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Cognition is affected early in Huntington's disease, and in HD animal models there is evidence that this reflects abnormal synaptic plasticity. We investigated whether there is evidence for abnormal synaptic plasticity using the human motor cortex-rTMS model, and if so, if there is any difference between premanifest HD gene carriers and very early manifest HD patients or any relationship with ratings of the severity of motor signs.
Fifteen HD gene carriers (7 premanifest, 8 very early manifest) and 14 control participants were given a continuous train of 100 bursts of theta burst stimulation (cTBS: three pulses at 50 Hz and 80% AMT repeated every 200ms). The size of the motor evoked potential was measured at regular intervals until 21 minutes after cTBS.
HD gene carriers and controls responded differently to theta burst stimulation (F4.9,131.9=1.37, p=0.048) with controls having more inhibition than HD gene carriers (F1,27=13.3, p=0.001). Across all time points mean inhibition differed between the groups (F2,26=6.32, p=0.006); controls had more inhibition than either HD gene carrier subgroup (p=0.006 for premanifest and p=0.009 for early symptomatic) whereas there was no difference between premanifest and early symptomatic HD gene carriers. The measure of cortical plasticity was not associated with any clinical ratings (UHDRS motor score, estimate of age at onset).
Motor cortex plasticity is abnormal in HD gene carriers but is not closely linked to the development of motor signs of HD.
Huntington's disease (HD) is caused by a CAG repeat expansion in the IT15 gene that leads to abnormal movements, behavioural and psychiatric problems, and declining powers of cognition . Several of the symptoms of HD involve deficits in learning and memory. These encompass difficulties with attention, working and verbal episodic memory and behaviour inhibition, as well as recognition of emotion and explicit learning [2-4]. Interestingly, incidental learning seems relatively spared, at least in the premanifest and early manifest stages of the disease . Given the importance of synaptic changes such as long term potentiation and depression (LTP/LTD) in these processes , it is perhaps not surprising that mouse models of HD show abnormal plasticity even before these animals have signs of the disease or neurone loss [6-8]. The animals have behavioural abnormalities with concomitant deficits in hippocampal LTP and LTD, and mossy fibre potentiation [9-13]. While many studies in HD mouse models examined the hippocampus and the striatum there is also evidence for altered cortical plasticity .
The aim of the present study was to explore whether similar abnormalities of synaptic plasticity can be observed in human carriers of the HD mutation. Although HD is often seen as a disease of the basal ganglia, there is good evidence from structural imaging and microarray analysis to suggest that cortical areas, in particular the motor cortex, are involved early in HD [14-18]. Thus we used a recently designed measure of LTD-like plasticity in the motor cortex involving repetitive transcranial magnetic stimulation (TMS) given in a theta burst pattern (theta burst stimulation, TBS) which has after-effects on the excitability of corticospinal projections that last for 30-60min after the end of stimulation . Given that these disappear after pretreatment with drugs that interfere with NMDA receptor function , they are thought to be produced by changes in synaptic plasticity similar to those observed in animal experiments. In the present study we used this method to investigate motor cortex plasticity in a homogeneous group of premanifest and very early manifest HD patients. We were interested in two questions: is there evidence for abnormal synaptic plasticity in the motor cortex, and if so, is there any difference between premanifest HD gene carriers and very early manifest HD patients or any relationship with ratings of the severity of motor signs.
Fifteen HD gene carriers (12 women, mean age 41.6 years, range 28-64; for CAG repeat length see table 1) and 14 control participants (9 women, mean age 42.4 years, range 28-62) were recruited. The same clinician (SJT) with long standing experience in HD examined patients clinically; motor, psychiatric and cognitive signs were scored using the Unified Huntington's Disease Rating Scale (UHDRS) . Seven HD gene carriers were clinically premanifest according to the UHDRS (diagnostic confidence score of less than 4), and 8 HD gene carriers were early manifest (clinical stage 1 ). The predicted time to symptom onset was estimated according to Langbehn et al . All HD gene carriers apart from two scored > 27 on the Mini-Mental State Examination (MMSE) . Two early HD patients scored 26 on the MMSE, possibly suggesting mild cognitive impairment. Neurological examination was normal in controls, and there was no history of psychiatric disorders or substance abuse. HD gene carriers and controls were unmedicated at the time of the study. Participants gave informed written consent according to the Declaration of Helsinki, and the local ethics committee approved the study protocol.
