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Motor cortical dysfunction develops in spinocerebellar ataxia type 3
Clinical Neurophysiology 127 (2016) 3418–3424
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Motor cortical dysfunction develops in spinocerebellar ataxia type 3 Michelle A. Farrar a,⇑, Steve Vucic b, Garth Nicholson c, Matthew C. Kiernan d a
Discipline of Paediatrics, School of Women’s and Children’s Health, UNSW Medicine, The University of New South Wales, Sydney, Australia Department of Neurology, Westmead Hospital and Western Clinical School, University of Sydney, Sydney, Australia c ANZAC Research Institute, University of Sydney, Concord Hospital, New South Wales, Australia d Sydney Medical School, Brain & Mind Centre, University of Sydney, Sydney, Australia b
a r t i c l e
i n f o
Article history: Accepted 7 September 2016 Available online 15 September 2016 Keywords: Spinocerebellar ataxia type 3 Corticomotoneuron Transcranial magnetic stimulation
h i g h l i g h t s Cortical dysfunction is an early feature of SCA3 and associated with motor symptoms and ataxia. Identifying cortical neuronal dysfunction in SCA3 may be of therapeutic significance. SCA3 onset is not confined to the cerebellum and brainstem.
a b s t r a c t Objective: Spinocerebellar ataxia type 3 (SCA3) is an inherited neurodegenerative disorder characterized by cerebellar ataxia and variable expression of clinical features beyond the cerebellum. To gain further insights into disease pathophysiology, the present study explored motor cortex function in SCA3 to determine whether cortical dysfunction was present and if this contributed to the development of clinical manifestations. Methods: Clinical phenotyping and longitudinal assessments were combined with central (thresholdtracking transcranial magnetic stimulation) and peripheral (nerve excitability) techniques in 11 genetically characterized SCA3 patients. Results: Short-interval intracortical inhibition was significantly reduced in presymptomatic and symptomatic SCA3 patients (1.3 ± 1.4%) compared to healthy controls (10.3 ± 0.7%, P < 0.0005), with changes evident prior to clinical onset of ataxia and related to worsening severity (R = 0.78, P < 0.005). Central motor conduction time was also significantly prolonged in presymptomatic and symptomatic SCA3 patients (7.5 ± 0.4 ms) compared to healthy controls (5.3 ± 0.2 ms, P < 0.0005) and related to clinical severity (R = 0.81, P < 0.005). Markers of peripheral motor neurodegeneration and excitability did not correlate with cortical hyperexcitability or ataxia. Conclusions: Simultaneous investigation of clinical status, and central and peripheral nerve function has identified progressive cortical dysfunction in SCA3 patients related to the development of ataxia. Significance: These findings suggest alteration in cortical activity is associated with SCA3 pathogenesis and neurodegeneration. Ó 2016 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease and the most common hereditary spinocerebellar ataxia. Also known as Machado–Joseph disease, there is a wide spectrum of clinical expression in addition to ataxia, that may include progressive external ophthalmoplegia, dysarthria, dysphagia, pyramidal disturbances, dystonia, parkinsonism, ⇑ Corresponding author at: Department of Neurology, Sydney Children’s Hospital, High St., Randwick, NSW 2031, Australia. Fax: +61 2 93821580. E-mail address: [email protected] (M.A. Farrar).
sleep disorders, rigidity, peripheral neuropathy and distal muscle atrophy, suggestive of neurodegeneration beyond that originally described in the cerebellum and brainstem (Boller and Segarra, 1969; Pogacar et al., 1978; Rosenberg, 1992). Symptoms usually begin between the ages of 20 and 50 years and loss of motor control is ultimately so severe that survival is limited to 15–30 years following clinical onset. SCA3 presents clinically when neuronal loss within the cerebellum and brainstem has become advanced and irreversible, emphasizing the need to further understand pathophysiological processes, particularly the detection of early neuronal dysfunction, such that therapeutic strategies may be directed at earlier stages of disease.
http://dx.doi.org/10.1016/j.clinph.2016.09.005 1388-2457/Ó 2016 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
M.A. Farrar et al. / Clinical Neurophysiology 127 (2016) 3418–3424
SCA3 is caused by an unstable expansion of CAG triplet repeats in the ataxin-3 (ATXN3) gene on chromosome 14, resulting in an abnormal expanded polyglutamine repeat in the ubiquitously expressed ATXN3 protein, which makes the protein highly susceptible to misfolding and aggregation (Kawaguchi et al., 1994). Abnormalities beyond the cerebellum and brainstem have been demonstrated with neuroimaging and neuropathological studies to reveal neurodegeneration affecting the thalamus, and frontal, temporal, limbic, parietal and occipital lobes (D’Abreu et al., 2012; de Rezende et al., 2015). In addition, loss of motor neurons in the spinal cord and has been established, and may be associated with muscle cramps, amyotrophy and fasciculations, such that SCA3 may be regarded as a chronic motor neurone disorder (Kinoshita et al., 1995; Franca et al., 2008). Although the mechanisms by which the misfolded ATXN3 protein induces motor neuron toxicity in SCA3 remain elusive, impairment of axonal transport, oxidative stress, neuronal signaling and RNA toxicity have all been reported (Li et al., 2015). Importantly, the mutant ATXN3 protein, which appears to be sequestered ubiquitously within the central nervous system, may induce neuronal hyperexcitability, which in turn may mediate the underlying pathogenesis in SCA3 (Jeub et al., 2006; Chen et al., 2008; Shakkottai et al., 2011). The impact of mutant ATXN3 on motor neurone function throughout the cortex and spinal cord remains to be fully elucidated and is of pathophysiological and therapeutic importance. Perhaps of relevance, cortical dysfunction has been linked to neurodegeneration in amyotrophic lateral sclerosis (Vucic and Kiernan, 2006; Vucic et al., 2008). Recently, the application of threshold tracking transcranial magnetic stimulation (TMS) techniques has enabled the early detection of upper motor neuron dysfunction (Menon et al., 2015). As such, the present study utilized clinical assessments combined with cortical and peripheral nerve assessment to further clarify the processes involved in neurodegeneration in SCA3.
2. Methods Clinical phenotyping and assessment was combined with conventional and specialized neurophysiological assessments in 11 patients with genetically confirmed SCA3. A subset of presymptomatic SCA3 patients and symptomatic SCA3 patients with mild to moderate disease and duration less than 10 years were followed longitudinally for up to 36 months to characterize progression. Diagnosis was confirmed by DNA testing showing an expanded CAG repeat in the ATXN3 gene on chromosome 14q32.1 in all patients. All patients gave informed consent to the procedures, which were approved by the South Eastern Sydney and Illawarra Area Health Service Human Research Ethics Committee. SCA3 patients underwent clinical assessments of cerebellar ataxia using the International Cooperative Ataxia Rating Scale (ICARS), a 100-point semi quantitative scale that provides a total score (0–100) and four subscores: posture and stance (0–34), kinetic cerebellar function (0–52), dysarthria (0–8) and oculomotor dysfunction (0–6) (Trouillas et al., 1997). Greater scores indicate worsening cerebellar ataxia. The Total Neuropathy Scale-clinical version (TNSc) (Cornblath et al., 1999), was also assessed for each patient. The TNSc combines information obtained from grading of symptoms and signs to clinically quantify neuropathy and includes seven categories with severity rankings from 0 (none) to 4 (very severe): sensory symptoms; motor symptoms; autonomic symptoms; pinprick sensibility; vibration sensibility (128-Hz tuning fork); strength; and deep tendon reflexes (total score 0–28). Muscle strength was clinically assessed using the MRC for abductor pollicis brevis (APB), as this muscle was utilised for excitability testing.
