Francesco Mori

Silvia Rossi

Giulia Sancesario

Claudia Codecà

Giorgia Mataluni

Fabrizia Monteleone

Fabio Buttari

Hajime Kusayanagi

Maura Castelli

Caterina Motta

Valeria Studer

Giorgio Bernardi

Giacomo Koch

Sergio Bernardini

Diego Centonze
email: centonze@uniroma2.it

Cognitive and cortical plasticity deficits correlate with altered amyloid-β CSF levels in Multiple Sclerosis

Keywords: cognition, inflammation, LTP, tau protein, transcranial magnetic stimulation

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INTRODUCTION

Multiple Sclerosis (MS) causes cognitive deficits since the early stages of the disease, by altering memory, attention and executive functions [START_REF] Amato | Neocortical volume decrease in relapsing-remitting MS patients with mild cognitive impairment[END_REF](Amato et al, , 2006a(Amato et al, , 2008a,b;,b;[START_REF] Chiaravalloti | Cognitive impairment in multiple sclerosis[END_REF][START_REF] Genova | Cognition in multiple sclerosis: a review of neuropsychological and fMRI research[END_REF].

The synaptic and molecular mechanisms at the basis of cognitive impairment in MS are still poorly understood. A large body of evidence converges in indicating that learning and memory processes are encoded in brain circuits as experience-and activity-dependent long-term synaptic changes. In particular, long-term potentiation (LTP) is a form of synaptic plasticity in which repeated stimulation connects neurons by making synapses more responsive to future activation, and is commonly considered a major candidate for representing the substrate of learning and memory processes [START_REF] Bliss | A synaptic model of memory: long-term potentiation in the hippocampus[END_REF][START_REF] Malenka | The long-term potential of LTP[END_REF].

LTP induction is regulated by a variety of factors, able to interfere with critical receptor and postreceptor events at excitatory synapses [START_REF] Bliss | A synaptic model of memory: long-term potentiation in the hippocampus[END_REF][START_REF] Malenka | The long-term potential of LTP[END_REF][START_REF] Feldman | Synaptic mechanisms for plasticity in neocortex[END_REF][START_REF] Kessels | Synaptic AMPA receptor plasticity and behavior[END_REF][START_REF] Minichiello | TrkB signalling pathways in LTP and learning[END_REF]. Among these factors, amyloid-β, particularly the isoform amyloid-ß 1-42 , that was consistently found to be reduced in the cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients, plays a major role in LTP deficits and cognitive impairment in AD. More specifically, amyloid-ß 1-42 , aggregates in oligomers that impair synaptic plasticity mechanisms [START_REF] Klyubin | Amyloid beta protein immunotherapy neutralizes Abeta oligomers that disrupt synaptic plasticity in vivo[END_REF][START_REF] Shankar | Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory[END_REF][START_REF] Walther | Improved learning and memory in aged mice deficient in amyloid beta-degrading neutral endopeptidase[END_REF][START_REF] Townsend | Oral treatment with a {gamma}-secretase inhibitor improves long-term potentiation in a mouse model of Alzheimer's disease[END_REF]. Its role in other disorders associated with mental deterioration is however still unclear.

Amyloid-β peptides are formed by proteolytic cleavage of a transmembrane protein, the amyloid precursor protein (APP) by β-site APP-cleaving enzyme 1 (BACE). BACE cleaves APP, causing the secretion of an extracellular soluble fragment of APP, termed sAPP-β, and the retention of a 99 residue C-terminal fragment. This fragment can undergo further cleavage by γ-secretase to release 40 or 42 amino acid long amyloid-β fragments into the cytoplasm. However, APP may undergo at least two and most likely several different processing pathways. In the non-amyloidogenic pathway, APP is cleaved at the α-site in the middle of the amyloid-β domain. This cleavage obliterates amyloid-β formation, instead yielding an N-terminal sAPP-α fragment [START_REF] Andreasson | Aspects of beta-amyloid as a biomarker for Alzheimer's disease[END_REF].

We postulated that, in MS, acute inflammation is able to interfere with amyloid-β 1-42 or tau metabolism, and that, like in AD, this abnormal metabolism could be associated with cognitive dysfunction and synaptic plasticity. Notably, although both amyloid-β 1-42 and tau protein alter LTP induction and cognitive performances in animal models [START_REF] Oddo | Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction[END_REF][START_REF] Klyubin | Amyloid beta protein immunotherapy neutralizes Abeta oligomers that disrupt synaptic plasticity in vivo[END_REF][START_REF] Rosenmann | A novel transgenic mouse expressing double mutant tau driven by its natural promoter exhibits tauopathy characteristics[END_REF][START_REF] Shankar | Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory[END_REF][START_REF] Polydoro | Age-dependent impairment of cognitive and synaptic function in the htau mouse model of tau pathology[END_REF][START_REF] Walther | Improved learning and memory in aged mice deficient in amyloid beta-degrading neutral endopeptidase[END_REF][START_REF] Townsend | Oral treatment with a {gamma}-secretase inhibitor improves long-term potentiation in a mouse model of Alzheimer's disease[END_REF], their role in human synaptic plasticity has never been explored.