Surface electromyograms (EMG) were recorded from the right first dorsal interossoeus (FDI) muscle using silver/silver-chloride disc surface electrodes (1 cm diameter) in a belly tendon montage. The EMG signal was amplified and analogue filtered (30Hz to 1kHz) with a Digitimer D150 amplifier (Digitimer Ltd., Welwyn Garden City, UK). Data (sampling rate 4kHz) was digitised for off-line analysis using Signal software (Cambridge Electronic Devices, Cambridge, UK).
HD gene carriers and controls were seated relaxed in a comfortable chair. Magnetic stimuli were given with a hand-held figure-of-eight coil (outer winding diameter 9cm) connected to a High Power Magstim 200 stimulator (Magstim Co., Whitland, Dyfed, UK). This stimulator induces a pulse with monophasic waveform. For repetitive transcranial magnetic stimulation we used a Super Rapid Magstim stimulator (Magstim Co., Whitland, Dyfed, UK). This stimulator generates a magnetic pulse with a biphasic (anterior posterior/posterior-anterior/) waveform. Both stimulators induce a current in the brain with posterior-anterior flow when the coil is placed tangentially to the scalp with the handle positioned at an angle of 45° pointing backwards. The optimal spot for right FDI stimulation was marked with a felt pen.
Resting motor threshold (RMT) was defined as the minimum intensity needed to evoke an MEP of >50μV in 5 out of 10 consecutive trials in the relaxed FDI. Active motor threshold (AMT) was defined as the minimum intensity (in % of maximum stimulator output) needed to evoke a MEP of >200μV in 5 out of 10 trials in the tonically active FDI (~10% of maximal contraction as assessed visually on an oscilloscope). Thresholds were approached from above threshold in steps of 1% stimulator output. Once no MEPs could be elicited the intensity was increased in steps of 1% stimulator output until a minimal MEP was observed. This intensity was taken as motor threshold.
Theta burst stimulation (TBS) followed the protocol described in detail by Huang et al . In brief, repetitive transcranial magnetic stimulation consisted of three pulses at a frequency of 50 Hz and an intensity of 80% AMT repeated with a frequency of 5 Hz (i.e. every 200ms). A continuous train of 100 bursts (300 stimuli) was given (continuous TBS, cTBS).
Two researchers blinded to HD gene carriers' clinical characteristics collected and analysed the TMS data. Peak to peak amplitude of MEP was measured with in-house software. To assess the effects of cTBS we examined whether there was a main effect of ‘time’ on MEP amplitude. This was tested using repeated measures analysis of variance (ANOVA). To test whether HD gene carriers, either premanifest or early manifest, differed from controls or from each other we introduced ‘group’ as between-subjects factor. In addition, we tested whether the time course of the after effects of cTBS differed between HD gene carriers and controls (interaction of ‘time’ and ‘group’ in the repeated measures ANOVAs).
A statistical difference in the ANOVAs was followed by a post-hoc paired t-test analysis. Mauchly's test tested for sphericity in the repeated measures ANOVAs, and the Greenhouse-Geisser correction was applied to the DFs if necessary. Statistical significance levels were set to p=0.05. All statistical analyses were performed using SPSS 16 for Windows software package.
Thresholds were similar in controls (AMT 30.8 (SD 5.5); RMT 40.6 (6.9); rapid AMT 44.8 (6.8)) and HD gene carriers (AMT 32.7 (5.7); RMT43 (7.1); rapid AMT 46.4 (8.8)).
Theta burst stimulation induced inhibition in the controls that lasted for the 21 minutes observation period after the end of stimulation (repeated measures ANOVA, main effect of ‘time’, F4.3,56=4.3, p=0.003). In the group of HD gene carriers and controls together this effect was lost; both groups responded differently to theta burst stimulation (repeated measures ANOVA, interaction ‘time*group’, F4.9,131.9=1.37, p=0.048, Figure 1A) with controls having more inhibition than HD gene carriers (ANOVA on data in Figure 1A, main effect of ‘group’, F1,27=13.3, p=0.001). Looking at the time course of the effect (0-9 versus 11-21 minutes after the end of cTBS) it appeared that there was less inhibition at the later time points but this was not significant (repeated measures ANOVA, no main effect of ‘time’). We next compared all 3 groups (controls, premanifest and early manifest) using the mean inhibition across all time points. This showed a difference between the groups (ANOVA, main effect of ‘group’, F2,26=6.32, p=0.006); controls had more inhibition than either HD gene carrier subgroup (p=0.006 for premanifest and p=0.009 for early symptomatic, Figure 1B) whereas there was no difference between premanifest and early symptomatic HD gene carriers.
