CO1 - The meaning of TDP- 43 in ALS
Tibor Hortobagyi - Institute of Psychiatry, King’s College London
The transactive response DNA-binding protein 43
(TDP-43) is a major ubiquitinated protein both in ALS
and in the major subtype of frontotemporal lobar degeneration (FTLD-U). This recent discovery triggered
the re-classification of neurodegenerative diseases with
a new class of ‘TDP-43 proteinopathies’, which includes the majority of ALS cases with and without
dementia.
The presence of TDP-43-positive inclusions and the
pathologic signature with hyperphosphorilation and
ubiquitination in the affected brain and spinal cord
suggested that these diseases share a common
pathomechanism with FTLD-U and ALS representing
two extremes of a clinicopathological spectrum. TDP43 has been detected both in sporadic and familial
forms of ALS and FTLD. There were differences in
distribution of neuronal pathologies consistent with the
earlier characterised four FTLD subtypes, colocalisation with ubiquitin and p62, and previously
unrecognised glial pathology. Only rare cases of SOD1
familial ALS revealed TDP-43 pathology.
The function of TDP-43 in the nervous system is uncertain, and the pathomechanistic role in neurodegeneration remained speculative. A causative role as a
disease protein had been challenged by studies that
failed to identify TARDP mutations and claimed TDP43 expression in tau- and synucleinopathies. However,
the discovery of mutations of the TDP-43 gene both in
sporadic and familial cases strongly suggested a link to
ALS pathogenesis. Interestingly, dementia was conspicuously absent in all mutant cases.
Although further studies are required to understand the
physiological and pathological significance of TDP-43,
findings suggest that abnormalities in transcriptional
regulation and protein trafficking from the nucleus to
the cytosol may represent a major process in the pathogenesis of ALS and related dementias. Furthermore,
TDP-43 is a candidate biomarker of the disease, its
progression, and hopefully cure following targeted
therapeutic interventions.
CO2 - Muscle NOGO and Motor Neuron Disease
Pierre-François Pradat - Paris ALS Center, Paris,
France
Nogo proteins, which belong to the reticulon family,
inhibit neurite growth and nerve regeneration. A study
in the SOD1 transgenic mouse model showed an increase in the muscle expression of isoforms A and B
and a reduction in the expression of isoform C. Subsequently, Nogo-A upregulation in muscle was detected
in a series of ALS patients compared to normal control
subjects and patients with myopathy or peripheral neuropathy. In ALS patients, Nogo-A expression levels
correlated with both the degree of atrophy in muscle
fibres and the level of disability (ALSFRS-R). We
have recently shown that Nogo-A upregulation in
muscle predicted progression to an ALS phenotype
with 94% sensitivity and 88% specificity in a series of
patients presenting with lower motor neurone syndrome. Jokic et al (2006) have demonstrated that the
genetic ablation of Nogo-A extended survival and reduced muscle denervation in SOD1 transgenic mice. In
turn, overexpression of Nogo-A in wild-type muscle
fibers led to destabilization of the neuromuscular junctions with retraction of the nerve terminal and shrinkage of the post-synaptic structures. These findings,
together with other data suggesting that motor neuron
pathology begins at the distal axon and proceeds in a
"dying back" pattern, suggest that Nogo-A play a role
in the pathophysiology of ALS and may represent a
new therapeutic target. Nogo-A test is also a promising
diagnostic marker but further studies assessing its accuracy are needed before its use in clinical practice.
CO3 - Isolation and Proteomic Characterization of
Mutant SOD1-containing Inclusion Bodies
Daniel Bergemalm, Karin Nilsson, Anders Olofsson,
Per Zetterström, Karin S Graffmo, Thomas
Brännström, Stefan Marklund
Department of medical biosciences, Clinical chemistry
and Pathology. Department of molecular biology.
Umeå University
daniel.bergemalm@medbio.umu.se
Introduction: A characteristic finding in ALS patients
and transgenic murine models carrying SOD1 mutations are inclusion bodies displaying SOD1immunoreactivity. A possible toxic property of mutant
SOD1 is interaction with proteins that become inactivated, depleted or erroneously activated. These proteins could co-aggregate with SOD1 and become part
of cellular inclusion bodies.
Aim: To identify proteins in SOD1 inclusion bodies.
Methods/Results: Spinal cord homogenates from terminal G127X and G85R mice were subjected to ultracentrifugation in density gradients. From these separations fractions containing SOD1-inclusions, free from
other organelles, could be isolated. There were no inclusions in corresponding fractions of presymptomatic
or control animals. By atomic force microscopy, the
inclusions were essentially round shaped with a large
heterogeneity in size. Relevant fractions were further
evaluated by 2-dimensional gel analysis/MALDI-TOF
and by LC-MS/MS. From control animals only a few
or no spots could be found on 2D gels. In G85R and
G127X animals about 20-30 spots could be seen, most
overlapping between the models. SOD1 accounted for
about 50% of the total amount of protein. About ten
1
proteins have so far been identified, some previously
known from immunohistochemistry to be constituents
of SOD1-inclusion bodies.
Discussion: By this method we are able to find previously unknown SOD1-aggregation partners as well as
other proteins that might be trapped in these inclusions. Knowledge of the identity of such proteins
might be valuable for the development of understanding the toxic mechanism of mutant SOD1s and the
pathogenesis of ALS.
CO 4 - Novel Antibodies Reveal Inclusions Containing Misfolded SoD1 in ALS Patients
Karin Nilsson, P Andreas Jonsson, Peter M Andersen,
Karin S Graffmo, Stefan L Marklund & Thomas
Brannstrom
Departments of Medical Biosciences (Pathology and
Clinical Chemistry), Umea University, Sweden
karin.nilsson@medbio.umu.se
Background: Mutations in CuZn-superoxide dismutase
(SOD1) cause amyotrophic lateral sclerosis (ALS) and
are found in 6% of ALS patients. The cause(s) of the
disease in the reminder is largely unknown. Misfolded
and aggregation-prone forms of mutant SOD1s are
thought to trigger the disease. In several other neurodegenerative conditions such as Alzheimer´s, Parkinson´s and Creutzfeldt-Jacob´s diseases, proteins that
are mutated in some of the familial patients are also
involved in the pathogenesis in patients lacking such
mutations.
Aim: Could wild-type SOD1, by analogy, be involved
in ALS patients lacking SOD1 mutations?
Methods: Two sets of novel antibodies, raised in rabbits and chicken, against peptides spaced along the
human SOD1 sequence, were developed and shown to
be specific for denatured SOD1. These were used to
examine SOD1 in spinal cords of ALS patients lacking
mutations in the enzyme.
Results: Small granular SOD1-immunoreactive inclusions were found in spinal motoneurons of all 37 sporadic and familial ALS patients studied, but only
sparsely in 3 of 27 neurodegenerative and 2 of 19 nonneurological control patients.The granular inclusions
were by confocal microscopy found to partly colocalize with markers for lysosomes but not with inclusions
containing TAR DNA binding protein-43, ubiquitin or
markers for endoplasmatic reticulum or mitochondria.
Granular inclusions were also found in carriers of
SOD1 mutations and they were the major type of inclusions detected in ALS patients homozygous for the
wild type-like D90A mutation.
Conclusion: The findings suggest that SOD1 may be
involved in ALS pathogenesis in patients lacking mutations in the enzyme.
CO5 - Proteomic Identification of Amyotrophic
Lateral Sclerosis-Specific Biomolecules in Peripheral Fluids
Alessandra Giuliano Albo§, Chiara Abrescia§, Davide
Corpillo§, Lorenza Franciosi§, Katarzyna Lis§,
Massimo Natale§, Paolo Bongioanni*, Vincenzo De
Tata$, Luisa Martino$, Enrico M. Bucci§.
§ Laboratorio Integrato Metodologie Avanzate,
BioIndustry Park Canavese S.p.A., Colleretto Giacosa,
Italy.
giulianoalbo@bipcamail.it
Amyotrophic Lateral Sclerosis (ALS) is a complex
disease for which more than one molecular mechanism
has been hypothesized. The diagnosis is presently clinical and does not discriminate between different ALS
subtypes at the molecular level. Our aim is to identify
by proteomic strategies specific biomarkers of ALS
presence and progression. The achievement of this
goal will not only provide tools for a better diagnosis
and classification of patients, but will also contribute
to elucidate ALS mechanisms and biochemical pathways, possibly leading to new therapeutic targets.
In order to find ALS-related molecules, we chose to
analyze plasma. In addition to plasma-specific biomarkers directly correlated to ALS, blood-brain barrier damage reported in patients may cause an abnormal interchange of soluble substances between the two
compartments and therefore allow the detection of
brain-specific biomolecules in plasma. Moreover, from
a clinical point of view, blood screening is not very
invasive and is easy to perform. To reach our goal,
bidimensional electrophoresis maps were obtained
from plasma of healthy, inflamed (post-stroke) and
ALS-affected individuals and analyzed using the Image Master 2D Platinum software.
Spots whose differences in volume percentage between
ALS and controls were statistically significant
(P<0.001) were submitted to trypsin digestion and
identified by mass spectrometry. By subsequently performing an ANOVA test, we built a panel of biological
indicators of ALS, able to separate patients from controls with a predictability of about 80%. We will next
assess the biochemical significance of our candidate
biomarkers in ALS pathogenesis.
CO6 - Proteomics of plasma from Portuguese patients with familial Amyotrophic Lateral Sclerosis
Angelina S. Palma1, Mamede de Carvalho2,3, Nicolas
Grammel4, Susana Pinto2,3, Nuno Barata1, Harald S.
Conradt4, Júlia Costa1,5
2
1
Instituto de Tecnologia Química e Biológica, Oeiras,
Portugal, 2Department of Neurology, Hospital de Santa
Maria, Lisboa, 3Neuromuscular Unit, Instituto de
Medicina Molecular, Faculdade de Medicina de
Lisboa, 4GlycoThera GmbH, Germany, 5Instituto de
Biologia Experimental e Tecnológica, Oeiras
jcosta@itqb.unl.pt
Introduction: In ALS, the identification of abnormal
proteins in biological fluids might be useful for the
understanding of the ethiopathogenesis of the disease.
Furthermore, it can provide biomarkers useful for diagnosis, to monitor disease progression and to study
the effect of drugs. Plasma is a suitable fluid for
screening such targets since blood collection is a relatively simple procedure.
Aims: To analyse proteins from the plasma of a group
of Portuguese familial ALS (FALS) patients not carrying SOD1 mutations, age-matched healthy controls,
sporadic ALS patients and controls with other muscular disorders.
Methods: Proteomic techniques consisting of twodimensional gel electrophoresis and matrix-assisted
laser desorption time-of-flight mass spectrometry
(MALDI-TOF MS) have been used.
Results: Most relevant was the finding in the FALS
patients of an isoform of vitamin D-binding protein
(DBP) at pI 5.2, identified as GC2 by liquid chromatography electrospray ionization-TOF MS. GC2 was
absent from the healthy controls. Concomitantly, decrease of more acidic isoforms of DBP were observed
for the FALS patients.
Conclusions: The results suggested that the GC2 polymorphism of DBP could constitute a risk factor for
ALS.
CO9 - Testing Therapies in Animal Models: New
Guidelines.
C. Bendotti1 and Albert Ludolph2
1. Mario Negri Institute for Pharmacological Research,
Milan, Italy; 2. University of Ulm, Germany
The recent negative outcome of a large randomized
clinical trials of minocycline in ALS patients (Gordon
et al. 2007), as opposed to the positive results from
preclinical trials in SOD1 mutant mice, once again
raises debate about the reliability of testing therapies in
these standard animal models. There are many potential reasons accounting for such disparity, not last is
the lack of application in animal studies of a rigorous
study design and statistical analysis as those used for
the clinical trials in patients. A recent study from the
ALS Therapy Development Institute (Scott et al. 2008)
has questioned the efficacy of treatments reported in
the majority of published studies using SOD1G93A
mouse model. In fact, using a computer modelling and
statistical analysis of the survival length of a wide
populations of SOD1G93A mice tested, Scott and collaborators have demonstrated that the positive results
in this animal model among different studies are most
likely measurements of noise in the distributions of
survival means as opposed to actual drug effect due to
uncontrolled confounding variables in the screening
system. The most confounding variables are the occurrence of non ALS death, the incidence of low-copy
transgenes due to recombinations events and the genetic background. In fact, when they used an optimized
study design to retest different compounds in
SOD1G93A mice, including minocycline, they found
no effect.
Hence, the necessity of standardizing the methods for
the design and the interpretation of preclinical trials. In
this presentation we will discuss and up-date the guidelines established during the 142nd ENMC report (Ludolph et al. 2007), in the light of the informations derived from ALSTDI study and of other suggestions
from the literature.
References:
Scott et al. (2008) Design,power and interpretation of
studies in the standard murine model of ALS. Amyotrophic Lateral Sclerosis, 9:1,4-15
Ludolph et al. (2007). Guidelines for the preclinical in
vivo evaluation of pharmacological active drugs for
ALS/MND: Report on the 142nd ENMC international
workshop. Amyotrophic Lateral Sclerosis 2007, 1–7,
iFirst article
Gordon et al. (2007) Efficacy of minocycline in patients with amyotrophic Lateral Sclerosis: a phase iii
randomised trial. Lancet Neurology 6: 1045-53
CO10 - Study of an in Vitro Model of ALS: Role of
TNFalpha, Glutamate and p38MAPK in the Selective Death of Motorneurons.
M. Tortarolo, D. Lidonnici, C. Daleno, E. Calcagno,
G. Spaltro, C. Bendotti
Mario Negri Institute for Pharmacological Research
tortarolo@marionegri.it
Background: The study of animal models of ALS has
provided clues about factors underlying selective motoneuron death, suggesting the role of neuroinflammation, excitotoxicity and p38MAPK. However in vivo
approach often gives unclear information because of
the variety of the cell types and the complexity of
pathways involved. Thus, the use of simplified in vitro
models can help in deciphering the mechanisms implicated. Recent works have pointed out the role of the
3
astrocytes, showing their toxic influence on motoneurons when they express SOD1 mutants.
Aims: Using astrocyte-spinal neuron cocultures as
cellular model of motoneuron death we studied the role
of TNFalpha, AMPA receptors and p38MAPK in the
harmful astrocyte-motoneuron cross-talk.
