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Repetitive transcranial magnetic stimulation (rTMS) is emerging as a non-invasive therapeutic strategy in the battle against Alzheimer’s disease. Alzheimer’s disease patients primarily show alterations of the default mode network for which the precuneus is a key node. Here, we hypothesized that targeting the precuneus with TMS represents a promising strategy to slow down cognitive and functional decline in Alzheimer’s disease patients. We performed a randomized, double-blind, sham-controlled, phase 2, 24-week trial to determine the safety and efficacy of precuneus stimulation in patients with mild-to-moderate Alzheimer’s disease. Fifty Alzheimer’s disease patients were randomly assigned in a 1:1 ratio to either receive precuneus or sham rTMS (mean age 73.7 years; 52% female). The trial included a 24-week treatment, with a 2-week intensive course in which rTMS (or sham) was applied daily five times per week, followed by a 22-week maintenance phase in which stimulation was applied once weekly. The Clinical Dementia Rating Scale–Sum of Boxes was selected as the primary outcome measure, in which post-treatment scores were compared to baseline. Secondary outcomes included score changes in the Alzheimer’s Disease Assessment Scale–Cognitive Subscale, Mini-Mental State Examination and Alzheimer’s Disease Cooperative Study–Activities of Daily Living scale. Moreover, single-pulse TMS in combination with EEG was used to assess neurophysiological changes in precuneus cortical excitability and oscillatory activity. Our findings show that patients that received precuneus repetitive magnetic stimulation presented a stable performance of the Clinical Dementia Rating Scale–Sum of Boxes score, whereas patients treated with sham showed a worsening of their score. Compared with the sham stimulation, patients in the precuneus stimulation group also showed also significantly better performances for the secondary outcome measures, including the Alzheimer’s Disease Assessment Scale–Cognitive Subscale, Mini-Mental State Examination and Alzheimer’s Disease Cooperative Study–Activities of Daily Living scale. Neurophysiological results showed that precuneus cortical excitability remained unchanged after 24 weeks in the precuneus stimulation group, whereas it was significantly reduced in the sham group. Finally, we found an enhancement of local gamma oscillations in the group treated with precuneus stimulation but not in patients treated with sham. We conclude that 24 weeks of precuneus rTMS may slow down cognitive and functional decline in Alzheimer’s disease. Repetitive TMS targeting the default mode network could represent a novel therapeutic approach in Alzheimer’s disease patients.

In Alzheimer disease (AD) animal models, synaptic dysfunction has recently been linked to a disorder of high-frequency neuronal activity. In patients, a clear relation between AD and oscillatory activity remains elusive. Here, we attempt to shed light on this relation by using a novel approach combining transcranial magnetic stimulation and electroencephalography (TMS-EEG) to probe oscillatory activity in specific hubs of the frontoparietal network in a sample of 60 mild-to-moderate AD patients.Sixty mild-to-moderate AD patients and 21 age-matched healthy volunteers (HVs) underwent 3 TMS-EEG sessions to assess cortical oscillations over the left dorsolateral prefrontal cortex, the precuneus, and the left posterior parietal cortex. To investigate the relations between oscillatory activity, cortical plasticity, and cognitive decline, AD patients underwent a TMS-based neurophysiological characterization and a cognitive evaluation at baseline. The latter was repeated after 24 weeks to monitor clinical evolution.AD patients showed a significant reduction of frontal gamma activity as compared to age-matched HVs. In addition, AD patients with a more prominent decrease of frontal gamma activity showed a stronger impairment of long-term potentiation-like plasticity and a more pronounced cognitive decline at subsequent follow-up evaluation at 24 weeks.Our data provide novel evidence that frontal lobe gamma activity is dampened in AD patients. The current results point to the TMS-EEG approach as a promising technique to measure individual frontal gamma activity in patients with AD. This index could represent a useful biomarker to predict disease progression and to evaluate response to novel pharmacological therapies. ANN NEUROL 2022;92:464-475.

