doi:10.1093/brain/awn071
Brain (2008), 131, 1540 ^1550
Cortical hyperexcitability may precede the onset of
familial amyotrophic lateral sclerosis
Steve Vucic,1 Garth A. Nicholson2 and Matthew C. Kiernan1
1
Prince of Wales Medical Research Institute and Prince of Wales Clinical School, University of New South Wales
and 2University of Sydney, Northcott Neurobiology Laboratory, ANZAC Research Institute, Sydney, New South Wales,
Australia
Correspondence to: Assoc. Prof. Matthew C. Kiernan, Prince of Wales Medical Research Institute, Barker Street, Randwick,
Sydney, NSW 2031, Australia
E-mail: [email protected]
Familial amyotrophic lateral sclerosis (FALS) is an inherited neurodegenerative disorder of the motor neurons.
While 10^15% of cases are caused by mutations in the copper/zinc superoxide-dismutase-1 (SOD-1) gene,
the dying-forward hypothesis, in which corticomotoneurons induce anterograde excitotoxic motoneuron degeneration, has been proposed as a potential mechanism. The present study applied novel threshold tracking
transcranial magnetic stimulation techniques to investigate the mechanisms underlying neurodegeneration in
FALS. Studies were undertaken in 14 asymptomatic and 3 pre-symptomatic SOD-1 mutation carriers, followed
longitudinally for up to 3 -years. The pre-symptomatic subjects were asymptomatic at the time of their initial
study but developed symptoms during the follow-up period. Results were compared to 7 SOD-1 FALS patients,
50 sporadic ALS patients and 55 normal controls. Short-interval intracortical inhibition (SICI) was
significantly reduced in SOD-1FALS (21.2 0.6%) and sporadic ALS patients (20.7 0.3%) compared to asymptomatic SOD-1mutation carriers (9.8 1.5%, P_0.00001) and normalcontrols (8.5 1.0%, P_0.00001). SICIreduction
was accompanied by increases in intracortical facilitation, motor evoked potential amplitudes and the slope of the
magnetic stimulus-response curve. In two pre-symptomatic SOD-1mutation carriers SICI was completely absent
(SICI patient 1, 23.2%; patients 2, 21.3%), while in one subject there was a 32% reduction in SICI prior to symptom
onset. These three individuals subsequently developed clinical features of ALS. Simultaneous investigation of
central and peripheral excitability has established that cortical hyperexcitability develops in clinically affected
SOD-1FALS patients, similar to that seen in sporadic ALS patients, thereby suggesting that a similar pathophysiological process in evident in both familial and sporadic ALS patients. In addition, the present study has established
that cortical hyperexcitability precedes the development of clinical symptoms in pre-symptomatic carriers of
the SOD1mutation, thereby suggesting that cortical hyperexcitability underlies neurodegeneration in FALS.
Keywords: familial ALS; cortical hyperexcitability; SOD-1
Abbreviations: ALSFRS-R = amyotrophic lateral sclerosis function rating scale revised; ANOVA = analysis of variance;
CMAP = compound muscle action potentials; CMCT = central motor conduction time; CSP = cortical silent period;
FALS = familial amyotrophic lateral sclerosis; ICF = intracortical facilitation; ISI = interstimulus interval; MEP = motor
evoked potential; MRC = Medical Research Council; NI = neurophysiological index; RMT = resting motor threshold;
SICI = short-interval intracortical inhibition; SOD-1 = copper/zinc superoxide-dismutase-1 gene; TMS = transcranial
magnetic stimulation
Received December 10, 2007. Revised March 12, 2008. Accepted March 25, 2008. Advance Access publication May 9, 2008
Introduction
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive
neurodegenerative disorder of the motor neurons in the
cortex, brainstem and spinal cord (Borasio and Miller,
2001; Rowland and Shneider, 2001). Degeneration of motor
neurons results in progressive paresis of limb, bulbar and
respiratory muscles leading to death typically 3–5 years after
symptom onset (Cudkowicz et al., 2004).
Ten percent of all ALS cases are familial (FALS), in
which two or more family members are clinically affected
(Andersen, 2006a). Autosomal dominant and recessive modes
of inheritance have been reported and eight genetic loci
ß The Author (2008). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
Cortical hyperexcitability in FALS
have been identified in FALS to date (Andersen, 2006a).
Mutations in one of these genes, the copper/zinc superoxidedismutase-1 gene (SOD-1), result in a typical ALS phenotype,
called ALS 1 (Rosen et al., 1993). The SOD-1 gene is located
on chromosome 21 q22.1, spans 11 kilobases of genomic DNA
and comprises 5 exons and 4 introns (Levanon et al., 1985).
To date over 122 different SOD-1 mutations have been
reported in FALS, the majority being missense mutations with
an autosomal-dominant mode of inheritance evident in most
mutations (Gros-Louis et al., 2006; Andersen, 2006a). The
penetrance in SOD-1 FALS, which refers to the proportion of
individuals with a particular genotype expressing the disease,
is age dependent, with 50% of patients expressing the
disease by age 43 years and 490% by 70 years (Cudkowicz
et al., 1997; Robberecht, 2002; Aggarwal and Nicholson,
2005). Only 17 mutations are associated with this high degree
of penetrance (Andersen, 2006a). Some mutations, such
as the I113T may be transmitted asymptomatically from
grandparents to grandchildren (Suthers et al., 1994; Jones
et al., 1995).