Methods: Primary astrocyte-spinal neuron cocolture
was prepared obtaining the expression of SOD1-G93A
only in one cell type or in both. The motoneuron survival was evaluated by cell count. Immunocitochemistry, RT-PCR, HPLC were used.
Results: In astrocyte-spinal neuron coculture a significant and selective motoneuron death was observed
when SOD1-G93A was expressed in astrocytes or
motoneurons or in both suggesting that the only presence of mutant SOD1 in one cell type is enough to
trigger motoneuron loss. Functional studies and expression evaluation ruled out a primary role of TNFalpha and AMPA receptors in this phenomenon but suggested a pivotal role of p38MAPK pathway in motoneuron death.
Conclusion: We obtain an in vitro model that reproduce the selective motoneuron death triggered by
SOD1-G93A. The results indicate a key role of astroglial cell in this event and the involvement of
p38MAPK pathway.
Study supported by Telethon Foundation
CO11 - Electrophysiological Studies and Toxicity
Test of Primary Motoneuron Cultures.
D. Ragancokova, K.Jahn, A. Kotsiari, S. Petri, R.
Dengler and K. Krampfl
Department of Neurology, Medizinische Hochschule
Hannover
Ragancokova.Daniela@mh-hannover.de
Amyotrophic lateral sclerosis (ALS) is a devastating
disease selectively affecting motoneurons with no current effective treatment. Chronic dysregulation of the
intracellular calcium homeostasis (excitotoxicity) is
thought to be one of the major molecular mechanisms
in the pathophysiology of this fatal neurodegeneration.
We investigated neuroprotective effects of VPA and its
three analogous 3-propylheptanoic acid (3-PHA), R
(+) - and S (-) -2-n-propyl-4-pentynoic acid (R and S4-yn-VPA) on a motoneuron-enriched cell culture that
was obtained from rat embryonic spinal cord by OptiPrep density centrifugation and seeded on poly-DLornithin/ laminin or on highly purified neonatal rat
Schwann cells or astrocytes .
We tested VPA and the analogues as well as kainate (a
toxic glutamate receptor agonist eliciting excitotoxicity) plus combination of the compounds together with
kainate.
In calcium imaging experiments high intracellular
calcium transients were shown after short pulses of
kainate in motoneuron cultures 7 DIV. However, 3
DIV did not react to Kainate at all. The immunocytochemistry demonstrated that kainate caused 10% neuronal cell death in cocultures after 10 DIV even in a
low concentration such as 30µM and was dose dependent to motoneuron vulnerability. Neuroprotective
effect was studied by applying kainate with VPA, 3PHA and S-4-yn-VPA, respectively. There was no
significant effect except for a slightly decreased
amount of the cells with the analogue R-4-yn-VPA.
But surprisingly it turned out to act neuroprotective
against kainate. The amplitude of calcium transients
was reduced in the presence of VPA. However, by
patch clamp technique with a fast application system
we ruled out a direct effect of VPA on AMPA receptors.
To sum up, neuroprotective effect of the tested compounds is mild what should allow to prospect for further strategies and correlations to the pharmacological
effects on synaptic activity to protect motoneurons
against excitotoxicity.
CO12 - Glutamate AMPA Receptors and their Associated Trafficking Proteins (ABP, NSF, PICK-1,
GRIP-1 and ALSIN) in the Spinal Cord of
SOD1G93A Mice.
Veglianese P., Caron I., Marino M., Bendotti C.
1.Mario Negri Institute for Pharmacological Research,
Milan , Italy;
bendotti@marionegri.it
Among different hypotheses, the excitotoxicity is considered to play a major role in the ALS. Dysfunction of
the AMPA receptors (a subtype of the glutamate receptors) can lead to an increased calcium influx in the
motor neurons leading them to death. Protein able to
modulate the intracellular trafficking of the AMPA
receptors subunits from cytosol to membrane have
been recently characterized: N-ethylmaleimidesensitive fusion protein (NSF), AMPA binding protein
(ABP), protein interacting C-Kinase-1 (PICK-1), glutamate receptor interacting protein (GRIP-1) and
Alsin. The latter is involved in a juvenile form of ALS.
The aim of this study is to analyse the distribution and
expression of the AMPA receptor subunits, in particular GluR2, and their associated trafficking proteins in
the spinal cord of a transgenic mouse model of ALS
(TgSOD-1G93A) at different stages of the disease
progression. Results obtained by confocal microscopy
and biochemistry have revealed an early decreased
expression of GluR2 and a concomitant increase ex-
4
pression of NSF, GRIP-1 and Alsin selectively in the
motor neurons of presymptomatic mice as compared to
age matched non transgenic mice. Analysis of mice
spinal cord at the symptomatic and end stage of disease, characterized by remarkable loss of motor neurons and reactive gliosis, showed increase expression
for both GluR1 and NSF in the reactive astrocytes. We
suggest that an early deregulated trafficking of the
GluR2 subunits may alter the calcium permeability of
motor neurons making them more susceptible to excitotoxicity.
Supported by Telethon GP06141
CO13 - Impaired Secretion of Human beta-Trace
Protein in NSC-34/hSOD1G93A Cells
Catarina Gomes, Eckart Grabenhorst, Harald S.
Conradt, Júlia Costa
Instituto de Tecnologia Química e Biológica,
GlycoThera
cgomes@itqb.unl.pt
Introduction: Amyotrophic Lateral Sclerosis (ALS) is a
neurodegenerative disease. Some familial ALS cases
result from mutations in superoxide dismutase 1
(SOD1). Golgi apparatus (GA) has been reported to
appear disrupted in MN from tissues of ALS patients
and mouse models. The molecular mechanism underlying GA fragmentation in ALS is not known at present. These alterations might have consequences to the
function of the secretory pathway of MN, and the
transport and secretion of essential proteins is compromised, as well as their glycosylation. Beta-Trace
(beta-TP) is a 24 kDa secretory glycoprotein known as
prostaglandin D synthase is a major polypeptide constituent of human CSF.
Aims: We aimed at elucidating the effects of mutant
SOD1-related Golgi disruption on the secretion and
glycosylation profile of beta-TP.
Methods: In this work different cell lines of NSC-34
cells, mouse hybrid cell line, which has characteristics
of MN, stably overexpressing human mutant
hSOD1G93A and beta-TP have been constructed as an
ALS cell model.
Results: We analysed beta-TP produced by the different cell lines for 16 and 24 hours. We observed that
cell lines expressing hSOD1G93A produced lower
amounts of beta-TP when compared with the levels of
the protein produced from the cell lines expressing
hSOD1wt. The protein was resistant to Endo H and
sensitive to PNGase F digestion, but the pattern was
the same for each cell line.
Discussion/Conclusions: The impaired secretion of
beta-TP for the cells expressing mutant protein could
be related with alterations in the mechanisms underlying secretion in the cells caused by overexpression of
mutant hSOD1G93A.
C016 - Two Novel TARDBP Mutations in Familial
Amyotrophic Lateral Sclerosis
Peter Kühnlein, Anne-Dorte Sperfeld, Hans-Jürgen
Gdynia, Manuela Neumann, Albert C. Ludolph
Department of Neurology, University of Ulm, Ulm,
Germany
peter.kuehnlein@uni-ulm.de
Background: Abnormal neuronal inclusions composed
of the TAR DNA binding protein 43 (TDP-43) are the
characteristic neuropathological lesions in sporadic
and familial forms of amyotrophic lateral sclerosis
(ALS). This makes TARDBP an interesting candidate
gene for genetic screening in ALS.
Methods: All coding exons of TARDBP were sequenced in 134 sporadic ALS and 31 index patients
with familial forms of non-SOD1-ALS.
Results: We report the identification of two novel missense mutations in TARDBP (p.Gly348Cys and
p.Asn352Ser) in two small kindreds with a hereditary
form of ALS with early spinal onset resulting in fatal
respiratory insufficiency without clinical relevant
bulbar symptoms or signs of frontotemporal dementia.
The mutations are located in the C-terminus of TDP-43
and were absent control individuals.
Conclusion: Mutations in TARDBP are a rare cause of
familial ALS. The fact that all identified TARDBP
mutations are located at the C-terminus of TDP-43,
which is involved in gene expression regulation and
protein-protein interaction, suggests that they may all
cause disease through similar mechanisms.
CO17 - Lack of Association Between VEGF Polymorphisms and ALS in an Italian Population
Ricci C.(1), Penco S.(2), Corrado L.(3), Abballe C.(1),
Greco G.(1), Cozzi L.(2), Causarano R. (4), D'Alfonso
S.(3), Mazzini L.(3), Giannini F.(1), Battistini S.(1)
1)Department of Neuroscience, Neurology Section,
University of Siena, Italy; 2) Laboratory of Medical
Genetics, Azienda Ospedaliera Niguarda Ca' Granda,
Milan, Italy; 3)Laboratory of Human Genetics, Department of Medical Sciences, Eastern Piedmont University, Novara , Italy; 4)Department of Neurology,
Azienda Ospedaliera Niguarda Ca' Granda, Milan,
Italy.
ricci6@unisi.it
5
Background: Increasing evidence from the literature
points to a role of the vascular endothelial growth
factor (VEGF), a gene induced by hypoxia to elicit
angiogenesis, in ALS pathogenesis. Initial studies
showed that homozygosity for the AAG and AGG
haplotypes at positions -2578C/A, - 1154G/A and 634G/C significantly increased the risk for ALS in
some populations. However subsequent researches
have produced conflicting results. Thus, independent
replication is strongly recommended.
Aims: Aim of the present study was to investigate the
possible association between VEGF "at risk" haplotypes and ALS and to search for possible genotypephenotype correlation in a large cohort of Italian patients.
Methods: Three hundred and seventeen Italian patients
and 360 age-and sex-matched controls were studied.
All patients have been previously screened for SOD1
mutations and those positive were not included in the
analysis. Genotyping for the -2578, -1154 and -634
polymorphisms was performed by PCR and subsequent restricted enzyme digestion or direct sequencing.
Results: VEGF genotyping of the -2578C/A, 1154G/A and - 634G/C polymorphisms showed no
significant difference in allele frequencies between
patients and control groups. Within the patient group,
genotype and haplotype frequencies of VEGF polymorphisms did not show significant correlation with
gender, age at onset (> or < 58 years) and site of onset
(bulbar or spinal).
Conclusions: We could not find an association between the polymorphisms in the VEGF-promoter and
ALS susceptibility in an Italian population.
CO18 - Genetic Variation in DPP6 Gene is Not Associated with Susceptibility to Amyotrophic Lateral
Sclerosis in Two European Populations
Isabella Fogh1, Sandra D’Alfonso2, Cinzia Gellera3,
Ana Beleza1, Antonia Ratti4, Cristina Cereda5, Lucia
Corrado2, Silvana Penco6, Nicola Ticozzi4, Gianni
Sorarù7, Barbara Castellotti3, Stella Gagliardi5,
Lorenza Cozzi6, Ammar Al-Chalabi1, John Powell1,
Vincenzo Silani4
1
King’s College, London; 2Università del Piemonte
Orientale A. Avogadro, Novara; 3IRCCS Istituto
Nazionale Neurologico Carlo Besta, Milano; 4IRCCS
Istituto Auxologico Italiano, Università di Milano;
5
IRCCS Istituto Neurologico C. Mondino, Pavia;
6
Ospedale Niguarda Ca'Granda, Milano; 7Università
degli Studi di Padova
i.fogh@iop.kcl.ac.uk
Background: Only 15% of amyotrophic lateral sclerosis (ALS) patients have a family history, the remaining
cases occurring sporadically. While familial ALS is
well characterized with several causative genes identified to date, the genetics of sporadic ALS is poorly
understood. Last year three independent genome-wide
association (GWA) studies (Schymick, 2007; Dunckley, 2007; van Es, 2007) found no single gene strongly
associated with susceptibility to ALS; suggesting that a
complex interaction between environmental factors
and many susceptibility genes of small effect best describes this disease. One such susceptibility gene of
small effect recently nominated by a GWA study from
different populations of European origin is dipeptidylpeptidase 6 (DPP6) which slightly increases the risk of
developing ALS (OR 1.3) (van Es, 2008; Cronin,
2008).
Aims: To confirm the association of DPP6 with ALS
phenotype we tested the candidate polymorphism
rs10260404 in two other European populations, Italian
and British.
Methods: The Italian cohort included 980 cases and
1000 healthy controls while the British cohort consisted of 500 cases and 500 controls. The Italian cohort
tested in this study has been collected by the Italian
ALS Consortium created from the collaboration of 7
different institutions located in North Italy. We are
recruiting more cases in other Italian neurological institutions for the first large-scale Italian SALS Screen.
The British cohort was collected at King’s College
London. Individuals were genotyped following standard procedures (TaqMan, Applied Biosystems) and
allelic discrimination was scored using an ABI799HT.
Results: Assuming an odds ratio of 1.3 with a causative allele frequency of 0.4, as described by the authors, we have > 99% power to detect an association at
p = 0.05. Preliminary data show no evidence of association with susceptibility to ALS (Fisher's exact chisquared test p = 0.9).
Conclusion: The polymorphism rs10260404, as with
other variants reported to be associated with sporadic
ALS, failed to be replicated in a different population.
CO19 - Latent Cluster Analysis in the Phenotypic
Classification of ALS
J. Ganesalingam, D. Stahl, L. Wijesekera, CE Shaw,
PN Leigh, A. Al-Chalabi
Institute of Psychiatry, Kings College London
jeban.ganesalingam@iop.kcl.ac.uk
Background: Amyotrophic lateral sclerosis is a heterogenous disorder. It has traditionally been categorised
6
according to type of motor neuron affected (upper or
lower). Correct classification is important for the design of clinical trials where drugs may be more effective for one particular sub-group. We used latent class
analysis, a model based cluster analysis method, to
identify phenotypic groups on an ALS database consisting of over 1500.
Aims: To test the hypothesis that latent cluster analysis
will identify patient groups that predict measureable
outcomes such as survival times.
Methods: Clinical details of over 1500 cases seen in
the Motor Nerve Clinic in King's College Hospital
were entered into a database. A latent cluster analysis
was carried out using M-plus software. The Bayesian
Information Criterion (BIC) was used to estimate the
best fit for the number of clusters. To describe characterics of the different clusters, clusters were compared
using univariate analysis (ANOVA for continuous
variables and chi-squared for categorical variables).