In humans, LTP can be explored in a safe and non-invasive manner by using paradigms of repetitive transcranial magnetic stimulation (rTMS) [START_REF] Ziemann | TMS induced plasticity in human cortex[END_REF][START_REF] Cooke | Plasticity in the human central nervous system[END_REF][START_REF] Lazzaro | The physiological basis of the effects of intermittent theta burst stimulation of the human motor cortex[END_REF]. A novel LTP-inducing protocol has been recently developed, in the attempt to mimic the physiological activity of hippocampal neurons during learning episodes [START_REF] Huang | Theta burst stimulation of the human motor cortex[END_REF][START_REF] Lazzaro | The physiological basis of the effects of intermittent theta burst stimulation of the human motor cortex[END_REF]. This new protocol has been termed theta burst stimulation (TBS) and, according to in vitro experiments in hippocampal preparations, it produces a NMDA receptordependent LTP in the human cortex, by enhancing corticospinal excitability for several minutes after the end of the stimulation [START_REF] Huang | Theta burst stimulation of the human motor cortex[END_REF].

Thus, in the current study we investigated whether altered CSF concentrations of amyloid-β 1-42 metabolism are associated with abnormal patterns of cognitive performances and of cortical plasticity in a sample of MS patients that were submitted to an extensive neuropsychological examination and to neurophysiological investigations of cortical plasticity by means of TBS.

SUBJECTS AND METHODS

The study was approved by the Ethics Committee of the University Hospital Tor Vergata, Rome.

Abbreviations used throughout the text are provided in the Supplementary Text.

MS patients

We collected CSF from 42 patients (30 females and 12 males, aged 23-54 years), admitted to the neurological clinic of the University Hospital Tor Vergata of Rome, and later diagnosed as suffering from clinically isolated syndrome (CIS) (n=7), relapsing-remitting (RR) MS (n=30), or primary progressive (PP) MS (n=5). After their admittance, all patients underwent, in sequence, brain (and in selected cases also spinal) magnetic resonance imaging (MRI) scan, neuropsychological assessment, TMS and CSF withdrawal within 24 hours. Corticosteroids or other MS-specific immunoactive therapies were initiated later when appropriate.

The diagnosis of CIS or MS was established by clinical, laboratory and MRI parameters, and matched published criteria [START_REF] Mcdonald | Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis[END_REF][START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF]. In all instances, patients underwent detection of oligoclonal banding in the CSF (positive in 92% of cases). Expanded Disability Status Scale (EDSS) scores were between 0 and 5.

As controls, we used CSF from 12 age-and gender-matched individuals (8 females and 4 males, aged 25-52 years) without inflammatory or degenerative diseases of the central or peripheral nervous system. These subjects underwent lumbar puncture because of a clinical suspect of acute peripheral neuropathy, meningitis, or subarachnoidal hemorrhage, which were not confirmed. All the subjects gave their written informed consent to the study.

MRI acquisition and analysis

Three Tesla MRI scan consisted of dual-echo proton density, FLAIR, T2-weighted spin-echo images and pre-contrast and post-contrast T1-weighted spin-echo images. All images were acquired in the axial orientation with 3 mm-thick contiguous slices. T2 lesion volume was determined by manual tracing and the number of gadolinium-enhancing (Gd+) (0.2 ml/Kg e.v.) lesions was counted by a neuroradiologist who was unaware of the patient's clinical details [START_REF] Centonze | The endocannabinoid system is dysregulated in multiple sclerosis and in experimental autoimmune encephalomyelitis[END_REF].

CSF determination of amyloid-β 1-42 and total tau

Immediately after its collection, CSF was centrifuged to eliminate cells and cellular debris and stored at -80°C until analyzed [START_REF] Centonze | The endocannabinoid system is dysregulated in multiple sclerosis and in experimental autoimmune encephalomyelitis[END_REF]. Levels of both amyloid-β 1-42 and total tau were determined according to standard procedures, using commercially available sandwich enzyme-linked immunosorbent assays (Innotest β-Amyloid 1-42, Innotest h-tau Ag, Innogenetics, Ghent, Belgium) [START_REF] Sancesario | Abeta1-42 Detection in CSF of Alzheimer's disease is influenced by temperature: Indication of reversible Abeta1-42 aggregation?[END_REF]. Briefly, 25 μl of the CSF from each patient were dispensed into corresponding 96-well ELISA plates, pre-coated either with the monoclonal antibody 21F12 for amyloid-β 1-42 , or AT120 for total tau, and incubated respectively with the biotinylated antibody 3D6 or HT7. Bound antibodies were then detected by a peroxidase-labeled streptavidin, after addition of a substrate solution. The reaction was stopped by sulphuric acid. The absorbance of the reaction product was read at 450 nm. The biomarker concentrations in the samples were calculated based on the amyloid-β 1-42 and tau standard sigmoid curve equation.