Mutations in the SOD-1 gene result in acquisition of
new cytotoxic activity by the SOD-1 protein, referred to
as a ‘toxic gain of function’ (Andersen, 2006b). Although
the mechanisms by which the SOD-1 protein results in
neurodegeneration remain as yet undefined, from the
transgenic SOD-1 mouse model there is increasing evidence
of glutamate-mediated excitotoxicity (Van Den Bosch et al.,
2006). Specifically, impaired glutamate clearance and
increased glutamate concentration within the cortex have
been reported in the SOD-1 (G93A) mice (Roy et al., 1998).
The presence of SOD-1 mutations also increases motor
neuron sensitivity to glutamatergic excitotoxicity by postsynaptic calcium-dependent mechanisms (Van Den Bosch
et al., 2006). Of further relevance, SOD-1 mutations may
result in mitochondrial dysfunction, further increasing the
susceptibility of motor neurons to glutamate-mediated cell
death (Hand and Rouleau, 2002).
Cortical hyperexcitability is mediated by glutamate, the
principle excitatory neurotransmitter in the brain (Hand and
Rouleau, 2002; Stys, 2005), which causes neurotoxicity by
activating Ca2+-dependent enzyme systems (Stys, 2005). Of
clinical relevance, cortical excitability may be assessed
by transcranial magnetic stimulation (TMS) techniques
(Kujirai et al., 1993; Vucic et al., 2006). TMS studies in
sporadic ALS patients, have provided evidence for increased
excitability early in the course of disease (Eisen et al., 1993;
Prout and Eisen, 1994; Mills and Nithi, 1997; Vucic and
Kiernan, 2006; Ziemann et al., 1997b). Conversely, in SOD-1
FALS patients the TMS findings have been contradictory, with
some reporting cortical hyperexcitability (Weber et al., 2000;
Turner et al., 2005b), while others have reported normal
cortical excitability (Stewart et al., 2006). This discrepancy
may be explained in part by marked variability in the motor
evoked potential (MEP) amplitude with consecutive stimuli
resulting from spontaneous fluctuations in the resting
threshold of cortical neurons (Kiers et al., 1993). In order to
Brain (2008), 131, 1540 ^1550
1541
clarify these discordant findings in FALS, and to further
investigate the site of disease onset in ALS, novel threshold
tracking TMS techniques were applied longitudinally in
asymptomatic SOD-1 mutation carriers with the results
compared to FALS patients manifesting the disease and to
sporadic ALS patients.
Methods
Patients
Subjects were recruited from the SOD-1 database at the Molecular
Genetic Laboratory, Concord Hospital and from the multidisciplinary motor neuron disease clinic at Prince of Wales Hospital (Sydney,
Australia). In total 17 asymptomatic and pre-symptomatic SOD-1
mutation carriers (7 males, 10 females: age range 23–60 years, mean
age: 40 years) were followed longitudinally for up 3 years (2.2 0.2
years), in addition to seven clinically definite familial ALS patients as
per the revised El Escorial criteria (Brooks et al., 2000) who also
expressed SOD-1 mutation (4 males, 3 females: age range 25–70
years, mean age 47 years). The pre-symptomatic subjects were also
asymptomatic at the time of their initial study but developed
symptoms during the follow-up period. Families with three different
SOD-1 mutations were studied including: valine to glycine mutation
in exon 5 at codon position 148 (V148G); isoleucine to threonine
mutation in exon 4 at codon position 113 (I113T); glutamic acid to
glycine mutation in exon 4 at codon position 100 (E100G). The
pattern of inheritance was autosomal dominant and all subjects were
heterozygous for the SOD-1 mutation. Fifty sporadic ALS patients
were also studied (32 males, 18 females: age range 26–78 years, mean
age 58 years).
ALS patients were clinically reviewed on a regular basis through
the multidisciplinary MND clinic at Prince of Wales Hospital.
This incorporated assessment using the amyotrophic lateral
sclerosis function rating scale revised (ALSFRS-R) (Cedarbaum
et al., 1999). Strength was assessed using the Medical Research
Council (MRC) rating scale (Medical Research Council, 1976),
and a separate hand function score was also recorded (Triggs
et al., 1999). Asymptomatic SOD-1 mutation carriers also underwent clinical examination at the time of formal neurophysiological assessment. The results of cortical excitability for familial
and sporadic ALS patients were compared to 55 healthy controls
(28 men; 27 women, aged 23–83 years, mean: 47 years). All
subjects gave informed consent to the procedures, which were
approved by the South East Sydney Area Health Service Human
Research Ethics Committee.
Peripheral nerve testing
The median nerve was stimulated electrically at the wrist in all
studies. The resultant compound muscle action potential (CMAP)
was recorded from the abductor pollicis brevis monolaterally in
most cases, and was measured from baseline to negative peak.
From peripheral nerve conduction studies, the neurophysiological
index (NI), a marker of ALS progression, was derived according to
a previously reported formula (de Carvalho and Swash, 2000):
NI ¼ CMAP amplitude ðmVÞ
F-wave frequency=distal motor latency ðmsÞ;
where F-wave frequency was expressed as the number of F
responses recorded in 20 trials.
1542
Brain (2008), 131, 1540 ^1550
Cortical excitability
Subsequently, cortical excitability was assessed by applying TMS
to the motor cortex by means of a 90 mm circular coil oriented to
induce current flow in a posterior–anterior direction. The coil was
adjusted until the optimal position for an evoked response was
obtained from the abductor pollicis brevis muscle monolaterally.
Currents were generated by two high-power magnetic stimulators
which were connected via a BiStim (Magstim Co., Whitland,
South West Wales, UK) such that conditioning and test stimuli
could be independently set and delivered through the one coil.
Threshold tracking paired-pulse TMS was performed according
to a previously reported method (Vucic et al., 2006). The threshold tracking strategy used a target response of 0.2 mV ( 20%),
located in the middle of the established linear relationship between
the logarithm of the MEP amplitude and the stimulus intensity
(Vucic et al., 2006). Resting motor threshold (RMT) was defined
as the stimulus intensity required to produce and maintain this
target MEP response.