Results: The BIC was minimized at five clusters. The
proportions belonging to each group were in a
517:44:786:115:4 ratio. There were significant differences between groups for age of onset, diagnostic delay, gender, family history, bulbar onset and phenotype. No significant differences were found for ethnicity or number of anatomical regions affected.
Conclusions: We have found that multiple ALS phenotypic groups can be identified using latent cluster analysis. These may be useful in the design of clinical drug
trials with the aim of finding more specific therapy,
and also for predicting prognosis.
CO 20 - Overlap of ALS and Dementia
Paul Wicks
For many years, ALS was thought to affect only the
body, with the mind and senses remaining intact. Indeed, much of the patient literature and information in
medical textbooks continues to promote this myth. In
reality, at least a subgroup of ALS patients experience
personality changes and cognitive problems consistent
with frontotemporal dementia (ALS-FTD). Neuropsychology, neuropathology, and more recently genetic
studies have started to unravel an underyling shared
mechanism beneath ALS and FTD. In addition, a larger subgroup of ALS patients experience milder cognitive deficits that might go completely unnoticed (ALS
with Cognitive Impairment, ALSCi). Both ALS-FTD
and ALSCi are caused by extra-motor neuronal degeneration although other factors such as premorbid intelligence, disability, and hypoxia should all be considered. It is important that we tell patients the truth about
cognitive change and the fact that it can occur in ALS;
otherwise when it does happen families are left without
any support, and the relationship with medical professionals is left damaged. Patients want to be informed
about their disease, especially in light of the ease of
information access provided by the internet.
CO21 - Cognitive Dysfunction in Patients with Amyotrophic Lateral Sclerosis: The Relevance for Every Day Living
Vita Stukovnik, Grega Repovs, Janez Zidar
Institute of Clinical Neurophysiology, Division of
Neurology, University Medical Centre Ljubljana, Slovenia
vita.stukovnik@kclj.si
Amyotrophic lateral sclerosis (ALS) was traditionally
thought to affect solely the lower motor neurons and
corticospinal tracts. Recent studies suggest that the
pathogenic processes of ALS are more extensive, involving dysfunction of cortical grey and white matter
with clinical correlates of impairment in cognition and
language. Until recently cognitive deterioration was
associated almost exclusively with a subgroup of 3-5%
ALS patients with frontotemporal dementia. However,
a number of neuropsychological investigations recently demonstrated selective impairments in executive
and memory function in nondemented ALS patients
(25-75%). The relevance of these results for every day
living though still remains in question, exposing the
need for the development and use of ecologically valid
test. To address the question of prevalence of cognitive
dysfunction and also its impact on patients daily activities we used a battery of standardised neuropsychological tests as well as a specially designed ecologically
valid test of executive functions. We are presenting our
preliminary results on 12 bulbar and spinal ALS patients showing diminished capacity to cope with every
day problems that depend upon intact executive cognitive abilities.
CO22 - Demential Form of Amyotrophic Lateral
Sclerosis (ALS): Is It a Clinical Entity?
J.M.B.Lima, M.Pernes, C.H.Gress
Instituto de Neurologia Deolindo Couto/ Universidade
Federal do Rio de Janeiro, Brazil
Introduction: Studies of the cognitive disorders in ALS
have been reported quite often. Taking into account
that structural lesions of frontal cortex must be associated to the basic pathogenic process, as well as alterations in the cortical and sub-cortical regions in clinical
trials and on neuroimaging tests of 16 patients, mental
alterations, seen in some ALS patients, can be a clinical announcement of the disease, usually the onset of
ALS.
7
Objectives: To demonstrate that structural alterations
in the cortical and sub-cortical regions of the brain,
found in some ALS patients, may be related to the
pathogenic process of the disease. These alterations
may sometimes lead to mental problems (demential
form).
Methodology: Clinical and anatomical – pathological
study of 9 patients, plus evaluation of 16 patients submitted to neuroimaging tests.
Results: On the anatomical/pathological study of 9
patients (8 of necropsy and 1 of biopsy), signs of brain
atrophy in 7 cases were observed; in 4 of those, there
were spongiform alterations, of the 3 cases with dementia, 2 presented alterations. Regarding the neurological imaging study of 16 patients with cognitive/behavioral disorders, signs of cortical/sub cortical
atrophy were found in 5 of them; non-specific gliose
lesions and microangiopathy in the other patients. Six
patients were submitted to the MRI with functional
study. Neuron “disappearance” was noticed in the motor cortex and alterations in the pyramidal via (significant to ALS).
Conclusion: This study shows that brain compromising
in ALS may be associated to mental disorders regarded
in many patients. This can be an anatomical/pathological substratum of dementia in some patients (Demential form of ALS?). A more consistent
anatomical/clinical connection was not found, yet the
findings lead to the necessity of further investigation
on this matter.
CO23 - Ceftriaxone Treatment is able to Reduce
Oxidative Stress in ALS Patients
Carlesi C, Lo Gerfo A, Piazza S, Molesti E, Serradori
M^, Siciliano G
Department of Neuroscience and ^Toracocardiac Department, University of Pisa, Pisa
cecilia.carlesi@tiscalinet.it
Introduction: Glutamate-mediated neurotoxicity has
been suggested as a mechanism of motor neuron death.
Ceftriaxone has been considered putative therapeutic
agent for ALS, by a modulation of astroglial GLT1
glutamate transporter expression.
Aims: To report laboratory results of blood oxidative
stress markers in ALS patients treated with a first
course of ceftriaxone (2 g/die, for 14 days) within a
planned one year study with bimonthly drug cycles.
Methods: The study has included 38 patients. 14 patients presented with the bulbar form of disease, 10 the
classic form and 14 a predominant lower motorneuron
onset form. Disease was evaluated by ALS-FRS-r,
MRC scale, dynamometric test and respiratory functionally parameters. Blood levels of oxidative stress
markers as glutathione (GSH), advanced oxidation
protein products (AOPP) and ferric reducing ability of
plasma (FRAP) were evaluated with spectrometric
methods.
Results: Before therapy, no correlation was found between oxidative stress marker blood levels, the duration and the severity of the disease. After therapy
AOPP levels decreased significantly (p<0.001), while
GSH and FRAP showed a slight not significant decrement. Respiratory functionally parameters, were unchanged.
Discussion: Based on the results, short term therapy
with ceftriaxone is able to reduce circulating levels of
some markers of oxidative stress in ALS. Whether or
not this finding is related to a direct effect of this drug
on pathogenic mechanism of the disease or to an indirect effect mediated by its antibacteric action is still an
open question, to be addressed with long term studies
in conjunction with the assessment of the clinical effects.
CO24- A Pilot Trial of G-CSF in Amyotrophic Lateral Sclerosis
Adriano Chiò, Gabriele Mora, Claudia Caponnetto,
Gabriele Siciliano, Mario Sabatelli, Vincenzo La
Bella, Vincenzo Silani, Cristina Moglia, Gianluigi
Mancardi, Piero Tonali, Andrea Calvo, Roberto
Mutani, Massimo Corbo, Rosanna Scimè, Giuseppe
D'Onofrio, Mario Petrini, Mario Melazzini, Francesca
Gualandi, Corrado Tarella
Department of Neurology and Hematology, University
of Turin, Pavia, Milan, Genua, Pisa, Rome and
Parlermo
achio@usa.net
Background: Granulocyte-colony stimulating factor
(G-CSF) stimulates the mobilization of BMCs and is
used for the treatment of hematological disorders. In
the ALS mouse model BMCs have protective effects
on motor neurons. G-CSF has also direct neuroprotective effects on motor neurons.
Objective: To evaluate feasibility, safety, tolerability,
and clinical effect of repeated procedures G-CSF in
ALS (EudraCT-number 2005-03248-75).
Design/Methods: The trial was performed in 7 ALS
centers. Inclusion criteria were: age 40-65, disease
duration 12 months, FVC 80%. After a 4-month runin period, patients underwent 4 cycles of treatment
with G-CSF at 3-month intervals. G-CSF was administered s.c., 5 g/kg b.i.d., for 4 days. BBB was permeabilized with 18% mannitol i.v., 125 cc q.i.d., for 5
days. CSF was examined at entry and during the first
and third treatment. Complete blood cells and circulat-
8
ing CD34+ cells were determined, day 0 through day
6. Progression rates of ALS-FRS-R and FVC were
compared between the pre-treatment and the treatment
period.
Results: 26 patients were recruited (15 men, 11 women; mean age, 54.2 years). The median baseline
CD34+level was 2.15/ L. During the treatment there
was an increase of CD34+ up to a peak of 45.6/ L at
the 4th day. A comparable hematological response was
observed at each of the four cycles of treatment.
CD34+ cells were found in CSF of 3 patients. The only
adverse effects were a transient increase of growth
hormone level (1 patient), and a deep venous thrombosis (1 patient).
Conclusions/Relevance: This trial indicates that (a) GCSF use to induce BMCs mobilization is safe, well
tolerated, and feasible in ALS; (b) it determines few
and reversible adverse effects; (c) BMCs mobilization
in ALS parallels that commonly observed in the
healthy population (d) there are no signs of impaired
mobilization after repeated courses of G-CSF. A placebo-controlled trial is required to verify the possible
benefits of the repeated BMCs mobilization in ALS.
Study supported by Istituto Superiore di Sanità and MURST
CO25 - What do we know after 15 years of noninvasive ventilation in ALS?
Anabela Pinto, MD PhD
Clínica Universitária de MFR, FML, IMM, HSM
Lisboa- Portugal
There is compelling evidence that non invasive ventilation (NIV) prolongs survival by several months and
improves QOL more than any other currently available
treatment. Although promising, NIV is currently used
with lack of appropriate standardization and the absence of reference values. The large number of published papers and the diversity of the used methodologies are some of the points that hamper its wide clinical application. Most of the conventional strategies are
reactive rather than proactive, essentially ignoring the
problem until the inevitable occurrence of respiratory
failure. However, this procedure is now rapidly evolving since the publication of the AAN Practice Parameter that was associated with improvement in the standard of care as well as with the increasing recognition of
the potential advantages of early intervention.
In fact, long-term ventilation has evolved from a concept of necessity ventilation to a more satisfactory
approach of preventive LTV, enabled by the impressive development of noninvasive mechanical ventilation (NIV), that due to its convenience, efficacy, and
its safety compared with invasive ventilation, rapidly
gained popularity. This review focuses mainly on the
presentation of efficacy of different modes of ventila-
tion, the importance of interface selection and adequate
control of compliance to treatment in order to reduce
the likelihood of intolerance, usually associated to
respiratory complications or failure representing the
most common cause of death in ALS.
In addition in an effort to delineate future directions
that may lead to the establishment of reference values,
we report published and unpublished personal data
clarifying predictors of survival with NIV among demographic, clinical and ventilator settings of ALS
patients.
CO26 - Muscle Mitochondrial Energy Metabolism,
Neuromuscular Junction and Amyotrophic Lateral
Sclerosis
Luc Dupuis
INSERM U692/ Université Louis Pasteur, Faculté de
Médecine, 11 rue Humann, 67085 STRASBOURG,
France
The pathophysiology of ALS and its animal models
features profound disturbances of energy homeostasis,
most notably muscle hypermetabolism. We previously
documented in both patients (1) and mSOD1 mice (2,
3), that these energy homeostasis defects contribute to
the overall survival. To determine whether muscle
hypermetabolism is per se sufficient to drive motor
neuron disease in mice, we used transgenic mice genetically engineered to display post-natal muscle restricted hypermetabolism. In this talk, the complete
neuromuscular characterization of these mice will be
presented. These results are consistent with muscle
metabolism being a strong modifyer of ALS pathology.
References:
Dupuis et al., Neurology, 2008.
Fergani et al., J Lipid Res, 2007.
Dupuis et al., Proc Natl Acad Sci USA, 2004
CO27 - Nutritional Assessment and Management in
Patients with Amyotrophic Lateral Sclerosis
M. Brugnani, G.D. Oggioni*, L. Antonelli, L. Testa*,
P. Greco, L E. Franzon**, S. Caldano**, N.
Nasuelli*, F. D'Andrea, L.Mazzini*
Clinical Nutrition Unit, "Maggiore della Carità"
Hospital, *Department of Neurology, Piemonte
Orientale
University,
**Rehabilitation
Unit,
'Maggiore della Carita` Hospital, Novara, Italy
Background: The nutritional status is an important
topic in ALS patients care and it is an independent
prognostic factor for survival. Nutritional assessment
is recommended every 3-6 months but there are no
evidence about the sort of dietary intervention required.
9
Objectives: To evaluate the patients' nutritional status
and nutritional problems at first evaluation in a third
level ALS centre. To assess the effects of the individualized nutritional therapy on disease progression and
nutritional status.
Methods: 129 ALS patients (74M;54F) referring to our
centre were recruited from 2004 to 2007. Nutritional
assessment included: height, weight and BMI, skinfold
thickness, mid-upper arm circumference and midupper arm muscle area, food intake, ability to eating,
chewing and swallowing. All patients were also evaluated by neurological, functional and respiratory assessment. 56 patients were followed every 4 months
for at least 1 year with the same procedures. Nutrition
counselling and/or individualized nutritional therapy
had been provided to patients and carers.
Results: At the first visit 18 patients manifested severe
(>10% of usual) and 28 mild (>5%; <10%) weight
loss. Cox's analysis did not show any statistically significant correlation between severity of the weight loss
and clinical or demographic variables. A significant
(ANOVA) slowing down of the progression of the
weight loss, more evident in patients with severe basal
weight loss and with disease duration <1 year, was
observed in the following 12 months.
Conclusions: Individualized nutrition counselling is
effective to maintain body weight also in patients who
manifest severe weight loss early in the course of the
disease.
CO28 - Identifying the Role of Diaphragm Pacing
Stimulation (DPS) in Amyotrophic Lateral Sclerosis: Experience from over 40 Cumulative Years of
Utilization
Raymond Onders, MaryJo Elmo, Bashir Katirji, Robert Schilz, Athony Ignagni
University Hospitals Case Medical Center, Cleveland,
Ohio
raymond.onders@uhhospitals.org
Background: Respiratory failure is a major cause of
mortality in ALS patients. Diaphragm pacing stimulation (DPS) is being utilized worldwide to provide natural diaphragm ventilation.