Background New diagnostic criteria consider Alzheimer’s disease (AD) as a clinico-biological entity identifiable in vivo on the presence of specific patterns of CSF biomarkers. Objective Here we used transcranial magnetic stimulation to investigate the mechanisms of cortical plasticity and sensory-motor integration in patients with hippocampal-type memory impairment admitted for the first time in the memory clinic stratified according to CSF biomarkers profile. Methods Seventy-three patients were recruited and divided in three groups according to the new diagnostic criteria: 1) Mild Cognitive Impaired (MCI) patients (n = 21); Prodromal AD (PROAD) patients (n = 24); AD with manifest dementia (ADD) patients (n = 28). At time of recruitment all patients underwent CSF sampling for diagnostic purposes. Repetitive and paired-pulse transcranial magnetic stimulation protocols were performed to investigate LTP-like and LTD-like cortical plasticity, short intracortical inhibition (SICI) and short afferent inhibition (SAI). Patients were the followed up during three years to monitor the clinical progression or the conversion to dementia. Results MCI patients showed a moderate but significant impairment of LTP-like cortical plasticity, while ADD and PROAD groups showed a more severe loss of LTP-like cortical plasticity. No differences were observed for LTD-like cortical plasticity, SICI and SAI protocols. Kaplan-Meyer analyses showed that PROAD and MCI patients converting to dementia had weaker LTP-like plasticity at time of first evaluation. Conclusion LTP-like cortical plasticity could be a novel biomarker to predict the clinical progression to dementia in patients with memory impairment at prodromal stages of AD identifiable with the new diagnostic criteria based on CSF biomarkers.

Although the cerebellum is not among the most renowned brain structures affected in Alzheimer`s disease (AD), recent evidence suggest that it undergoes degenerative changes during the course of the disease. A main neurophysiological feature of AD patients is the remarkable impairment of long term potentiation (LTP)-like cortical plasticity assessed in the primary motor cortex (M1) using theta burst stimulation (TBS) protocols. In healthy conditions, continuous (cTBS) and intermittent TBS (iTBS) of the cerebellum induce respectively long term depression (LTD)-like and LTP-like after effects in the contralateral M1. Here we aimed at examining the effects of cerebellar TBS on contralateral M1 excitability in a sample of 15 AD patients and 12 healthy age matched controls (HS). Motor evoked potentials (MEPs) were obtained in the contralateral M1 before and after cerebellar cTBS and iTBS protocols. As compared to HS, AD patients showed an impairment of LTP-like cortical plasticity mechanisms following cerebellar iTBS. No difference was observed for the cTBS protocol, in which both populations exhibited the expected LTD-like after effect. This study shows that mechanisms of cerebellar-cortical plasticity are impaired in AD. Given its role in high order cognitive functions, new potential therapeutic strategies could be built up in the future to modulate neural activity in the cerebellum in AD.

Biobanking has emerged as a strategic challenge to promote knowledge on neurological diseases, by the application of translational research. Due to the inaccessibility of the central nervous system, the advent of biobanks, as structure collecting biospecimens and associated data, are essential to turn experimental results into clinical practice. Findings from basic research, omics sciences, and in silico studies, definitely require validation in clinically well-defined cohorts of patients, even more valuable when longitudinal, or including preclinical and asymptomatic individuals. Finally, collecting biological samples requires a great effort to guarantee respect for transparency and protection of sensitive data of patients and donors. Since the European General Data Protection Regulation 2016/679 has been approved, concerns about the use of data in biomedical research have emerged. In this narrative review, we focus on the essential role of biobanking for translational research on neurodegenerative diseases. Moreover, we address considerations for biological samples and data collection, the importance of standardization in the preanalytical phase, data protection (ethical and legal) and the role of donors in improving research in this field.

BACKGROUND AND PURPOSE Diabetes mellitus (DM) is considered a risk factor for Alzheimer's disease (AD) and shares some pathological pathways, such as activation of amyloid cascade and tau phosphorylation. The aim of the present study was to investigate to what extent DM could impact on neurodegeneration within the AD continuum, using β amyloid (A: Aβ1-42 ) and phosphorylated tau (T: p-tau) biomarkers to discriminate patients by Alzheimer's pathological change (A+/T-) and AD (A+/T+), according to the National Institute on Aging and Alzheimer's Association classification. In addition, we aimed to evaluate whether APOE genotype interacts with tau protein and glucose metabolism dysfunction to affect the pathological process. METHODS For this retrospective observational study, 1350 patients were recruited. The patients underwent a complete clinical investigation, neuropsychological assessment, lumbar puncture for cerebrospinal fluid (CSF) biomarkers analysis and APOE genotyping. RESULTS A total of 607 patients fulfilled the clinical criteria of mild cognitive impairment or early dementia. In A+T- patients (n = 350), DM did not influence CSF biomarker levels, while among A+T+ patients (n = 257) those with DM showed increased total tau (t-tau) levels compared to non-DM patients (DM: 919.4 ± 444 vs. non-DM: 773.1 ± 348.2; p = 0.04), but similar p-tau (p = 0.72) and Aβ1-42 levels (p = 0.83). Furthermore, multivariable regression analyses showed a significant association between DM and t-tau CSF levels, adjusting for age and sex, in APOE E4+ carriers (coefficient 222.83, 95% confidence interval 47.49-398.1; p = 0.01), but not in APOE E4- (p = 0.53). CONCLUSIONS The present study shows a clear dependency of CSF t-tau levels on DM for APOE E4+ AD patients, suggesting important differences between APOE E4-related and non-related disease, with key implications for AD pathophysiology and treatment.