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2.1. Neurophysiological studies In addition to clinical examination, cortical function was assessed using previously described threshold tracking transcranial magnetic stimulation (TMS) techniques (Vucic et al., 2006). Two high-power magnetic stimulators connected to a BiStim device (Magstim Co., Whitland, South West Wales, UK) produced magnetic currents and enabled conditioning and test stimuli to be independently set and administered through the one coil. These were applied over the motor cortex utilizing a 90 mm circular coil, oriented to induce current flow in a posterior-anterior direction and the resultant motor evoked potential (MEP) was recorded from APB. The optimal position for a motor evoked potential (MEP) from the right APB muscle was obtained by adjusting the coil position. The MEP was measured from peak to peak and the threshold tracking target set to 0.2 mV, the midpoint of the steepest portion of the logarithmic stimulus response curve (Fisher et al., 2002). This threshold tracking technique overcomes the marked variability in the MEP amplitude with successive stimuli related to spontaneous variations in the resting threshold of cortical neurons, as may occur with conventional constant paired test stimuli TMS techniques in which changes in the amplitude of the test response is measured (Kiers et al., 1993; Weber and Eisen, 2002). The APB muscle was at rest for the recording of all neurophysiological parameters (except the cortical silent period) and the surface EMG closely observed for the presence of voluntary activity. Single-stimulus TMS was used to record stimulus–response (SR) curves, resting motor threshold (RMT), central motor conduction time (CMCT) and cortical silent period (CSP) duration. Three stimuli were delivered at each level of stimulus intensity. RMT was the stimulus intensity that maintained a target MEP of 0.2 mV, which was the middle of the steepest portion of the logarithmic stimulus response curve and the MEP was measured from peak to peak. The maximum MEP amplitude (MEP/CMAP, %) and minimum MEP onset latency (ms) was calculated from the average of three stimuli delivered at 150% RMT. CMCT (ms) was calculated according to the F-wave method (Claus, 1990). CSP was produced by a singlepulse TMS while subjects performed a weak voluntary contraction. The duration of the silent period was determined from the beginning of the MEP to the return of EMG activity. Threshold tracking paired-pulse TMS studies were performed (Vucic et al., 2006). In the paired-pulse paradigm a subthreshold conditioning stimulus preceded a suprathreshold test stimulus at increasing interstimulus intervals (ISIs) as follows: 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 7, 10, 15, 20, and 30 ms. Three stimulus combinations were delivered sequentially and monitored: (i) the intensity required to produce the unconditioned test response (RMT); (ii) the subthreshold conditioning stimulus alone, verifying that the subject remained relaxed and no MEP response was produced; (iii) conditioning and test stimuli in combination. When two consecutive MEP responses were within 20% of the target response (0.2 mV), tracking was deemed acceptable and the computer advanced to the next ISI. The subthreshold conditioning stimulus (70% RMT) was such that it did not evoke a response. Intracortical inhibition induced by a conditioning stimulus was measured as the increase in the test stimulus intensity required to produce the target MEP and calculated with the following formula:
Inhibition ¼ ðConditioned test stimulus intensity RMTÞ=RMT 100 Two distinct physiological phases of SICI have been recognized in healthy subjects, occurring at ISIs 6 1 ms and 3 ms, ascribed to axonal refractoriness and activation of different inhibitory circuits, and both are reported (Vucic et al., 2006; Fisher et al., 2002). Facilitation was measured as the decrease in the conditioned test stimulus intensity required to produce the target MEP. In normal
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subjects, a subthreshold conditioning stimulus inhibits the response of the test stimulus over intervals of 1–7 ms, termed short intracortical inhibition (SICI). The response is facilitated at intervals from about 10 to 20 ms, termed intracortical facilitation (ICF). Peripheral nerve function was also assessed during the same sitting using the TROND protocols of the multiple nerve excitability QTRACs software (Institute of Neurology, London, England), a fully automated sequence of tests (stimulus–response, strength–duration, threshold electrotonus, recovery cycle, current-threshold relationship) (Kiernan et al., 2000). The median nerve was stimulated electrically at the wrist and the resultant CMAP was recorded from APB. MEP and CMAP recordings were filtered (3 Hz–3 kHz) and amplified with a GRASS ICP511 AC amplifier (Grass-Telefactor, Astro-Med Inc., West Warwick, RI, USA) and sampled with a 12bit data acquisition card at 10 kHz (National Instruments PCIMIO-16E-4). QTRACS software (Institute of Neurology, Queen Square, London, UK) controlled data acquisition and stimulation delivery (magnetic and electrical). 2.2. Data analysis Cortical excitability in symptomatic and pre-symptomatic SCA3 patients was compared with 62 age-matched controls (31 males; age range 23–83 years; mean 45.8 years). Peripheral nerve excitability parameters were compared with 40 age-matched controls (22 males; age range 23– 81 years; mean 39.5 years). The patients and controls did not differ significantly in skin temperature (SCA3; 32.1 ± 0.8 °C; controls 32.5 ± 0.1 °C) or gender ratio. All results were expressed as mean ± standard error of the mean (SEM). Mann–Whitney U tests were used to compare mean differences between SCA3 patients and controls with post hoc testing (sequential Bonferroni correction) used for multiple comparisons. A probability (P) value of < 0.005 was considered statistically significant. Measurements between pre-symptomatic SCA3 patients were compared to the 95% confidence intervals of healthy controls (and considered significant if outside this range). Paired Student’s t tests were used to compare longitudinal paired excitability recordings and clinical assessments to characterize progression and P < 0.05 was considered statistically significant. Correlations between neurophysiological indices and clinical measurements of disease severity were analysed using the Spearman’s rank test. 3. Results The study population included 5 males and 6 females (mean age 46 years, range 30–64 years). Clinical severity varied, ranging from presymptomatic to severe as reflected by the broad spectrum in ICARS score (range 0–59, median 27), with the latter having profound ataxia and inability to stand or ambulate (Table 1). Neurological examination of presymptomatic individuals was normal. The first reported symptom universally related to gait ataxia. In the present cohort, a slowly progressive ‘‘ataxia-plus’’ syndrome evolved indicative of central and peripheral neurodegeneration, with progressive disturbances of gait and balance, cramps of the arms, legs, face, chest and neck, numbness and paraesthesias of the hands and feet, and fasciculations commonly reported. Distal muscle atrophy, dysarthria, pyramidal signs (spasticity and rigidity) and external ophthalmoplegia were common clinical signs in manifesting patients. While clinical features of peripheral neuropathy were evident in 54% of patients (TNS 3.0 ± 1.5), APB muscle strength was similar between groups (MRC APB: SCA3 4.7 ± 0.2; controls 5.0 ± 0.0, P = 0.1). Amyotrophy was evident in 36%, all with disease onset aged greater than 50 years. Pathological muscle cramps were experienced in 83% of patients. Disease dura-