Neuropsychological assessment

Cognitive functions were assessed through the Brief Repeatable Neuropsychological Battery (BRB) [START_REF] Rao | A Manual for the Brief Repeatable Battery of Neuropsychological Tests in Multiple Sclerosis[END_REF]) by an expert trained clinician in a subgroup of 21 MS patients (4 CIS and 17 RR MS) who gave consent to the examination. The BRB assesses the cognitive domains most frequently impaired in MS [START_REF] Amato | Neocortical volume decrease in relapsing-remitting MS patients with mild cognitive impairment[END_REF] (Amato et al, 2006b). Failure of a test was defined when the score was at least 2 standard deviations below the mean normative values.

Consistently with previous works (Amato et al, 2006a(Amato et al, , 2008b)), those patients who failed at least three tests were considered cognitively impaired (CI), and those who failed less than three tests were considered cognitively preserved (CP). No subjects were taking psychoactive drugs or substances that might interfere with neuropsychological performance.

TMS

TMS protocols were performed in a subgroup of 30 MS patients (5 CIS, 23 RR MS and 2 PP MS) who gave consent to the examination and were asymptomatic in the upper right limb. Electromyographic (EMG) traces were recorded from the right first dorsal interosseus muscle (FDI) with surface cup electrodes. The active electrode was placed over the muscle belly and the reference electrode over the metacarpophalangeal joint of the index finger. Responses were amplified with a Digitimer D360 amplifier (Digitimer, Welwyn Garden City, Hertfordshire, United Kingdom) through filters set at 20 Hz and 2 kHz with a sampling rate of 5 kHz, then recorded by a computer with SIGNAL software (Cambridge Electronic Devices, Cambridge, United Kingdom).

MEPs were evoked through a figure-of-eight coil with external loop diameter of 70 mm connected to a Magstim 200 2 magnetic stimulator (Magstim Company, Whitland, Wales, UK). Coil position was adjusted to find the optimal scalp site to evoke motor responses in the contralateral FDI, the motor "hot spot", at the beginning of each experimental session and marked over the patients scalp with a pencil. The coil was held tangentially to the scalp surface with the handle pointing posteriorly and laterally at about 45° with respect to the mid-sagittal axis of the head.

iTBS

Intermittent TBS (iTBS) was delivered over the motor "hot spot" of the right FDI through a Magstim Rapid 2 stimulator. The active motor threshold (AMT) was defined as the minimum stimulation intensity required to evoke a liminal motor potential from the FDI during voluntary contraction (about 200 µV in 50% of 10 trials). Stimulation intensity was 80% of AMT. The iTBS protocol consisted of 10 bursts, each burst composed of three stimuli at 50 Hz, repeated at a theta frequency of 5 Hz every 10 s for a total of 600 stimuli (200 s). Sixty MEPs were collected before iTBS (baseline) and at two different time points (0 and 15 minutes) after the end of iTBS.

Stimulation intensity was set to induce a stable MEP of approximately 1 mV peak to peak amplitude in the relaxed right FDI at baseline and remained unchanged until end of recordings. MEP's amplitudes were then averaged at each time point and normalized to the mean baseline amplitude.

Intracortical circuits in right M1

We also tested, through paired-pulse (pp) TMS, short interval intracortical inhibition (SICI, mediated by intrinsic GABAAergic circuits) [START_REF] Kujirai | Corticocortical inhibition in human motor cortex[END_REF], intracortical facilitation (ICF, believed to follow the preferential recruitment of intrinsic excitatory fibers) [START_REF] Kujirai | Corticocortical inhibition in human motor cortex[END_REF], short intracortical facilitation (SICF, likely mediated by excitatory cortical interneurons) [START_REF] Ziemann | Pharmacological control of facilitatory I-wave interaction in the human motor cortex. A paired transcranial magnetic stimulation study[END_REF] SICI and ICF were tested using ppTMS with a subthreshold CS preceding a suprathreshold TS [START_REF] Kujirai | Corticocortical inhibition in human motor cortex[END_REF][START_REF] Rothwell | Techniques and mechanisms of action of transcranial stimulation of the human motor cortex[END_REF]. CS stimulus was set at 80% AMT. Three conditions were presented in a random order: control (TS given alone) and two pp conditions (TS preceded by CS) at one of two different ISI (2 and 10 ms).

For SICF, the intensity of CS was set to 90% RMT. Six randomly intermixed conditions were presented in a random order: TS given alone and five conditions with the TS followed by CS at one of five different ISI (1.5, 2.1, 2.7, 3.7, 4.5 ms) [START_REF] Hanajima | Mechanisms of intracortical I-wave facilitation elicited with pairedpulse magnetic stimulation in humans[END_REF].

For LICI the intensity of CS was set at 120% RMT. Two conditions were presented in a random order: control (TS given alone) and one paired-pulse condition (TS preceded by CS) at 100 ms ISI [START_REF] Valls-Solé | Human motor evoked responses to paired transcranial magnetic stimuli[END_REF]. For each experiment ten responses were collected for the test stimulus alone and for conditioned MEPs at each ISI. Changes in MEP amplitude at each ISI were expressed as percentage of the mean unconditioned MEP amplitude.

