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Corticomotoneuronal function in asymptomatic SOD-1 mutation carriers
Clinical Neurophysiology 121 (2010) 1781–1785
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Clinical Neurophysiology journal homepage: www.elsevier.com/locate/clinph
Corticomotoneuronal function in asymptomatic SOD-1 mutation carriers Steve Vucic a,e, Jennica M.C. Winhammar b,c,e, Dominic B. Rowe b,c,d, Matthew C. Kiernan e,* a
Department of Neurology, Westmead Hospital, Western Clinical School, University of Sydney, Australia Department of Neurology, Royal North Shore Hospital, Sydney, NSW, Australia c Northern Clinical School, University of Sydney, Australia d Australian School of Advanced Medicine, Innovation Road, Macquarie University, Australia e Prince of Wales Medical Research Institute and Prince of Wales Clinical School, University of New South Wales, Australia b
See Editorial, pages 1709–1710
a r t i c l e
i n f o
Article history: Accepted 16 February 2010 Available online 1 April 2010 Keywords: Diffusion tensor tractography Familial ALS SOD-1 mutation carrier
a b s t r a c t Objective: Diffusion tensor imaging (DTI) recently identified structural abnormalities of corticomotoneurons in asymptomatic copper/zinc superoxide-dismutase-1 (SOD-1) gene mutation carriers. The potential existence of longstanding corticomotoneuronal dysfunction would clearly have consequences for the medical management of asymptomatic SOD-1 mutation carriers. To clarify this unexpected finding, DTI techniques were combined with threshold tracking transcranial magnetic stimulation (TMS) to assess the anatomical and functional integrity of corticomotoneurons in asymptomatic SOD-1 mutation carriers. Methods: TMS studies were undertaken using a 90 mm circular coil on seven asymptomatic SOD-1 mutation carriers and results were compared to 62 healthy controls. DTI studies were carried out using a 3 T magnetic resonance device in the same asymptomatic SOD-1 mutation carriers. Results were compared to age-matched healthy controls. Results: In contrast to previous findings, there were no significant differences in fractional anisotropy (SOD-1 mutation carriers, 0.62 ± 0.01; controls, 0.61 ± 0.02, P = 0.2) and trace apparent diffusion coefficient (SOD-1 mutation carriers, 0.003 ± 0.0001; controls, 0.003 ± 0.0001) in asymptomatic SOD-1 mutation carriers. Of further relevance, there were no significant differences in short-interval intracortical inhibition (SOD-1 mutation carriers, 7.9 ± 3.4%; controls, 8.5 ± 1.1%, P = 0.26), intracortical facilitation (P = 0.5), MEP amplitude (P = 0.44), resting motor threshold (P = 0.36) and cortical silent period duration (P = 0.29). Conclusions: Combined anatomical and functional modalities established normal integrity of corticomotoneurons in asymptomatic SOD-1 mutation carrier subjects. Significance: Additional factors other than simply SOD-1 mutation expression are required to trigger cortical hyperexcitability and neurodegeneration in FALS. Ó 2010 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder of the motor neurons in the motor cortex, brain stem and spinal cord, with median survival of 3–5 years (Winhammar et al., 2005). Ten percent of ALS cases are familial (FALS), with mutations in the copper/zinc superoxide-dismutase1 gene (SOD-1) the most frequent cause (Rosen et al., 1993). The pathophysiological processes underlying ALS have been linked to
* Corresponding author. Address: Prince of Wales Medical Research Institute, Barker Street, Randwick, Sydney, NSW 2031, Australia. Tel.: +61 2 9382 2422; fax: +61 2 9382 2437. E-mail address: [email protected] (M.C. Kiernan).