Initially, the stimulus response curve for cortical stimulation was
determined by increasing the intensity of the magnetic stimulus to
the following levels: 60, 80, 90, 100, 110, 120, 130, 140 and 150%
RMT. Three stimuli were delivered at each level of stimulus intensity.
The maximum MEP amplitude (mV) and MEP onset latency (ms)
were recorded. Central motor conduction time (CMCT, ms) was
calculated according to the F-wave method (Cros et al., 1990).
Subsequently, the cortical silent period (CSP) induced by singlepulse TMS was recorded while ALS patients performed a voluntary
contraction, set to 30% of maximal voluntary contraction (Vucic
et al., 2006). The level of contraction was monitored by recording the
surface EMG of the abductor pollicis brevis. While performing the
task, patients were provided with visual feedback information. As for
the stimulus response curves, CSP duration curves were determined
by increasing the intensity of the magnetic stimulus to the following
levels: 60, 80, 90, 100, 110, 120, 130, 140 and 150% RMT. The CSP
duration was measured from the beginning of MEP to the return of
EMG activity (Cantello et al., 1992).
Short-interval intracortical inhibition (SICI) and intracortical
fascilitation (ICF) were determined by using subthreshold conditioning stimuli (70% RMT) at increasing interstimulus intervals
(ISIs) delivered in a fixed order as follows: 1, 1.5, 2, 2.5, 3, 3.5, 4,
5, 7, 10, 15, 20 and 30 ms (Vucic et al., 2006). Stimuli were
delivered sequentially as a series of three channels: channel 1
tracked the stimulus intensity required to produce the unconditioned test response (i.e. RMT); channel 2 monitored the response
to the subthreshold conditioning stimulus; and channel 3 tracked
the stimulus required to produce the target MEP when conditioned by a stimulus equal in intensity to that on channel 2.
Stimuli were delivered every 5–10 s and the computer advanced to
the next ISI only when tracking was stable.
SICI was measured as the increase in the test stimulus intensity
required to evoke the target MEP. Inhibition was calculated using
the following formula:
S. Vucic et al.
at 10 kHz using a 16-bit data acquisition card (National Instruments PCI-MIO-16E-4). Data acquisition and stimulation delivery
(both electrical and magnetic) were controlled by QTRACS software (ß Professor Hugh Bostock, Institute of Neurology, Queen
Square, London, UK).
Statistical analysis
Student’s t-test was used to compare mean differences between
groups for averaged SICI (1–7 ms), ICF (10–30 ms) and RMT.
Analysis of variance (ANOVA) was used for multiple comparisons
between magnetic stimulus response and CSP curves generated
by increasing the test stimulus intensity from 60% to 150% RMT.
A probability (P) value of 50.05 was considered statistically significant. Results are expressed as mean standard error of the mean.
Results
The clinical and genetic features for 17 asymptomatic SOD-1
mutation carriers and 7 clinically affected FALS patients are
summarized in Table 1. The asymptomatic SOD-1 mutation
carriers underwent annual TMS testing at which time they
were clinically reviewed by the same examiner. The physical
examination in all the asymptomatic SOD-1 mutation
carriers, including the three pre-symptomatic carriers was
normal. Specifically, there were no upper motor neuron
features, such as increased muscle tone, hyper-reflexia, extensor plantar responses, positive Hoffman sign or the presence
of a jaw-jerk in any of the subjects at the time of testing. Three
SOD-1 mutation carrier subjects were pre-symptomatic,
i.e. asymptomatic at the time of TMS, and their case histories
are outlined.
Case histories
Case 1 (Table 1)
Inhibition ¼ ðConditioned test stimulus intensity RMTÞ
100
RMT
A 43-year-old male who underwent initial threshold tracking
TMS in June 2005. At the time of testing, the subject was
asymptomatic and formal neurological examination was
normal. Furthermore, the CMAP amplitude (9.7 mV) and
neurophysiological index (2.2) were within normal limits.
Although surface EMG, including testing over the target
muscle was unremarkable, formal needle EMG testing was
not undertaken at time of cortical excitability testing. In
September of 2005, this subject developed fasciculations,
muscle cramps and weakness of the right foot and hip. Subsequently, he developed wasting of the muscles in the right leg,
including calf, anterior leg and thigh muscles. By December
2006, he developed fasciculations in the proximal upper limb
muscles along with weakness of the shoulder girdle muscles.
Neurophysiological testing in September 2005 established
features of denervation in the lower limbs accompanied by
fasciculations.
Facilitation was measured as the decrease in the conditioned test
stimulus intensity required to evoke a target MEP.