Data analysis

Differences in tau and amyloid-β 1-42 CSF levels were assessed using analysis of variance, followed by post-hoc comparison using the Tukey highest significant difference procedure, to account for multiple comparisons and by T Test or Mann-Whitney test for comparisons between two groups.

Relationships between tau and amyloid-β 1-42 CSF levels and MRI findings were assessed using Spearman correlation test for nonparametric values and Pearson's correlation test for parametric values. Correlation analysis between amyloid-β 1-42 CSF levels and cognitive variables was performed by calculating Spearman correlation coefficients.

Correlations between tau and amyloid-β 1-42 CSF levels with iTBS at each timepoint, SICI, ICF, LICI and SICF at each ISI were calculated using Pearson's correlation coefficient. Differences between groups were assessed using repeated measures analysis of variance for iTBS, SICF, SICI, ICF and LICI. Bonferroni correction was used to correct for multiple comparisons. Data, expressed as mean ± SEM, were considered significant at the 0.05 level.

RESULTS

CSF contents of amyloid-β 1-42 and total tau protein in MS patients and their correlation with neuropsychological parameters

We first measured amyloid-β 1-42 and tau protein levels in the CSF of MS patients. Amyloid-β 1-42 levels were significantly reduced in the population of MS subjects, compared to control individuals (MS group: 267±22 pg/ml, n = 42; control group: 390±45 pg/ml, n = 12, p < 0.05) (Fig. 1A).

Conversely, tau protein levels were not altered in the CSF of MS patients (MS group: 128±11 pg/ml, n = 42; control group: 143±14 pg/ml, n = 12, NS) (Fig. 1B).

MS patients were, then, classified as cognitively impaired (CI, n = 11) or cognitively preserved (CP, n = 10) on the basis of their neuropsychological performance, without differences in terms of demographic characteristics (CI: 7 females and 4 males, aged 25-40 years with 11±7 years of education; CP: 6 females and 4 males, aged 23-37 years with 11 ± 3 years of education).

The reduction of amyloid-β 1-42 levels was related to cognitive dysfunction (CI MS group: 145±25 pg/ml, n=11; CP MS group: 297±46 pg/ml, n=10; control group: 390±45 pg/ml, n = 12; F = 9.81, p = 0.0005), since in CP MS amyloid-β 1-42 levels were higher than in CI MS patients (p < 0.05), and indistinguishable from controls (NS) (Fig. 1C). In CI MS patients, tau protein CSF levels were conversely still indistinguishable from those measured in controls and in CP patients (CI MS group: 110±18 pg/ml, CP MS group: 176±37 pg/ml, control group: 143±14 pg/ml, F = 2.22, p = 0.12) (Fig. 1D).

The most frequently failed tests by examined MS patients were those assessing sustained attention and concentration (SDMT, PASAT) and verbal memory (SRT-LTS, SRT-CLTR). Interestingly, a moderate positive correlation was found between amyloid-β 1-42 CSF levels and scores on measures of attention/concentration (SDMT: n = 21, r = 0.51, p = 0.02; PASAT: n = 21, r = 0.54, p = 0.01) (Fig. 1E,F). The scores on verbal memory tests were, conversely, not significantly related to amyloid-β contents (SRT-LTS: n = 21, r = -0.08, p = 0.61; SRT-CLTR: n = 21, r = -0.11, p = 0.58).

None of the neuropsychological measurements correlated to tau CSF levels in MS patients (n = 21, p = NS for all parameters, not shown).

Since pathology of corpus callosum (CC) may play a major role in cognitive impairment of MS patients [START_REF] Ozturk | MRI of the corpus callosum in multiple sclerosis: association with disability[END_REF], we further compared scores in all neuropsychological tests between patients showing lesions within the CC (n=6) and patients without CC lesions (n=15) at conventional MRI. Comparison of neuropsychological scores between CIS and RRMS was also performed. No significant differences emerged between groups (n = 21, all p = NS) (not shown).

Correlation between amyloid-β 1-42 and total tau protein with Gd+ lesions in MS patients

Upon grouping of MS patients according to the presence of Gd+ lesions at the MRI (Gd+ MS group: n = 22 (14 patients complaining new symptoms since 10 days or less), Gd-MS group: n = 20 (4 patients with new symptoms since 10 days or less), control group: n = 12, F = 7.98, p = 0.0009), amyloid-β 1-42 levels were lower in Gd+ MS patients (209±26 pg/ml) than in both Gd-MS patients (331±25 pg/ml, p < 0.05) and in controls (390±45 pg/ml, p < 0.01) (Fig. 2A). No significant alteration of tau protein CSF content was revealed by grouping Gd-and Gd+ patients (Gd+ MS group: 129±11 pg/ml, Gd-MS group: 124±20 pg/ml, control group: 143±14 pg/ml F = 0.3, p = 0.74) (Fig. 2B).