ObjectiveTo determine the ability of transcranial magnetic stimulation (TMS) in detecting synaptic impairment in patients with Alzheimer’s disease (AD) and predicting cognitive decline since the early phases of the disease.MethodsWe used TMS-based parameters to evaluate long-term potentiation (LTP)-like cortical plasticity and cholinergic activity as measured by short afferent inhibition (SAI) in 60 newly diagnosed patients with AD and 30 healthy age-matched subjects (HS). Receiver operating characteristic (ROC) curves were used to assess TMS ability in discriminating patients with AD from HS. Regression analyses examined the association between TMS-based parameters and cognitive decline. Multivariable regression model revealed the best parameters able to predict disease progression.ResultsArea under the ROC curve was 0.90 for LTP-like cortical plasticity, indicating an excellent accuracy of this parameter in detecting AD pathology. In contrast, area under the curve was only 0.64 for SAI, indicating a poor diagnostic accuracy. Notably, LTP-like cortical plasticity was a significant predictor of disease progression (p=0.02), while no other neurophysiological, neuropsychological and demographic parameters were associated with cognitive decline. Multivariable analysis then promoted LTP-like cortical plasticity as the best significant predictor of cognitive decline (p=0.01). Finally, LTP-like cortical plasticity was found to be strongly associated with the probability of rapid cognitive decline (delta Mini-Mental State Examination score ≤−4 points at 18 months) (p=0.04); patients with AD with lower LTP-like cortical plasticity values showed faster disease progression.ConclusionsTMS-based assessment of LTP-like cortical plasticity could be a viable biomarker to assess synaptic impairment and predict subsequent cognitive decline progression in patients with ADs.

The cerebellum plays a critical role in promoting learning of new motor tasks, which is an essential function for motor recovery. Repetitive transcranial magnetic stimulation (rTMS) of the cerebellum can be used to enhance learning. In this study, we investigated the effects of cerebellar intermittent theta burst stimulation (c-iTBS), a high-frequency rTMS protocol, on visuo-motor learning in a sample of hemiparetic patients due to recent stroke in the territory of the contralateral middle cerebral artery. Eight stroke patients were enrolled for the purposes of the study in the chronic stage of recovery (i.e., at least 6 months after stroke). In two sessions, Patients were randomly assigned to treatment with real or sham c-iTBS applied over the cerebellar hemisphere ipsilateral to the affected body side. c-iTBS was applied immediately before the learning phase of a visuo-motor adaptation task. Real, but not sham, c-iTBS improved visuo-motor learning as revealed by an increased performance in of the learning phase of the visuo-moto adaptation task. Moreover, we also found that real but not sham c-iTBS induced a sustained improvement in the re-adaptation of the recently learned skill (i.e., when patients were re-tested after 30 min). Taken together, these data point to c-iTBS as a potential novel strategy to promote motor learning in patients with stroke.

OBJECTIVE Alzheimer's disease (AD) is considered an age-related disorder. However, it is unclear whether AD induces the same pathological and neurophysiological modifications in synaptic functions independently from age of disease onset. We used transcranial magnetic stimulation tools to investigate the mechanisms of cortical plasticity and sensory-motor integration in AD patients with a wide range of disease onset. METHODS We evaluated newly diagnosed sporadic AD (n = 54) in comparison with healthy age-matched controls (HS; n = 24). Cortical plasticity mechanisms of long-term potentiation (LTP) or of long-term depression (LTD) were assessed using respectively intermittent (iTBS) or continuous theta burst stimulation (cTBS) protocols. Sensory-motor integration was evaluated by means of short afferent inhibition (SAI) protocol. RESULTS AD patients show after iTBS an impairment of LTP-like cortical plasticity forming a paradoxical LTD in comparison to HS. LTD-like cortical plasticity is similar between AD and HS. LTP-like cortical plasticity is not associated with age, but AD patients presenting with more altered LTP-like cortical plasticity have more-severe cognitive decline at 18 months. SAI is impaired in AD and shows a strong association with the individual age of subjects rather than with disease age of onset. INTERPRETATION Cortical LTP disruption is a central mechanism of AD that is independent from age of onset. AD can be described primarily as a disorder of LTP-like cortical plasticity not influenced by physiological aging and associated with a more-severe cognitive decline. Ann Neurol 2016;80:202-210.