For subsequent analyses with clinical parameters we used the mean inhibition across all time points.
We first tested whether the response to cTBS was related to UHDRS motor score. To this end we used backward logistic regression analysis with UHDRS motor score as the dependent variable and age, CAG repeat length, and the overall average of inhibition induced by cTBS as the dependent variables. Only age and CAG repeat length predicted UHDRS motor score (F2,12=10.64, p=0.002, adjusted R2=0.58). We next examined if the response to theta burst stimulation changed when HD gene carriers approach the onset of motor signs. To this end we assessed how the estimate of predicted age at onset was predicted using age, CAG repeat length, and cTBS data. The cTBS data did not improve prediction over age and CAG repeat length (F2,12=29.6, p<0.001, adjusted R2=0.8). We then examined in the same way how the response to cTBS was predicted. There was no association with any of the variables.
We used theta burst stimulation, a novel form of repetitive transcranial magnetic stimulation, to investigate motor cortical plasticity. In controls, we were able to reproduce the inhibitory effects shown previously . However, cTBS did not induce any changes in HD expansion mutation carriers, both premanifest or very early motor manifest. Thus our data in human HD are in agreement with evidence for abnormal plasticity in HD mouse models [9-13].
Since HD gene carriers differed from controls we next distinguished between clearly premanifest HD gene carriers and those that had very early motor signs. Reduced motor cortex plasticity was not associated with the stage of HD – there was no difference between premanifest and early symptomatic patients – or with clinical ratings of motor signs. This is similar to a previous study that found that MEP recruitment curves at rest were also reduced to the same extent in premanifest and manifest HD gene carriers suggesting that both are likely to be an intrinsic consequence of the abnormal genotype rather than a consequence of the symptoms . In the context of life-long expression of mutant huntingtin it is interesting to note that mutant huntingtin decreases expression of brain-derived neurotrophic factor (BDNF) and its tropomyosin-related kinase B receptor in neocortex and hippocampus of humans [27, 28] and mice [28-32]. In the healthy animal, BDNF is almost exclusively produced by the cortex from where it is transported in cortico-striatal axons to the striatum, which does not express BDNF itself . Transgenic mice with reduced cortical BDNF expression develop striatal degeneration and expression profiles very similar to R6/2 transgenics and human HD. This suggeststhat the cortical generation of BDNF and its axonal transport to the striatum provides important trophic support for the striatum even though so far BDNF has not been shown to be a causative factor in humans . BDNF is also a potent, positive modulator of LTP induced in slice preparations by naturalistic TBS . Reduced levels of BDNF in cortex could therefore be one factor that contributes to loss of response to the cTBS protocol in the present data even though we acknowledge that hippocampal neurones and motor cortical neurones are likely to differ electrophysiologically. Consistent with this, in a recent study we found that a common BDNF polymorphism (BDNF Val66Met) in healthy subjects was also predictive of a loss of response to cTBS . Indeed, BDNF has been shown to rescue synaptic plasticity in hippocampal slices of a knock-in mouse model of HD  .
In summary we demonstrate that motor cortex plasticity is abnormal in HD expansion mutation carriers.Premanifest and early motor manifest HD gene carriers were similar, and the amount of plasticity was not associated with age, CAG repeat length or UHDRS motor score. This suggests that abnormal motor cortex plasticity may not be closely linked to the development of motor signs of HD. However, one of the limitations of our study is the small numbers of participants in the premanifest and very early manifest group. What determines the development of motor signs, and the possible contribution of abnormal motor cortex function in the premanifest phase of HD needs to be the subject of further research.
We thank all our patients, and the controls, for participating in this study. We are grateful to Dr Ed Wild and Ms Susie Henley for helping with patient recruitment. SJT is funded by the Medical Research Council, the Wellcome Trust, the Brain Research Trust, the UK Huntington's disease association and the High Q Foundation.
Disclosure: The authors report no conflict of interest