These results might indicate that acute brain inflammation is associated with alterations of amyloidβ metabolism, leading to decreased CSF detection of this biologically active peptide. We further explored this possibility by correlating both amyloid-β 1-42 and tau protein levels with the number of Gd+ lesions seen at the MRI. These analyses uncovered a significant negative correlation between amyloid-β 1-42 content and Gd+ lesions (n = 22, r = -0.45, p = 0.03) (Fig. 2C), while no correlation was seen between amyloid-β 1-42 and total lesion load in T2 MRI sequences (n = 42, r = 0.06, p = 0.69) (Fig. 2D). No correlation was also found between tau levels in the CSF of MS patients and both Gd+ lesions (n = 22, r = -0.21, p = 0.34) and T2 lesion load (n = 42, r = -0.08, p = 0.57) (Fig. 2E,F).

Correlation between TBS-induced LTP with amyloid-β 1-42 and total tau levels in MS patients

Amyloid-β dimers isolated from AD brains completely block LTP induction in the rodent hippocampus, and consequently cause cognitive defects [START_REF] Shankar | Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory[END_REF]. In an attempt to see if also in MS brains amyloid-β 1-42 is able to alter LTP induction, we explored whether any correlation could be uncovered between CSF content of amyloid-β 1-42 and the magnitude of LTP induced in MS patients with TBS. A significant positive correlation was found between the magnitude of LTP measured 15 min after the TBS protocol and the levels of amyloid-β 1-42 in the CSF (r = 0.48, p = 0.007), a finding which may suggest that also in MS, as in AD, reduced CSF contents of amyloid-β 1-42 may be associated with defective memory-related synaptic plasticity (Fig. 3A,B).

Correlation between neuropsychological assessments and TBS-induced LTP

To investigate whether cognitive impairment was associated with reduced cortical LTP in MS subjects, we compared the mean percentage values of peak to peak MEP amplitudes before and after TBS in CI and CP MS patients. A repeated measures ANOVA with TIME (3 levels: baseline, 0 and 15 min post TBS) as within subjects and GROUP (2 levels: CI and CP) as between subjects main factors showed a significant effect of time (F = 8.04, p = 0.002), a significant effect of GROUP (F = 6.82, p = 0.023) and a significant TIME x GROUP interaction (F = 5.96, p = 0.031).

At post-hoc contrasts CI patients showed a lower effect of TBS at both timepoints (0 minutes post TBS: 1.09 ± 0.14; 15 minutes post: 1.18 ± 0.12; all p < 0.05) compared to CP patients (0 minutes post TBS: 1.67 ± 0.16; 15 minutes post: 1.71 ± 0.16) (Fig. 4). We also performed a T-Test for independent data on the peak effect of TBS represented by the post TBS time-point with the highest mean MEP amplitude. A significantly lower peak effect of TBS in CI (n = 10, mean ± SE = 1.39 ± 0.15) compared to CP (n = 10, mean ± SE = 2,32 ± 0.23) MS patients (p = 0.008) was observed (not shown). These findings, showing a lower effect of TBS in CI patients, are in line with previous observations that rTMS has an altered effect in AD patients [START_REF] Inghilleri | Altered response to rTMS in patients with Alzheimer's disease[END_REF][START_REF] Battaglia | Cortical plasticity in Alzheimer's disease in humans and rodents[END_REF] and show for the first time that cognitive impairment in MS patients is paralleled by an alteration of NMDAR dependent synaptic plasticity.

Comparison of TBS effect between CIS, RRMS and PPMS did not show any significant difference.

Role of amyloid-β 1-42 in the modulation of excitatory and inhibitory synaptic transmission in MS

mediated inhibition alter its induction. Accordingly, blockade of NMDA glutamate receptors prevents LTP [START_REF] Bliss | A synaptic model of memory: long-term potentiation in the hippocampus[END_REF][START_REF] Cooke | Plasticity in the human central nervous system[END_REF], as also does the enhancement of GABA-mediated inhibition [START_REF] Matsuyama | Long-term potentiation-like facilitation through GABAA receptor blockade in the mouse dentate gyrus in vivo[END_REF][START_REF] Gong | GABA transporter-1 activity modulates hippocampal theta oscillation and theta burst stimulationinduced long-term potentiation[END_REF][START_REF] Pan | Selective gating of glutamatergic inputs to excitatory neurons of amygdala by presynaptic GABAb receptor[END_REF]. Thus, we tried to investigate whether amyloid-β 1-42 -mediated inhibition of LTP could be secondary to impaired basal synaptic transmission in cortical neurons of MS patients. In ppTMS experiments, however, we found that the degree of SICI, ICF, SICF, and LICI was not related to the CSF levels of amyloid-β 1-42 in MS patients, suggesting that this protein affects synaptic plasticity but not basal excitatory or inhibitory transmission (Fig. 5). Comparisons between CI and CP MS patients also did not reveal any significant difference (not shown).