Recordings of CMAP and MEP were amplified and filtered
(3 Hz–3 kHz) using a GRASS ICP511 AC amplifier (GrassTelefactor, Astro-Med Inc., West Warwick, RI, USA) and sampled
A 34-year-old male who underwent threshold tracking TMS
testing in June 2006. Three months later, he reported
Case 2 (Table 1)
Cortical hyperexcitability in FALS
Brain (2008), 131, 1540 ^1550
1543
Table 1 Clinical details for the 17 asymptomatic superoxide dismutase-1 (SOD-1) mutation carriers and 7 clinically affected
familial amyotrophic lateral sclerosis (FALS) with SOD-1 mutation
Asymptomatic
SOD-1
Age (years)/
Sex
SOD-1
mutation
Follow-up
period (years)
ALS
onset
1a
2a
3a
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Mean
SEM
43/M
34/M
41/F
25/F
60/F
47/F
37/M
25/F
44/F
43/F
49/M
23/F
45/F
57/M
31/M
54M
31/F
40
3.1
V148G
E100G
V148G
V148G
I113T
V148G
V148G
I113T
V148G
V148G
V148G
V148G
E100G
I113T
V148G
I113T
V148G
3
2
3
3
1
3
3
3
3
3
1
2
1
2
3
2
1
2.2
0.2
UL, LL
UL, LL
UL, LL
Symptomatic
SOD1 patients
Age (years)/
Sex
SOD-1
mutation
ALS
onset
Disease
duration
(months)
I113T
V148B
E100G
V148G
I113T
V148G
V148G
UL
LL
LL
LL
UL
LL
LL
9
33
14
6
7
13
1.5
11.9
3.9
21.6
3.4
18
70/F
19
35/M
20
49/F
21
44/M
22
53/M
23
50/M
24b
25/F
Mean
47
SEM
5.4
Sporadic ALS patients (N = 50)
Mean
58.7
SEM
1.8
Time to
ALS onset
(months)
3
3
8
4.7
1.7
ALSFRS-R
Triggs
hand
score
MRC
(46) 48
(47) 48
(44) 48
48
48
48
48
48
48
48
48
48
48
48
48
48
48
48
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
0
ALSFRS-R
Triggs
hand
score
MRC
41
42
37
45
46
38
48
42.4
1.6
2
0
1
0
1
0
0
0.6
0.3
4
4
4
5
4
5
5
4.4
0.2
38.2
0.9
1.2
0.1
4.2
0.1
a
Although the ALSFRS-R scores in three pre-symptomatic SOD-1 mutation carriers were normal at the time of cortical excitability testing,
the ALSFRS-R reduced with the onset of clinical features of ALS some time after cortical excitability testing (ALSFRS-R for case#1, 46;
case#2, 47; case#3, 44). In addition, for case 3, the MRC score was normal at the time of cortical excitability testing in May 2005 and
April 2006. However, at last testing, in 2007, the MRC score was reduced as measured from the weak left abductor pollicis brevis muscle
(see case history #3 in Results). bPatient 24 reported difficulty with running and endurance training. Physical examination disclosed a
generalized hyperreflexia and neurophysiological testing revealed neurogenic changes, consistent with ALS. Data for 50 sporadic ALS
patients (32 males and 18 females) is also presented for comparison. Twenty-nine ALS patients reported upper limb onset, 12 lower limb,
8 bulbar and 1 respiratory onset of disease.
Three different SOD-1 mutations were present in the patients studied; GTA to GGA sequence change resulting in valine to glycine
substitution in exon 5 at the 148 position (V148G), GAA to GGA sequence change resulting in a glutamic acid to glycine substitution in
exon 4 at the 100 position (E100G), and ATT to ACT sequence change resulting in an isoleucine to threonine substitution in exon 4 at the
113 position (I113T). The follow-up period refers to the time period between first assessment at time of cortical excitability testing to last
review, measured in years. The site of disease onset was classified as either upper limb (UL), lower limb (LL) or bulbar (B). Disease duration
refers to the period from symptom onset to date of testing, while time to ALS onset refers to time to development of clinical features
of ALS after the last testing. The patients were clinically graded using the amyotrophic lateral sclerosis functional rating scale revised
(ALSFRS-R), with a maximum score of 48 when there is no disability. Muscle strength was clinically assessed using the Medical Research
Council (MRC) for the abductor pollicis brevis, as this muscle was utilized for excitability testing.
generalized fasciculations and difficulty running. Although
the neurological examination was normal at the time of TMS
testing, re-assessment at the time of symptom onset disclosed
widespread fasciculations with a generalized hyperreflexia.
At the time of cortical excitability testing, the CMAP amplitude (9.1 mV) and NI (2.3) were unremarkable. Formal needle
EMG testing was not undertaken at the time of cortical
excitability testing. However, subsequent needle EMG testing
1544
Brain (2008), 131, 1540 ^1550
S. Vucic et al.
disclosed fasciculations of complex morphology, with associated neurogenic abnormalities.
Case 3 (Table 1)
A 41-year-old female who underwent TMS testing in May
2005 and April 2006. Peripheral neurophysiological testing,
in the form of CMAP amplitude and NI, in May 2005
(CMAP amplitude, 13 mV; NI, 3.3) and April 2006 (CMAP
amplitude, 12.8 mV; NI, 3.0) were normal, as was surface
EMG sampling. In December 2006, the subject reported
lower-limb weakness manifesting as difficulty with walking
and standing on her toes. By late January 2007, the subject
developed exertional dyspnoea, left-hand weakness (MRC
grade 4, see Table 1) and generalized fasciculations. Neurophysiological testing, nerve conduction studies and needle
electromyography (EMG), disclosed widespread neurogenic changes consistent with ALS. The subject underwent
further TMS testing in April 2007. Currently, the patient is
wheelchair-dependent, with global weakness, and requires
nocturnal pressure support ventilation. The SOD-1 mutation (V148G) in this family results in an aggressive ALS
phenotype with median survival from disease onset being
typically 512 months.
Peripheral nerve studies
The CMAP amplitude (symptomatic FALS 6.1 1.6 mV;
sporadic ALS 5.9 0.6 mV; controls 10.3 0.5 mV,
P50.001) and neurophysiological index (symptomatic
FALS 1.3 0.4; sporadic ALS, 0.8 0.1; controls 2.6 0.2,
P50.005), both quantitative markers of peripheral disease
burden, were significantly reduced in symptomatic FALS and
sporadic ALS patients compared to controls. There were no
differences in the CMAP amplitude (asymptomatic SOD-1
mutation carriers, 9.9 0.8 mV; controls 10.3 0.5 mV) and
neurophysiological index (asymptomatic SOD-1 mutation
carriers, 2.3 0.3; controls 2.6 0.2) between asymptomatic SOD-1 mutation carriers and normal controls.
