Lovastatin improves impaired synaptic plasticity and phasic alertness in patients with neurofibromatosis type 1

In this experiment we investigated PAS-induced LTP-like plasticity in healthy controls and patients with NF1. Motor evoked potentials increased in amplitudes in healthy controls (baseline: 1.00 ± 0.17; POST 1: 1.44 ± 0.47; POST 2: 1.60 ± 0.71; POST 3: 1.71 ± 0.48), but not in patients with NF1 after PAS (baseline: 1.03 ± 0.15; POST 1: 0.92 ± 0.43; POST 2: 0.97 ± 0.33; POST 3: 0.98 ± 0.51; see Figure 1). We performed rmANOVA on untransformed MEP data, which revealed significant main effects (TIME: F _[3;60] = 4.269, p = 0.011; GROUP: F _[1;20] = 10.243, p = 0.004), and significant interaction (F _[3;60] = 5.854, p = 0.002). Post hoc analysis demonstrated that the increase in MEP amplitudes was significant in the control group for all points in time (POST 1 (p = 0.009), POST 2 (p = 0.016) and POST 3 (p = 0.001)), but not in patients group and that there were significant differences between both groups at POST 1 (p = 0.014), POST 2 (p = 0.015) and POST 3 (p = 0.002). For more detail see also Figure 1.

There was no significant difference in baseline MEP amplitudes between groups (NF1: 1.03 ± 0.15 mV; healthy controls: 1.00 ± 0.17 mV; p = 0.782). Healthy controls and patients did not differ in regard to resting motor threshold (NF1: 39.55 ± 7.42%; healthy controls: 38.36 ± 6.02%; p = 0.686) or perceptual threshold (NF1: 0.54 ± 0.22 mA; controls: 0.61 ± 0.17 mA; p = 0.430). RmANOVA of resting motor threshold data revealed no significant main effects or interaction (TIME: F _[3;60] = 0.263, p = 0.844; GROUP: F _[1;20] = 0.612, p = 0.444; TIME*GROUP: F _[3;60] = 0.819, p = 0.484).

In experiment 1b we tested intracortical inhibition in patients with NF1 and healthy controls. Patients with NF1 showed an increased SICI compared to healthy controls (ISI of 2 ms: 0.75 ± 0.33 to 0.51 ± 0.22 (p = 0.190), 3 ms: 0.77 ± 0.46 to 0.51 ± 0.24 (p = 0.190), 5 ms: 0.95 ± 0.41 to 0.69 ± 0.20, (p = 0.089), and over all ISI: 0.82 ± 0.40 to 0.57 ± 0.23, (p = 0.012; see Figure 2)).

In experiment 1c attentional performance was examined in patients with NF1 and healthy controls. In most subscales of the test of attentional performance task (TAP) patients with NF1 scored in lower ranges than healthy controls (see Figure 3). Notably, deficits became manifest in the subscales alertness (-WT, +WT) and Visual Scanning (critical stimulus) where 10.33% (-WT), 9.53% (+WT) respectively 23.86% (critical stimulus). Here, the present sample scores were significantly (Alertness: -WT: 0.003; +WT: 0.012; Visual Scanning: critical stimulus: 0.047) lower than in healthy controls. In both conditions of alertness, with and without a warning tone, we observed significant faster reaction times in the healthy control group than in the patients group (without warning tone (-WT): HC: 216.9 ± 16.02 ms; NF1: 239.3 ± 12.3 ms; unpaired t-test: p = 0.003, with warning tone (+WT): HC: 218.2 ± 17.87 ms; NF1: 239 ± 15.26; unpaired t-test: p = 0.012).

Patients with clinically diagnosed neurofibromatosis type 1 (Exp. 1a: n = 11, mean age 28.0 (range 17–44) years, Exp. 1b: n = 10, 27.8 years (range 19–44), Exp. 1c: n = 10, 27.8 years (range 19–44)) according to the criteria of the National Institutes of Health (NIH, 1988) participated in this study. Patients with NF1 and healthy controls were not treated with psychotropic medications. Inclusion criteria were age 18 to 45 years and right-handedness. Exclusion criteria were segmental NF1, deafness, severely impaired vision, use of central active drugs (e.g. methylphenidate). The control group in Exp. 1 consisted of age-matched healthy volunteers (Exp. 1a: n = 11, 24.73 ± 3.58 years, 5 female, 6 male (range 18–31 years) Exp. 1b: n = 10, 23.3 ± 3.77, 6 female, 4 male (range 19–30 years) aged; Exp. 1c: 22.0 ± 2.31, 6 female, 4 male (range 20–26 years). All subjects were self-reported right-handed and did not fulfil any exclusion criteria concerning the safety of TMS [33].