tion, the period from symptom onset to the date of assessment, correlated with clinical measures of disease burden, including ICARS posture and gait subscale (R = 0.75, P < 0.01), reflecting neurodegeneration. In terms of axonal excitability studies, the threshold currents required to elicit a response were similar in SCA3 patients and controls (SCA3, 3.2 ± 1.1 mV; controls, 3.7 ± 1.1 mV). Strength duration time constant (sSD), an indirect measure of nodal persistent Na+ conductances was significantly prolonged in SCA3 patients compared to controls (SCA3, 0.51 ± 0.03 ms; controls, 0.43 ± 0.01 ms, P < 0.01, Fig. 1), but was within normal limits in presymptomatic SCA3 patients (Patient 1 0.44 ms; Patient 2 0.42 ms; controls 95%CI 0.31–0.45 ms). In contrast, there were no significant differences in rheobase, threshold electrotonus, recovery cycle or current–threshold relationship in SCA3 patients when compared with controls. Motor amplitudes were relatively preserved in all SCA3 patients (SCA3 7.1 ± 1.1 mV; controls 6.9 ± 1.1 mV, P = 0.8). 3.1. Cortical function in pre-symptomatic and symptomatic SCA3 patients The motor cortex was excitable in all SCA3 patients (Table 2). Paired-pulse TMS, disclosed that SICI was significantly reduced in symptomatic SCA3 patients and pre-symptomatic individuals when compared with controls (P < 0.0005, Fig. 2, Table 2). Mean SICI (between ISI 1 and 7 ms) was significantly reduced (symptomatic SCA3 1.3 ± 1.4%, controls 10.3 ± 0.7%, P < 0.0005; presymptomatic SCA3 patient 1 4.8%, patient 2 5.7%, controls 95% CI 9.0–11.7), as was peak SICI at ISI 1 ms (symptomatic SCA3 2.0 ± 1.9%, controls 7.2 ± 0.8%, P < 0.0005; pre-symptomatic SCA3 patient 1 1.0%, patient 2 5.5%, controls 95% CI 5.6–8.8) and 3 ms (symptomatic SCA3 0.2 ± 2.2%, controls 15.2 ± 1.3%, P = 0.001; pre-symptomatic SCA3 patient 1 9.5%, patient 2 10.6%, controls 95% CI 12.7–17.6) Notably SICI was absent in 4 SCA3 patients. ICF was significantly increased in symptomatic SCA3 patients when compared with controls (SCA3 5.6 ± 1.7%, controls 0.5 ± 0.8%, P = 0.005, Fig. 2). In contrast, ICF was normal in presymptomatic SCA3 subjects (patient 1 1.3%, patient 2 1.2%, controls 95% CI 2.0 to 1.3%). Central motor conduction time was significantly prolonged in all SCA3 patients when compared to controls (SCA3 7.5 ± 0.4 ms; controls 5.3 ± 0.2 ms, P < 0.0005; pre-symptomatic SCA3 Patient 1 6.7 ms; Patient 2 5.7 ms, controls 95% CI 4.9–5.7 ms). The RMT remained stable throughout the testing period and was similar between symptomatic SCA3 patients and controls (SCA3 62.9 ± 3.2%; controls 59.5 ± 1.0%, P = 0.2). Interestingly RMT was reduced in pre-symptomatic patients (pre-symptomatic SCA3 patient 1 58.7%; Patient 2 47.5%, controls 95% CI 57.5–61.5%). Of further relevance, the MEP amplitude, expressed as percentage of CMAP response, was maintained in SCA3 and presymptomatic patients when compared to healthy controls (symptomatic SCA3 34.5 ± 8.9%; controls 24.9 ± 1.8%, P = 0.2; pre-symptomatic SCA3 patient 1 17.9%; Patient 2 22.2%; controls 95% CI 21.3–28.3 ms). In addition, the CSP in symptomatic SCA3 patients was similar to healthy controls (symptomatic SCA3 222.7 ± 14.0 ms; controls 210.2 ± 3.1 ms, P = 0.2). In contrast, the CSP was reduced in presymptomatic SCA3 patients (pre-symptomatic SCA3 patient 1 158.6 ms; Patient 2 173.4 ms, controls 95% CI 204.1–216.8 ms). 3.2. Correlation with clinical parameters Combining measures of cortical and peripheral nerve function with clinical assessment, it was evident that cerebellar ataxia (total ICARS) was significantly associated with a greater reduction in SICI (mean SICI R = 0.78, P < 0.005; Peak SICI R = 0.88, P < 0.005,
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M.A. Farrar et al. / Clinical Neurophysiology 127 (2016) 3418–3424 Table 1 Clinical details for 11 patients with Spinocerebellar ataxia type 3 (SCA3). SCA3 patients
Age (years)
Age of clinical onset, disease duration (years)
Number of CAG repeats
ICARS score
TNS score
Current medications
Additional clinical features
1 2 3 4 5 6 7 8* 9 10 11 Median
34 40 30 33 35 40 64 52 59 60 47 40.0
Presymptomatic Presymptomatic 26, 4 30, 3 28, 7 31, 9 60, 4 38, 14 45, 14 50, 10 24, 23 31, 9
63 70 74 73 74 73 72 69 72 69 71 71.5
0 1 16 16 20 27 27 35 49 49 59 27
0 0 0 0 0 8 9 9 11 10 2 3.0
None Venlafaxine# None Gabapentin,# Amitriptyline# None None None Temazepam,# Biperiden, baclofen,# tramadol# None None Temazepam#
None None Pyramidal Pyramidal Pyramidal Pyramidal Amyotrophy Pyramidal, hand dystonia, amyotrophy Amyotrophy Amyotrophy Bradykinesia
The patients with SCA3 were clinically graded using the International Cooperative Ataxia Rating Scale (ICARS), a 100-point semi quantitative scale that provides a total score (0–100) and four sub-scores: posture and stance (0–34), kinetic cerebellar function (0–52), dysarthria (0–8) and oculomotor dysfunction (0–6). Greater scores indicate worsening cerebellar ataxia. Peripheral neuropathy was assessed using the Total Neuropathy Scale-clinical version (TNSc) with a higher score reflecting severity of neuropathy, range 0–28 see Section 2. Pyramidal signs include rigidity and spasticity. * Hand dystonia precluded assessment of cortical excitability by means of TTMS in patient 8. # This medication may be expected to affect cortical excitability with enhancement of SICI and minimal effect on RMT (i.e. contrary to present results).
Fig. 1. Strength duration time constant in symptomatic and pre-symptomatic SCA3 patients. The error bars represent standard error of the mean. **P < 0.01 compared to control.
Table 2 Cortical excitability in SCA3 patients.#
Central motor conduction time (ms) Resting motor threshold (%) MEP:CMAP (%) Cortical silent period (ms) Average SICI 1–7 ms (%) Peak SICI at 3 ms (%) SICI at 1 ms (%) Average ICF 10–30 ms (%)
SCA3 patients
Controls
P value
7.5 ± 0.4 62.9 ± 3.2 34.5 ± 8.9 222.7 ± 14.0 1.3 ± 1.4 0.2 ± 2.2 2.0 ± 1.9 5.6 ± 1.7
5.3 ± 0.2 59.5 ± 1.0 24.9 ± 1.8 210.2 ± 3.1 10.3 ± 0.7 15.2 ± 1.3 7.2 ± 0.8 -0.5 ± 0.8
<0.0005 0.2 0.2 0.2 <0.0005 0.001 <0.0005 0.005
SICI: short interval intracortical inhibition; ICF: intracortical facilitation. Values are represented as mean ± SEM. # Reproducible MEPs were obtained in 8 symptomatic patients. Hand dystonia precluded threshold tracking TMS in one patient.
Fig. 3) and increases in CMCT (R = 0.81, P < 0.005). Markers of peripheral motor neurodegeneration, including APB MRC, TNS, CMAP amplitude and sSD were not associated with ataxia. Taken together, these correlations suggest that alterations in cortical excitability are an early feature that evolves with ataxia and may reflect global dysfunction, reflecting neurodegeneration. 3.3. Longitudinal studies in symptomatic and pre-symptomatic SCA3 patients Three SCA3 patients with mild to moderate disease (ICARS < 20) and disease duration less than 10 years were followed longitudi-
nally for up 18 months with clinical and neurophysiological assessments. At initial assessment, all 3 patients ambulated without support, however over a study interval of 12–18 months, deterioration in gait and balance was evident (ICARS baseline 14.7.0 ± 2.8, ICARS 12–18 months 20.6 ± 3.6, P = 0.01). Cortical excitability studies demonstrated a reduction in mean SICI (SICI baseline 4.2 ± 3.6%, longitudinal 3.6 ± 2.8%, P = 0.01) and peak SICI ISI 3 ms (SICI baseline 9.3 ± 6.3%, longitudinal 2.3 ± 2.0%, P < 0.05) over the follow-up period. In contrast, other measures of cortical excitability remained stable. Two pre-symptomatic SCA3 patients were followed longitudinally for 24 and 36 months and did not develop clinical symptoms or changes in ICARS score over this time. Measures of cortical excitability, in particular resting motor threshold and SICI were also stable (average SICI patient 1 study 1 3.3%, study 2 5.7%; patient 2 study 1 4.8%, Study 2 4.1%: Peak SICI, patient 1 study 1 8.3%, study 2 10.6%; patient 2 study 1 9.5%, study 2 9.7%).