Although the MEP onset latency was longer in FALS and
sporadic ALS patients (symptomatic FALS 22.6 1.1 ms;
ALS 22.1 0.3 ms; asymptomatic SOD-1 mutation carriers
20.9 0.7 ms; controls 20.4 0.3 ms, P50.01), there was
no significant difference in the CMCT between FALS and
sporadic ALS patients compared to asymptomatic SOD-1
mutation carriers and controls (symptomatic FALS
5.0 0.5 ms; ALS 5.1 0.3 ms; asymptomatic SOD-1 mutation carriers 5.2 0.4 ms; controls 5.1 0.2 ms). The MEP
onset latency in the three pre-symptomatic subjects (case #1,
19.2 ms; case #2, 20.3 ms; case #3, 20.3 ms) and the CMCT
(case #1, 5.7 ms; case #2, 5.3 ms; case #3, 5.3 ms) were
unremarkable.
Cortical excitability
The full sequence of cortical excitability was well tolerated and successfully completed in all asymptomatic
SOD-1 mutation carriers, FALS and sporadic ALS patients.
Fig. 1 (A) SICI, defined as the stimulus intensity required to
maintain a target output of 0.2 mV (see ‘Methods’ section) was
reduced in clinically affected familial amyotrophic lateral sclerosis
(FALS) and sporadic ALS patients when compared to normal
controls (P50.001). (B) Averaged SICI, between interstimulus
interval (ISI) 1^7 ms was also reduced in FALS and sporadic ALS
patients compared to asymptomatic superoxide dismutase-1
(SOD-1) mutation carriers and controls (P50.001). The error bars
represent standard error of the mean. The asymptomatic SOD-1
mutation carrier data point does not include values from
pre-symptomatic patients.
Short-interval intracortical inhibition, as reflected by an
increase in the conditioned stimulus intensity required
to track a constant target MEP of 0.2 mV, was significantly
reduced in symptomatic FALS (1.2 0.6%) and sporadic
ALS (0.8 1.2%) patients compared to asymptomatic
SOD-1 mutation carriers (9.8 1.5%, N = 14) and controls
(8.5 1.1%, P50.001, Fig. 1A and B). In two pre-symptomatic SOD-1 mutation carriers who developed clinical
features of ALS within 3-months of assessment of cortical
excitability, SICI was absent (averaged SIC 1–7 ms case #1,
3.2%; case #2, 1.3%, Table 1, Fig. 2), in sharp contrast
to the remaining asymptomatic SOD-1 mutation carriers
(Fig. 2). In addition, peak SICI at ISIs 51 and 3 ms were
reduced in these two pre-symptomatic SOD-1 mutation
carriers (case#1 SICI ISI 51 ms = 4.8%, ISI 3 ms = 3.8%;
case #2 SICI ISI 51 ms = 8.4%, ISI 3 ms = 1.4%) when
compared to asymptomatic SOD-1 mutation carriers (SICI
ISI 51 ms = 6.8 0.7%, ISI 3 ms = 11.8 0.6%) and controls
(SICI ISI 51 ms = 7.0 0.9%, ISI 3 ms = 13.7 1.3%) In the
third pre-symptomatic SOD-1 mutation carrier (case #3),
there was a 32% reduction in averaged SICI prior to the
development of clinical features of ALS and SICI was reduced
in the motor cortex contralateral to side of development
Cortical hyperexcitability in FALS
Brain (2008), 131, 1540 ^1550
1545
Fig. 3 Averaged SICI is depicted for three pre-symptomatic
subjects (case #1^3, from Table 1) and 14 asymptomatic superoxide
dismutase-1 (SOD-1) mutation carriers (subjects 4 ^17, Table 1).
Averaged SICI was absent in two pre-symptomatic subjects, but
was present in all the asymptomatic SOD-1 mutation carriers.
The numbers on the x-axis, correspond to the number ascribed to
each subject in Table 1. Although subject #3 appeared to have
normal SICI, there was reduction of 32% between studies 1 and 2,
as depicted in Fig. 2A, suggesting that a relative reduction in
SICI may herald cortical hyperexcitability.
Fig. 2 (A) Averaged SICI, between 1 and 7 ms was reduced in a
pre-symptomatic SOD-1 mutation carrier (case #3, see Table 1)
prior to the development of ALS. SICI in the follow-up studies is
expressed as a fraction of the SICI value measured at the first
study, that is the SICI values were normalized. (B) In case #3,
SICI was reduced in the right motor cortex contralateral to
the side of muscle weakness. Stimulation of both cortices was
undertaken in only one subject (case #3) and performed at the
time of study 3 in 2007. (C) Biopsy of the left vastus lateralis
muscle, obtained in March 2007 and stained with ATPase pH 9.4.
Type I fibres are light and type II fibres are dark. The biopsy
demonstrates grouped atrophic fibres with both type I and type II
fibres (mixed-type fibres, illustrated by red arrow,
magnification 10). The asymptomatic SOD-1 mutation carrier
data point does not include values from case #1^3. The error
bars represent standard error of the mean.
of muscle weakness (Fig. 3A and B). In addition, there
was a marked reduction in SICI at ISI 51 (study 1 SICI
51 ms = 4.5%, study 2 SICI 51 ms = 0%, study 3 SICI
51 ms = 0%) and at ISI 3 ms (study 1 SICI 3 ms = 18.6%,
study 2 SICI 3 ms = 8.7%, study 3 SICI 3 ms = 6.7%) prior to
development of symptoms. As part of a subsequent diagnostic
work-up, this subject underwent a muscle biopsy which
revealed the presence of groups of atrophic muscle fibres with
mixed-type fibres, a finding distinctive of motor neuron
disease (Fig. 3C) (Baloh et al., 2007). In contrast to these three
subjects, in the remaining asymptomatic SOD-1 mutation
carriers, there was a non-significant increase of SICI over the
follow-up period (averaged SICI year 2, 11.0 1.5%, P = 0.3,
N = 10; year 3, 11.9 2.7%, P = 0.24, N = 7).
