R
Raoul
Raiteri L.
Raiteri M.
Rakhit
Ratti
René
Restagno
Righi
Riso
Rizzardini
Robberecht
Robertson
Ronchi
Rosenthal
Rossant
Rossi
Rotilio
Rusmini
Ruth
S27
SO7, P35
P35
SO3
P13, P26
S26
P28
P38
P8
P7, P9
S16, P34, P40
SO3
P24
S24
P31
P4
S1, S8, S18
S19, P6
SO3
Salani
Salemme
Salmona
Salvatore
Sandri
Santoro
Sau
Sbaiz
Schiavo
Severini
Shaw C.
Shaw P.
Siddique
Siklos
Silani
Simonini
Smith
Sobue
Sogayar
Sone
Spaltro
Spano
Stewart
Strange
Stroem
P24
P27
P33
P27
S24
P24
S19, P6, P8
P28
P31
P14
S14
S6
P21, P25
P39
S29, P13, P26
S19, P6, P8
P2
S7
P29
S7
S20, P33
P32
S9
P2
SO5, P22
T
Takahashi
Tanaka
Taroni
Tartari
Tedeschi
Testa
Ticozzi
Tortarolo
Towne
Trojsi
Trotti
S7
S7
P26
P7, P9
P27
P23, P26
P26
S20, P33
S27
P27
S23
U
Uccelli
Ulbrich
Urushitani
P41
P10
S10, P3
V
Valentine
Van Damme
Van Den Bosch
Vande Velde
Vasso
Veglianese
Vitellaro-Zuccarello
Volta
S2, S8
P34, P40
P34, P40
SO3
P13
S20, P9
S19, P8
P13
105
W
Wade
Ward
Wilcox
Witan
Wroe
SO1
P16, P17
P1
SO1
SO4
X
Xu
S28
Y
Yamada
Yan
Yee
Yong
P21
P11
P2
Z
Zappettini
Zaragoza
Zetterstroem
Zhai
Zhang
Zhu
Zona
SO7, P35, P41
P12
S9
P21
SO5, P22
SO5, P22
P14
S7
106
This Meeting has been made possible by the generous contribution
of our sponsors (in alphabetical order):
Associazione Italiana per la Sclerosi Laterale Amiotrofica (AISLA)
Bancaintesa San Paolo
Bio-Rad
Fondazione Cariplo
GE Healthcare
Gilson
Harlan
International Brain Research Organization (IBRO)
International Copper Association (ICA)
International Society for Neurochemistry (ISN)
Italian Society for Biochemistry (SIB)
M-MEDICAL
Motor Neuron Disease Association (MND Association)
Olympus
Space Import-Export s.r.l.
We thank for their patronage:
EALSC (European ALS Consortium)
SIN (Società Italiana di Neurologia)
SINS (Società Italiana di Neuroscienze)
The abstract book is a generous gift of Punzo&Colombo Canon
Business Center, Bergamo
107