4. Discussion The present series, incorporating cross-sectional and longitudinal approaches to investigate cortical function, combined with clinical and functional measures in presymptomatic and symptomatic SCA3 patients, has identified cortical dysfunction in SCA3 patients. Measures of cortical dysfunction including reduction in resting motor threshold, short-interval intracortical inhibition and cortical silent period duration developed in presymptomatic individuals before the manifestation of ataxia or pyramidal tract signs. With clinical progression of motor symptoms and ataxia ongoing reductions in SICI appeared, accompanied by increases in CMCT, reflecting neurodegeneration of motor pathways. These findings suggest that alteration in cortical activity is associated with SCA3 pathogenesis and evident early in clinical disease, likely to provide a window of therapeutic significance. Importantly, the present study demonstrates that SCA3 onset is not confined to the cerebellum and brainstem, with cortical neuronal dysfunction also evident early in the disease. The threshold tracking TMS technique utilised in the present study identified motor-cortical disinhibition in SCA3 patients, in contrast with previous studies (Schwenkreis et al., 2002). While the apparent causes for the discordant findings remain to be elucidated, methodological differences in assessing cortical excitability, namely use of threshold tracking versus constant stimulus paired
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Fig. 2. Cortical excitability in symptomatic and pre-symptomatic SCA3 patients. In healthy control subjects short-interval intracortical inhibition (SICI) arises between interstimulus intervals of 1–7 ms. Intracortical facilitation (ICF) follows, signified by a decrease in the test stimulus intensity (A). SICI was reduced in symptomatic and pre-symptomatic SCA3 patients compared with normal controls (B). Averaged SICI, between interstimulus intervals 1–7 ms was also reduced in symptomatic and presymptomatic SCA3 patients compared with normal controls. (C) ICF was similar between pre-symptomatic SCA3 patients and healthy controls but significantly smaller compared with symptomatic SCA3 patients. The error bars represent standard error of the mean. **P < 0.005, ***P < 0.0005, for symptomatic SCA3 patients compared with normal values. àMeasurement for pre-symptomatic SCA3 patients outside the 95% confidence interval for healthy controls.
pulse technique, could in part account for the findings. Another possibility for differences between the present and previous studies, may relate to phenotypic variability of SCA3 patients across the studies, as perhaps reflected by differences in CMCT between various SCA3 cohorts. SICI is considered to be produced by GABAsecreting inhibitory cortical interneurons and in various disorders cortical disinhibition may be due to primary pathology within the motor cortex or secondary to abnormalities within more extensive neural circuits, for example the cerebello-thalamo-cortical pathways in SCA3 (Kujirai et al., 1993; Hanajima et al., 1996, 2008). In addition SICI is modulated by cortical neurotransmitter systems, including glutamate, dopamine and noradrenaline (Ziemann, 2004). Previous cellular and neuroimaging studies in SCA3 provide insights that assist in interpreting these potential pathogenic mechanisms and are discussed below. Taken together, these suggest cortical disinhibition reflects complex interactions between neural networks together with cortical pathology. Importantly, mutant ATXN3 may induce abnormalities of neuronal excitability, via Kv channel dysfunction, along with impair-
Fig. 3. The relationship between clinical measures of severity and cortical excitability parameters. (A) Average short interval intracortical inhibition (SICI) and International Cooperative Ataxia Rating Scale (ICARS, total score 0–100, greater scores indicate worsening cerebellar ataxia). (B) Central motor conduction time and ICARS.
ment of mRNA expression of proteins involved in glutamatergic neurotransmission, intracellular Ca2+ signaling and GABA (A/B) receptor subunit formation (Chen et al., 2008; Chou et al., 2008; Konno et al., 2014). The subsequent modulation in cortical neurotransmitter systems may lead to cortical dysfunction and provide further understanding of the impact of mutant ATXN3 beyond the cerebellum. The regulatory role of ATXN3 in superoxide dismutatase 2 (SOD-2) expression also increases susceptibility towards oxidative stress and cell death in SCA3, similar to SOD-1 mutations in familial ALS (Vucic and Kiernan, 2009; Araujo et al., 2011). Significantly, the formation of ATXN3 containing aggregates, the pathological characteristic of SCA3, has been shown to be produced by L-glutamate induced excitation of neurons and dependent on functional Na+ and K+ channels as well as ionotropic and voltage gated Ca2+ channels, providing an explanation for selective loss of neurons (Koch et al., 2011). The cerebellum may modulate cortical inhibitory and excitatory circuits through the cerebellothalamocortical pathway, such that alterations in cerebellar excitability may provide an alternative explanation for changes in TMS parameters in SCA3 patients (Ugawa et al., 1991). Specifically activation of cerebellar purkinje cells produces inhibition of deep cerebellar nuclei which have a disynpatic excitatory pathway to the motor cortex through the ventral thalamus (Ugawa et al., 1991; Pinto and Chen, 2001). Some previous studies in degenerative ataxia patients have reported an absence of cerebellar inhibition with reduction in ICF and normal SICI (Ugawa et al., 1994; Liepert et al., 1998), thereby suggesting
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that cerebellar modulation of SICI seems improbable. Subsequent studies, however, have reported on variability in cortical excitability across the different ataxia genotypes (Schwenkreis et al., 2002), with reduced SICI documented in SCA 14 patients, reflecting abnormalities of intracortical inhibitory circuits (Ganos et al., 2014). Consequently, differences in TMS induced measures of motor cortex activation are likely to be primary in the disease process and reflect the widespread effect of mutant ATXN3 in the cerebral cortex in SCA3 patients. Further evidence of cortical involvement in SCA3 is provided from neuroimaging and neuropathological studies. Volumetric MRI revealed motor regions of the frontal lobe were particularly affected by atrophy and correlated with clinical measures of ataxia severity while PET and SPECT scans identified cortical areas of hypoperfusion (Etchebehere et al., 2001; de Rezende et al., 2015). Further, diffusion tensor imaging showed abnormal radial diffusivity in the frontal lobe white matter (Guimaraes et al., 2013). There are reports of predominant motor neuron degeneration in SCA3 with initial presentations as motor neurone disease, which also displays early cortical dysfunction with reductions in SICI (Pinto and De Carvalho, 2008; Vucic et al., 2008; Vucic and Kiernan, 2009). Furthermore, SCA3 and ALS possess common mechanistic themes, including RNA toxicity and dysfunction in channel signaling (Hekman and Gomez, 2015). In addition, cerebellar ataxia has recently been viewed in a similar way to ALS, with dysfunction of networks within the cerebellum and CNS (Tada et al., 2015). As such, the identification of cortical dysfunction in SCA3 patients in the present study may serve to further unite these concepts in SCA3 with the current understanding of ALS. Previous studies have investigated mechanisms underlying fasciculations and cramps in SCA3 patients (Kanai et al., 2003; Franca et al., 2008). The present study confirmed peripheral nerve hyperexcitability in SCA3, characterized by markedly prolonged sSD. While sSD is prolonged in SCA3, ALS and spinal muscular atrophy (SMA) patients, muscle cramps and fasciculations are not particularly prominent in SMA and cortical function is conserved (Farrar et al., 2011, 2012). These comparisons suggest cortical dysfunction may also drive muscle cramps and fasciculations. 4.1. Therapeutic implications Findings from the present studies support significant pathophysiological effects of mutant ATXN3 within the motor cortex and descending pathways in SCA3. These changes may be mediated by oxidative stress, alterations in neuronal signaling and biophysical properties of ion channels, suggesting several treatment approaches to improve motor function and reduce neurodegeneration in SCA3. Importantly, riluzole, the only disease modifying treatment for ALS (Miller et al., 2012), has been shown to partially normalize cortical excitability in ALS patients with proposed mechanisms including inhibition of glutamatergic transmission and modulation of Na+ channel function (Vucic et al., 2013). Moreover, riluzole has recently been shown to reduce ataxia after 12 months in a double blind placebo controlled trial in patients with ataxia of different aetiologies, supporting further pursuit of this approach in SCA3 (Romano et al., 2015). If cortical abnormalities may be consistently identified early in the disease course, and thereby prompt introduction of neuroprotective agents to improve motor function, it may reasonably be expected that disease progression could be stabilized. Funding source This work was supported by funding to Forefront, a collaborative research group dedicated to the study of motor neurone
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disease, from the National Health and Medical Research Council of Australia program grant (#1037746).