Aim: Review of results from the initial site implanting
DPS in ALS patients.
Methods: From 2005 to 2008, patients were evaluated
and then underwent diaphragm motor point mapping
with percutaneous intra-muscular electrode implantation. Diaphragm conditioning ensued.
Results: 47 patients were implanted with DPS. No
significant peri-operative adverse events occurred even
with forced vital capacity(FVC) as low as 20% predicted. Feeding tubes were safely implanted simultaneously with a 12 month survival >75%. The mean
time from implant was 10 months (range 1-26
months). After DPS, results show an average rate of
decline in FVC of 0.87 % per month from the preimplantation decline of 2.4% per month, extrapolating
to 24 additional ventilator free months. For 26 patients
implanted greater than 12 months there is a mean survival from diagnosis of 45 months. DPS can continuously assess diaphragm EMG activity. It was found
that ALS patients develop instability of respiratory
control and even acquired central hypoventilation. 23
patients (49%) utilized DPS during sleep. Additional
findings include: increased muscle thickness; increase
of respiratory compliance by 20%; maintenance of
diaphragm Type I slow twitch oxidative muscle fibers;
and greater fluoroscopically observed diaphragm excursion with DPS.
Conclusion: DPS decreases the decline of respiratory
failure leading to increased survival in ALS. DPS
overcomes upper motor neuron involvement and the
instability of respiratory control.
CO29 - Sternocleidomastoid Muscle Stregth and its
Impact on the Respiratory Tests
Susana Pinto, Mamede de Carvalho
Institute of Molecular Medicine, Lisbon Faculty of
Medicine (Lisbon, PT), Santa Maria Hospital. (Lisbon,
PT)
Introduction: Sternocleidomastoid muscle (SCM) is an
accessory inspiratory muscle, which activation in the
respiratory tests is easily observed. However, the real
impact of its degree of contraction in the respiratory
test results has not been addressed, in particular in
ALS. This is relevant as forced vital capacity (FVC)
and maximal inspiratory pressure (PImax) are expected
to represent diaphragmatic function.
Objectives: The authors aimed to study the involvement of SCM and to evaluate the role of diaphragm
and SCM on the respiratory tests performed in ALS.
Methodology: We studied 45 patients (mean age  SD:
60.6  13 years). In all patients we evaluated: neck
flexion strength; FVC; PImax; sniff nasal inspiratory
pressure (SNIP); ALS functional scale (ALS-FRS-R);
motor responses of SCM and diaphragm - amplitude
(SCM Ampl and Diaphr Ampl) and latencies (SCM
Lat and Diaphr Lat).
Results: In ALS patients, SCM Lat is increased and
SCM Ampl is smaller in patients with weak neck.
There is a significant correlation between SCM Amp
10
and Diaphr Ampl, FVC, PImax and SNIP. On multiple
regression analysis both PImax and SNIP are dependent on SCM Ampl and Diaphr Ampl, but not FVC.
Discussion: Hence, there is a parallel loss of motor
units in SCM and diaphragm. PImax and SNIP determination mostly depends on SCM and diaphragm
function, but FVC value is also dependent on expiratory muscles function. The results from the conventional
respiratory tests represent the synergist power of both
diaphragm and SCM. As the lost of motor units is parallel between these 2 muscles, SCM does not seem
able to replace a weak diaphragm.
CO30 - Percutaneous nocturnal oximetry in amyotrophic lateral sclerosis: periodical desaturation
João Costa, Susana Pinto, Anabela Pinto, Mamede de
Carvalho
Institute of Molecular Medicine. Lisbon Faculty of
Medicine.
joaoncosta@sapo.pt
Introduction: Respiratory function is critical in ALS
prognosis. Percutaneous nocturnal oximetry (PNO) is a
useful screening tool of respiratory function in ALS.
PNO recordings of some ALS patients disclose a periodical pattern of O2 desaturation (PP), the clinical
meaning of which is not fully understood.
Aims: We aimed to further characterize these changes
and the relations with the respiratory muscles status.
Methods: Prospective study of 261 consecutive ALS
patients. Clinical, pulmonary and neurophysiological
tests performed were: ALS functional rating scale,
forced vital capacity (FVC), maximal inspiratory pressure (Pimax), mouth occlusion pressure (MOP), phrenic nerve motor response, diaphragm needle EMG, and
PNO. A subgroup of patients underwent polysomnography. Patients with PP were selected for further analysis.
Results: A total of 837 PNO recordings were analyzed
independently by 2 authors (mean nº of recordings per
patient: 3.2). Forty-five patients (17.2%) showed typical PP. Four patients were excluded, 13 had normal
diaphragm (Group 1), and 28 had abnormal diaphragm
(Group 2). Group 1 and 2 were comparable, except for
respiratory functional score, FVC and PImax, which
were significantly lower in Group 2. In Group 1, REM
sleep was absent and hypoventilation occurred at slowwave sleep. Five patients in Group 1 were very spastic,
had low MOP/FVC and a short survival.
Conclusion: This study identified a subgroup of ALS
patients (Group 1) with marked signs of upper motor
neuron lesion, strong respiratory muscles, periodical
hypoventilation, low MOP/FVC ratio and poor prog-
nosis. We speculate that they have a central respiratory
dysfunction and deserve special care.
CO31 - The New Set of Criteria for Neurophysiological Diagnosis in ALS (Awaji criteria)
A Eisen on behalf of the Consensus of an International
Symposium sponsored by IFCN, December 3-5 2006,
Awaji-shima, Japan: Mamede de Carvalho; Reinhard
Dengler; Andrew Eisen; John D England; Ryuji Kaji;
Jun Kimura; Kerry Mills; Hiroshi Mitsumoto; Hiroyuki Nodera; Jeremy Shefner; Michael Swash
A consensus meeting was held to determine the best
use and interpretation of electrophysiological data in
the diagnosis of ALS. The utility of needle EMG and
nerve conduction studies was affirmed. It is recommended that electrophysiological evidence for chronic
neurogenic change should be taken as equivalent to
clinical information in the recognition of involvement
of individual muscles in a limb. In addition, in the
context of a suspected clinical diagnosis of ALS, fasciculation potentials should be taken as equivalent to
fibrillation potentials and positive sharp waves in recognising denervation. The importance of searching for
instability in fasciculation potentials and in motor unit
potentials in ALS is stressed. These changes in the
interpretation of electrophysiological data render obsolete the category Probable Laboratory-Supported ALS
in the modified El Escorial diagnostic criteria for ALS.
Methods for detection of upper motor neuron abnormality appear sensitive but require further study, particularly regarding their value when clinical signs of
upper motor neuron lesion are uncertain.
CO33 - Evaluation of transverse relaxation time in
motor brainstem nuclei as potential in-vivo biomarker for disease progression in the G93A-SOD1
mouse model
S Bucher 1, KE Braunstein 1, B Schwalenstöcker 1, T
Kaulisch 2, M Neumaier 2, HG Niessen 2, AC Ludolph 1 and D Stiller 2
1 Department of Neurology, Ulm University, Germany; 2 In-Vivo-Imaging laboratory, Department of Drug
Discovery Support, Boehringer Ingelheim Pharma
GmbH & Co. KG, Biberach an der Riss, Germany
birgit.schwalenstoecker@uni-ulm.de
Magnetic resonance imaging (MRI) provides an excellent tool to study disease progression in the G93ASOD1 mouse model in vivo. Recently, we reported an
age-dependent increase of transverse relaxation time
(T2 relaxation time) in the motor brainstem nuclei
facial, hypoglossal and trigeminal nucleus long before
first clinical symptoms of the disease are present. The
enhancement in the brainstem nuclei was correlated
11
with the development of dentritic vacuoles in the motor neurons, a characteristic sign of neurodegeneration
in ALS. These vacuoles are often associated with degenerating mitochondria. However, the underlying
mechanisms for the vacuolisation are still unclear but
seem to be induced by the mutant SOD1.
The aim of our study was to influence the T2 relaxation time with preclinical antioxidant treatment and to
evaluate the transverse relaxation time as potential
non-invasive biomarker for the disease progression in
an ALS mouse model.
In our study, we applied T2-weighted MRI at 4.7 Tesla
in the brainstem of vitamin E-treated G93A-SOD1
mice in comparison with untreated transgenic mice and
wild-type mice. Scanning was performed every 20
days between day 40 and 120 after birth.
T2-weighted MRI of vitamin E-treated and untreated
mice revealed a significant increase of transversal relaxation time in the facial, trigeminal, hypoglossal
nucleus from day 80 on in comparison with the untreated mice. However, the comparison of treated and
untreated transgenic mice exhibited a statistically significant difference in the age-dependent increase of T2
relaxation time during the progression of the disease in
favour of preclinical intervention with vitamin E.
Our results suggest that T2 relaxation time can be used
as biomarker for quantitative longitudinal evaluation of
the disease progression in the G93A-SOD1 mouse
during experimental drug therapy in-vivo.
CO34 - Use of the Compound Muscle Action Potential Scan Curve to Estimate the Degree of Denervation in Amyotrophic Lateral Sclerosis
M B Bromberg
University of Utah, Salt Lake City, UT, USA
mbromberg@hsc.utah.edu
Background: Amyotrophic lateral sclerosis (ALS) is
characterized by loss of upper and lower motor neurons. Clinical neurophysiology is used to detect lower
motor neuron loss, but mild loss may not be evident by
the amplitude of the compound muscle action potential
(CMAP) or may be underestimated by electromyography (EMG).
Aims: We are investigating the use of a CMAP scan
curve as a non-invasive technique to identify denervation.
Methods: Normal and ALS subjects received a CMAP
scan curve of the first dorsal interosseous muscle consisting of 250 stimuli with currents from CMAP
threshold to a maximal response. They also receive
both routine and quantitative EMG studies and motor
unit number estimation (MUNE) by the spike triggered
averaging techniques using a decomposition algorithm.
Results: Stimuli for scan curves were well tolerated. Normal subjects had smooth sigmoidal shaped
scan curves, while ALS subjects had breaks or jumps
in their scan curves. Routine needle EMG studies
were mildly abnormal in ALS subjects and quantitative
EMG metrics showed increased motor unit amplitude,
minimally increased waveform complexity, and normal or mildly increased duration. MUNE values were
reduced in ALS subjects, and at times to greater degrees than expected from both routine and quantitative
EMG results.
Conclusions: These data support the notion that the
CMAP scan curve is sensitive to denervation in hand
muscles, and is well tolerated. It may be a useful technique to apply to multiple muscles to supplement clinical and EMG data in making the diagnosis of ALS.
CO35 - ALS and TMS II: Central Silence Period
C. Civardi MD, A. Collini MD, L. Mazzini MD, L.
Testa MD, G. Oggioni MD, N. Nasuelli MD, F.
Monaco MD, R. Cantello MD.
Department of Neurology. Università del Piemonte
Orientale "A. Avogadro", Novara, Italy.
c_civardi@yahoo.com
Introduction: Suppression of the volitional EMG activity due to a transcranial magnetic stimulus (TMS) is
called central silent period (CSP). In amyotrophic lateral sclerosis (ALS) CSP was usually reported reduced
in length.
Aims: We studied the CSP in a sample of consecutive
ALS.
Methods: We studied 38 ALS patients (23 males; men
age 54 + 13; spinal form=28). Based on the El Escorial
criteria we had 18 clinically definite; 17 clinically
probable and 3 clinically possible. TMS was delivered
with a large round coil over the vertex; intensity of
stimulation was set=1,5x relaxed threshold. Motor
evoked potentials and CSP were recorded bilaterally
from the first dorsal interosseous muscle (FDI) while
patients performed their maximum effort. For each
muscle we recorded at least eight trails. We correlated
the CSP duration with clinical variables. We compared
data of ALS patients with a group of 25 normal controls.
Results: In patients, the CSP duration was not different
as compared with the controls(199,9 ms vs 185,8 ms),
while the standard deviation was much higher(+69,7
ms vs +20,5 ms). A cluster analysis defined three CSP
homogeneous groups, termed "short CSP", mean value=135 ms, which was the most frequent (42,7 %);
12
"normal"(mean value 218 ms; 40,3%), and "long"
(mean value 311 ms; 17%). The CSP length was significantly correlated to the Norris score (p=0.025) and
to the Babinski sign (p=0.047), while did not correlate
with the ALS duration.
Conclusions: In our patients, the CSP showed a heterogeneous pattern. The most frequent was represented
by a "short" CSP, although "normal" and "long" durations were not so infrequent. CSP length was correlated with ALS clinical severity.
CO36 - Quantitative Sensory Testing in Motor
Neuron Disease
Matthias Ponfick, Regina Gastl, Albert C.
Ludolph, Hans-Jürgen Gdynia, Anne-Dorte Sperfeld,
Roland Klug
University of Ulm, Department of Neurology, Oberer
Eselsberg 45, 89081 Ulm, Germany
Objective: Motor neuron diseases (MND) are a heterogeneous group of neurodegenerative disorders. Although the main disease characteristic is the degeneration of the motor system, an affection of the sensory
nervous system has occasionally been described. In
this study we examined 21 patients with MND (1 PLS,
3 PMA and 17 ALS; 14 women, 7 men) with quantitative sensory testing to evaluate sensory abnormalities.
Methods: The mean age of the collective was 62.4
years (+/- 10,67 years, range: 40-83 years), the mean
disease duration was 41,9 months (+/-34,55 months).
To detect sensory impairments we used the quantitative sensory testing as described in the DFNS protocol.
There are 13 parameters which are bilaterally testing
all types of sensory nerve fibres in three locations
(face, dorsum of the hand and foot). Age, gender and
region were eliminated by using the z transformation.
Then we created a variable which calls damage points
to evaluate all abnormal values which were higher or
lower than 2 standard deviations assuming the normative data from the DFNS.
Results: 19 out of 21 patients had sensory deficits (6499%, 99%CI). As a result of the z-transformation there
were no differences between the tested region or gender, only the confounder age kept influence on the
data. We found that the C and A-delta fibres are more
often damaged then other nerve fibre types (84 damage
points for C fibres vs. 60 A-delta vs. 37 A-beta vs. 63
non specific fibres). Furthermore, patients with a bulbar disease onset demonstrated an higher damage point
status than patients with spinal disease onset (upper/
lower extremities). (12,5 vs. 10,7 vs. 10,5).