Background Alzheimer's disease (AD) is characterized by a primary impairment of long-term declarative memory caused by deposition of misfolded protein aggregates. Experimental studies showed that AD neuropathological alterations impair synaptic plasticity and memory performance. Transcranial Magnetic Stimulation protocols have been recently introduced to investigate altered mechanisms of cortical plasticity in AD patients. Aim To investigate relationship between Long-Term Potentiation (LTP)-like cortical plasticity and patients’ neuropsychological performance. Methods We applied intermittent theta burst stimulation and extensive neuropshycological battery in 75 newly diagnosed AD patients. Results We found that LTP-like cortical plasticity impairment is selectively associated to a less efficient verbal memory (r = 0.45; p = 0.002), but not to other cognitive functions, independently from biomarkers and other demographic and clinical factors. Conclusion These findings suggest that LTP-like cortical plasticity may represent a neurophysiological surrogate of memory in AD patients by evaluating the weight of pathological changes responsible for cognitive dysfunction.

BackgroundFew data exist on malignant middle cerebral artery infarction (MMI) among patients with acute ischemic stroke (AIS) after endovascular treatment (ET). Numerous predictors of MMI evolution have been proposed, but a comprehensive research of patients undergoing ET has never been performed. Our purpose was to find a practical model to determine robust predictors of MMI in patients undergoing ET.MethodsPatients from a prospective single-center database with AIS secondary to large intracranial vessel occlusion of the anterior circulation, treated with ET, were retrospectively analyzed. We investigated demographic, clinical, and radiological data. Multivariate regression analysis was used to identify clinical and imaging predictors of MMI.Results98 patients were included in the analysis, 35 of whom developed MMI (35.7%). No differences in the rate of successful reperfusion and time from stroke onset to reperfusion were found between the MMI and non-MMI groups. The following parameters were identified as independent predictors of MMI: systolic blood pressure (SBP) on admission (p=0.008), blood glucose (BG) on admission (p=0.024), and the CTangiography (CTA) Alberta Stroke Program Early CT Score (ASPECTS) (p=0.001). A scoreof ≤5 in CTA ASPECTS was the best cut-off to predict MMI evolution (sensitivity 46%; specificity 97%; positive predictive value 78%; negative predictive value 65%).Conclusionsin our study a clinical and radiological features-based model was strongly predictive of MMI evolution in AIS. High SBP and BG on admission and, especially, a CTA ASPECTS ≤5 may help to make decisions quickly, regardless of time to treatment and successful reperfusion.

Background: Subtle diffuse intrathecal inflammation is undetectable by conventional neuroimaging, and could influence multiple sclerosis (MS) disease course. Objective: To explore the role of subclinical persisting intrathecal inflammation in radiologically isolated syndrome (RIS) or clinically isolated syndrome (CIS) conversion to MS, and in early MS disease reactivation. Methods: One-hundred ninety-three subjects with RIS, CIS, relapsing–remitting (RR), or primary progressive (PP) MS were included, along with 76 matched controls. Cerebrospinal fluid (CSF) levels of interleukin-8 (IL-8), a major proinflammatory cytokine, were measured as a biomarker of intrathecal inflammation. Patients were followed up for 2 years. Clinical and imaging measures of disease progression were recorded. Results: High central contents of IL-8 were associated to clinical progression in subjects with RIS, and to the risk of conversion to MS in subjects with CIS. Asymptomatic intrathecal inflammation placed subjects at risk for MS conversion, even regardless lesion load. CSF IL-8 levels were higher in RR MS with high disease activity. Higher number of relapses in the first two years since diagnosis and shorter first inter-attack intervals were observed in patients with high levels of IL-8. Conclusion: IL-8 might provide utility in determining the presence of active intrathecal inflammation, and could be important in diagnostically undefined cases.