a complex interaction between genetic factors and molecular pathways including oxidative stress, glutamate excitotoxicity, mitochondrial dysfunction, axonal transport systems, and glial cell dysfunction with resultant damage of critical target proteins and organelles within the motor neuron, thereby underlying neurodegeneration (Vucic and Kiernan, 2009). Cortical hyperexcitability and excitotoxicity have been linked to the process of motor neuron degeneration (Boillee et al., 2006; Bruijn et al., 2004; Eisen et al., 1992; Shaw and Kuncl, 2002; Shaw, 2005), with further support provided by pharmacological studies that have established the clinical effectiveness of riluzole, an inhibitor of glutamate release, in ALS (Bensimon et al., 1994; Kiernan, 2005; Lacomblez et al., 1996; Zoing et al., 2006). In addition, transcranial magnetic stimulation (TMS) studies have identified that
1388-2457/$36.00 Ó 2010 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2010.02.164
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cortical hyperexcitability appears as an early feature of ALS, preceding the development of clinical weakness in familial ALS (Desiato et al., 2002; Eisen et al., 1993; Mills and Nithi, 1997; Prout and Eisen, 1994; Vucic and Kiernan, 2006; Vucic et al., 2008). The presence of cortical hyperexcitability in sporadic ALS and familial ALS patients was identified by establishing the presence of reduced short-interval intracortical inhibition, resting motor threshold and cortical silent period duration, as well as increased motor evoked potential amplitude and intracortical facilitation. Identifying the presence of corticomotoneuronal degeneration in asymptomatic SOD-1 mutation carriers would clearly be of therapeutic significance. Diffusion tensor imaging (DTI) is a magnetic resonance imaging (MRI) technique capable of assessing the anatomical integrity of corticomotoneurons by quantifying the strength (trace apparent diffusion coefficient, trace ADC) and direction (fractional anisotropy, FA) of diffusion of water molecules within white matter tracts (Alexander et al., 2007). In normal brain, the diffusion of water is restricted by the spatial organization of axons, such that subcortical white matter tracts of the internal capsule have a higher anisotropy (Alexander et al., 2007). Of relevance, degeneration of axons within white matter tracts, as may be evident in manifesting ALS patients, has been associated with abnormalities of diffusion, resulting in increased trace ADC and reduced FA (Abe et al., 2004; Ellis et al., 1999; Graham et al., 2004; Sach et al., 2004; Toosy et al., 2003). Recently, DTI techniques identified the presence of increased trace ADC and reduced FA within the posterior limb of the internal capsule in asymptomatic SOD-1 mutation carriers (Ng et al., 2008), thereby suggesting that a loss of corticomotoneurons may precede the clinical onset of weakness in familial ALS. The existence of longstanding structural and functional abnormalities would have critically important consequences for the medical management of asymptomatic SOD-1 mutation carriers. In the least, such a finding would suggest that affected individuals should be commenced on neuroprotective agents, such as riluzole, prior to clinical disease onset. To consider this important issue, the present study used a novel combination of DTI and threshold tracking TMS techniques in asymptomatic SOD-1 mutation carriers to assess corticomotoneuronal integrity and function.
(Table 1). Asymptomatic SOD-1 mutation carriers underwent clinical examination and staging with amyotrophic lateral sclerosis function rating scale revised (ALSFRS-R) (Cedarbaum et al., 1999), Medical Research Council (MRC) rating scale (Medical Research Council, 1976) and Trigg’s hand function score (Triggs et al., 1999) at the time of assessment. All subjects gave informed consent, which were approved by the South East Sydney Area Health Service Human Research Ethics Committee.
2.1. DTI image acquisition All studies were performed using a 3 T Philips Intera System (Philips Medical Systems, Best, The Netherlands) with an eight channel, phased array head coil and gradient coils (0–33 mT/m). Head motion was restricted by placing pads on both sides of the subject’s head. A single shot spin echo–Echo Planar Imaging diffusion weighted sequence was performed for DTI acquisition. Fifteen directional scans were used with an isotropic voxel resolution of 2 mm (field of view 256 mm, matrix of 128 128, slice thickness of 2 mm, slice gap 0.2). Sixty slices were collected with an echo time of 68 ms and a b value of 800 s/mm2.
2.2. Diffusion tensor image data processing DTIStudio (Dr. Susumu Mori, Johns Hopkins University, Baltimore, MD) was used to post-process the DTI sequences. Quantitative diffusion parameter maps were created to calculate fractional anisotropy (FA), trace apparent diffusion coefficient (Trace ADC), colour maps, Parallel Diffusion (Para D – Ev1), and Perpendicular Diffusion (Perp D – [Ev2 + Ev3]/2), where Ev1 represents eigenvalue-1, Ev2, eigenvalue-2 and Ev3, eigenvalue-3. Fibre tracking was performed according to a previously reported protocol (Sage et al., 2007). Fibre tracking was carried out using DTIStudio and colour maps were used to locate the cerebral peduncle and internal capsule.
2.3. Neurophysiological studies 2. Materials and methods Studies were undertaken on seven asymptomatic SOD-1 mutation carriers (six females, one male: mean age 33 years, age range 25–47 years) from families with three different SOD-1 mutations
Table 1 Clinical details for the seven asymptomatic superoxide-dismutase-1 (SOD-1) mutation carriers. 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 subjects 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) scale for the abductor pollicis brevis, as this muscle was utilized for excitability testing as well as the Triggs hand score. Asymptomatic SOD-1
Age (years)/sex
SOD-1 mutation
ALSFRSR
Triggs hand score
MRC
1 2 3 4 5 6 7
25/F 47/F 37/M 25/F 23/F 45/F 31/F
V148G V148G V148G I113T V148G E100G V148G
48 48 48 48 48 48 48
0 0 0 0 0 0 0
5 5 5 5 5 5 5
Peripheral nerve studies were performed by stimulating the median nerve electrically at the wrist. The resultant compound muscle action potential (CMAP) was recorded from the abductor pollicis brevis and was measured from baseline to negative peak. In addition to the CMAP amplitude, a marker of peripheral disease burden that correlates with muscle weakness, the neurophysiological index (NI), was derived according to a previously reported formula (De Carvalho and Swash, 2000):
Neurophysiological index ¼ ½CMAP amplitude=DML F-wave frequency
where F-wave frequency refers to the number of F responses recorded in 20 trials and DML refers to the distal motor latency. Cortical excitability studies were performed by applying TMS to the motor cortex by means of a 90 mm circular coil according to a previously reported technique (Vucic et al., 2006). Using a threshold tracking paired-pulse TMS paradigm resting motor threshold (RMT), short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) were recorded (Vucic et al., 2006). The motor evoked potential (MEP) amplitude, MEP onset latency (ms) and cortical silent period (CSP) duration were recorded using single TMS pulses (Vucic et al., 2006). Central motor conduction time (CMCT, ms) was calculated according to the F-wave method (Cros et al., 1990).