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Clinical Neurophysiology journal homepage: www.elsevier.com/locate/clinph
Motor cortical dysfunction develops in spinocerebellar ataxia type 3 Michelle A. Farrar a,⇑, Steve Vucic b, Garth Nicholson c, Matthew C. Kiernan d a
Discipline of Paediatrics, School of Women’s and Children’s Health, UNSW Medicine, The University of New South Wales, Sydney, Australia Department of Neurology, Westmead Hospital and Western Clinical School, University of Sydney, Sydney, Australia c ANZAC Research Institute, University of Sydney, Concord Hospital, New South Wales, Australia d Sydney Medical School, Brain & Mind Centre, University of Sydney, Sydney, Australia b
a r t i c l e
i n f o
Article history: Accepted 7 September 2016 Available online 15 September 2016 Keywords: Spinocerebellar ataxia type 3 Corticomotoneuron Transcranial magnetic stimulation
h i g h l i g h t s Cortical dysfunction is an early feature of SCA3 and associated with motor symptoms and ataxia. Identifying cortical neuronal dysfunction in SCA3 may be of therapeutic significance. SCA3 onset is not confined to the cerebellum and brainstem.
a b s t r a c t Objective: Spinocerebellar ataxia type 3 (SCA3) is an inherited neurodegenerative disorder characterized by cerebellar ataxia and variable expression of clinical features beyond the cerebellum. To gain further insights into disease pathophysiology, the present study explored motor cortex function in SCA3 to determine whether cortical dysfunction was present and if this contributed to the development of clinical manifestations. Methods: Clinical phenotyping and longitudinal assessments were combined with central (thresholdtracking transcranial magnetic stimulation) and peripheral (nerve excitability) techniques in 11 genetically characterized SCA3 patients. Results: Short-interval intracortical inhibition was significantly reduced in presymptomatic and symptomatic SCA3 patients (1.3 ± 1.4%) compared to healthy controls (10.3 ± 0.7%, P < 0.0005), with changes evident prior to clinical onset of ataxia and related to worsening severity (R = 0.78, P < 0.005). Central motor conduction time was also significantly prolonged in presymptomatic and symptomatic SCA3 patients (7.5 ± 0.4 ms) compared to healthy controls (5.3 ± 0.2 ms, P < 0.0005) and related to clinical severity (R = 0.81, P < 0.005). Markers of peripheral motor neurodegeneration and excitability did not correlate with cortical hyperexcitability or ataxia. Conclusions: Simultaneous investigation of clinical status, and central and peripheral nerve function has identified progressive cortical dysfunction in SCA3 patients related to the development of ataxia. Significance: These findings suggest alteration in cortical activity is associated with SCA3 pathogenesis and neurodegeneration. Ó 2016 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease and the most common hereditary spinocerebellar ataxia. Also known as Machado–Joseph disease, there is a wide spectrum of clinical expression in addition to ataxia, that may include progressive external ophthalmoplegia, dysarthria, dysphagia, pyramidal disturbances, dystonia, parkinsonism, ⇑ Corresponding author at: Department of Neurology, Sydney Children’s Hospital, High St., Randwick, NSW 2031, Australia. Fax: +61 2 93821580. E-mail address: [email protected] (M.A. Farrar).
sleep disorders, rigidity, peripheral neuropathy and distal muscle atrophy, suggestive of neurodegeneration beyond that originally described in the cerebellum and brainstem (Boller and Segarra, 1969; Pogacar et al., 1978; Rosenberg, 1992). Symptoms usually begin between the ages of 20 and 50 years and loss of motor control is ultimately so severe that survival is limited to 15–30 years following clinical onset. SCA3 presents clinically when neuronal loss within the cerebellum and brainstem has become advanced and irreversible, emphasizing the need to further understand pathophysiological processes, particularly the detection of early neuronal dysfunction, such that therapeutic strategies may be directed at earlier stages of disease.
http://dx.doi.org/10.1016/j.clinph.2016.09.005 1388-2457/Ó 2016 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
M.A. Farrar et al. / Clinical Neurophysiology 127 (2016) 3418–3424
SCA3 is caused by an unstable expansion of CAG triplet repeats in the ataxin-3 (ATXN3) gene on chromosome 14, resulting in an abnormal expanded polyglutamine repeat in the ubiquitously expressed ATXN3 protein, which makes the protein highly susceptible to misfolding and aggregation (Kawaguchi et al., 1994). Abnormalities beyond the cerebellum and brainstem have been demonstrated with neuroimaging and neuropathological studies to reveal neurodegeneration affecting the thalamus, and frontal, temporal, limbic, parietal and occipital lobes (D’Abreu et al., 2012; de Rezende et al., 2015). In addition, loss of motor neurons in the spinal cord and has been established, and may be associated with muscle cramps, amyotrophy and fasciculations, such that SCA3 may be regarded as a chronic motor neurone disorder (Kinoshita et al., 1995; Franca et al., 2008). Although the mechanisms by which the misfolded ATXN3 protein induces motor neuron toxicity in SCA3 remain elusive, impairment of axonal transport, oxidative stress, neuronal signaling and RNA toxicity have all been reported (Li et al., 2015). Importantly, the mutant ATXN3 protein, which appears to be sequestered ubiquitously within the central nervous system, may induce neuronal hyperexcitability, which in turn may mediate the underlying pathogenesis in SCA3 (Jeub et al., 2006; Chen et al., 2008; Shakkottai et al., 2011). The impact of mutant ATXN3 on motor neurone function throughout the cortex and spinal cord remains to be fully elucidated and is of pathophysiological and therapeutic importance. Perhaps of relevance, cortical dysfunction has been linked to neurodegeneration in amyotrophic lateral sclerosis (Vucic and Kiernan, 2006; Vucic et al., 2008). Recently, the application of threshold tracking transcranial magnetic stimulation (TMS) techniques has enabled the early detection of upper motor neuron dysfunction (Menon et al., 2015). As such, the present study utilized clinical assessments combined with cortical and peripheral nerve assessment to further clarify the processes involved in neurodegeneration in SCA3.
2. Methods Clinical phenotyping and assessment was combined with conventional and specialized neurophysiological assessments in 11 patients with genetically confirmed SCA3. A subset of presymptomatic SCA3 patients and symptomatic SCA3 patients with mild to moderate disease and duration less than 10 years were followed longitudinally for up to 36 months to characterize progression. Diagnosis was confirmed by DNA testing showing an expanded CAG repeat in the ATXN3 gene on chromosome 14q32.1 in all patients. All patients gave informed consent to the procedures, which were approved by the South Eastern Sydney and Illawarra Area Health Service Human Research Ethics Committee. SCA3 patients underwent clinical assessments of cerebellar ataxia using the International Cooperative Ataxia Rating Scale (ICARS), a 100-point semi quantitative scale that provides a total score (0–100) and four subscores: posture and stance (0–34), kinetic cerebellar function (0–52), dysarthria (0–8) and oculomotor dysfunction (0–6) (Trouillas et al., 1997). Greater scores indicate worsening cerebellar ataxia. The Total Neuropathy Scale-clinical version (TNSc) (Cornblath et al., 1999), was also assessed for each patient. The TNSc combines information obtained from grading of symptoms and signs to clinically quantify neuropathy and includes seven categories with severity rankings from 0 (none) to 4 (very severe): sensory symptoms; motor symptoms; autonomic symptoms; pinprick sensibility; vibration sensibility (128-Hz tuning fork); strength; and deep tendon reflexes (total score 0–28). Muscle strength was clinically assessed using the MRC for abductor pollicis brevis (APB), as this muscle was utilised for excitability testing.