Conclusions: Our data are demonstrating that sensory
deficits are a frequent feature in motor neuron diseases; the quantitative sensory testing is a sufficient meth-
od to detect sensory impairments in patients with
MND. Aetiologically, we found the C and A-delta
fibres to be the most affected.
CO37 - Assistive Technology for Augmentative
Communication in ALS
Luis Azevedo
Biomedical/Rehabilitation Engineer,
ANDITEC, Lisbon – Portugal
anditec@mail.telepac.pt
MSc.,
PhD.
This presentation will focus on how Assistive Technology (AT) for Alternative and Augmentative Communication (AAC) can contribute to a better quality of
life to people with Amyotrophic Lateral Sclerosis
(ALS). People affected by this disease experience a
range of speech symptoms as the disease progresses,
with a gradual loss of their natural speech abilities.
Most of those affected by ALS are unable to talk for an
extended period of time before their deaths. AAC
strategies together with adequate assistive technology
equipments, namely speech generating devices (SGD),
can be of the utmost importance to keep persons with
ALS communicating and interacting with their natural
contexts. Case studies of people with ALS using AT
devices will be presented, as well special interfaces
relying on biosensors, brain computer interfaces, eye
tracking systems, etc,. Finally, a novel “Integrated
System for Augmentative Communication, Manipulation and Mobility” with a multimodal access system
will also be demonstrated.
CO38 - Interventions for Patients with Amyotrophic Lateral Sclerosis
M B Bromberg - University of Utah, Salt Lake City,
UT, USA
mbromberg@hsc.utah.edu
Background: Amyotrophic lateral sclerosis (ALS) is a
progressive disorder leading to weakness and loss of
function and increasing need for durable medical
equipment (braces, walkers, wheelchairs) and interventions (gastric feeding tubes, non-invasive ventilation).
Aims: We performed a chart review of deceased ALS
patients to determine the time course of need for durable medical equipment and interventions.
Methods: ALS patients were grouped into site of onset:
bulbar (n = 26), upper extremity (n = 27) and lower
extremity (n = 25). A time line of needs was based on
when clinic personnel felt an item or intervention was
necessary. The time of need for each item for each
patient was expressed as a percent of the patient's total
disease duration.
13
Results: We found that 96% of patients with lower
extremity onset needed a wheelchair by 56% of their
disease duration compared to 46% of bulbar onset
patients who needed a wheel chair by78% of disease
duration. While 100% of bulbar onset patients need a
gastric feeding tube by 62% of disease duration, 48%
of patients with upper or lower extremity onset need a
feeding tube by 76% of their disease onset. A higher
percentage of lower extremity onset patients need noninvasive ventilation than bulbar onset patients (68%
versus 50%). Not all patients needed equipment or
interventions which is likely related to different patterns and rates of progression within the site of onset. The cost in US dollars for equipment for a large
percentage of patients is $30,000, and for interventions
is $5,000.
C onclusions: these data support the notion that 37% to
100% of als patients will need major items of equipment and interventions
CO39 - Proximity with Telemetry for the Control of
Compliance to NIV in ALS Patients
José Pedro Almeida1, Susana Pinto2, Mamede de
Carvalho2, Anabela Pinto1
1
University Clinics of PMR, Institute of Molecular
Medicine, Lisbon Faculty of Medicine, Santa Maria
Hospital. (Lisbon, PT); 2Institute of Molecular Medicine, Lisbon Faculty of Medicine (Lisbon, PT), Santa
Maria Hospital. (Lisbon, PT)
Background: Non-Invasive Ventilation (NIV) is an
efficient method to treat respiratory insufficiency in
ALS, it improves survival and quality of life. However, it requires a process of adaptation not always attained due to intolerance or poor compliance frequently attributed to the bulbar-onset type of disease. Since
tolerance and comfort may be obtained with individualized parameters settings usually by trial and error
procedures in multiple office visits, we therefore aimed
to increase levels of compliance by remote controlling
the ventilator parameters settings.
Objectives: Evaluate the adaptation process in two
conditions either with a close follow-up in office visits
or with a proximity control of NIV adaptation through
modem communication, and find out which parameters
settings are the most useful in predicting long-term
satisfaction as well as survival.
Methods: 54 probable or definite ALS consecutive and
ventilated patients in the last three years were randomized according to the local of residence to check compliance and parameters settings in office visits (G1) or
to receive a device for remote controlling the Goodknight 425 ST from Lindemed (G2). We registered
throughout the clinical evolution, the number of office
visits, the parameters settings at initial adaptation, the
number and type of each parameters changing, the
total ventilator usage in days, the percentage of days
with use  6 hours, the percentage of spontaneous respiratory breathing. In addition, we recorded survival
from symptoms onset and from NIV adaptation. We
compared both groups and data was analysed to find
differences at admission in order to control for the
confounding variables and subsequently identify possible predictors of long-term satisfaction (compliance)
or survival.
Results: G1 included 28 pts 9 bulbar-onset and 19
spinal-onset with mean agesSD (611.6), G2 included 26 ALS pts, 8 bulbar-onset and 18 spinal onset with
mean ages  SD (592).There were not significant
differences at admission regarding age, gender, type of
disease-onset and disease duration. The amount of
non-compliant patients was lower in G2 though not
statistically significant. The number of changes in parameters settings was not different although the number of days to full compliance was significantly lower
in G2. Total survival from symptoms onset till January
2008, as well as the total number of ventilator usage in
days was significantly increased in G2. There were
significant differences regarding initial parameters
settings, showing lower inspiratory pressures (IPAP)
and breath frequencies in G2, and lower percentages of
spontaneous breathing in G1.
Conclusions: This study shows that remote controlling
is able to reduce the trial and errors procedures, fastens
the process of NIV adaptation and therefore may contribute to the increased survival observed.
CO40 - Age and Gender Differences in Caregivers’
Burden
Tramonti F, Davitti S, Tuccio MC, Bongioanni P.
paolo.bongioanni@tin.it
Introduction: Caregivers of patients with Amyotrophic
Lateral Sclerosis (ALS) have to face several problems
related to ALS onset and progression. Patients’ gradual
lack of independence creates patterns of close relationships, in which caregivers are strongly involved in
difficulties and suffering of their relatives with ALS
Aims: Aim of our study was the evaluation of different
aspects of caregivers’ burden, a topic widely recognised as one of the most relevant, not only for caregivers’ own conditions, but also for the effects that caregiver’s psychological wellbeing can exert on patient’s
quality of life.
Methods: We used the Caregiver Burden Inventory
(CBI), a 24-item questionnaire with 5 subscales that
measure different dimensions of burden. We administered it to 31 caregivers of ALS patients. We divided
the sample in three age groups (I: 26-45; II: 46-59; III:
14
60-70) and we correlated different dimensions of caregivers’ burden with age and gender.
Results: We found significant differences in gender
comparison: female caregivers claim higher levels of
burden in terms of time dedicated to the assistance. As
far as age groups are concerned, we found only a significant difference in physical burden, that is higher in
the eldest caregivers (Group III) than in the youngest
(Group I).
Discussion: Our findings might be a first step in the
assessment of possible differences among caregivers in
burden perception. Further studies are needed to evaluate also other relevant variables in such complex situations.
CO41 - Analysis of Co-Morbidities in a Database of
ALS Patients
Ilsemann JH, Kollewe K, Dengler R, Krampfl K, Petri
S
Department of Neurology, Hannover Medical School
julia-165@gmx.de
Background/Introduction: Amyotrophic lateral sclerosis (ALS) is characterized by a rapidly progressive loss
of upper motor neurons in the primary motor cortex
and lower motor neurons in brainstem and spinal cord.
Many different pathomechanisms such as excitotoxicity, oxidative stress, disruption of axonal transport, lack
of neurotrophic factors and oxidative stress are discussed in sporadic ALS.
Aims/ Methods: In the present study we evaluated a
cohort of 565 ALS patients of the ALS outpatient clinic of Hannover Medical School with respect to concomitant diseases. Disease characteristics (region of
onset, time course, progression of upper and lower
motor neuron symptoms) of patients with comorbidities were assessed.
Results: In our cohort of 565 ALS patients the prevalence of concomitant cardiovascular diseases was most
frequent (> 60%), followed by other neurological diseases. Manifest psychiatric syndromes (mainly depression, to a much lower extent anxiety or insomnia) were
present in approximately 10% of our ALS patients.
Movement disorders were present with slightly higher
incidence than in the normal population as well as the
percentage of patients having undergone trauma with
CNS involvement. Malignant conditions had occurred
in 12%. 1.2% of the patients in our cohort suffered
from epilepsy and had therefore been treated for years
before ALS-onset with anticonvulsant drugs which did
not seem to have an influence on disease manifestation
or progression rate.
Discussion/Conclusion: The analysis of co morbidities
in large ALS databases could provide hints for ALS
risk factors or conditions modifying the disease course.
CO44 - Challenges for future ALS Trials.
P Nigel Leigh
MRC Centre for Neurodegeneration Research, King’s
College London and King’s MND Care and Research
Centre, Department of Clinical Neuroscience, PO 41,
Academic Neurosciences Building, Institute of Psychiatry, London SE58AF UK.
ALS (MND) appears now a more complex disease
than it did two decades -or even one decade- back, but
new insights have been plentiful. Clinical trials should
be seen more as experiments on pathogenic mechanisms. We must learn more from failures and from
successes. So what have we learnt from ALS trials?
Only one phase 3 trial (the riluzole study published in
1996) has been convincingly positive, with only a
small effect on survival. Meta-analyses indicate that
the effect was real, and that there may have been a
small effect on function. Yet we still do not know how
riluzole works. We do know through the NNIPPS
(Neuroprotection and Natural History in Parkinson
Plus Syndromes- PS and MSA) trial that the effect on
survival in ALS is not translated to PSP or MSA. So
riluzole does not have a generic neuroprotective effect.
We might conclude that we would be unwise to abandon survival as an arbiter of efficacy. Paradoxically,
we should beware of equating survival with function in
trials. They are related, of course, but the relationship
is complex. ALS patients (and thus functional
measures of deterioration) are very susceptible to adverse drug effects. A recent example may be the North
American minocycline trial- maximum tolerated dose
may not be the best option for neuroprotection. What
might be key elements of a strategy to find effective
therapies? The selection of agents for trials has been
idiosyncratic, and inevitably (given our ignorance of
mechanisms) determined more by the enthusiasm or
bias of particular investigators or companies than rational processes. More rigorous criteria for moving
from cellular and animal models to man should be
agreed to avoid time-wasting studies. We urgently
need a new approach to phase 2 studies that includes
dose finding. There are advocates of various alternative designs, including futility designs, but there are
major drawbacks to these and it is far from certain that
this approach will expedite the search for new treatments. Normally, dose-finding relies on functional
measures that are relatively insensitive to change. Ideally, we need to identify biomarkers that will indicate
whether the supposed target has been modified. Examples would include inflammatory markers as indicators
of the actions of minocycline and other putative anti-
15
inflammatory agents, and proteomic or metabonomic
markers of cell damage. Most likely, CSF analyses will
be required. New imaging techniques may contribute.
We are on the threshold of new phase in ALS research.
Understanding of the mechanisms of SOD1 ALS may
at last be crystallising into more focused hypotheses,
and treatments are being developed to modify this
form of the disease. The identification of TDP-43 gene
mutations and molecular pathology offers new insights
into disease mechanisms, and the prospects for success
in sporadic ALS through genome wide association
studies - although far from certain-are encouraging.
Biomarker work is taking off, and even imaging may
catch up with mechanisms in the next decade. We
need more tools to study the human brain in life, and to
develop an effective experimental medicine approach
to neurodegenerative disorders if we are to integrate
laboratory science with clinical practice to find effective treatments.
Acknowledgements: The King’s MND Care and Research Centre is supported by the MND Association
and by King’s College London and King’s College
Hospital NHS Foundation Trust. PNL has received
research grant support from the MRC, Wellcome
Trust, ALS Association (ALSA), UK Department of
Health, MND Association, and Tim Perkins family
(MND Research Fund).
In the last 5 years PNL has held consultancies with
and/or has received research grants in relation to clinical trials from Sanofi-Aventis, GlaxoSmithKline,
ONOPharma, Novartis, Exonhit, Teva, Trophos,
Roche, Oxford Biomedica, Evotec. Neuronova, and
Phytopharm. .
P1 - Interaction Between the PON1 L55M Polymorphism and Population Density in ALS: A Population-Based Case-Only Gene-Environment Study
Diekstra FP; Beleza-Meireles A; Tripathi VB; van den
Berg LH; Leigh PN; Shaw CE; Al-Chalabi A
Institute of Psychiatry, King's College London
ana.beleza@iop.kcl.ac.uk
Introduction: Paraoxonase (PON) polymorphisms have
been implicated in several genetic association studies
of ALS. Paraoxonases are detoxifying enzymes involved in the metabolism of organophosphates, drugs
and oxidised LDL. Environmental toxins are likely to
vary between urban and rural environments and population density will therefore act as a surrogate marker
of environmental exposure to PON substrates. We
therefore tested the hypothesis that genetic variation
within PON1 or PON2 would interact with population
density in the location of residence of ALS patients.
Methods: Genotyped patients were those in the South
East England ALS (SEALS) population-based register
for whom DNA was available (n=98). PON genotypes
were obtained at previously associated SNPs rs662
(PON1 Q192R), rs854560 (PON1 L55M), rs6954345
(PON2 C311S) and rs11981433 (PON2 intronic). Statistical analyses were carried out using SPSS v15.0,
PLINK and Haploview. Median population density
was used to categorise patients into rural or urban environments. A case-only analysis was carried out using
Fisher's exact test and the Cochran-Armitage trend test.
Results: There was a significant interaction with population density for rs854560 (L55M) at the genotypic
level (genotypic p=0.00048, Cochran-Armitage
p=0.0047). We therefore tested other models and
found the most significant association was for the recessive model (p=0.00023). There was a weaker signal
from rs662 (genotypic p=0.043, Cochran-Armitage
p=0.0099). Haplotype analysis did not improve the
association.
Conclusion: There is a significant interaction between
population density and PON genotype in ALS.