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2.4. Statistical analysis The results of cortical excitability testing for asymptomatic SOD-1 mutation carriers was compared to 62 healthy controls (31 men; 31 women, aged 23–83 years, mean: 45 years). Further, cortical excitability data from the SOD-1 mutation carriers were compared to a sub-group of 26 younger controls (12 men; 14 women, aged 23–39 years, mean age: 31 years). Further, DTI data from asymptomatic SOD-1 mutation carriers was compared to seven normal controls (five males, two females; mean age: 56 years; age range: 42–65 years). Mann–Whitney U test or Student’s t-test was used to assess differences between group means. A probability value (P) of <0.05 was considered statistically significant. 3. Results The clinical and genetic features for the seven asymptomatic SOD-1 mutation carriers are summarized in Table 1. The neurological examination in all the asymptomatic SOD-1 mutation carriers remained normal. 3.1. Diffusion tensor imaging Fibre tracking was undertaken on the left corticospinal tract along four points: point 1, the cerebral peduncle; point 4, internal capsule; and points 2 and 3 were equidistant between points 1 and 4 (Fig. 1). There was no significant difference in FA between asymptomatic SOD-1 mutation carriers (point 1, 0.5 ± 0.02; point 2, 0.7 ± 0.04; point 3, 0.6 ± 0.03; point 4, 0.5 ± 0.02) and controls (point 1, 0.5 ± 0.05, P = 0.4; point 2, 0.7 ± 0.03, P = 0.2; point 3, 0.7 ± 0.04, P = 0.08; point 4, 0.5 ± 0.04, P = 0.4) at any level of the CST. Further, there was also no significant difference in the FA for whole of the CST between asymptomatic SOD-1 mutation carriers (0.62 ± 0.01) and controls (0.61 ± 0.02, P = 0.2). In addition, there was no significant difference in Trace ADC, Parallel Diffusivity (Para D) or Perpendicular Diffusivity (Perp D) for the whole of CST between asymptomatic SOD-1 mutation carriers (Trace ADC, 0.003 ± 0.0001; Para D, 0.002 ± 6.8 105; Perp D, 0.0005 ± 2.6 105) and controls (Trace ADC, 0.003 ± 0.0001; Para D, 0.001 ± 6.1 105, Perp D, 0.0005 ± 2.9 105). Further, the Trace ADC, Perp D and Para D at each point along to CST were not significantly different between asymptomatic SOD-1 mutation carriers and controls.
Internal capsule Point 3 Point 2
Cerebral peduncle
Fig. 1. A colour map of the corticospinal tract (CST) using diffusion tensor imaging (DTI). The CST was selected by placing two regions of interest on the cerebral peduncle and the internal capsule. Once the corticospinal tract (in red) was located, four regions of interest were placed along the CST. Point 1 – cerebral peduncle, point 4 – internal capsule, points 2 and 3 – slices located exactly in between these points.
3.2. Neurophysiological studies The CMAP amplitude (asymptomatic SOD-1 mutation carriers, 10.7 ± 1.2 mV; controls, 10.3 ± 0.5 mV, P = 0.34) and NI (asymptomatic SOD-1 mutation carriers, 2.6 ± 0.3; controls, 2.6 ± 0.2, P = 0.35) were similar between groups. Further, there was no significant difference in the CMCT between asymptomatic SOD-1 mutation carriers (5.1 ± 0.6 ms) and controls (5.1 ± 0.2 ms, P = 0.40, Fig. 2A). Short-interval intracortical inhibition is reflected by an increase in the conditioned stimulus intensity required to track a constant target MEP of 0.2 mV (see Section 2). There was no significant difference in averaged SICI, over interstimulus intervals 1–7 ms, between asymptomatic SOD-1 mutation carriers (7.9 ± 3.4%) and controls (8.5 ± 1.1%, Fig. 2B, P = 0.26). Following SICI, a period of intracortical facilitation develops, marked by a decrease in the test stimulus intensity required to maintain the target MEP (Vucic et al., 2006). There was no significant difference in ICF between asymptomatic SOD-1 mutation carriers (0.3 ± 1.3%) and controls (0.4 ± 0.8%, P = 0.5). Further, the MEP amplitude (asymptomatic SOD-1 mutation carriers, 27.5 ± 7.8%; controls, 26.2 ± 2.4%,
Fig. 2C, P = 0.44) and resting motor threshold (asymptomatic SOD-1 mutation carriers, 58.1 ± 3.4%; controls, 60.6 ± 1.3%, P = 0.36) were comparable between groups, as was the CSP duration (asymptomatic SOD-1 mutation carriers, 0–219.0 ± 15.1 ms; controls, 0–209.0 ± 4.7 ms, P = 0.29).