Background Sympathovagal imbalance has been associated with poor prognosis in chronic diseases, but there is conflicting evidence in multiple sclerosis. Objectives The objective of this study was to investigate the autonomic nervous system dysfunction correlation with inflammation and progression in multiple sclerosis. Methods Heart rate variability was analysed in 120 multiple sclerosis patients and 60 healthy controls during supine rest and head-up tilt test; the normalised units of low frequency and high frequency power were considered to assess sympathetic and vagal components, respectively. Correlation analyses with clinical and radiological markers of disease activity and progression were performed. Results Sympathetic dysfunction was closely related to the progression of disability in multiple sclerosis: progressive patients showed altered heart rate variability with respect to healthy controls and relapsing–remitting patients, with higher rest low frequency power and lacking the expected low frequency power increase during the head-up tilt test. In relapsing–remitting patients, disease activity, even subclinical, was associated with lower rest low frequency power, whereas stable relapsing–remitting patients did not differ from healthy controls. Less sympathetic reactivity and higher low frequency power at rest were associated with incomplete recovery from relapse. Conclusions Autonomic balance appears to be intimately linked with both the inflammatory activity of multiple sclerosis, which is featured by an overall hypoactivity of the sympathetic nervous system, and its compensatory plastic processes, which appear inefficient in case of worsening and progressive multiple sclerosis.

Background: Predictive markers of cardiac side effects would be helpful for the stratification and individualized monitoring of multiple sclerosis (MS) patients prescribed with fingolimod. Objective: To test whether the autonomic balance predicts a cardiac response after the first dose of fingolimod. Methods: A total of 55 consecutive relapsing–remitting MS (RRMS) patients underwent ‘head-up tilt’, Valsalva maneuver, deep breathing and handgrip tests before their first dose of fingolimod. The normalized unit of the high frequency (HF) component (HF normalized units; HFnu), reflecting mostly vagal activity; and the low frequency (LF) component (LF normalized units; LFnu) reflecting mostly sympathetic activity, were considered for the analysis of heart rate (HR) variability. The patients’ HR and electrocardiographic parameters ((the interval between P wave and ventricular depolarization (PR); the interval between Q and T waves (QT)) were recorded during 6-hour post-dose monitoring. Results: We found significant correlations between measures of parasympathetic function and fingolimod-induced bradycardia. Subjects with higher Valsalva ratio and HR variation during deep breathing had, in fact, nadir HR ≤ 50 beats/minute (bpm) after the first fingolimod dose. Conversely, significant negative correlations were found between measures of sympathetic function and fingolimod-induced PR interval increase. Subjects with lower LFnu at rest and less increase of blood pressure on the handgrip test showed a PR interval increase > 20 ms after fingolimod. Conclusions: Assessing autonomic control of cardiovascular functions can be useful to predict cardiac effects after the first fingolimod dose.

Background: Chronic inflammation leads to gray matter damage in progressive multiple sclerosis (MS), but the mechanism linking inflammation and neurodegeneration is unclear. Objective: The objective of this paper is to investigate the synaptic mechanism of inflammatory neurodegeneration in progressive forms of MS. Methods: Cytokine and neurofilament-light were determined in cerebrospinal fluid (CSF) of MS patients. In vitro electrophysiology and cell swelling experiments were performed to measure the effects of inflammatory cytokines in the CSF of MS patients on synaptic transmission and neuronal integrity. Results: Tumor necrosis factor-α (TNF) was higher in CSF of progressive MS subjects, and caused excitotoxic neuronal death in vitro. In murine brain slices incubated in the presence of CSF from progressive MS, in fact, we observed increased spontaneous excitatory postsynaptic currents (sEPSCs) and glutamate-mediated neuronal swelling through a mechanism dependent on enhanced TNF signaling. We also suggested a pathogenic role of B cells in TNF CSF increase, exacerbation of glutamatergic transmission and neuronal damage, since CNS depletion of B cells with intrathecal rituximab caused a dramatic reduction of TNF levels, of TNF-induced sEPSC alterations, and of neurofilament CSF concentrations in a patient with progressive MS. Conclusion: Our results point to TNF as a primary neurotoxic molecule in progressive forms of MS.