P2 - Combining the Evidence for PON Polymorphisms in ALS: Weak Evidence for Association
Diekstra FP; Beleza-Meireles A; Tripathi VB; van den
Berg LH; Leigh PN; Shaw CE; Al-Chalabi A
Institute of Psychiatry, King's College London
fdiekstra@gmail.com
Introduction: Paraoxonases are detoxifying enzymes
involved in the metabolism of organophosphates,
drugs and lipid peroxides. Polymorphisms in the
paraoxonase (PON) gene family have been implicated
in several genetic association studies of ALS. However, almost each of the studies found different variants
to be associated or found no association at all.
Aims: In the present study we aimed to assess the evidence for association between PON polymorphisms
and ALS.
Methods: A literature search was performed in order to
find studies that investigated paraoxonase gene polymorphisms in ALS. Results for allelic case-control
association tests were extracted from the retrieved
articles. Only those SNPs that had either been associated or had been investigated in more than one study
were included. Evidence for association for each of the
SNPs was assessed using Fisher's method for combining p values.
Results: Six studies reported PON gene variants in
ALS. A total of forty-four SNPs had been studied, of
which eleven were included in the present study.
Combining p values for allelic tests resulted in a significant association for rs6954345 (p=0.014), rs854560
(p=0.024), rs10487132 (p=0.03). However, none of
16
these associations withstood Bonferroni correction for
multiple testing. The combined p value for the rs662
(Q192R) variant, which has been implicated in ALS
most often, did not reach statistical significance
(p=0.051).
Conclusion: The current literature provides weak evidence for an association between PON polymorphisms
and ALS.
P3 - Hemochromatosis Gene Polymorphisms and
Sporadic Amyotrophic Lateral Sclerosis
Golenia A, Zawislak D, Ostrowska M, Slowik A,
Tomik B, Szczudlik A.
Department of Neurology, Jagiellonian University,
Medical College, Krakow, Poland
agolenia@gmail.com
Background: The pathogenesis of amyotrophic lateral
sclerosis (ALS) is still unknown, the oxidative stress
seems to play a great role in ALS pathogenesis. Iron
misregulation and accumulation may contribute to
oxidative damage. In ALS patients, elevated iron level
were demonstrated in the central nervous system,
which could imply a change in iron exposure of motor
neurons.
The iron homeostasis pathways in sporadic
ALS(sALS) were studied by association between the
risk of the disease and H63D,C282Y polymorphisms
of the hemochromatosis (HFE) gene.
Objective: We have assessed the relationship between
H63D and C282Y polymorphisms of the HFE gene
and the increasing risk of sALS.
Material & Methods: We have included 225 unrelated
patients with sALS and 303 unrelated healthy controls
matched for age and sex. The definite or probable diagnosis of ALS was established according to El Escorial Criteria (1998) in Department of Neurology, Jagiellonian University, Medical College in Krakow. The
polymorphisms were studied by polymerase chain
reaction (PCR) and restricted enzyme digestion.
Results: Genotype frequency of the HFE gene polymorphisms in the patients with sALS did not differ
significantly from genotype frequency of the controls
and did not influence the risk of occurrence of sALS
(H63D p=0.081; C282Y p=0.626).
Conclusion: Although some previous studies discovered an association with H63D polymorphism of HFE
gene and greater risk of sALS in a few European populations, we demonstrated that H63D and C282Y polymorphisms of the HFE gene are not connected with an
increasing risk of sALS in the Polish population. Our
results are in line with data from American population.
P4 - Expression of histone deacetylases 1-11 in ALS
post mortem tissue
Janssen C, Sarlette A, Dengler R, Krampfl K, Petri S
Department of Neurology, Hannover Medical School
clja105@stud.mh-hannover.de
Background/Introduction: Transcriptional dysregulation has been shown both in human sporadic ALS and
in the G93A mouse model. Histone deacetylases
(HDAC) catalyze the deacetylation of lysine residues
in the amino terminals of the highly conserved core
histones H2A, H2B, H3, and H4. Acetylation of histones correlates with transcriptional activity and determines specific temporal and spatial gene expression
patterns. HDAC enzymes have become potential therapeutic targets for several diseases, among them malignant, cardiovascular and neurodegenerative disorders. In the G93A-ALS-mouse model, HDAC inhibitors have already been shown to have neuroprotective
capacities. HDAC can be grouped into four classes,
class I (HDAC 1-3, 8), class II (HDAC 4-7, 9, 10),
class II (syn. SIRT 1-7) and class IV (HDAC 11) with
distinct localization, substrates and physiological roles.
Aims: The mRNA expression levels of HDAC 1-11
have previously been characterized in rat brain but not
yet studied in human tissue. In the present study we
assessed the distribution and expression levels of the
HDAC isoforms HDAC 1 - 11 in post mortem ALS
and control brain and spinal cord.
Methods: We performed in situ hybridization histochemistry (ISH) on human post mortem motor cortex
sections of ALS patients (n=3) and age matched controls with no history of neurological disease (n=3).
mRNA expression was quantified macroscopically by
densitometric analysis of digitized film autoradiograms, the expression at the cellular level was studied
by liquid emulsion autoradiography.
Results: In contrast to rat brain, where HDAC 3, 5, and
11 were predominant, in human motor cortex and spinal cord, HDAC 2 and HDAC11 showed highest
mRNA levels. Comparison between ALS and controll
tissues will enable the detection of disease specific
expression patterns.
Discussion/Conclusion: The precise characterization of
ALS-related alterations in HDAC expression levels or
distribution patterns could allow for further improvement of novel pharmacotherapeutic approaches.
P5 - Construction of a Large Human scFv Antibody
Phage Llibrary and Isolation of Tumor-Specific
17
Internalizing Human scFv by Selection of Library
Using Tum
Hoda Ayat, Ali Mohammad Ahadia, Oscar Burrone ,
Mehdi Arbabi.
Shahrekord University
hoda.ayat@gmail.com
Introduction: Antibody internalization into the cancer
cell is required for many targeted therapeutics, such as
immunotoxins, immunoliposomes, antibody-drug conjugates and for targeted delivery of genes into cells. In
addition, Phage display is a powerful technique that
can be used to develop antibodies to target molecules.
Aims: Because of the extracellular accessibility of the
Her-2 make this marker a suitable target for tumor
therapy, the aims of this study was finding of molecules capable of binding to cancer cells expressing
Her-2 and internalized them.
Methods: A human scFv library as fusion to phage was
constructed from lymph nodes of breast cancer patients
with anti Her-2 antibodies. The library was subjected
to four rounds of selection on a Her-2 expressing human breast cancer cell line SKBR3. Internalized phage
were recovered from within the cells and used for the
next round of selection. After selection, most of clones
analyzed bound SKBR3 and other tumor cells but did
not bind normal human cells.
Results: We isolated human scFv antibody fragments
and their binding characteristics have been studied by
ELISA, FACS, Immunofluorescence and western blotting. Both in soluble and phage format, they bind specifically to cells transfected with the ErbB2 gene.
Discussion: These scfvs are potentially immunoreagent
for diagnostics and therapeutics of certain cancers and
are able to introduce toxic agents to cells or directly
inhibit tumor cell growth that needs more studies.
P6 - Effect of the Iron Chelator Desferoxamine on
Myelomonocytes from Sporadic ALS Patients Subjected to Hypoxic Stress.
Moreau Caroline1,; Gosset Philippe 2, MD, PhD; Brunaud-Danel Véronique1, MD; Lassalle Philippe2, PhD
; Destée Alain1, MD,PhD; Defebvre Luc1, MD, PhD.
Devos David1 MD, PhD.
1 Neurology Department, EA 2683, Lille University
Medical Center, Lille, France
c-moreau@chru-lille.fr
Background: Acute intermittent hypoxia frequently
occurs early in ALS and may contribute to motorneu-
ron degeneration. Myelomonocytes are involved in the
progression of ALS.
We have observed functional abnormalities in the regulation of angiogenic factors (especially in the HIF-1/
VEGF pathway) during hypoxia in the CSF and monocytes from sporadic ALS patients. HIF-1 is physiologically targeted for degradation by the ubiquitin proteasome pathway, involving PHDs.
Objectives: To assess the in vitro effect of a prolyl
hydroxylase (PHD) inhibitor (desferoxamine, DFO) on
sporadic ALS patients' monocytes exposed to hypoxic
stress.
Method: Monocytes from 10 sporadic ALS patients
and 10 controls were isolated and placed in culture
chambers. Four conditions were tested: normoxia,
normoxia with DFO, 24 hours of hypoxia, followed by
re-oxygenationwith and without DFO. Cell's vitality
and VEGF levels were measured in cytoplasma and
supernatants (ELISA).
Results: Preliminary results confirmed the lack of
VEGF up-regulation in ALS monocytes, compared
with controls. A 2-fold increase in VEGF levels was
observed in cytoplasma and supernatants after addition
of DFO in ALS and controls during normoxia. No
elevation of NF-KB was observed in ALS in the DFO
condition, compared with baseline.
Conclusion: This study confirms the potential utility of
an iron chelator (DFO) in the restoration of HIF-1 mediated VEGF neuroprotection in the very early stage of
the disease.
P7 - Misfolded SOD1 in Transgenic Mice is Mostly
Oligomeric
Per Zetterström(1), Karin Sixtensdotter-Graffmo(2),
Thomas Brännström(2) and Stefan L Marklund(1)
Umeå University,Department of Medical Bioscences,
Clinical Chemistry(1) and Pathology(2)
per.zetterstrom@medbio.umu.se
Introduction: 140 different mutations in SOD1 have
been linked to ALS and all should share a common
neurotoxic mechanism. Our group has previously
shown that soluble misfolded subfractions of mutant
SOD1 enriched in the affected spinal cord of transgenic ALS mouse models are a common denominator for
mutant SOD1s with widely different molecular characteristics.
Aims: To determine the molecular structure of misfolded SOD1 in murine spinal cords. Antibodies specific for misfolded forms of SOD1 were used for the
purpose.
18
Methods: A sandwich ELISA (misELISA) was developed using antibodies specific for misfolded SOD1.
Tissue extracts from transgenic mice were subjected to
gel chromatography. Eluting fractions were analyzed
for total SOD1 with western immunoblots. Misfolded
SOD1 in the fractions was analyzed with the
misELISA.
Results: In G93A transgenic mice two peaks were seen
in the chromatography when analysed for total SOD1.
One large peak of 32 kDa composed of dimeric SOD1
and a smaller of 16 kDa with monomeric SOD1. The
misELISA showed two peaks, one at 140 kDa and one
at 45 kDa. This would correspond to SOD1 nonamers
and trimers. Less than 2% of the total SOD1 was found
to react in the misELISA. Analysis of D90A and G85R
mice with the misELISA show similar nonamer-trimer
patterns with an additional monomeric peak.
Discussion: In many neurological diseases large protein aggregates are found late in the disease process. If
the aggregates are toxic or if precursor molecular
forms exerts the toxicity is not known. We show here
that misfolded SOD1 in tissues adopts several conformations including monomeric, tri and nonameric
forms. Whether these are toxic remains to be shown.
P8 - Decreased mRNA and Protein Expression of
the Transcriptional Co-Activator PGC-1α in ALS
Post Mortem Tissue and G93A Mice
Sarlette A, Krampfl K, von Neuhoff N, Dengler R,
Petri S
Department of Neurology, Hannover Medical School
sarlette.alexander@mh-hannover.de
Background/Introduction: There is ample evidence that
oxidative damage by reactive oxygen species (ROS)
plays an important role in ALS pathogenesis. Mitochondria are the major source of cellular ROS production, which further increases if mitochondria are damaged (Beal, 2005, Hervias et al., 2006). The transcriptional co-activator peroxisome proliferator-activated
receptor-γ co-activator 1α (PGC-1α) plays a pivotal
role in the regulation of mitochondrial metabolism and
biogenesis via activation of transcription factors such
as nuclear respiratory factor-1 (NRF-1) (Scarpulla,
2006). Alterations in PGC-1α expression and function
have previously been described in models of Huntington’s and Alzheimer’s disease (Weydt et al., 2006, Shi
and Gibson, 2007).
Aims: In the present study, we investigated the mRNA
and protein expression of PGC-1α and NRF-1 in human post mortem tissue of ALS patients as well as in
the G93A-ALS mouse model in presymptomatic, early
–and late symptomatic stages.
Methods: We performed in situ hybridization histochemistry, quantitative real time PCR, immunohistochemistry, and Western blot experiments on human
post mortem tissue of ALS patients and age matched
controls and on spinal cord tissue from the G93A
mouse model.
Results: Both in post mortem ALS tissue and in G93A
mice, we observed a reduction of PGC-1α and NRF-1
at the mRNA and protein level, in the animal model
already detectable before symptom onset.
Discussion/Conclusion: We therefore conclude that a
decrease in PGC-1α could contribute to reduced antioxidant defense mechanisms in familial and sporadic
ALS and represent an interesting therapeutic target.
References:
Beal MF (Mitochondria take center stage in aging and neurodegeneration. Ann Neurol 58:495-505.2005); Hervias I,
Beal MF, Manfredi G (Mitochondrial dysfunction and amyotrophic lateral sclerosis. Muscle Nerve 33:598-608.2006);
Scarpulla RC (Nuclear control of respiratory gene expression in mammalian cells. J Cell Biochem 97:673-683.2006);
Shi Q, Gibson GE (Oxidative stress and transcriptional regulation in Alzheimer disease. Alzheimer Dis Assoc Disord
21:276-291.2007); Weydt P, Pineda VV, Torrence AE,
Libby RT, Satterfield TF, Lazarowski ER, Gilbert ML,
Morton GJ, Bammler TK, Strand AD, Cui L, Beyer RP,
Easley CN, Smith AC, Krainc D, Luquet S, Sweet IR,
Schwartz MW, La Spada AR (Thermoregulatory and metabolic defects in Huntington's disease transgenic mice implicate PGC-1alpha in Huntington's disease neurodegeneration.
Cell Metab 4:349-362.2006).