4. Discussion Using a combination of structural (DTI) and functional (threshold tracking TMS) techniques, findings from the present study would suggest normal anatomical and functional integrity of corticomotoneurons in asymptomatic SOD-1 mutation carriers. Specifically, fractional anisotropy, trace ADC, parallel diffusivity and perpendicular diffusivity were comparable to controls, thereby indicating that the corticomotoneurons appeared anatomically intact. Further, the functional integrity of corticomotoneurons remained intact, as confirmed by finding normal cortical excitability in asymptomatic SOD-1 mutation carriers. Although these findings do not absolutely prove the integrity of corticomot-
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Central motor conduction time (ms)
A
6
4
2
0
Averaged SICI (%)
B
12
8
4
C
40
MEP amplitude (% CMAP)
0
30
Asymptomatic SOD-1 Controls
20
subjects from one family with SOD-1 mutations that were different to mutations expressed in the present cohort. Lastly, the inherent variability of DTI results stemming from differences in the quality of spatial normalization of the DTI images may also contribute to the observed differences in part (Sage et al., 2009). A potential limitation of the present study is that the SOD-1 mutation carriers were younger than normal controls. Given that the FA decreases and trace ADC increases with age (Abe et al., 2002; Pfefferbaum et al., 2005; Sullivan and Pfefferbaum, 2006), it remains possible that differences between the groups were masked by age effects. However, this seems unlikely as previous age-related changes were reported predominantly in the frontal white matter, corpus callosum, lentiform nucleus and thalamus, but not the internal capsule, a region of interest in the present study. Further support for corticomotoneuronal integrity in asymptomatic SOD-1 subjects was established using the threshold tracking TMS technique. Specifically, measures of cortical excitability previously reported to be abnormal in sporadic ALS and clinically affected FALS patients (Vucic and Kiernan, 2006; Vucic et al., 2008), were normal in the present study. Further, the central motor conduction time, which correlated with reduced FA in sporadic ALS patients (Sach et al., 2004), was also normal, thereby reaffirming the integrity of corticomotoneurons in asymptomatic SOD-1 mutation carriers. In conclusion, the present study has established normal anatomical and functional corticomotoneuronal integrity in asymptomatic SOD-1 mutation carriers, although pre-symptomatic biomolecular abnormalities are yet to be excluded. Taken together, the findings in the present study suggest that acquisition of as yet unidentified co-factors, in addition to SOD-1 mutations, are required to develop ALS, and identification of these co-factors may yet prove useful in the development of more effective therapeutic strategies in ALS.
10
Acknowledgements 0 Fig. 2. (A) Central motor conduction time, determined using the F-wave method (see Section 2) was similar in asymptomatic superoxide-dismutase-1 (SOD-1) mutation carriers and controls. (B) Short-interval intracortical inhibition (SICI), defined as the stimulus intensity required to maintain a target output of 0.2 mV (see Section 2), was also similar asymptomatic SOD-1 mutation carriers and controls. (C) The motor evoked potentials (MEP) amplitude, expressed as a percentage of the compound muscle action potential response amplitude, was not significantly different between asymptomatic SOD-1 mutation carriers and controls.