BackgroundCollateral flow (CF) is an effective predictor of outcome in acute ischemic stroke (AIS) with potential to sustain the ischemic penumbra. However, the clinical prognostic value of CF in patients with AIS undergoing mechanical thrombectomy has not been clearly established. We evaluated the relationship of CF with clinical outcomes in patients with large artery anterior circulation AIS treated with mechanical thrombectomy.MethodsBaseline collaterals of patients with AIS (n=135) undergoing mechanical thrombectomy were independently evaluated by CT angiography (CTA) and conventional angiography and dichotomized into poor and good CF. Multivariable analyses were performed to evaluate the predictive effect of CF on outcome and the effect of time to reperfusion on outcome based on adequacy of the collaterals.ResultsEvaluation of CF was consistent by both CTA and conventional angiography (pConclusionsCTA is a valid tool for assessing the ability of CF to predict clinical outcome in patients with AIS treated with mechanical thrombectomy. Limiting time to reperfusion is of definite value in patients with good collaterals and also to some extent in those with poor collaterals.

BACKGROUND AND PURPOSE: Noncontrast CT ASPECTS has been investigated as a predictor of outcome in patients with acute ischemic stroke. Our purpose was to investigate whether CTA source images are a better predictor of clinical and radiologic outcomes than NCCT ASPECTS in candidates for endovascular stroke therapy. MATERIALS AND METHODS: CT scans of patients (n = 124) were independently evaluated by 2 readers for baseline NCCT and CTA source image ASPECTS and for follow-up ASPECTS. An mRS of ≤2 at 3 months was considered a favorable outcome. Receiver operating characteristic curve analysis was used to assess the ability of NCCT and CTA source image ASPECTS to identify patients with favorable outcomes. A stepwise multiple regression analysis was performed to find independent predictors of outcome. RESULTS: Baseline CTA source image ASPECTS correlated better than NCCT ASPECTS with follow-up ASPECTS (r = 0.76 versus r = 0.51; P for comparison of the 2 coefficients < .001). Receiver operating characteristic curve analysis showed that baseline CTA source image ASPECTS compared with NCCT ASPECTS can better identify patients with favorable outcome (CTA source image area under the curve = 0.83; 95% CI, 0.76–0.91; NCCT area under the curve = 0.67; 95% CI, 0.58–0.77; P < .001). Finally, the stepwise regression analysis showed that lower age, good recanalization, lower time to recanalization, and good baseline CTA source image ASPECTS, not NCCT ASPECTS, were independent predictors of favorable outcome. CONCLUSIONS: CTA source image ASPECTS predicts outcome better than NCCT ASPECTS; this finding suggests CTA rather than NCCT as a main step in the decision-making process for patients with acute ischemic stroke.

Objective:To explore the inflammatory processes in the pathogenesis of psychiatric symptoms and the prognostic value of psychiatric comorbidities in multiple sclerosis (MS).Methods:Four hundred five patients with relapsing-remitting (RR) MS underwent psychiatric evaluation by means of Beck Depression Inventory II (BDI-II) and State/Trait Anxiety Inventory (STAI-Y). The inflammatory activity level was assessed by MRI. In a subset of 111 treatment-naive patients, CSF levels of proinflammatory cytokines were determined. Correlation and regression analyses were performed to determine associations between variables.Results:Relapsing patients demonstrated greater values of STAI-state and BDI-II compared with remitting patients but comparable trait-anxiety scores. There were no significant differences in psychometric parameters between relapsing and asymptomatic MRI-active patients, highlighting the effect of subclinical inflammation on mood disturbances. A significant reduction of STAI-state and BDI-II scores was recorded, along with the subsiding of neuroinflammation. Interleukin-2 CSF levels were found to correlate with STAI-state, while tumor necrosis factor-α and interleukin-1β correlated with BDI-II. Because emotional disorders were associated with subclinical inflammation, variations of the psychometric profile were able to detect subclinical reactivation earlier. In line with this, high STAI-state values considerably predicted the possibility of disease reactivation.Conclusions:Mood alterations are induced by intrathecal inflammation, even though not clinically apparent, and are able to predict inflammatory reactivations in RRMS. Inflammation is therefore a biological event, not less important than the traditional psychosocial factors, involved in mood disorders.