Subjects were seated comfortably in an armchair with their stimulated hand resting on a cushion. Motor-evoked potentials (MEPs) were recorded from the left abductor pollicis brevis (APB) muscle at rest by surface electromyography (EMG) using silver/silver chloride electrodes with a surface area of 263 mm² (AMBU, Ballerup, Denmark) in belly-tendon recording technique. Data were band-pass filtered (20 – 2000 Hz) and amplified using an Ekida DC universal amplifier (EKIDA GmbH, Helmstadt, Germany), digitised at 5 kHz sampling rate using a MICRO1401mkII data acquisition unit (Cambridge Electronic Design Ltd, Cambridge, UK) and stored on a standard personal computer for online visual display and later offline analysis using Signal Software version 3 (CED Ltd, UK).

For focal transcranial magnetic stimulation the intersection of a figure eight shaped stimulation coil with an external wing diameter of 90 mm was centred tangentially on the scalp over the motor cortex with its handle pointing in a posterior direction and laterally at an angle of approximately 45° away from the midline. Thus the current in the brain induced by magnetic stimulation was in a posterior-anterior direction, roughly perpendicular to the central sulcus and was therefore optimal for activating the neurons of the corticospinal pathways transsynaptically [34, 35]. The coil was connected to a Magstim 200 Stimulator (The Magstim Company Ltd, Whitland, UK) which outputs a monophasic current waveform. By moving the coil over the motor cortex while administering stimuli of suprathreshold intensity, we could identify the optimal position for eliciting MEPs of maximum amplitudes from the target muscle (“hotspot”). The individual hotspot was recorded using an optically tracked navigation system (camera: NDI, Waterloo, Ontario, Canada; software: Fraunhofer Institute (IPA), Stuttgart, Germany) described previously [36] and thus kept constant throughout investigations. Magnetic stimuli were administered to the cortex at a frequency of 0.1 Hz, except stimuli used to identify the hotspot and motor threshold, which were administered at 0.25 Hz. We determined the resting motor threshold using a maximum-likelihood threshold-hunting procedure [37, 38]. We used 16 TMS stimuli starting at 45% of maximum stimulator output. A positive MEP was defined as a muscle activation of >50 μV. The initial stimulator output for evaluation was chosen to target a mean MEP amplitude of 800–1200 μV (SI_1mV) and was then kept constant throughout the investigations to assess changes in MEPs. MEP size was determined by measuring the two highest peaks of opposite polarity [29, 39, 40] and then averaged over 20 trials for each point of investigation. Subjects were asked to relax the target muscle during all measurements and relaxation was monitored by visual feedback via EMG-baseline.

Paired associative stimulation consisted of 200 pairs at a frequency of 0.25 Hz of peripheral electric stimulation of the left median nerve at the wrist, followed by TMS of the right M1 at the optimal site to elicit MEPs in the left APB muscle [41]. Electrical stimulation was applied through a Digitimer DS7 electrical stimulator (Digitimer Ltd, Welwyn Garden City, Hertfortshire, UK) using a bipolar electrode with the cathode proximal. We identified the optimal stimulation site at the wrist, affixed the electrode, and determined the threshold of perception. During PAS, constant current square wave pulses with duration of 1000 μs were applied at an intensity of three times the perceptual threshold. We employed the intensity for TMS which produced MEP amplitudes of, on average, 1 mV before the intervention (SI_1mV). The same intensity was used for evaluation. The ISI between electrical and transcranial magnetic stimulation was 25 ms as this interval induces an increase in MEP amplitudes [19].

One crucial point when applying PAS is the subject’s attention level. It may influence the PAS effect’s magnitude. To avoid potential influence of attention on the results, we constantly reminded all the subjects to focus their attention on the stimulated hand and mentally count the number of electric stimuli. This assured a comparable level of attention between both groups (i.e. HC and NF1). Additionally, none of the patients reported of any diagnosed or relevant attention deficit in daily life.

As application of a high number of TMS stimuli might influence subsequent induction of plasticity [21], we limited the number of pre-interventional TMS stimuli in all experiments to 200. Each subject participated in studies of PAS where we measured MEP amplitudes and resting motor threshold before (PRE) as well as at three points in time after PAS (POST 1, POST 2 and POST 3). For experimental design see Figure 6.

All experiments were performed by TMS with the target muscle at rest. To evoke SICI, a subthreshold conditioning stimulation was delivered 2, 3 and 5 ms before a test stimulus [23]. The stimulator output for test stimulus was adjusted to evoke mean MEP amplitude of 1 mV peak-to-peak.

The standard protocol for SICI has been optimized to maximize inhibitory effects of the preceding, conditioning stimulus using an intensity of 80% of resting motor threshold [42]. Using this paradigm increased inhibition in patients with NF1 compared to healthy subjects may not be detected because of a ceiling effect. Because it is known, that decreasing the conditioning stimulus intensity is lowering the inhibitory effect, we first created an intensity curve for SICI by using randomized conditioning stimulus intensities in trials of 0.5, 0.55, 0.6, 0.65, 0.7, 0.75 and 0.8 resting motor threshold [43, 44]. Then we selected the stimulus intensity with lowest but still significant inhibition in the control group. This was the case using a conditioning stimulus intensity of 0.60 resting motor threshold (60%: 0.83 ± 0.42 to 0.58 ± 0.27, p = 0.017; 55%: 1.03 ± 0.51 to 1.0 ± 0.57, p = 0.575). This intensity was used to test the hypothesis (i) whether inhibition in patients with NF1 is increased and (ii) whether lovastatin leads to a disinhibition in patients with NF1.