DISCUSSION

Grey matter pathology, brain atrophy, and loss of anatomical connectivity are the most accredited determinants of progressive cognitive decline in MS [START_REF] Calabrese | Cortical lesions and atrophy associated with cognitive impairment in relapsing-remitting multiple sclerosis[END_REF][START_REF] Calabrese | Widespread cortical thinning characterizes patients with MS with mild cognitive impairment[END_REF][START_REF] Staff | Multiple sclerosis with predominant, severe cognitive impairment[END_REF][START_REF] Tiemann | Cognitive decline in multiple sclerosis: impact of topographic lesion distribution on differential cognitive deficit patterns[END_REF]. Acute and reversible mental deterioration also occurs during clinical relapses, independently of the primary functional system involved [START_REF] Foong | Neuropsychological deficits in multiple sclerosis after acute relapse[END_REF]. The presence of Gd+ lesions at the brain MRI also correlates with poor cognitive performance in asymptomatic and symptomatic MS patients [START_REF] Foong | Neuropsychological deficits in multiple sclerosis after acute relapse[END_REF][START_REF] Bellmann-Strobl | Poor PASAT performance correlates with MRI contrast enhancement in multiple sclerosis[END_REF], suggesting that focal inflammation is able to diffusely disrupt the neuronal substrate of information processing, leading to acute cognitive impairment. Given that amyloid-β 1-42 metabolism is regulated by inflammation [START_REF] Griffin | Interleukin-1 mediates Alzheimer and Lewy body pathologies[END_REF][START_REF] Hickman | Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer's disease mice[END_REF][START_REF] Schmidt | Interrelation of inflammation and APP in sIBM: IL-1 beta induces accumulation of beta-amyloid in skeletal muscle[END_REF] a recent study performed in a large sample of subjects, reported that in MS patients there is an overall decrease of CSF amyloid-ß 1-42 in comparison with healthy controls [START_REF] Mattsson | Reduced cerebrospinal fluid BACE1 activity in multiple sclerosis[END_REF]. These results seem to indicate that in MS there could be an altered expression of amyloid-β 1-42 , possibly related to the underlying inflammatory processes.

The present study shows that during the inflammatory phase of MS, amyloid-β 1-42 metabolism is altered leading to its reduced detection in the CSF. We found, in fact, that amyloid-β 1-42 levels were significantly lower in subjects with Gd+ lesions at the MRI. Interestingly, these findings are in good agreement with previous studies showing that IL-1β, an important pro-inflammatory cytokine, alters amyloid-β metabolism in the cerebral cortex, increasing APP levels in rat brain homogenate [START_REF] Fan | Interleukin-1beta-induced brain injury in the neonatal rat can be ameliorated by alpha-phenyl-n-tert-butyl-nitrone[END_REF]. There is no information, conversely, about IL-1β effect on CSF amyloid β 1-42 levels. However, inflammation increases BACE activity [START_REF] Hong | Interferon gamma stimulates beta-secretase expression and sAPPbeta production in astrocytes[END_REF], and BACE activity levels were found to be higher in patients with low amyloid β 1-42 levels in the CSF [START_REF] Mulder | BACE1 activity in cerebrospinal fluid and its relation to markers of AD pathology[END_REF]. Anti-inflammatory cytokines, on the other hand, enhance the activity of amyloid-β degrading enzymes, thus favouring amyloid-β tissue deposit clearance [START_REF] Shimizu | IL-4-induced selective clearance of oligomeric beta-amyloid peptide(1-42) by rat primary type 2 microglia[END_REF].

The present study shows, for the first time, a difference in cognitive profile of MS patients related to CSF levels of amyloid-β 1-42 . Interestingly, the cognitive domains that seemed to be affected in association with low amyloid-β CSF levels were those regarding attention, concentration and information-processing speed, the same influenced by the presence of radiological disease activity (Bellmann- [START_REF] Bellmann-Strobl | Poor PASAT performance correlates with MRI contrast enhancement in multiple sclerosis[END_REF]. Gd enhancement has been associated with poor PASAT performance in otherwise physically stable MS patients (Bellmann- [START_REF] Bellmann-Strobl | Poor PASAT performance correlates with MRI contrast enhancement in multiple sclerosis[END_REF] and to CSF amyloid-β 1-42 levels (present work), in line with the idea that CSF amyloid-β 1-42 expression, altered by brain inflammation, may cause a diffuse impairment of cerebral connectivity with a negative impact on cognitive functioning. Conversely, neuropsychological test scores examining memory did not correlate with amyloid-β 1-42 levels.