oneurons, when taken in total, the present study would suggest that corticomotoneuronal function in asymptomatic SOD-1 mutation carriers appears intact. Reduced FA and increased tensor ADC were recently reported within the posterior limb of the internal capsule in a cohort of asymptomatic SOD-1 mutation carriers from one family (Ng et al., 2008). These abnormalities were thought to be indicative of neurodegeneration within the white matter tracts and were similar to that reported in sporadic ALS (Ellis et al., 1999; Sach et al., 2004; Sage et al., 2007). In contrast, the present study has established normal corticomotoneuronal integrity in asymptomatic SOD-1 mutation carriers, thereby arguing against pre-clinical axonal degeneration within the white matter tracts. A possible explanation for the observed discrepancy may relate to differences in acquisition and analysis of DTI. Although Ng et al. (2008) used 25 diffusion directions, as opposed to 15 directions used in the present study, their analysis was based on a voxelbased technique and data acquisition carried out using a 1.5 T machine. In contrast, the present study used the more sensitive 3 T machine and performed data analysis on the more precise fibretracking programme. Of further relevance, Ng et al. (2008) studied
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Contents lists available at ScienceDirect
Clinical Neurophysiology journal homepage: www.elsevier.com/locate/clinph
Corticomotoneuronal function in asymptomatic SOD-1 mutation carriers Steve Vucic a,e, Jennica M.C. Winhammar b,c,e, Dominic B. Rowe b,c,d, Matthew C. Kiernan e,* a
Department of Neurology, Westmead Hospital, Western Clinical School, University of Sydney, Australia Department of Neurology, Royal North Shore Hospital, Sydney, NSW, Australia c Northern Clinical School, University of Sydney, Australia d Australian School of Advanced Medicine, Innovation Road, Macquarie University, Australia e Prince of Wales Medical Research Institute and Prince of Wales Clinical School, University of New South Wales, Australia b
See Editorial, pages 1709–1710
a r t i c l e
i n f o
Article history: Accepted 16 February 2010 Available online 1 April 2010 Keywords: Diffusion tensor tractography Familial ALS SOD-1 mutation carrier
a b s t r a c t Objective: Diffusion tensor imaging (DTI) recently identified structural abnormalities of corticomotoneurons in asymptomatic copper/zinc superoxide-dismutase-1 (SOD-1) gene mutation carriers. The potential existence of longstanding corticomotoneuronal dysfunction would clearly have consequences for the medical management of asymptomatic SOD-1 mutation carriers. To clarify this unexpected finding, DTI techniques were combined with threshold tracking transcranial magnetic stimulation (TMS) to assess the anatomical and functional integrity of corticomotoneurons in asymptomatic SOD-1 mutation carriers. Methods: TMS studies were undertaken using a 90 mm circular coil on seven asymptomatic SOD-1 mutation carriers and results were compared to 62 healthy controls. DTI studies were carried out using a 3 T magnetic resonance device in the same asymptomatic SOD-1 mutation carriers. Results were compared to age-matched healthy controls. Results: In contrast to previous findings, there were no significant differences in fractional anisotropy (SOD-1 mutation carriers, 0.62 ± 0.01; controls, 0.61 ± 0.02, P = 0.2) and trace apparent diffusion coefficient (SOD-1 mutation carriers, 0.003 ± 0.0001; controls, 0.003 ± 0.0001) in asymptomatic SOD-1 mutation carriers. Of further relevance, there were no significant differences in short-interval intracortical inhibition (SOD-1 mutation carriers, 7.9 ± 3.4%; controls, 8.5 ± 1.1%, P = 0.26), intracortical facilitation (P = 0.5), MEP amplitude (P = 0.44), resting motor threshold (P = 0.36) and cortical silent period duration (P = 0.29). Conclusions: Combined anatomical and functional modalities established normal integrity of corticomotoneurons in asymptomatic SOD-1 mutation carrier subjects. Significance: Additional factors other than simply SOD-1 mutation expression are required to trigger cortical hyperexcitability and neurodegeneration in FALS. Ó 2010 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder of the motor neurons in the motor cortex, brain stem and spinal cord, with median survival of 3–5 years (Winhammar et al., 2005). Ten percent of ALS cases are familial (FALS), with mutations in the copper/zinc superoxide-dismutase1 gene (SOD-1) the most frequent cause (Rosen et al., 1993). The pathophysiological processes underlying ALS have been linked to
* Corresponding author. Address: Prince of Wales Medical Research Institute, Barker Street, Randwick, Sydney, NSW 2031, Australia. Tel.: +61 2 9382 2422; fax: +61 2 9382 2437. E-mail address: [email protected] (M.C. Kiernan).