P9 - Learning from Yeast: Simple Models of Neurodegenerative Disorders
Tiago Fleming Outeiro
Cell and Molecular Neuroscience Unit, Instituto de
Medicina Molecular, Instituto de Fisiologia, Faculdade
de Medicina da Universidade de Lisboa, Lisboa,
Portugal and Massachusetts General Hospital, Harvard
Medical School, Boston, USA
touteiro@gmail.com
Introduction: Protein misfolding and aggregation are
central events in many disorders including several
neurodegenerative diseases. This suggests that alterations in normal protein homeostasis may contribute to
pathogenesis, but the exact molecular mechanisms
involved are still poorly understood. The budding yeast
Saccharomyces cerevisiae is one of the model systems
of choice for studies in molecular medicine. Modeling
human diseases in this simple organism has already
shown the incredible power of yeast to unravel the
complex mechanisms and pathways underlying these
pathologies.
19
Aims: Our goal was to uncover molecular pathways
involved in Parkinson’s and Huntington’s disease
through the use of yeast as a model organism.
death was observed using TUNEL assay. Immunohistochemistry of neuronal markers was carried out in
order to investigate the types of neurons affected.
Methods: In this study we used yeast genetics to model
neurodegenerative disorders such as Parkinson's or
Huntington's disease. We generated yeast strains expressing human proteins (alpha-synuclein and huntingtin) involved in these disorders and conducted genetic
enhancer and suppressor screens. These studies were
coupled with cell and molecular biology techniques to
learn about the molecular basis of the diseases.
Results: SOD1G93A-dsRed transfected neurons showed
cell death as demonstrated by TUNEL staining (no. of
embryos analysed = 36/36). We also observed a decrease in the population of motor neurons (stained for
Islet1/2) on the transfected side of the embryo as compared to the non-transfected control side. Embryos
expressing mutant TDP43 (n = 49) showed a dramatic
reduction in maturation as observed by a failure to
develop normal limb and tail buds. The percentage of
mature embryos electroporated with TDP-43Q331K was
11.66% and for TDP-43M337V was 15% as compared to
97.66% for embryos electroporated with TDP-43WT.
TUNEL staining demonstrated a significant increase in
the number of apoptotic nuclei in embryos expressing
either mutant TDP-43 when compared to TDP-43WT
(TDP-43Q331K n = 29.5; TDP-43M337V n = 21.6, TDP43WT n = 5.6, where n is mean of TUNEL positive
cells/section for 5 sections analysed from 3 embryos).
Results: This work led to the identification of several
molecular mechanisms associated with the pathological roles of alpha-synuclein and huntingtin. Interestingly, we found that alpha-synuclein disrupts intracellular trafficking and increases the sensitivity to oxidative stress. We are now validating these pathways as
potential therapeutic targets for therapeutic intervention.
Discussion: The use of yeast has proven fruitful in the
study of many neurodegenerative diseases. Several
features intrinsically associated with these diseases,
such as the formation of protein aggregates, the cellular toxicity mediated by these misfolded proteins, oxidative stress and hallmarks of apoptosis have been
faithfully recapitulated in yeast, enabling us to take
advantage of this powerful model to rapidly perform
powerful genetic screens with the goal of identifying
novel candidate therapeutic targets.
P10 - The Chick Embryo as a Toxicity Screen for
Genes in Amyotrophic Lateral Sclerosis
Vineeta Tripathi, Jemeen Sreedharan, Sarah Guthrie,
Christopher Shaw, Ammar Al-Chalabi
Department of Clinical Neuroscience, King's College
London, Medical Research Council (MRC) Centre for
Vineeta.Tripathi@iop.kcl.ac.uk
Background: Genetic technology has advanced to the
point that large numbers of people are being tested for
gene variants contributing to ALS. The chick embryo
provides a model system in which effects on intact
tissues can be examined quickly and simply in a cell
and stage-specific way. We have used the chick embryo model in order to study the effects of gene mutations found by linkage studies.
Methods: We electroporated SOD1G93A-dsRed,
SOD1WT-dsRed and HA and Myc tagged TDP-43WT,
TDP-43Q331K and TDP-43M337V DNA into the spinal
cords of HH stage 14 chick embryos using in ovo electroporation. After 24 hours the embryos were processed for frozen sectioning and then apoptotic cell
Conclusions: The chick model offers a relatively quick
and cost-effective means of validating genetic discoveries and will provide valuable insights into disease
mechanisms.
P11 - Human NMDA Receptors Modulation by
Sera from Amyotrophic Lateral Sclerosis Patients
and Mutated Superoxide Dismutase Transgenic
Rats
Laura Texidó1, Sara Hernández2, Mireia Martin-Satué1,
Mònica Povedano3, Carles Solsona1, Josep Esqueda2,
Jordi Marsal1.
1. CIBERNED. Laboratory of Cellular and Molecular
Neurobiology, Department of Pathology and Experimental Therapeutics, Medical School-Bellvitge Campus, IDIBELL-University of Barcelona. L’Hospitalet
de Llobregat, Spain; 2. Department of Cellular Neurobiology. Medical School, University of Lleida, Spain;
3. Neurology Unit, Bellvitge Hospital, L’Hospitalet de
Llobregat, Spain.
lteixido@ub.edu
Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease, characterized by the selective degeneration
of the motor neurons in the motor cortex, brain-stem
and spinal cord. We tested the effect of sera from sporadic ALS patients and mutated human SOD-1
(mSOD1 G93A) transgenic rats on human NMDA
receptors (NMDAR). We hypothesize that an endogenous excitotoxic factor is implicated in neuronal death
in ALS, mediated by the activation of NMDA noncanonical signalling pathways. Sera from ALS patients
or healthy subjects were pretreated to inactivate com-
20
plement pathways and dialysed to remove glutamate.
Sera from mSOD1 G93A rats were obtained at different stages of the neurodegenerative progression. Sera
from transgenic rats were also pretreated to eliminate
complement system and glutamate. Human NMDAR
were expressed in Xenopus laevis oocytes, and glutamate-induced currents were recorded using the two
electrode voltage clamp technique. We observed that
sera from sporadic ALS patients induced transient
oscillatory currents in Xenopus oocytes expressing
NMDAR significantly higher than the electric charge
carried by currents induced by sera from healthy subjects. The currents were inhibited by MK-801, a noncompetitive blocker of NMDAR. Results of sera from
mSOD1 G93A transgenic rats were similar to those of
sera from ALS patients; samples from patients with
another type of neuromuscular disease did not exert
this effect. The oscillatory currents recorded are due to
internal calcium mobilization, our data therefore agree
with the view that ALS patients sera contain some
soluble factor/s that activates intracellular calcium
concentration.
the cases when based on TA and in 48% of the cases
when based on FDI recordings. Considering all muscles, the CMCT was impaired in 98% of patients. Considered trapezius and TA recordings, the pathological
CMCT emerged in 92% of the cases. Although we had
only 3 patients with clinically possible ALS, trapezius
CMCTs were prolonged in all of them, while were
normal for the FDI.
P12 - ALS and TMS I: Trapezius Muscle Evaluation
Kantonsspital St.Gallen,
Muskelzentrum/ALS clinic, Switzerland
christoph.neuwirth@kssg.ch
A. Collini MD, C. Civardi MD, L. Mazzini MD, L.
Testa MD, G. Oggioni MD, N. Nasuelli MD, F.
Monaco MD, R. Cantello MD.
Department of Neurology. Università del Piemonte
Orientale "A. Avogadro", Novara, Italy.
alessandracollini@virgilio.it
Introduction: Transcranial magnetic stimulation (TMS)
is a useful and safe method to test the corticospinal
system integrity. In ALS the early detection of the
upper motor neuron (UMN) involvement represents an
important marker of the disease.
Aims: We applied TMS in a sample of consecutive
ALS.
Methods: We studied 38 ALS patients (23 males; 54 +
13 yrs; spinal onset, n= 28). Based on the El Escorial
criteria we had 18 clinically definite, 17 clinically
probable and 3 clinically possible. With a large round
coil over the vertex we tested the bilateral trapezius,
first dorsal interosseous (FDI) and tibialis anterior
(TA) muscles. For all we determined the relaxed
threshold, the total, peripheral and central motor conduction time (CMCT). We compared ALS data with a
group of 25 normal controls.
Results: Average relaxed threshold was increased in
the ALS group(54, + 13,5 vs 47,6 + 6; p=0,01). In
ALS patients the CMCT was prolonged: in 80% of the
cases when based on trapezius recordings; in 66% of
Conclusions: TMS evaluation of the trapezius muscle
can represent a promising target of study. It can be
expected to be able to distinguish ALS from cervical
spondylotic myelopathy, and to detect the early involvement of UMN. The combined evaluation of trapezius and TA muscles appears to increase this probability.
P13 - Motor Unit Number Index (MUNIX):A novel
neurophysiological technique to follow disease progression in ALS
Ch. Neuwirth, S. Nandedkar, E. Stalberg, M. Weber
Background: Several motor unit number estimation
(MUNE) techniques have been applied to quantify
motoneuron loss in ALS. Disadvantages of the existing
methods are that they are time consuming (20-30 min.
per muscle) and/or invasive. MUNIX is based on surface-EMG recordings, requiring only a few minutes
per muscle.
Objective: To evaluate the feasibility of MUNIX as a
marker to objectively measure disease progression in
ALS.
Methods: As of summer 2007 seven patients (5 males,
2 females, mean age 61, disease duration less than 14
months) were enrolled in a randomized controlled clinical trial (SIRONA) At 2-monthly follow-up visits
clinical data, CMAPs and MUNIX from 8 muscles
(APB, ADM, AH, EDB bilaterally) were obtained.
After measuring one single supramaximal CMAP,
surface EMG was recorded during voluntary contraction of muscles at increasing force levels (minimal to
maximal, 9 times).
Results: By the end of March data were available over
a 6-month-period from each patient. The method was
very well tolerated, no participant retired from the trial.
Technically no major problems occurred. At study
entry mean MUNIX per muscle was 56.5 ± 20.4, mean
CMAP per muscle 4.3 ± 1.3 and mean ALSFRSR 43.6
± 2.4. After 6 months MUNIX had dropped to 53.1 ±
18.0, CMAP to 3.9 ± 1.6 and ALSFRSR to 39.0 ± 5.2.
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Changes were only significant for the ALSFRSR
(p<0.019 paired t-test).
Discussion: MUNIX analysis is quick to perform and
well tolerated, but patient cooperation is necessary.
Changes over a 6-month-period were not significant.
One-year data and their statistical analysis will show
whether MUNIX is superior to standard neurophysiological measures (CMAP) and clinical scores.
P14 - Recovery of the Blink Reflex in ALS and Endophenotypes
Van der Graaff MM*, Brugman F**, Koelman
JHTM*, Bour LJ*, De Jong JMBV*, Van den Berg
LH**, Van Beilen I**, Van Doorn PA#, De Rijk M# #,
De Visser M*
* Department of Neurology/Clinical neurophysiology,
Academic Medical Centre, Amsterdam ; ** Department of Neurology, University Medical Centre
Utrecht, the Netherlands; # Department of Neurology,
Erasmus Medical Centre, Rotterdam, the Netherlands;
##
Department of Neurology, Catharina Hospital, Eindhoven, the Netherlands
mm.vandergraaff@amc.uva.nl
Introduction: Signs of upper and lower motor neuron
lesion (UMN and LMN) are mandatory to diagnose
ALS, but the LMN lesion often dominates the clinical
picture. Whether the primary target of disease is the
UMN, LMN, or both has not been settled. The blink
reflex consists of an early ipsilateral reflex (R1) and a
late bilateral reflex (R2). R2 recovery curves are obtained after application of paired stimuli. The recovery
is expressed as the size of the test response as a percentage of the conditioning stimulus. Enhanced recovery is a measure of hyperexcitability of facial nerve
motor neurons and brainstem interneurons.
Aims: To investigate whether an enhanced recovery
curve of the blink reflex in ALS and endophenotypes
corresponds with phenotypes carrying clinical UMN
features.
Methods: Patients with bulbar-onset ALS, limb-onset
ALS, progressive spinal muscular atrophy (PMA),
primary lateral sclerosis (PLS) and healthy controls (8
subjects per group) were enrolled. All patients had
weakness for < 1.5 year, except for the PLS group.
Follow up visit was at 6 months. Paired stimuli were
delivered at interstimulus intervals (ISI) of 200, 300
and 500 ms. Latency of R1 and latency, area and peak
amplitude of R2 were measured.
Results: At baseline we observed a significantly longer
R2 latency only in PLS compared to controls
(35.0±1.51 vs. 32.4±1.20 ms.,p=0.003). We found a
trend towards enhanced recovery of R2 amplitude at
all stimulus intervals in PLS compared to controls
(resp. 39±17% vs. 26±7% at ISI 200ms., 51±25% vs.
35± 11% at ISI 300ms. and 60±24% vs.51± 9% at ISI
500ms.), which reached significance for the area under
the curve at ISI 300ms.(34±10% vs 20±9%, p=0.021).
At follow up no trends or significant changes were
found in any of the groups.
Discussion: Only in PLS we found evidence of hyperexcitability of facial motor neurons and brainstem interneurons. The recovery of the blink reflex is not
helpful in demonstrating UMN involvement in ALS or
PMA at an early stage of disease.
P15 - New Aspects on Depression and Quality of
Life in ALS
Lulé, Dorothée 1,2; Häcker, Sonja 2; Ludolph, Albert
C 1; Birbaumer, Niels 2,3; Kübler, Andrea 2,4
1 University of Ulm, Department of Neurology, Germany; 2 Eberhard-Karls-University of Tübingen, Institute of Medical Psychology and Behavioural Neurobiology, Germany; 3 National Institutes of Health (NIH),
NINDS, Human Cortical Physiology, Section Bethesda, USA; 4 Clinical and Health Psychology Research
Centre, School of Human and Life Sciences, Roehampton University, London, UK
dorothee.lule@uni-ulm.de
Introduction: A good understanding of circumstances
of life is a prerequisite for end-of-life decisions in ALS
patients.
Aims: Our studies aimed at exploring the circumstances of life in patients with severe physical restrictions
like those with ALS.
Methods: In two different studies depression and quality of life were investigated in ALS patients longitudinally and in comparison to healthy controls, respectively.
Results: Our studies indicate that there is no correlation of physical disability and depression or quality of
life (QoL) in ALS. Depression rate correlated negatively with education. Experiencing a high QoL was
possible at any stage of the disease.
Discussion: The rationale against live-prolonging
treatment in severely disabled patients is an expected
poor QoL. Our data confirmed that a positive life experience is possible in severely physically impaired patients even in the terminal phase as shown here by the
example of patients with Amyotrophic Lateral Sclerosis (ALS).