Cortical atrophy was not measured in the present study since imaging was performed for clinical purposes, thus only conventional MRI scans were collected. However CSF tau levels, already reported to be associated with neurodegenerative damage [START_REF] Vemuri | MRI and CSF biomarkers in normal, MCI, and AD subjects. Diagnostic discrimination and cognitive correlations[END_REF][START_REF] Ghoshal | Tau conformational changes correspond to impairments of episodic memory in mild cognitive impairment and Alzheimer's disease[END_REF] were found to be normal in our patients. A further limitation of the present study may be represented by anxiety, due to diagnosis communication or expectancy for the disease. Anxiety is known to influence the performance in the neuropsychological tests [START_REF] Peretti | Anxiety and cognition disorders[END_REF][START_REF] Tombaugh | A comprehensive review of the Paced Auditory Serial Addition Test (PASAT)[END_REF], especially those addressing concentration and attention. However, all participants were diagnosed as suffering from CIS or MS during hospitalization, and diagnosis was communicated after all data collection.

The synaptotoxic properties of amyloid-β 1-42 have long been recognized, as well as its ability to interfere with LTP and with cognitive abilities [START_REF] Klyubin | Amyloid beta protein immunotherapy neutralizes Abeta oligomers that disrupt synaptic plasticity in vivo[END_REF][START_REF] Battaglia | Cortical plasticity in Alzheimer's disease in humans and rodents[END_REF][START_REF] Shankar | Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory[END_REF][START_REF] Nygaard | Cellular prion protein mediates the toxicity of beta-amyloid oligomers: implications for Alzheimer disease[END_REF][START_REF] Walther | Improved learning and memory in aged mice deficient in amyloid beta-degrading neutral endopeptidase[END_REF][START_REF] Townsend | Oral treatment with a {gamma}-secretase inhibitor improves long-term potentiation in a mouse model of Alzheimer's disease[END_REF]. A recent study, reported that amyloid-β dimers isolated from AD patients can impair hippocampal LTP and memory in mice, and induce dendritic spine retraction in neurons [START_REF] Shankar | Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory[END_REF]. Of note, while the role of amyloid-β in LTP disruption has been demonstrated in a variety of experimental models of AD [START_REF] Klyubin | Amyloid beta protein immunotherapy neutralizes Abeta oligomers that disrupt synaptic plasticity in vivo[END_REF][START_REF] Battaglia | Cortical plasticity in Alzheimer's disease in humans and rodents[END_REF][START_REF] Shankar | Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory[END_REF][START_REF] Walther | Improved learning and memory in aged mice deficient in amyloid beta-degrading neutral endopeptidase[END_REF][START_REF] Townsend | Oral treatment with a {gamma}-secretase inhibitor improves long-term potentiation in a mouse model of Alzheimer's disease[END_REF], its correlates with LTP measured in humans have never been demonstrated before. Thus, the correlation between amyloid-β 1-42 CSF levels and LTP amplitude reported in the present study is a first indication that amyloid-β is a potent regulator of synaptic plasticity also in the human brain, with potentially relevant consequences for understanding the mechanisms of cognitive deficits not only in MS subjects but also in other neurological disorders, obviously including AD. Importantly, although both altered cognitive performances and LTP induction was found in mice overexpressing tau protein [START_REF] Polydoro | Age-dependent impairment of cognitive and synaptic function in the htau mouse model of tau pathology[END_REF], tau levels did not correlate with TBS-induced LTP amplitude in MS patients (Fig. 3C,D). In this respect, it is noteworthy that only LTP induced by high-frequency stimulation but not that induced by TBS was altered in mice overexpressing tau [START_REF] Polydoro | Age-dependent impairment of cognitive and synaptic function in the htau mouse model of tau pathology[END_REF].

Previous investigations found that in MS patients SICI was lower in secondary progressive MS (SPMS) in comparison with RRMS patients in the remitting phase, and that SICI correlated significantly with EDSS scores [START_REF] Conte | Intracortical excitability in patients with relapsing-remitting and secondary progressive multiple sclerosis[END_REF]. These results are in apparent contrast with our findings. However our study group did not include patients with SPMS and the mean age and disease duration of these patients (age: 51 ± 0, disease duration: 21.0 ± 2) are also very different in comparison with our study group (age: 34.3 ± 1.4 years, disease duration: 2.7±0.8 years). Other investigators found that fatigue in MS is associated with lower SICI compared to MS patients without fatigue and healthy subjects [START_REF] Liepert | Motor cortex excitability and fatigue in multiple sclerosis: a transcranial magnetic stimulation study[END_REF]. In our study group only very few patients complained fatigue and also in this case our group differed in terms of mean age (41.4 ± 4.75 years). Moreover one of the inclusion criteria in the study from Liepert was that patients had to be in the remitting phase of the disease, thus presumably also had longer disease durations. Finally, a similar reduction of SICI was found in the relapsing phase of the disease in comparison with remitting patients in a study by [START_REF] Caramia | Brain excitability changes in the relapsing and remitting phases of multiple sclerosis: a study with transcranial magnetic stimulation[END_REF]. Also in this case the study group had longer disease duration (mean 4.29 ± 3.26 years), included only patients with a definite diagnosis of RRMS. Thus differences in the results may be explained by the different clinical characteristics of the samples studied.