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2.1. Neurophysiological studies In addition to clinical examination, cortical function was assessed using previously described threshold tracking transcranial magnetic stimulation (TMS) techniques (Vucic et al., 2006). Two high-power magnetic stimulators connected to a BiStim device (Magstim Co., Whitland, South West Wales, UK) produced magnetic currents and enabled conditioning and test stimuli to be independently set and administered through the one coil. These were applied over the motor cortex utilizing a 90 mm circular coil, oriented to induce current flow in a posterior-anterior direction and the resultant motor evoked potential (MEP) was recorded from APB. The optimal position for a motor evoked potential (MEP) from the right APB muscle was obtained by adjusting the coil position. The MEP was measured from peak to peak and the threshold tracking target set to 0.2 mV, the midpoint of the steepest portion of the logarithmic stimulus response curve (Fisher et al., 2002). This threshold tracking technique overcomes the marked variability in the MEP amplitude with successive stimuli related to spontaneous variations in the resting threshold of cortical neurons, as may occur with conventional constant paired test stimuli TMS techniques in which changes in the amplitude of the test response is measured (Kiers et al., 1993; Weber and Eisen, 2002). The APB muscle was at rest for the recording of all neurophysiological parameters (except the cortical silent period) and the surface EMG closely observed for the presence of voluntary activity. Single-stimulus TMS was used to record stimulus–response (SR) curves, resting motor threshold (RMT), central motor conduction time (CMCT) and cortical silent period (CSP) duration. Three stimuli were delivered at each level of stimulus intensity. RMT was the stimulus intensity that maintained a target MEP of 0.2 mV, which was the middle of the steepest portion of the logarithmic stimulus response curve and the MEP was measured from peak to peak. The maximum MEP amplitude (MEP/CMAP, %) and minimum MEP onset latency (ms) was calculated from the average of three stimuli delivered at 150% RMT. CMCT (ms) was calculated according to the F-wave method (Claus, 1990). CSP was produced by a singlepulse TMS while subjects performed a weak voluntary contraction. The duration of the silent period was determined from the beginning of the MEP to the return of EMG activity. Threshold tracking paired-pulse TMS studies were performed (Vucic et al., 2006). In the paired-pulse paradigm a subthreshold conditioning stimulus preceded a suprathreshold test stimulus at increasing interstimulus intervals (ISIs) as follows: 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 7, 10, 15, 20, and 30 ms. Three stimulus combinations were delivered sequentially and monitored: (i) the intensity required to produce the unconditioned test response (RMT); (ii) the subthreshold conditioning stimulus alone, verifying that the subject remained relaxed and no MEP response was produced; (iii) conditioning and test stimuli in combination. When two consecutive MEP responses were within 20% of the target response (0.2 mV), tracking was deemed acceptable and the computer advanced to the next ISI. The subthreshold conditioning stimulus (70% RMT) was such that it did not evoke a response. Intracortical inhibition induced by a conditioning stimulus was measured as the increase in the test stimulus intensity required to produce the target MEP and calculated with the following formula:
Inhibition ¼ ðConditioned test stimulus intensity RMTÞ=RMT 100 Two distinct physiological phases of SICI have been recognized in healthy subjects, occurring at ISIs 6 1 ms and 3 ms, ascribed to axonal refractoriness and activation of different inhibitory circuits, and both are reported (Vucic et al., 2006; Fisher et al., 2002). Facilitation was measured as the decrease in the conditioned test stimulus intensity required to produce the target MEP. In normal
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subjects, a subthreshold conditioning stimulus inhibits the response of the test stimulus over intervals of 1–7 ms, termed short intracortical inhibition (SICI). The response is facilitated at intervals from about 10 to 20 ms, termed intracortical facilitation (ICF). Peripheral nerve function was also assessed during the same sitting using the TROND protocols of the multiple nerve excitability QTRACs software (Institute of Neurology, London, England), a fully automated sequence of tests (stimulus–response, strength–duration, threshold electrotonus, recovery cycle, current-threshold relationship) (Kiernan et al., 2000). The median nerve was stimulated electrically at the wrist and the resultant CMAP was recorded from APB. MEP and CMAP recordings were filtered (3 Hz–3 kHz) and amplified with a GRASS ICP511 AC amplifier (Grass-Telefactor, Astro-Med Inc., West Warwick, RI, USA) and sampled with a 12bit data acquisition card at 10 kHz (National Instruments PCIMIO-16E-4). QTRACS software (Institute of Neurology, Queen Square, London, UK) controlled data acquisition and stimulation delivery (magnetic and electrical). 2.2. Data analysis Cortical excitability in symptomatic and pre-symptomatic SCA3 patients was compared with 62 age-matched controls (31 males; age range 23–83 years; mean 45.8 years). Peripheral nerve excitability parameters were compared with 40 age-matched controls (22 males; age range 23– 81 years; mean 39.5 years). The patients and controls did not differ significantly in skin temperature (SCA3; 32.1 ± 0.8 °C; controls 32.5 ± 0.1 °C) or gender ratio. All results were expressed as mean ± standard error of the mean (SEM). Mann–Whitney U tests were used to compare mean differences between SCA3 patients and controls with post hoc testing (sequential Bonferroni correction) used for multiple comparisons. A probability (P) value of < 0.005 was considered statistically significant. Measurements between pre-symptomatic SCA3 patients were compared to the 95% confidence intervals of healthy controls (and considered significant if outside this range). Paired Student’s t tests were used to compare longitudinal paired excitability recordings and clinical assessments to characterize progression and P < 0.05 was considered statistically significant. Correlations between neurophysiological indices and clinical measurements of disease severity were analysed using the Spearman’s rank test. 3. Results The study population included 5 males and 6 females (mean age 46 years, range 30–64 years). Clinical severity varied, ranging from presymptomatic to severe as reflected by the broad spectrum in ICARS score (range 0–59, median 27), with the latter having profound ataxia and inability to stand or ambulate (Table 1). Neurological examination of presymptomatic individuals was normal. The first reported symptom universally related to gait ataxia. In the present cohort, a slowly progressive ‘‘ataxia-plus’’ syndrome evolved indicative of central and peripheral neurodegeneration, with progressive disturbances of gait and balance, cramps of the arms, legs, face, chest and neck, numbness and paraesthesias of the hands and feet, and fasciculations commonly reported. Distal muscle atrophy, dysarthria, pyramidal signs (spasticity and rigidity) and external ophthalmoplegia were common clinical signs in manifesting patients. While clinical features of peripheral neuropathy were evident in 54% of patients (TNS 3.0 ± 1.5), APB muscle strength was similar between groups (MRC APB: SCA3 4.7 ± 0.2; controls 5.0 ± 0.0, P = 0.1). Amyotrophy was evident in 36%, all with disease onset aged greater than 50 years. Pathological muscle cramps were experienced in 83% of patients. Disease dura-