Objective: The frequency of inflammatory episodes in the early stages of multiple sclerosis (MS) has been correlated with late neurodegeneration, but the mechanism by which inflammation gives rise to delayed neuronal damage is unknown. Increased activity of the neurotransmitter glutamate is thought to play a role in the inflammation-driven neurodegenerative process of MS, and therefore we tested whether inflammatory cytokines released during acute MS attacks have the property of enhancing glutamate-mediated transmission and excitotoxicity in central neurons. Methods: We compared the effect of cerebrospinal fluid (CSF) from active and quiescent MS patients on glutamate-mediated excitatory postsynaptic currents (EPSCs) and excitotoxic damage in rodent brain slices. We also measured CSF concentrations of tumor necrosis factor-α, of interleukin-1β (IL-1β), and of IL-1 receptor antagonist (IL-1ra), and correlated cytokine levels with cortical excitability assessed in MS patients by means of paired-pulse transcranial magnetic stimulation (TMS). Results: CSF from MS patients with enhanced brain lesions at magnetic resonance imaging was able to increase spontaneous EPSC frequency and glutamate-mediated neuronal swelling in vitro, through a mechanism dependent on enhanced IL-1β signaling and increased glutamate α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor stimulation. Furthermore, IL-1β/IL-1ra ratio was significantly higher in the CSF of active MS subjects, and correlated with intracortical facilitation, an accredited TMS measure of glutamate transmission. Finally, we identified for the first time transient receptor potential vanilloid 1 channels as essential intermediates for the synaptic action of IL-1β on central glutamatergic synapses. Interpretation: Our results provide compelling evidence of the synaptic mechanism linking inflammation and excitotoxic neurodegeneration in MS. ANN NEUROL 2012;71:76–83

Objective: It is still unclear whether chronic cerebrospinal venous insufficiency (CCSVI) is associated with multiple sclerosis (MS), because substantial methodological differences have been claimed by Zamboni to account for the lack of results of other groups. Furthermore, the potential role of venous malformations in influencing MS severity has not been fully explored. This information is particularly relevant, because uncontrolled surgical procedures are increasingly offered to MS patients to treat their venous stenoses. Methods: In the present study, CCSVI was studied in 84 MS patients and in 56 healthy subjects by applying the Zamboni method for CCSVI identification. Results: We found no significant differences (p = 0.12) in CCSVI frequency between MS and control subjects. Furthermore, no differences were found between CCSVI-positive and CCSVI-negative patients in terms of relevant clinical variables such as disease duration, time between onset and first relapse, relapsing or progressive disease course, and risk of secondary progression course. Statistically significant differences were not found between CCSVI-positive and CCSVI-negative MS subjects by analyzing direct measures of disability such as mean Expanded Disability Status Scale (EDSS) (p = 0.07), mean progression index (p > 0.1), and mean MS severity score (p > 0.1). The percentage of subjects who reached EDSS 4.0 and 6.0 milestones was not different among CCSVI-negative and CCSVI-positive subjects, and no significant correlation was found between severity of disability and number of positive CCSVI criteria. Interpretation: Our results indicate that CCSVI has no role in either MS risk or MS severity. Ann Neurol 2011

Background: Genetic and pharmacological inactivation of cannabinoid CB1 receptors (CB1Rs) exacerbates disease course in experimental autoimmune encephalomyelitis, suggesting that CB1Rs might play a role in the neurodegenerative damage associated with multiple sclerosis (MS). Objectives: To see whether CNR1 gene polymorphism could influence disease progression in relapsing–remitting MS. Methods: The genotype of 350 patients for the number of AAT repeats was characterized and correlation studies were performed with measures of disease severity and progression. Results: MS patients with the homozygous genotype for long AAT repeats in the CNR1 gene had more severe disease and higher risk of progression. These subjects had significantly higher scores on both the progression index and the MS severity scale. Furthermore, the percentage of patients with MS functional composite score progression or Bayesian Risk Estimate for MS (BREMS) score ≥2 (considered at very high risk of secondary progression) was significantly higher in the AAT long group than in the short group, while the frequency of patients with BREMS score ≤−0.63 (very likely to remain progression-free) was not significantly different between the two groups, although lower in the long group. Finally, the frequency of patients prescribed a second-line treatment was significantly higher among subjects of the AAT long group, providing a further, indirect indication of higher disease severity. Conclusions: The results of the present investigation point to CB1R as an important modulator of disease severity in relapsing MS subjects.