a complex interaction between genetic factors and molecular pathways including oxidative stress, glutamate excitotoxicity, mitochondrial dysfunction, axonal transport systems, and glial cell dysfunction with resultant damage of critical target proteins and organelles within the motor neuron, thereby underlying neurodegeneration (Vucic and Kiernan, 2009). Cortical hyperexcitability and excitotoxicity have been linked to the process of motor neuron degeneration (Boillee et al., 2006; Bruijn et al., 2004; Eisen et al., 1992; Shaw and Kuncl, 2002; Shaw, 2005), with further support provided by pharmacological studies that have established the clinical effectiveness of riluzole, an inhibitor of glutamate release, in ALS (Bensimon et al., 1994; Kiernan, 2005; Lacomblez et al., 1996; Zoing et al., 2006). In addition, transcranial magnetic stimulation (TMS) studies have identified that
1388-2457/$36.00 Ó 2010 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2010.02.164
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cortical hyperexcitability appears as an early feature of ALS, preceding the development of clinical weakness in familial ALS (Desiato et al., 2002; Eisen et al., 1993; Mills and Nithi, 1997; Prout and Eisen, 1994; Vucic and Kiernan, 2006; Vucic et al., 2008). The presence of cortical hyperexcitability in sporadic ALS and familial ALS patients was identified by establishing the presence of reduced short-interval intracortical inhibition, resting motor threshold and cortical silent period duration, as well as increased motor evoked potential amplitude and intracortical facilitation. Identifying the presence of corticomotoneuronal degeneration in asymptomatic SOD-1 mutation carriers would clearly be of therapeutic significance. Diffusion tensor imaging (DTI) is a magnetic resonance imaging (MRI) technique capable of assessing the anatomical integrity of corticomotoneurons by quantifying the strength (trace apparent diffusion coefficient, trace ADC) and direction (fractional anisotropy, FA) of diffusion of water molecules within white matter tracts (Alexander et al., 2007). In normal brain, the diffusion of water is restricted by the spatial organization of axons, such that subcortical white matter tracts of the internal capsule have a higher anisotropy (Alexander et al., 2007). Of relevance, degeneration of axons within white matter tracts, as may be evident in manifesting ALS patients, has been associated with abnormalities of diffusion, resulting in increased trace ADC and reduced FA (Abe et al., 2004; Ellis et al., 1999; Graham et al., 2004; Sach et al., 2004; Toosy et al., 2003). Recently, DTI techniques identified the presence of increased trace ADC and reduced FA within the posterior limb of the internal capsule in asymptomatic SOD-1 mutation carriers (Ng et al., 2008), thereby suggesting that a loss of corticomotoneurons may precede the clinical onset of weakness in familial ALS. The existence of longstanding structural and functional abnormalities would have critically important consequences for the medical management of asymptomatic SOD-1 mutation carriers. In the least, such a finding would suggest that affected individuals should be commenced on neuroprotective agents, such as riluzole, prior to clinical disease onset. To consider this important issue, the present study used a novel combination of DTI and threshold tracking TMS techniques in asymptomatic SOD-1 mutation carriers to assess corticomotoneuronal integrity and function.
(Table 1). Asymptomatic SOD-1 mutation carriers underwent clinical examination and staging with amyotrophic lateral sclerosis function rating scale revised (ALSFRS-R) (Cedarbaum et al., 1999), Medical Research Council (MRC) rating scale (Medical Research Council, 1976) and Trigg’s hand function score (Triggs et al., 1999) at the time of assessment. All subjects gave informed consent, which were approved by the South East Sydney Area Health Service Human Research Ethics Committee.
2.1. DTI image acquisition All studies were performed using a 3 T Philips Intera System (Philips Medical Systems, Best, The Netherlands) with an eight channel, phased array head coil and gradient coils (0–33 mT/m). Head motion was restricted by placing pads on both sides of the subject’s head. A single shot spin echo–Echo Planar Imaging diffusion weighted sequence was performed for DTI acquisition. Fifteen directional scans were used with an isotropic voxel resolution of 2 mm (field of view 256 mm, matrix of 128 128, slice thickness of 2 mm, slice gap 0.2). Sixty slices were collected with an echo time of 68 ms and a b value of 800 s/mm2.
2.2. Diffusion tensor image data processing DTIStudio (Dr. Susumu Mori, Johns Hopkins University, Baltimore, MD) was used to post-process the DTI sequences. Quantitative diffusion parameter maps were created to calculate fractional anisotropy (FA), trace apparent diffusion coefficient (Trace ADC), colour maps, Parallel Diffusion (Para D – Ev1), and Perpendicular Diffusion (Perp D – [Ev2 + Ev3]/2), where Ev1 represents eigenvalue-1, Ev2, eigenvalue-2 and Ev3, eigenvalue-3. Fibre tracking was performed according to a previously reported protocol (Sage et al., 2007). Fibre tracking was carried out using DTIStudio and colour maps were used to locate the cerebral peduncle and internal capsule.
2.3. Neurophysiological studies 2. Materials and methods Studies were undertaken on seven asymptomatic SOD-1 mutation carriers (six females, one male: mean age 33 years, age range 25–47 years) from families with three different SOD-1 mutations
Table 1 Clinical details for the seven asymptomatic superoxide-dismutase-1 (SOD-1) mutation carriers. 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 subjects 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) scale for the abductor pollicis brevis, as this muscle was utilized for excitability testing as well as the Triggs hand score. Asymptomatic SOD-1
Age (years)/sex
SOD-1 mutation
ALSFRSR
Triggs hand score
MRC
1 2 3 4 5 6 7
25/F 47/F 37/M 25/F 23/F 45/F 31/F
V148G V148G V148G I113T V148G E100G V148G
48 48 48 48 48 48 48
0 0 0 0 0 0 0
5 5 5 5 5 5 5
Peripheral nerve studies were performed by stimulating the median nerve electrically at the wrist. The resultant compound muscle action potential (CMAP) was recorded from the abductor pollicis brevis and was measured from baseline to negative peak. In addition to the CMAP amplitude, a marker of peripheral disease burden that correlates with muscle weakness, the neurophysiological index (NI), was derived according to a previously reported formula (De Carvalho and Swash, 2000):
Neurophysiological index ¼ ½CMAP amplitude=DML F-wave frequency
where F-wave frequency refers to the number of F responses recorded in 20 trials and DML refers to the distal motor latency. Cortical excitability studies were performed by applying TMS to the motor cortex by means of a 90 mm circular coil according to a previously reported technique (Vucic et al., 2006). Using a threshold tracking paired-pulse TMS paradigm resting motor threshold (RMT), short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) were recorded (Vucic et al., 2006). The motor evoked potential (MEP) amplitude, MEP onset latency (ms) and cortical silent period (CSP) duration were recorded using single TMS pulses (Vucic et al., 2006). Central motor conduction time (CMCT, ms) was calculated according to the F-wave method (Cros et al., 1990).