tion, the period from symptom onset to the date of assessment, correlated with clinical measures of disease burden, including ICARS posture and gait subscale (R = 0.75, P < 0.01), reflecting neurodegeneration. In terms of axonal excitability studies, the threshold currents required to elicit a response were similar in SCA3 patients and controls (SCA3, 3.2 ± 1.1 mV; controls, 3.7 ± 1.1 mV). Strength duration time constant (sSD), an indirect measure of nodal persistent Na+ conductances was significantly prolonged in SCA3 patients compared to controls (SCA3, 0.51 ± 0.03 ms; controls, 0.43 ± 0.01 ms, P < 0.01, Fig. 1), but was within normal limits in presymptomatic SCA3 patients (Patient 1 0.44 ms; Patient 2 0.42 ms; controls 95%CI 0.31–0.45 ms). In contrast, there were no significant differences in rheobase, threshold electrotonus, recovery cycle or current–threshold relationship in SCA3 patients when compared with controls. Motor amplitudes were relatively preserved in all SCA3 patients (SCA3 7.1 ± 1.1 mV; controls 6.9 ± 1.1 mV, P = 0.8). 3.1. Cortical function in pre-symptomatic and symptomatic SCA3 patients The motor cortex was excitable in all SCA3 patients (Table 2). Paired-pulse TMS, disclosed that SICI was significantly reduced in symptomatic SCA3 patients and pre-symptomatic individuals when compared with controls (P < 0.0005, Fig. 2, Table 2). Mean SICI (between ISI 1 and 7 ms) was significantly reduced (symptomatic SCA3 1.3 ± 1.4%, controls 10.3 ± 0.7%, P < 0.0005; presymptomatic SCA3 patient 1 4.8%, patient 2 5.7%, controls 95% CI 9.0–11.7), as was peak SICI at ISI 1 ms (symptomatic SCA3 2.0 ± 1.9%, controls 7.2 ± 0.8%, P < 0.0005; pre-symptomatic SCA3 patient 1 1.0%, patient 2 5.5%, controls 95% CI 5.6–8.8) and 3 ms (symptomatic SCA3 0.2 ± 2.2%, controls 15.2 ± 1.3%, P = 0.001; pre-symptomatic SCA3 patient 1 9.5%, patient 2 10.6%, controls 95% CI 12.7–17.6) Notably SICI was absent in 4 SCA3 patients. ICF was significantly increased in symptomatic SCA3 patients when compared with controls (SCA3 5.6 ± 1.7%, controls 0.5 ± 0.8%, P = 0.005, Fig. 2). In contrast, ICF was normal in presymptomatic SCA3 subjects (patient 1 1.3%, patient 2 1.2%, controls 95% CI 2.0 to 1.3%). Central motor conduction time was significantly prolonged in all SCA3 patients when compared to controls (SCA3 7.5 ± 0.4 ms; controls 5.3 ± 0.2 ms, P < 0.0005; pre-symptomatic SCA3 Patient 1 6.7 ms; Patient 2 5.7 ms, controls 95% CI 4.9–5.7 ms). The RMT remained stable throughout the testing period and was similar between symptomatic SCA3 patients and controls (SCA3 62.9 ± 3.2%; controls 59.5 ± 1.0%, P = 0.2). Interestingly RMT was reduced in pre-symptomatic patients (pre-symptomatic SCA3 patient 1 58.7%; Patient 2 47.5%, controls 95% CI 57.5–61.5%). Of further relevance, the MEP amplitude, expressed as percentage of CMAP response, was maintained in SCA3 and presymptomatic patients when compared to healthy controls (symptomatic SCA3 34.5 ± 8.9%; controls 24.9 ± 1.8%, P = 0.2; pre-symptomatic SCA3 patient 1 17.9%; Patient 2 22.2%; controls 95% CI 21.3–28.3 ms). In addition, the CSP in symptomatic SCA3 patients was similar to healthy controls (symptomatic SCA3 222.7 ± 14.0 ms; controls 210.2 ± 3.1 ms, P = 0.2). In contrast, the CSP was reduced in presymptomatic SCA3 patients (pre-symptomatic SCA3 patient 1 158.6 ms; Patient 2 173.4 ms, controls 95% CI 204.1–216.8 ms). 3.2. Correlation with clinical parameters Combining measures of cortical and peripheral nerve function with clinical assessment, it was evident that cerebellar ataxia (total ICARS) was significantly associated with a greater reduction in SICI (mean SICI R = 0.78, P < 0.005; Peak SICI R = 0.88, P < 0.005,
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M.A. Farrar et al. / Clinical Neurophysiology 127 (2016) 3418–3424 Table 1 Clinical details for 11 patients with Spinocerebellar ataxia type 3 (SCA3). SCA3 patients
Age (years)
Age of clinical onset, disease duration (years)
Number of CAG repeats
ICARS score
TNS score
Current medications
Additional clinical features
1 2 3 4 5 6 7 8* 9 10 11 Median
34 40 30 33 35 40 64 52 59 60 47 40.0
Presymptomatic Presymptomatic 26, 4 30, 3 28, 7 31, 9 60, 4 38, 14 45, 14 50, 10 24, 23 31, 9
63 70 74 73 74 73 72 69 72 69 71 71.5
0 1 16 16 20 27 27 35 49 49 59 27
0 0 0 0 0 8 9 9 11 10 2 3.0
None Venlafaxine# None Gabapentin,# Amitriptyline# None None None Temazepam,# Biperiden, baclofen,# tramadol# None None Temazepam#
None None Pyramidal Pyramidal Pyramidal Pyramidal Amyotrophy Pyramidal, hand dystonia, amyotrophy Amyotrophy Amyotrophy Bradykinesia
The patients with SCA3 were clinically graded using the International Cooperative Ataxia Rating Scale (ICARS), a 100-point semi quantitative scale that provides a total score (0–100) and four sub-scores: posture and stance (0–34), kinetic cerebellar function (0–52), dysarthria (0–8) and oculomotor dysfunction (0–6). Greater scores indicate worsening cerebellar ataxia. Peripheral neuropathy was assessed using the Total Neuropathy Scale-clinical version (TNSc) with a higher score reflecting severity of neuropathy, range 0–28 see Section 2. Pyramidal signs include rigidity and spasticity. * Hand dystonia precluded assessment of cortical excitability by means of TTMS in patient 8. # This medication may be expected to affect cortical excitability with enhancement of SICI and minimal effect on RMT (i.e. contrary to present results).
Fig. 1. Strength duration time constant in symptomatic and pre-symptomatic SCA3 patients. The error bars represent standard error of the mean. **P < 0.01 compared to control.
Table 2 Cortical excitability in SCA3 patients.#
Central motor conduction time (ms) Resting motor threshold (%) MEP:CMAP (%) Cortical silent period (ms) Average SICI 1–7 ms (%) Peak SICI at 3 ms (%) SICI at 1 ms (%) Average ICF 10–30 ms (%)
SCA3 patients
Controls
P value
7.5 ± 0.4 62.9 ± 3.2 34.5 ± 8.9 222.7 ± 14.0 1.3 ± 1.4 0.2 ± 2.2 2.0 ± 1.9 5.6 ± 1.7
5.3 ± 0.2 59.5 ± 1.0 24.9 ± 1.8 210.2 ± 3.1 10.3 ± 0.7 15.2 ± 1.3 7.2 ± 0.8 -0.5 ± 0.8
<0.0005 0.2 0.2 0.2 <0.0005 0.001 <0.0005 0.005
SICI: short interval intracortical inhibition; ICF: intracortical facilitation. Values are represented as mean ± SEM. # Reproducible MEPs were obtained in 8 symptomatic patients. Hand dystonia precluded threshold tracking TMS in one patient.
Fig. 3) and increases in CMCT (R = 0.81, P < 0.005). Markers of peripheral motor neurodegeneration, including APB MRC, TNS, CMAP amplitude and sSD were not associated with ataxia. Taken together, these correlations suggest that alterations in cortical excitability are an early feature that evolves with ataxia and may reflect global dysfunction, reflecting neurodegeneration. 3.3. Longitudinal studies in symptomatic and pre-symptomatic SCA3 patients Three SCA3 patients with mild to moderate disease (ICARS < 20) and disease duration less than 10 years were followed longitudi-
nally for up 18 months with clinical and neurophysiological assessments. At initial assessment, all 3 patients ambulated without support, however over a study interval of 12–18 months, deterioration in gait and balance was evident (ICARS baseline 14.7.0 ± 2.8, ICARS 12–18 months 20.6 ± 3.6, P = 0.01). Cortical excitability studies demonstrated a reduction in mean SICI (SICI baseline 4.2 ± 3.6%, longitudinal 3.6 ± 2.8%, P = 0.01) and peak SICI ISI 3 ms (SICI baseline 9.3 ± 6.3%, longitudinal 2.3 ± 2.0%, P < 0.05) over the follow-up period. In contrast, other measures of cortical excitability remained stable. Two pre-symptomatic SCA3 patients were followed longitudinally for 24 and 36 months and did not develop clinical symptoms or changes in ICARS score over this time. Measures of cortical excitability, in particular resting motor threshold and SICI were also stable (average SICI patient 1 study 1 3.3%, study 2 5.7%; patient 2 study 1 4.8%, Study 2 4.1%: Peak SICI, patient 1 study 1 8.3%, study 2 10.6%; patient 2 study 1 9.5%, study 2 9.7%).