Short-interval intracortical inhibition is followed by a
period of ICF marked by a decrease in the test stimulus
intensity required to maintain the target MEP of 0.2 mV.
ICF was increased in symptomatic FALS (2.5 0.7%)
and sporadic ALS patients (3.0 1.2%) when compared
to asymptomatic SOD-1 mutation carriers (0.1 0.5%)
and controls (1 0.3%) over a period of testing from
10 to 30 ms (Fig. 4A, P50.05). ICF increased in two presymptomatic SOD-1 mutation carriers prior to the development of ALS (case #1, 9.3%; case #2, 6.1%) compared
to remaining asymptomatic SOD-1 mutation carriers and
controls (Fig. 4B). In the third pre-symptomatic SOD-1
mutation carrier (case #3), ICF again increased in the
motor cortex contralateral to the side of muscle weakness
(Fig. 4C and D).
Another measure of cortical excitability, the MEP amplitude which is expressed as a percentage of the CMAP
response, reflects the density of corticospinal projection onto
the anterior horn cells. The MEP amplitude was increased
in both clinically affected FALS patients and sporadic
ALS patients compared to asymptomatic SOD-1 mutation
carriers and controls (symptomatic FALS 42.8 13.0%;
ALS 37.7 4.5%; asymptomatic SOD-1 mutation carriers
20.6 4.2%; controls 26.2 2.4%, P50.05). Interestingly, in
the three pre-symptomatic subjects, the MEP amplitude was
not increased (case #1, 18.8%; case #2, 25%; case #3, 13%).
1546
Brain (2008), 131, 1540 ^1550
S. Vucic et al.
Fig. 4 (A) Intracortical facilitation (ICF), which develops at interstimulus intervals (ISI) of 10 ^30 ms, was increased in clinically affected
familial amyotrophic lateral sclerosis (FALS) and sporadic amyotrophic lateral sclerosis (SALS) patients compared to asymptomatic SOD-1
mutation carriers and controls. (B) In two pre-symptomatic superoxide dismutase-1 (SOD-1) mutation carriers (case #1 and #2),
ICF was increased compared to the remaining asymptomatic superoxide dismutase-1 (SOD-1) mutation carriers and controls prior to
development of clinical features of ALS. (C) In a further pre-symptomatic SOD-1 mutation carrier (case #3), ICF increased prior to
the development of ALS. (D) Intracortical facilitation was increased in the right motor cortex, which was contralateral to the side of
development of muscle weakness. Stimulation of both cortices was undertaken in only one subject (case #3). The error bars represent
standard error of the mean.
The absolute MEP amplitude in clinically affected FALS
was 1.5 0.3 mV, in sporadic ALS 2.1 0.3 mV, asymptomatic SOD-1 mutation carriers 2.1 0.6 mV and controls
2.6 0.7 mV. In the three pre-symptomatic subjects the
absolute MEP amplitude was as follows: case #1, 1.8 mV;
case #2, 0.5 mV; case #3, 1.6 mV. The slope of the tangent
for the steepest point of the stimulus-response curve was
greater in symptomatic FALS patients compared to asymptomatic SOD-1 mutation carriers and controls (ANOVA
P50.05, Fig. 5).
RMT, defined as the unconditioned stimulus intensity
required to produce and maintain a target MEP response, was
not significantly different between the groups (symptomatic
FALS 59.2 5.1%; sporadic ALS 57.9 1.6%; asymptomatic SOD-1 mutation carriers, 59.4 2.2%; controls,
60.6 1.3%). However, the RMT was significantly reduced
in sporadic ALS patients with limb-onset disease (sporadic
ALS limb-onset 56.1 1.7%; controls, 60.6 1.3%, P50.05).
Furthermore, the RMT in one pre-symptomatic patient was
reduced when compared with normal controls (case #1, 56%,
95% confidence interval 58–63.2%), while it was at the
lower limit of normal in a second pre-symptomatic patient
(case #2, 58%). In the third pre-symptomatic patient the RMT
Fig. 5 The motor evoked potentials (MEP) amplitude and the
gradient of the stimulus-response curve was increased in clinically
affected familial amyotrophic lateral sclerosis (FALS) and sporadic
ALS (SALS) patients when compared to asymptomatic superoxide
dismutase-1 (SOD-1) mutation carriers and normal controls
(ANOVA, P50.05). The error bars represent standard error of
the mean.
was comparable to that evident in normal controls
(case #3, 60%).
Changes in the CSP, defined as a period of electrical silence
that interferes with ongoing EMG activity following an MEP
Cortical hyperexcitability in FALS
in a contracting muscle, was also measured. In the present
series, the increase in the CSP recruitment curve were nonlinear in FALS patients similar to the relationship established
previously for control subjects (Vucic et al., 2006). The mean
CSP duration increased from 0 to 199.6 6.2 ms in affected
FALS and from 0 to 208.9 4.2 ms in asymptomatic SOD-1
mutation carriers as stimulus intensity increased from 60 to
150% RMT, and was not significantly different when compared to controls (0 to 209.0 4.7 ms). However in three
pre-symptomatic SOD-1 mutation carriers, CSP duration was
reduced compared to controls and other asymptomatic
SOD-1 mutation carriers, but comparable to sporadic ALS
patients, prior to the development of clinical features of ALS
(case #1 CSP duration, 0–180 ms; case #2, 0–192 ms; case #3,
0–141 ms; sporadic ALS 0–177 8.5 ms, 95% confidence
interval normal controls 199.6–218.4 ms). In sporadic ALS
patients, the mean CSP duration increased from 0 to
176.7 8.5 ms, and was significantly reduced when compared
to controls (P50.01).