A recent investigation based on a different TMS protocol to induce neuroplastic changes, suggested that motor plasticity in MS patients is comparable to that found in healthy subjects [START_REF] Zeller | Rapid-onset central motor plasticity in multiple sclerosis[END_REF]. Neuroplasticity in this study was however assessed only in patients in the remitting phase of the disease, with a stable clinical condition, while the alterations observed in our study were evident in patients with cognitive impairment and presence of Gd+ lesions.

The reduction in CSF amyloid-ß 1-42 in AD has been postulated to reflect deposition of the aggregated insoluble fibrillar amyloid-ß peptides in senile plaque, with lower levels of diffusion into the CSF. A limitation of the present study is that we do not provide any evidence of amyloid-β tissue deposition in our patients. We cannot argue, therefore, that the decrease in amyloid-β 1-42 CSF levels observed is consequent to tissue deposition. However, AD neuropathology apart from amyloid-ß deposition includes variable amounts of soluble species that show much stronger correlations with severity of dementia than fibrillar amyloid-ß [START_REF] Terry | The pathogenesis of Alzheimer disease: an alternative to the amyloid hypothesis[END_REF][START_REF] Lue | Soluble amyloid beta peptide concentration as a predictor of synaptic change in Alzheimer's disease[END_REF][START_REF] Mclean | Soluble pool of A beta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease[END_REF][START_REF] Gandy | Days to criterion as an indicator of toxicity associated with human Alzheimer amyloid-beta oligomers[END_REF]. Synthetic amyloid-β dimers are extremely potent inhibitors of synaptic plasticity both in vivo and in vitro whereas even relatively high concentrations of amyloid-β monomers are inactive [START_REF] Selkoe | Soluble oligomers of the amyloid beta-protein impair synaptic plasticity and behavior[END_REF][START_REF] Hu | Soluble amyloid-beta peptides potently disrupt hippocampal synaptic plasticity in the absence of cerebrovascular dysfunction in vivo[END_REF][START_REF] Shankar | Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory[END_REF]. Moreover,

  , and long interval intracortical inhibition (LICI, mediated by local GABAB pathways) (Valls-Solé et al, 1992) of the left M1. One figure of eight coil, external diameter 70 mm was held tangentially to the scalp over the motor "hot spot" for right FDI muscle. Stimulation intensity for TS was adjusted in each experiment to evoke a MEP of approximately 1 mV peak to peak amplitude in the relaxed right FDI.

Figure 1 .

 1 Figure 1. CSF contents of amyloid-β 1-42 are reduced in MS patients and correlate with their

Figure 2 .

 2 Figure 2. Disease activity at MRI correlates with reduction of amyloid-β 1-42 levels in the CSF of

Figure 3 .

 3 Figure 3. TBS induced LTP magnitude correlates with amyloid-β 1-42 levels in the CSF of MS

Figure 4 .Figure 5 .

 45 Figure 4. TBS induced LTP differs between cognitively impaired and cognitively preserved MS

, and incorporates tests of verbal memory (Selective Reminding

	Test [SRT]); visual memory (10/36 Spatial Recall Test); attention, concentration, and speed of
	information processing (Paced Auditory Serial Addition Test [PASAT], Symbol Digit Modalities
	Test [SDMT]); and verbal fluency (Word List Generation). Performance on each test of the BRB
	was assessed by applying the available Italian normative values

ACKNOWLEDGEMENTS

This investigation was supported by the Italian National Ministero dell'Università e della Ricerca to DC, by the Italian National Ministero della Salute, and by Fondazione TERCAS to DC.

human ex vivo samples of CSF that contain amyloid-β oligomers but not monomers, potently inhibit LTP [START_REF] Klyubin | Amyloid beta protein immunotherapy neutralizes Abeta oligomers that disrupt synaptic plasticity in vivo[END_REF]. The lowering of natively measured amyloid-β 1-42 in the CSF of our patients may be consequent to oligomerization as observed in AD [START_REF] Englund | Oligomerization partially explains the lowering of Abeta42 in Alzheimer's disease cerebrospinal fluid[END_REF]. Finally a transgenic mice, overexpressing APP E693Q develops learning and memory deficits in association with formation of amyloid beta oligomers without generating amyloid plaques in their brains at any age [START_REF] Gandy | Days to criterion as an indicator of toxicity associated with human Alzheimer amyloid-beta oligomers[END_REF]. These observations, even though only at a speculative stage, could help explaining the findings of our iTBS experiments. Another possibility is that the observed findings could be related to inflammation-induced downregulation of β-site APP cleavage. Accordingly, BACE activity is modulated by inflammation [START_REF] Mattsson | Reduced cerebrospinal fluid BACE1 activity in multiple sclerosis[END_REF], and is also involved in the promotion of myelination by oligodendrocytes [START_REF] Hu | Bace1 modulates myelination in the central and peripheral nervous system[END_REF]. Nevertheless, the mechanism by which amyloid-β 1-42 is reduced during inflammation in MS remains unclear and further studies are needed to better clarify this issue.

DISCLOSURE/CONFLICT OF INTERESTS

The authors have no financial interests or conflicts of interest to disclose.