With an interval of 4 s between trials, 10 conditioned MEPs were collected for each ISI, and in each experimental trial a total of 5 unconditioned test stimulus MEPs were recorded. Thus, for each conditioning pulse intensity 35 trials MEPs were recorded (3 ISIs with 10 MEPs each and 5 unconditioned MEPs). The order of data acquisition for each conditioning pulse intensity was randomized between subjects. The average of the amplitude of each conditioned MEP was expressed as a percentage of the average test stimulus MEP amplitude in the same session.

Attention was measured through a computerized, standardized neuropsychological test, the TAP [45]. This test is widely used to evaluate attentional deficits including NF1. In order to be analogous to statin-responsive tests in animal studies of NF1, we choose four subtests of TAP: alertness and Go/No Go for basic attention and general anticipation; visual scanning for visual-spacial perceptivity (analogous to the morris water maze task for visual-spatial learning); incompatibility for divided visuospacial attention (analogous to the lateralized reaction time task).

In this test, reaction time is examined under two conditions. The first condition concerns simple reaction time measurements, in which a cross appears on the monitor a randomly varying intervals and to which the subjects should respond as quickly as possible by pressing a key. Intrinsic alertness is measured in this condition. In a second condition, reaction time is measured in response to a critical stimulus preceded by a cue stimulus presented as warning tone (WT). Thus, alertness is equivalent to anticipation and a degree of impulsiveness. It is the pre-requisite for effective behaviour, and is in this respect the basis of every attention performance.

In the visual scanning task, a matrix-like arrangement of 5 × 5 stimuli is used. Subjects have to “scan” this matrix and decide whether this arrangement includes a critical stimulus or not. One reaction key is used for the answer “present” and another for the answer “not present”. Thus, visual scanning tests the exploration of the special environment, which is one of the basic abilities subserving the safe movement in space.

In the go/no-go tasks, the subject has to react selectively to one class of stimuli but not to others. For testing a go/no-go-task was realized with 2 stimuli, squares with different textures, where two were targets. The aim of this examination is an assessment of the capacity of focused attention (reject irrelevant information). Thus, the go/no-go task tests the ability to suppress an inappropriate reaction, that is, control of impulsive behaviour.

In the incompatibility task, a right or left pointing arrow appears in the left or right side of the visual field. Here, the subjects have to press the button for the direction of the arrow but not the site where the arrow appears. This procedure tests the interference tendency in terms of stimulus-reaction incompatibility.

We performed the study in a placebo-controlled, randomized and double blind design for pharmacological interventions. Patients obtained 5 tablets containing 40 mg lovastatin (Mevacor, 1 A Pharma®) or placebo (lactose-monohydrate, magnesium stearate, cellulose powder). Patients and all other investigators were blind to the treatment allocation. Tablets were given every day at the same time at the day of examination, 2.8 h before PAS started. This was done because the time to reach peak plasma concentration is 2.8 hours [46]. Because this was a short term administration and the aim of this study was “proof of principle”, a dosage exceeding the clinical routine dosage was administered. It has been shown that doses of 200 mg of lovastatin were proven to be save in adult human volunteers [32], moreover overdose up to 5–6 g have been tolerated with no specific symptoms and recovery without sequelae. None of the participants reported any side effects.

We ensured comparable relaxation in the recorded muscle while recording the TMS measurements visually. In addition, we investigated offline the EMG baseline of each trial. Pre-facilitated trials were excluded from further analyses. Trials that differed by over twice the standard deviation from the mean were left out of the analysis. We computed all statistical analyses using SPPS version 15.0 Software (SPSS Inc., Chicago, IL, USA). Before any further analysis, data were tested for normality. For experiments 1, statistical evaluation was performed via a repeated-measures analysis of variance (rmANOVA) with group (two levels: CONTROL and NF1) as between subject factor and time (four levels: PRE, POST 1, POST 2 and POST 3) as within-subjects factor. For experiment 2, we performed rmANOVA with drug (two levels: ON and OFF) and time (four levels: PRE, POST 1, POST 2 and POST 3) as within-subject factors. We used the Greenhouse-Geisser correction to adjust for violations of sphericity, if necessary. In case of significant main effects, we employed post-hoc two-tailed Student’s paired or unpaired t-tests without correction. This method was used for MEP and resting motor threshold values. To analyse baseline parameters, we used two-tailed Student’s paired t tests. We compared baseline MEP amplitudes using a one-way analysis of variance (ANOVA) with experiment as factor. MEP amplitudes of each subject in experiment 2 were analysed using an rmANOVA with TIME (four levels: PRE, POST 1, POST 2 and POST 3) as within-subject factor. The significance level was set at 0.05 in all analyses. All values given are mean group values ± standard deviation, if not indicated otherwise.