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P16 - Onset and Spreading Patterns of Upper and
Lower Motor Neuron Symptoms in ALS
Fahlbusch M, Kollewe K, Dengler R, Petri S, Krampfl
K
Department of Neurology, Hannover Medical School
marion_fahlbusch@web.de
Background/Introduction: Amyotrophic lateral sclerosis (ALS) is a fatal rapidly progressive adult-onset
neurodegenerative disorder characterized by the progressive degeneration of upper and lower motor neurons. It has been debated whether “dying forward”
mechanisms originating in the primary motor cortex
and/ or “dying back” phenomena starting in the lower
motor neurons occur in ALS since the disease has first
been described by Jean Martin Charcot in 1874.Ravits
et al. have previously analyzed the distribution of upper and lower motor neuron degeneration in the early
disease period and concluded that this is a focal process which advances contiguously. In a concomitant
neuropathological study, Ravits et al. could show that
the advancement of lower motor neuron degeneration
occured in a graded manner with the most severe loss
in the region of disease onset.
Aims/ methods: In the present study we performed a
retrospective analysis of 187 ALS patients from our
database to define the frequency of certain patterns of
disease progression after onset in a certain region
(bulbar/ cervical/ thoracic/ lumbar). ALS patients were
classified into subgroups for bulbar, cervical, thoracic
or lumbar onset and then attributed to distinct patterns
of disease spreading according to the occurrence of
subsequent symptoms in either ipsilateral or contralateral extremities or the bulbar region.
Results: We so could define whether the spreading of
symptoms typically occurred into contiguous or distant
regions and into caudal or rostral direction. We also
analyzed whether upper and lower motor neuron signs
occurred and progressed independently or simultaneously from each other.
Discussion/ Conclusion: Based on these observations,
conclusions on the underlying pathological anatomical
changes of motor neuron degeneration could be facilitated.
P17 - Phenotypic Spectrum of Juvenile and Adolescent Onset Sporadic Amyotrophic Lateral Sclerosis
Hans-Jürgen Gdynia, Stefan Waibel, Peter Kühnlein,
Larissa Arning, Albert C. Ludolph, Anne-Dorte Sperfeld
University of Ulm, Department of Neurology, Oberer
Eselsberg 45, 89081 Ulm, Germany
Objective: Amyotrophic lateral sclerosis is a fatal neurodegenerative disease which leads to permanent disability and premature death. Usually, it is on average
diagnosed in adults in their 5th decade of life, whereas
juvenile or adolescent disease onset is uncommon especially in sporadic forms. Here we describe detailed
phenotypic characteristics of 14 patients with sporadic
juvenile or adolescent onset ALS.
Methods: The patients were ascertained from our motor neuron outpatient clinic. The age at investigation
was 29.64 ± 5.11 years and the age of onset < 35 years
(26.79 ± 3.91 years). In all patients detailed clinical,
laboratory, electrophysiological, and imaging data
were analyzed. All patients underwent SOD1, Dynactin and Senataxin testing.
Results: The cohort consists of 12 Caucasian and 2
Turks. There were 10 male and 4 female patients.
None of them showed cognitive decline, impaired
hearing or vision. Due to the revisited El Escorial criteria, 1 patient was classified as possible ALS, 2 as
probable laboratory supported ALS, 4 as definite ALS,
and 6 as suspected ALS. The clinical symptoms and
the disease course of our patients were not different
from the hallmarks in typical ALS patients. All patients were SOD1, Dynactin and Senataxin (ALS4)
negative, although 3 patients appeared phenotypically
like ALS4.
Conclusion: The phenotypic spectrum of early onset
sporadic ALS did not differ from the classical adult
onset form. Although some phenotypic features of few
cases were comparable to ALS4 genetic testing was
negative. Furthermore, we did not find any additional
vulnerability factor or atypical symptoms in our patients.
P18 - Brain Compromising in Amyotrophic Lateral
Sclerosis (ALS): Clinical and Neurological Imaging
Study
J.M.B.Lima, M.Pernes, C.H.Gress
Instituto de Neurologia Deolindo Couto, Universidade
Federal do Rio de Janeiro, Brazil
Introduction: ALS is a neurodegenerative disease of
unknown etiology, one of its major characteristics is
the significant participation of motor neurons. Many
authors have been reporting brain compromising on a
regular basis recently. As neurological imaging tests
became more popular, brain alterations became more
visible. The authors show the relative high frequency
of the alterations, studying 65 ALS patients. They
highlight the possible clinical/pathological connection
between these alterations and cognitive/behavioral
disorders seen in many patients.
23
Objectives: To demonstrate that the anatomical/functional alterations are possibly connected to the
neurocognitive expressions found in ALS patients.
Methodology: The study is based on tests - Head CT,
MRI and MRI of head with functional study - of 65
ALS patients, of different ages and clinical types treated at the Institute of Neurology Deolindo Couto of the
Federal University of Rio de Janeiro.
Results: We have analyzed 65 neuroimaging tests: 26
Head CT; 44 MRI of head. We have evaluated 25
Head CT, of those, 19 presented no alterations and 6
presented diffuse brain atrophy. We have also studied
44 MRI of head, of those, 16 were normal and 37 with
the following alterations: hyper signal in T2 and
FLAIR, microangiopathy/gliose, brain atrophy.
ery was in the terminal stage of the disease while the
patient was already bed-ridden. The patient deceased
11 months after the birth of the second child. The two
siblings have developed without abnormalities to date.
Conclusion: It is rare that two pregnancies occur during the disease course of ALS. Delivery is usually
normal, except in the terminal stage of the disease
when caesarean section can become necessary. Neonatal outcome is reported to be good. However, regarding
the prognosis of ALS, the ethical aspects of pregnancy
in a fatal and rapidly progressive disease should be
discussed early in detail.
P20 - Contribution to the Clinical and Epidemiological Study of Amyotrophic Lateral Sclerosis (ALS):
Evaluation Within a Period of 30 years
J.M.B. Lima, M. Pernes, C.H. Gress
Conclusion: The findings suggest that probably there is
a connection between the cognitive/behavioral manifestations seen in some ALS patients and brain alterations regarded in the imaging tests. There is a need of
further investigation in order to establish the clinical/
pathological relationship of brain compromising in
ALS.
P19 - Pregnancy and Delivery in Terminal Stage of
ALS
S. Sarafov1, S. Petri2, M. Doitchinova3, Z.
Karagiozova4, B. Slancheva5, R. Dengler2, K.
Kollewe2
1.Medical University, Department of Neurology, Sofia, Bulgaria; 2. Medical School Hannover, Department of Neurology, Hannover, Germany; 3. Military
Medical Academy - Sofia, Department of Anesthesiology and Intensive Care, Department of Clinic of Gastroenterology; 4. University Hospital of Obstetric and
Gynecology - Clinic of Obstetrics "Fetal Medicine",
Sofia, Bulgaria; 5. University Hospital of Obstetric and
Gynecology - Clinic of Neonatology, Sofia, Bulgaria.
Kollewe.Katja@mh-hannover.de
Introduction: Amyotrophic Lateral Sclerosis (ALS) is a
neurodegenerative disease involving the upper and
lower motor neuron. The onset of the disease is usually
in the fourth to fifth decade. Occasionally the disease
develops in patients younger than 40 years and is very
uncommon in patients under 30 years. Therefore pregnancy in ALS-patients is rare, and only few sporadic
and familial ALS cases with pregnancy have been
described in the literature.
Case report: We report an additional case of a young
woman with sporadic ALS who delivered two healthy
children during the course of ALS. The second deliv-
Instituto de Neurologia Deolindo Couto/Universidade
Federal do Rio de Janeiro, Brazil
marli-pernes@yahoo.com.br
Introduction: ALS is a chronic, neurodegenerative
disease. The etiology is not known and its main characteristic is the significant compromising of motor
neuron cells, leading to total paralysis. In accordance
with several reports, the clinical profile is not uniform,
changing as the studied casuistry. Throughout the
years, however, some clinical and epidemiological
aspects have been reported. The authors report on the
study of two groups of patients evaluated in different
periods of time separated by 30 years.
Objectives: To evaluate the clinical and epidemical
profile of two groups of patients observed in two periods of time separated by 30 years, making comparisons of clinical/epidemiological parameters and showing variations of the clinical profile found in this evaluation.
Methodology: Descriptive-quantitative study of two
groups of ALS patients, enrolled in the ALS/MND
service of the Institute of Neurology Deolindo Couto
from the Federal University of Rio de Janeiro. Casuistry consisting of two groups: one of 136 and another of
164 patients. Several clinical aspects have been evaluated.
Conclusion: The authors came to the conclusion that,
among other clinical aspects, the variation of prevalence of age range: in the first group the predominance
was under 50, conversely, in the second group, evaluated 30 years later, the prevalence occurs after 50 years
old. Nevertheless, by comparing with the casuistry of
other authors, in Brazil, a significant number of occurrences among young people became clear. A peculiar
24
constitution of the Brazilian population or a clinical
characteristic of ALS in this country?
P21 - Isolated Continuous Rhythmic Lingual Myoclonus: Unusual Presentation of Amyotrophic Lateral Sclerosis
C. Civardi MD, A. Collini MD, L. Mazzini MD, L.
Testa MD, D. Mittino MD, F. Monaco MD, R.
Cantello MD.
Department of Neurology. Università del Piemonte
Orientale "A. Avogadro", Novara, Italy.
c_civardi@yahoo.com
Introduction: Isolated myoclonus of the tongue is an
exceptional entity, poorly documented and understood.
Recently we observed a case of isolated continuous
rhythmic lingual myoclonus as first symptom of amyotrophic
lateral
sclerosis
(ALS).
Case description: A 45-year-old woman suddenly manifested a continuous involuntary movement of tongue
and dysarthric speech. Neurological examination revealed continuous, rhythmic bilateral symmetric jerks
of the tongue that produced a narrowing of its anterior
portion, with a slight forward protrusion and without
lateral deviation. The movements were continuous,
apparently rhythmic, when the tongue was protruded
and when it was at rest. No other branchial muscles
involvement was observed. At the onset no other neurological signs were manifested. EEG, BAEP SEPs
and brain MR were normal. EMG recorded from the
bilateral genioglossus muscle showed continuous, lowfrequency (1-2 Hz), rhythmic bursts of bilaterally synchronous muscle activity. Six months later she developed a progressive bulbar and motor palsy typical of
ALS.
Conclusions: To our knowledge this represents the first
report of isolated continuous rhythmic lingual myoclonus as first sign of ALS. Imbalance of excitatory neurotransmission as reported at the pre-clinical phase of
bulbar-ALS could explain this rare form of myoclonus
P22 - Diaphragm pacing stimulation in ALS – the
Berlin experience with two patients
Thomas Meyer (1), Peter Linke (1), Robert Eisele (2),
Sven Schmidt (2)
Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum; Department of Neurology; Department of Abdominal Surgery
thomas.meyer@charite.de
Background: Respiratory failure remains the most
common cause of death in ALS. Non-invasive mechanical ventilation (NIV) may improve survival with
maintenance of quality of life. However, in a subgroup
of patients NIV shows several limitations. Diaphragm
pacing stimulation (DPS) is an additional method by
which the phrenic nerves can be activated through
laparoscopic placement of intramuscular diaphragm
electrodes.
Aim: to assess the feasibility of DPS in two ALS patients implanted in Berlin, Germany, in December
2007.
Methods: two patients were clinically evaluated who
underwent diaphragm motor point mapping with percutaneous intra-muscular electrode implantation and
DPS.
Results: Forced vital capacity (FVC) was 80% and
52% at time of the surgery, respectively. Propofol and
remifetanil without neuromuscular relaxants were used
for induction and maintenance of anesthesia. Duration
of surgery was 166 (patient #1) und 132 minutes (patient #2). Bilateral pneumothorax developed during
surgery in both patients. No other peri-operative adverse events occurred. The patients were discharged
from the hospital 5 and 2 days after surgery, respectively. Patient #1 used DPS for 30 minutes three times
per day, whereas patient #2 utilized DPS during 14
hours per day. In patient #1, one month after DPS,
there was a decline in FVC to 64%. However, after 3
month the decline of respiratory failure decreased
(FVC 64%). In patient #2, one month after DPS, there
was a decline in FVC to 34% (17% in the supine position). During the utilization of DPS, respiratory control
was more stabilized (FVC 40% in the upright, 32% in
the supine position). Two months after surgery, mechanical ventilation was required. Three months after
DPS, no significant response to stimulation was observed, most likely secondary to denervation atrophy.
Conclusion: DPS surgery and clinical use have performed reliably and safely. Clinical criteria have to be
defined for optimal patient selection and response to
DPS.
P23 - Behavioural changes in ALS/MND mouse
models
Kerstin E. Braunstein and Albert C. Ludolph
Ulm University, Department of Neurology, Germany
kerstin.braunstein@uni-ulm.de
Introduction: The phenotypes of animal models for
ALS/MND are mainly defined by motor neuron degeneration and muscle paralysis. The best characterized mouse model is the SOD1G93A model. In this
25
study we attempted to analyse several mouse models
which also develop phenotypes characterized by a
predominant motor neuron pattern.
Aims: Beyond the SOD1G93A model we investigated
the motor, behavioural and cognitive deficits of animals with a mutation in the protein tau (P301L-mice)
or in the motor protein dynein (Cra1-mice). By using a
specific testing battery we wanted to define the spectrum of functional changes in the behaviour of transgenic animals.
Methods: For the experiments we used male
SOD1G93A -, Cra1/+ -, Cra1/SODG93A -, P301L/+ mice and age-matched non-transgenic littermates at the
age of 3 months. The testing battery included experiments to measure general health, motor and cognitive
functions. For histological investigations, cryoslices of
the triceps and quadriceps were made and stained with
hematoxylin and eosin.
Results: Besides distinct motor deficits the present
results indicate also behavioural changes in Cra1/+ mice. In contrast to the SOD animals, these mice are
hyperactive and show coordination deficits. Memory
was normal in Cra1/+ -mice.
Discussion: The deficits in the behavioural tests cannot
be explained by an exclusive motor pattern of vulnerability. In contrast, we suggest that other regions of the
CNS are also affected by the disease process. To explain the complex deficits of Cra1/+ mice, further neuropathological studies are clearly warranted.
26