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2.4. Statistical analysis The results of cortical excitability testing for asymptomatic SOD-1 mutation carriers was compared to 62 healthy controls (31 men; 31 women, aged 23–83 years, mean: 45 years). Further, cortical excitability data from the SOD-1 mutation carriers were compared to a sub-group of 26 younger controls (12 men; 14 women, aged 23–39 years, mean age: 31 years). Further, DTI data from asymptomatic SOD-1 mutation carriers was compared to seven normal controls (five males, two females; mean age: 56 years; age range: 42–65 years). Mann–Whitney U test or Student’s t-test was used to assess differences between group means. A probability value (P) of <0.05 was considered statistically significant. 3. Results The clinical and genetic features for the seven asymptomatic SOD-1 mutation carriers are summarized in Table 1. The neurological examination in all the asymptomatic SOD-1 mutation carriers remained normal. 3.1. Diffusion tensor imaging Fibre tracking was undertaken on the left corticospinal tract along four points: point 1, the cerebral peduncle; point 4, internal capsule; and points 2 and 3 were equidistant between points 1 and 4 (Fig. 1). There was no significant difference in FA between asymptomatic SOD-1 mutation carriers (point 1, 0.5 ± 0.02; point 2, 0.7 ± 0.04; point 3, 0.6 ± 0.03; point 4, 0.5 ± 0.02) and controls (point 1, 0.5 ± 0.05, P = 0.4; point 2, 0.7 ± 0.03, P = 0.2; point 3, 0.7 ± 0.04, P = 0.08; point 4, 0.5 ± 0.04, P = 0.4) at any level of the CST. Further, there was also no significant difference in the FA for whole of the CST between asymptomatic SOD-1 mutation carriers (0.62 ± 0.01) and controls (0.61 ± 0.02, P = 0.2). In addition, there was no significant difference in Trace ADC, Parallel Diffusivity (Para D) or Perpendicular Diffusivity (Perp D) for the whole of CST between asymptomatic SOD-1 mutation carriers (Trace ADC, 0.003 ± 0.0001; Para D, 0.002 ± 6.8 105; Perp D, 0.0005 ± 2.6 105) and controls (Trace ADC, 0.003 ± 0.0001; Para D, 0.001 ± 6.1 105, Perp D, 0.0005 ± 2.9 105). Further, the Trace ADC, Perp D and Para D at each point along to CST were not significantly different between asymptomatic SOD-1 mutation carriers and controls.
Internal capsule Point 3 Point 2
Cerebral peduncle
Fig. 1. A colour map of the corticospinal tract (CST) using diffusion tensor imaging (DTI). The CST was selected by placing two regions of interest on the cerebral peduncle and the internal capsule. Once the corticospinal tract (in red) was located, four regions of interest were placed along the CST. Point 1 – cerebral peduncle, point 4 – internal capsule, points 2 and 3 – slices located exactly in between these points.
3.2. Neurophysiological studies The CMAP amplitude (asymptomatic SOD-1 mutation carriers, 10.7 ± 1.2 mV; controls, 10.3 ± 0.5 mV, P = 0.34) and NI (asymptomatic SOD-1 mutation carriers, 2.6 ± 0.3; controls, 2.6 ± 0.2, P = 0.35) were similar between groups. Further, there was no significant difference in the CMCT between asymptomatic SOD-1 mutation carriers (5.1 ± 0.6 ms) and controls (5.1 ± 0.2 ms, P = 0.40, Fig. 2A). Short-interval intracortical inhibition is reflected by an increase in the conditioned stimulus intensity required to track a constant target MEP of 0.2 mV (see Section 2). There was no significant difference in averaged SICI, over interstimulus intervals 1–7 ms, between asymptomatic SOD-1 mutation carriers (7.9 ± 3.4%) and controls (8.5 ± 1.1%, Fig. 2B, P = 0.26). Following SICI, a period of intracortical facilitation develops, marked by a decrease in the test stimulus intensity required to maintain the target MEP (Vucic et al., 2006). There was no significant difference in ICF between asymptomatic SOD-1 mutation carriers (0.3 ± 1.3%) and controls (0.4 ± 0.8%, P = 0.5). Further, the MEP amplitude (asymptomatic SOD-1 mutation carriers, 27.5 ± 7.8%; controls, 26.2 ± 2.4%,
Fig. 2C, P = 0.44) and resting motor threshold (asymptomatic SOD-1 mutation carriers, 58.1 ± 3.4%; controls, 60.6 ± 1.3%, P = 0.36) were comparable between groups, as was the CSP duration (asymptomatic SOD-1 mutation carriers, 0–219.0 ± 15.1 ms; controls, 0–209.0 ± 4.7 ms, P = 0.29).