4. Discussion The present series, incorporating cross-sectional and longitudinal approaches to investigate cortical function, combined with clinical and functional measures in presymptomatic and symptomatic SCA3 patients, has identified cortical dysfunction in SCA3 patients. Measures of cortical dysfunction including reduction in resting motor threshold, short-interval intracortical inhibition and cortical silent period duration developed in presymptomatic individuals before the manifestation of ataxia or pyramidal tract signs. With clinical progression of motor symptoms and ataxia ongoing reductions in SICI appeared, accompanied by increases in CMCT, reflecting neurodegeneration of motor pathways. These findings suggest that alteration in cortical activity is associated with SCA3 pathogenesis and evident early in clinical disease, likely to provide a window of therapeutic significance. Importantly, the present study demonstrates that SCA3 onset is not confined to the cerebellum and brainstem, with cortical neuronal dysfunction also evident early in the disease. The threshold tracking TMS technique utilised in the present study identified motor-cortical disinhibition in SCA3 patients, in contrast with previous studies (Schwenkreis et al., 2002). While the apparent causes for the discordant findings remain to be elucidated, methodological differences in assessing cortical excitability, namely use of threshold tracking versus constant stimulus paired
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Fig. 2. Cortical excitability in symptomatic and pre-symptomatic SCA3 patients. In healthy control subjects short-interval intracortical inhibition (SICI) arises between interstimulus intervals of 1–7 ms. Intracortical facilitation (ICF) follows, signified by a decrease in the test stimulus intensity (A). SICI was reduced in symptomatic and pre-symptomatic SCA3 patients compared with normal controls (B). Averaged SICI, between interstimulus intervals 1–7 ms was also reduced in symptomatic and presymptomatic SCA3 patients compared with normal controls. (C) ICF was similar between pre-symptomatic SCA3 patients and healthy controls but significantly smaller compared with symptomatic SCA3 patients. The error bars represent standard error of the mean. **P < 0.005, ***P < 0.0005, for symptomatic SCA3 patients compared with normal values. àMeasurement for pre-symptomatic SCA3 patients outside the 95% confidence interval for healthy controls.
pulse technique, could in part account for the findings. Another possibility for differences between the present and previous studies, may relate to phenotypic variability of SCA3 patients across the studies, as perhaps reflected by differences in CMCT between various SCA3 cohorts. SICI is considered to be produced by GABAsecreting inhibitory cortical interneurons and in various disorders cortical disinhibition may be due to primary pathology within the motor cortex or secondary to abnormalities within more extensive neural circuits, for example the cerebello-thalamo-cortical pathways in SCA3 (Kujirai et al., 1993; Hanajima et al., 1996, 2008). In addition SICI is modulated by cortical neurotransmitter systems, including glutamate, dopamine and noradrenaline (Ziemann, 2004). Previous cellular and neuroimaging studies in SCA3 provide insights that assist in interpreting these potential pathogenic mechanisms and are discussed below. Taken together, these suggest cortical disinhibition reflects complex interactions between neural networks together with cortical pathology. Importantly, mutant ATXN3 may induce abnormalities of neuronal excitability, via Kv channel dysfunction, along with impair-
Fig. 3. The relationship between clinical measures of severity and cortical excitability parameters. (A) Average short interval intracortical inhibition (SICI) and International Cooperative Ataxia Rating Scale (ICARS, total score 0–100, greater scores indicate worsening cerebellar ataxia). (B) Central motor conduction time and ICARS.
ment of mRNA expression of proteins involved in glutamatergic neurotransmission, intracellular Ca2+ signaling and GABA (A/B) receptor subunit formation (Chen et al., 2008; Chou et al., 2008; Konno et al., 2014). The subsequent modulation in cortical neurotransmitter systems may lead to cortical dysfunction and provide further understanding of the impact of mutant ATXN3 beyond the cerebellum. The regulatory role of ATXN3 in superoxide dismutatase 2 (SOD-2) expression also increases susceptibility towards oxidative stress and cell death in SCA3, similar to SOD-1 mutations in familial ALS (Vucic and Kiernan, 2009; Araujo et al., 2011). Significantly, the formation of ATXN3 containing aggregates, the pathological characteristic of SCA3, has been shown to be produced by L-glutamate induced excitation of neurons and dependent on functional Na+ and K+ channels as well as ionotropic and voltage gated Ca2+ channels, providing an explanation for selective loss of neurons (Koch et al., 2011). The cerebellum may modulate cortical inhibitory and excitatory circuits through the cerebellothalamocortical pathway, such that alterations in cerebellar excitability may provide an alternative explanation for changes in TMS parameters in SCA3 patients (Ugawa et al., 1991). Specifically activation of cerebellar purkinje cells produces inhibition of deep cerebellar nuclei which have a disynpatic excitatory pathway to the motor cortex through the ventral thalamus (Ugawa et al., 1991; Pinto and Chen, 2001). Some previous studies in degenerative ataxia patients have reported an absence of cerebellar inhibition with reduction in ICF and normal SICI (Ugawa et al., 1994; Liepert et al., 1998), thereby suggesting
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that cerebellar modulation of SICI seems improbable. Subsequent studies, however, have reported on variability in cortical excitability across the different ataxia genotypes (Schwenkreis et al., 2002), with reduced SICI documented in SCA 14 patients, reflecting abnormalities of intracortical inhibitory circuits (Ganos et al., 2014). Consequently, differences in TMS induced measures of motor cortex activation are likely to be primary in the disease process and reflect the widespread effect of mutant ATXN3 in the cerebral cortex in SCA3 patients. Further evidence of cortical involvement in SCA3 is provided from neuroimaging and neuropathological studies. Volumetric MRI revealed motor regions of the frontal lobe were particularly affected by atrophy and correlated with clinical measures of ataxia severity while PET and SPECT scans identified cortical areas of hypoperfusion (Etchebehere et al., 2001; de Rezende et al., 2015). Further, diffusion tensor imaging showed abnormal radial diffusivity in the frontal lobe white matter (Guimaraes et al., 2013). There are reports of predominant motor neuron degeneration in SCA3 with initial presentations as motor neurone disease, which also displays early cortical dysfunction with reductions in SICI (Pinto and De Carvalho, 2008; Vucic et al., 2008; Vucic and Kiernan, 2009). Furthermore, SCA3 and ALS possess common mechanistic themes, including RNA toxicity and dysfunction in channel signaling (Hekman and Gomez, 2015). In addition, cerebellar ataxia has recently been viewed in a similar way to ALS, with dysfunction of networks within the cerebellum and CNS (Tada et al., 2015). As such, the identification of cortical dysfunction in SCA3 patients in the present study may serve to further unite these concepts in SCA3 with the current understanding of ALS. Previous studies have investigated mechanisms underlying fasciculations and cramps in SCA3 patients (Kanai et al., 2003; Franca et al., 2008). The present study confirmed peripheral nerve hyperexcitability in SCA3, characterized by markedly prolonged sSD. While sSD is prolonged in SCA3, ALS and spinal muscular atrophy (SMA) patients, muscle cramps and fasciculations are not particularly prominent in SMA and cortical function is conserved (Farrar et al., 2011, 2012). These comparisons suggest cortical dysfunction may also drive muscle cramps and fasciculations. 4.1. Therapeutic implications Findings from the present studies support significant pathophysiological effects of mutant ATXN3 within the motor cortex and descending pathways in SCA3. These changes may be mediated by oxidative stress, alterations in neuronal signaling and biophysical properties of ion channels, suggesting several treatment approaches to improve motor function and reduce neurodegeneration in SCA3. Importantly, riluzole, the only disease modifying treatment for ALS (Miller et al., 2012), has been shown to partially normalize cortical excitability in ALS patients with proposed mechanisms including inhibition of glutamatergic transmission and modulation of Na+ channel function (Vucic et al., 2013). Moreover, riluzole has recently been shown to reduce ataxia after 12 months in a double blind placebo controlled trial in patients with ataxia of different aetiologies, supporting further pursuit of this approach in SCA3 (Romano et al., 2015). If cortical abnormalities may be consistently identified early in the disease course, and thereby prompt introduction of neuroprotective agents to improve motor function, it may reasonably be expected that disease progression could be stabilized. Funding source This work was supported by funding to Forefront, a collaborative research group dedicated to the study of motor neurone
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disease, from the National Health and Medical Research Council of Australia program grant (#1037746).
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