Discussion
The present longitudinal studies utilize novel threshold
tracking TMS techniques to explore cortical excitability
in familial SOD-1 positive ALS and sporadic ALS patients,
and have established the presence of cortical hyperexcitability in both symptomatic FALS and sporadic ALS
patients. Reduction in short-interval intracortical inhibition, associated with increases in intracortical facilitation and cortical stimulus-response curve gradient were
all indicative of cortical hyperexcitability. In contrast, in
asymptomatic SOD-1 mutation carriers cortical excitability
was normal. However, an increase in cortical excitability in
three pre-symptomatic SOD-1 mutation carriers preceded
the development of clinical features of ALS. Together, these
findings confirm that cortical hyperexcitability develops
prior to clinical symptoms in FALS.
Where does ALS begin?
Although the findings in the present study do not directly
support a pathogenic mechanism in ALS, namely excitotoxicity, they are compatible with the ‘dying forward’ hypothesis
that proposes that the initial abnormality in ALS occurs
within the corticomotorneurons or the motor cortex, with
anterograde excitotoxicity underlying subsequent anterior
horn cell degeneration. However, given that formal needle
EMG was not performed at the time of cortical excitability
studies, it remains possible that subtle, undetected changes
in anterior horn cells may have preceded or concurrently
developed with cortical hyperexcitability. The findings in the
present study, however, are in keeping with flumazenil PET
studies that demonstrated cortical abnormalities in two presymptomatic SOD1 patients (Turner et al., 2005a), and with
studies in a SOD-1 (G93A) mouse model suggesting that
degeneration of spinal cord motor neurons is secondary
Brain (2008), 131, 1540 ^1550
1547
to dysfunction within CNS motor pathways, i.e. within the
primary motor cortex, corticospinal tract and bulbospinal
pathways (Browne et al., 2006).
Evidence for cortical hyperexcitability in the present study
is provided by a significant reduction of SICI in both clinically
affected SOD-1 and sporadic ALS patients, and in longitudinal studies in three pre-symptomatic SOD-1 mutation
carriers who developed ALS within months of developing
cortical hyperexcitability. Interestingly, although case #3 exhibited SICI prior to and at the time of development of ALS,
there was a marked reduction of SICI 8 months prior to
development of ALS, illustrating the point that a relative
reduction in SICI may be indicative of the development of
cortical hyperexcitability. The mechanisms underlying reduction of SICI are complex and involve both degeneration
of GABA secreting inhibitory cortical interneurons, both in
the primary motor cortex and extramotor cortical areas, and
glutamate-mediated down-regulation of SICI (Nihei et al.,
1993; Stefan et al., 2001; Maekawa et al., 2004). In addition,
dysfunction of GABAergic synapses, whereby they become
excitatory, may be another mechanism underlying the reduction of SICI (Ben-Ari et al., 2004). Specifically, in the neonatal
brain, GABAergic synapses are excitatory. Conversion from
excitatory to inhibitory transmission is dependent on the
establishment of the chloride electrochemical gradient by
expression of a specific K+/Cl– co transporter (KCC2) on
post-synaptic membranes (Ben-Ari et al., 2004). Studies
assessing the function of KCC2 in FALS patients may yet
confirm this potential mechanism of SICI reduction.
Although the reduction of SICI in the present study was
interpreted as reflecting cortical hyperexcitability, other
interpretations remain possible. Given the fact that SICI is
reduced in other neurological and psychiatric disorders
(Ridding et al., 1995a, b; Abbruzzese et al., 1997; Chen
et al., 1997; Ziemann et al., 1997a, b; Hanajima et al., 1998;
Fitzgerald et al., 2002; Zanette et al., 2002; Rosenkranz
et al., 2005) it could be interpreted that SICI reduction
reflects a compensatory phenomenon related to brain injury
or a ‘bystander’ phenomena.
Alternatively, it may continue to be valid to interpret the
reduction of SICI in other neurological and psychiatric
disorders as cortical hyperexcitability, with the absence
of motor neuron degeneration in these other conditions
explained by a greater resistance of the motor neurons to the
stresses of cortical hyperexcitability. Specifically, a number
of cell-specific molecular features of motor neurons in ALS
patients render them vulnerable to cortical hyperexcitability,
mediated via glutamate excitotoxicity. First, motor neurons in
ALS patients preferentially express an AMPA receptor lacking
the functional GluR2 subunit, thereby rendering the motor
neurons more permeable to Ca2+ and thereby to activation of
Ca2+-dependent enzymatic pathways that mediate neuronal
death (Van Damme et al., 2002, 2005; Kawahara et al., 2004;
Kwak and Kawahara, 2005). Secondly, motor neurons vulnerable to degeneration lack the intracellular expression of Ca2+binding proteins parvalbumin and calbindin D28k, required
1548
Brain (2008), 131, 1540 ^1550
to buffer intracellular Ca2+ (Ince et al., 1993; Alexianu
et al., 1994). More recently, increased expression of the
inositol 1,4,5-triphosphate receptor 2 (ITPR2) gene was
reported in ALS (van Es et al., 2007). ITPR2 is involved in
glutamate-mediated neurotransmission, whereby stimulation of glutamate receptors results in binding of inositol
1,4,5-triphosphate to ITPR2, which subsequently increases
intracellular calcium (Choe and Ehrlich, 2006; van Es et al.,
2007). Aberrant activity of ITPR2 results in higher intracellular concentration of Ca2+ and ultimately apoptosis
(Gutstein and Marks, 1997).