4. Discussion Using a combination of structural (DTI) and functional (threshold tracking TMS) techniques, findings from the present study would suggest normal anatomical and functional integrity of corticomotoneurons in asymptomatic SOD-1 mutation carriers. Specifically, fractional anisotropy, trace ADC, parallel diffusivity and perpendicular diffusivity were comparable to controls, thereby indicating that the corticomotoneurons appeared anatomically intact. Further, the functional integrity of corticomotoneurons remained intact, as confirmed by finding normal cortical excitability in asymptomatic SOD-1 mutation carriers. Although these findings do not absolutely prove the integrity of corticomot-
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Central motor conduction time (ms)
A
6
4
2
0
Averaged SICI (%)
B
12
8
4
C
40
MEP amplitude (% CMAP)
0
30
Asymptomatic SOD-1 Controls
20
subjects from one family with SOD-1 mutations that were different to mutations expressed in the present cohort. Lastly, the inherent variability of DTI results stemming from differences in the quality of spatial normalization of the DTI images may also contribute to the observed differences in part (Sage et al., 2009). A potential limitation of the present study is that the SOD-1 mutation carriers were younger than normal controls. Given that the FA decreases and trace ADC increases with age (Abe et al., 2002; Pfefferbaum et al., 2005; Sullivan and Pfefferbaum, 2006), it remains possible that differences between the groups were masked by age effects. However, this seems unlikely as previous age-related changes were reported predominantly in the frontal white matter, corpus callosum, lentiform nucleus and thalamus, but not the internal capsule, a region of interest in the present study. Further support for corticomotoneuronal integrity in asymptomatic SOD-1 subjects was established using the threshold tracking TMS technique. Specifically, measures of cortical excitability previously reported to be abnormal in sporadic ALS and clinically affected FALS patients (Vucic and Kiernan, 2006; Vucic et al., 2008), were normal in the present study. Further, the central motor conduction time, which correlated with reduced FA in sporadic ALS patients (Sach et al., 2004), was also normal, thereby reaffirming the integrity of corticomotoneurons in asymptomatic SOD-1 mutation carriers. In conclusion, the present study has established normal anatomical and functional corticomotoneuronal integrity in asymptomatic SOD-1 mutation carriers, although pre-symptomatic biomolecular abnormalities are yet to be excluded. Taken together, the findings in the present study suggest that acquisition of as yet unidentified co-factors, in addition to SOD-1 mutations, are required to develop ALS, and identification of these co-factors may yet prove useful in the development of more effective therapeutic strategies in ALS.
10
Acknowledgements 0 Fig. 2. (A) Central motor conduction time, determined using the F-wave method (see Section 2) was similar in asymptomatic superoxide-dismutase-1 (SOD-1) mutation carriers and controls. (B) Short-interval intracortical inhibition (SICI), defined as the stimulus intensity required to maintain a target output of 0.2 mV (see Section 2), was also similar asymptomatic SOD-1 mutation carriers and controls. (C) The motor evoked potentials (MEP) amplitude, expressed as a percentage of the compound muscle action potential response amplitude, was not significantly different between asymptomatic SOD-1 mutation carriers and controls.
oneurons, when taken in total, the present study would suggest that corticomotoneuronal function in asymptomatic SOD-1 mutation carriers appears intact. Reduced FA and increased tensor ADC were recently reported within the posterior limb of the internal capsule in a cohort of asymptomatic SOD-1 mutation carriers from one family (Ng et al., 2008). These abnormalities were thought to be indicative of neurodegeneration within the white matter tracts and were similar to that reported in sporadic ALS (Ellis et al., 1999; Sach et al., 2004; Sage et al., 2007). In contrast, the present study has established normal corticomotoneuronal integrity in asymptomatic SOD-1 mutation carriers, thereby arguing against pre-clinical axonal degeneration within the white matter tracts. A possible explanation for the observed discrepancy may relate to differences in acquisition and analysis of DTI. Although Ng et al. (2008) used 25 diffusion directions, as opposed to 15 directions used in the present study, their analysis was based on a voxelbased technique and data acquisition carried out using a 1.5 T machine. In contrast, the present study used the more sensitive 3 T machine and performed data analysis on the more precise fibretracking programme. Of further relevance, Ng et al. (2008) studied
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