Another possible explanation for the SICI findings in the
pre-symptomatic SOD-1 mutation carriers is random fluctuation with a regression to the mean. Although the findings depicted in Fig. 3 would seem to argue against random
fluctuations, a larger cohort of pre-symptomatic SOD-1
mutation carriers would be required to definitively exclude
this possibility.
Although there was no significant reduction of SICI in
asymptomatic SOD-1 mutation carriers when compared
to controls, it remains possible that subtle abnormalities
of SICI may have gone undetected. Specifically, given that
the expression of SICI is stronger with higher test
stimulus intensities which generate larger MEP amplitudes
(Daskalakis et al., 2002), possibly due to recruitment of
later I-waves that are susceptible to inhibition by the
conditioning stimulus (Di Lazzaro et al., 1998), it remains
possible that selecting a tracking target of 0.2 mV may
have been suboptimal for identifying subtle deficient SICI
in the asymptomatic SOD-1 mutation carriers. In addition,
measurement of SICI using a conditioning stimulus intensity of 70% RMT, which usually results in maximum SICI
(Kujirai et al., 1993), may have resulted in saturated inhibition (floor effect). In order to detect subtle reduction of
SICI, lower conditioning stimuli intensities may be required
(MacKinnon et al., 2005). Given the fact that conditioning
stimulus intensity and the tracking target MEP amplitude
were not varied in the present study, subtle deficiencies
of SICI in asymtpomatic SOD-1 mutation carriers may
have passed undetected. Interestingly, in two asymptomatic SOD-1 mutation carriers (case #6 and case#10), who
continue to remain asymptomatic, SICI appeared to be
reduced. While this finding may raise issues regarding the
specificity of the link between cortical hyperexcitability and
neurodegeneration, longer-term follow-up will be essential
to clarify whether these subjects develop the clinical features
of ALS.
Previous cortical excitability studies in familial SOD-1
positive ALS patients have reported contradictory findings
(Weber et al., 2000; Turner et al., 2005b; Stewart et al.,
2006). While normal cortical excitability was reported in
patients with the D90A (Weber et al., 2000; Turner et al.,
2005b) and I113T mutations (Stewart et al., 2006), abnormalities of corticomotoneuronal function have been
recently reported in patients with the A4V mutations
(Stewart et al., 2006). The present findings establish the
S. Vucic et al.
presence of cortical hyperexcitability in patients with the
V148G, E100G and I113T SOD-1 mutations, in keeping
with the findings in the A4V mutation. The homozygous
D90A mutation is associated with a slowly progressive
phenotype, probably related to co-inheritance of protective
or modifying genes with the D90A alleles (Simpson and
Al-Chalabi, 2006). The inheritance of these protective
or modifying genes may have been responsible for normal
cortical excitability in the D90A SOD-1 positive patients.
In addition to reduction of SICI, ICF was increased in
clinically affected FALS, sporadic ALS and the three presymptomatic FALS subjects. The mechanisms underlying this
increase of ICF have not been elucidated. Studies describing
the relationship of descending corticospinal tract volleys to
ICF have established that the conditioning stimulus does not
alter the I-wave amplitude or number in the descending
corticospinal tract volley (Di Lazzaro et al., 2006). Rather,
it was postulated that ICF occurred as a result of either (i) the
conditioning stimulus increasing excitability at the spinal cord
level, such that motoneurons are more likely to be activated by
a descending corticospinal volley or (ii) the conditioning
stimulus altering the composition of the descending corticospinal volley such that a greater proportion of descending
corticospinal activity was destined for the target motor
neurons. Either of these two mechanisms could have been
responsible for the increased ICF in evident in the present
series. Interestingly the ICF in normal controls, was less than
that established by the constant stimulus method (Kujirai
et al., 1993). The reasons for this discrepancy remain unclear,
and may be related to technical factors, including less
variability in the MEP amplitude with the threshold tracking
technique.
In conjunction with reduction of SICI and increase in
ICF, the resting motor threshold was reduced in sporadic
ALS patients, although this reduction was only significant
in patients with limb onset-disease, as previously established (Vucic and Kiernan, 2006). Interestingly, although
the RMT was reduced in clinically affected FALS patients,
this reduction was not significant. While it remains possible
that cortical hyperexcitability is less prominent in clinically
affected FALS patients, more likely the lack of a significant
difference may reflect the smaller sample size in the
clinically affected FALS group.
In conclusion, the results from the present study complement previous findings in sporadic ALS patients, where
cortical hyperexcitability developed as an early feature (Eisen
and Weber, 2000; Vucic and Kiernan, 2006). Ultimately,
if earlier cortical abnormalities could be reliably identified,
institution of neuroprotective agents at that stage, including
anti-glutamatergic agents such as riluzole, may result in
a slower disease progression and prolonged survival.
Acknowledgements
S.V. was awarded the Clinical Fellowship of Motor Neuron
Disease Research Institute of Australia, with funding
Cortical hyperexcitability in FALS
provided by the Motor Neuron Disease Association of
NSW. Grant support was also received from the NSW
Ministry for Science and Medical Research Spinal Cord
Injury & Related Neurological Conditions Research Grant
Program and from the National Health and Medical
Research Council of Australia. The authors gratefully
acknowledge the assistance of Roger Pamphlett with the
muscle biopsy findings. Input from Professor Hugh
Bostock, particularly with threshold tracking software, is
again gratefully acknowledged.
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