Introduction To determine the ability of transcranial magnetic stimulation (TMS) in detecting synaptic impairment in Alzheimer’s disease (AD) patients and predicting cognitive decline since the early phases of the disease. Methods We evaluated long-term potentiation (LTP)-like cortical plasticity in 60 newly diagnosed AD patients. Pearson r correlation coefficient or Kruskal-Wallis runk sum test explored any relationship between LTP, demographics, cognition and AD-related biomarkers. Univariable analyses examined the association between LTP (respect to other AD-related biomarkers) and cognitive decline. Multivariable regression model revealed the best parameters able to predict disease progression. Results LTP plasticity was not significantly associated with sex (z = 0.89, p = 0.37), age (r = −0.02, p = 0.75) or APOE genotype (z = −0.81, p = 0.41). We confirm a significant association between LTP and both CSF t-tau (r = −0.34, p Higher values of LTP were associated with higher long-term verbal memory performances (CVLT delayed: r = 0.45; p = 0.002), while neither visual-spatial long-term memory (RCF delayed: r = 0.08; p = 0.53), general intelligence (RPM test: r = 0.11; p = 0.45), executive functions (FVF: r = −0.13; p = 0.36) or visual-spatial abilities (RCF copy: r = −0.08; p = 0.54) showed any association. Among AD patients, higher values of LTP were associated with better long-term verbal memory performances (r = 0.45; p = 0.002). Notably, LTP was a significant predictor of disease progression (p = 0.02), while no other neurophysiological, neuropsychological and demographic parameters, was associated with cognitive decline, except for a trend regarding sex (p = 0.07), long-term verbal memory (p = 0.07), visuospatial abilities (p = 0.10) and CSF total-tau levels (p = 0.09). Multivariable analysis then promoted LTP as the best significant predictor of cognitive decline (p = 0.01). Conclusion Synaptic dysfunction may be an early pathologic process in AD, and could be easily detected by means of TMS. Our data showed a significant correlation between episodic memory and LTP-like cortical plasticity, reinforcing the notion that this measure could be a neurophysiological surrogate of memory. Furthermore, our data show that LTP-like cortical plasticity is able alone to predict cognitive decline in AD patients. TMS could be a viable tool to assess synaptic impairment in AD patients, with LTP-like plasticity as the most sensitive marker of memory and predictor of cognitive decline progression.

Introduction Alzheimer‘s disease (AD) is characterized by loss of synaptic connections, cell death and disruption of structural and functional networks. One of the most consistent findings is the impairment of cortical plasticity, especially Long Term Potentiation (LTP) mechanisms. Recently, the use of a new lexicon and new diagnostic criteria allowed to considered AD as a clinico-biological entity identifiable in vivo on the presence of biomarkers. In light of the new lexicon and the new diagnostic criteria, aim of the current work is to investigate cortical plasticity in cognitive impaired (CI) patients admitted for the first time in the memory clinic and stratified according to CSF biomarker profile; moreover we followed patients up to a period of three years to explore the relationship between neurophysiological, neuropsychological and CSF biomarker and clinical progression. Methods Seventy-one consecutive patients recruited at the memory clinic admitted for their first visit for complaining memory symptoms and underwent CSF sampling. Then they undertake TMS examination, investigating mechanisms of long term potentiation (LTP) with intermittent Theta Busrt Stimluation (iTBS) and intracortical circuits. Patients were followed longitudinally with MMSE testing up to 36 months. According to the new criteria AD proposed we divided CI patients in basis of evidence ofin vivobiomarkers (as assessed by CSF analysis) and the presence of dementia. Resulting in three groups: (1) Mild Cognitive Impaired (MCI) patients (n = 22); Prodromal AD (PROAD) patients (n = 25); AD Dementia (ADD) patients (n = 25). Univariate regression analyses were performed to characterize the association between each clinical and neurophysiological variable with clinical progression (delta MMSE score at 36 months respect to baseline). A control group of 23 healthy subjects (HS) was recruited for control. Results For neurophysiological evaluations only iTBS protocol was different among the different groups showing a paradoxical reversal of LTP for ADD and PROAD and a poor response for MCI patients. ProAD worsened faster than MCI. Regression analyses showed that LTP impairment was related to clinical progression. Kaplan-Meyer analyses showed that CI patients expressing the worst LTP values were the ones to progress faster in a 3 year time. Conclusion the new criteria based on the presence of biomarkers and dementia allow us to identify CI patients at a prodromal stage that will develop dementia due to AD. LTP impairment drives the clinical progression in CI patients at prodromal stages even without evidence of biomarker positivity, confirming its pivotal role in determining cognitive decline.

Alterations of glutamate-mediated synaptic transmission occur in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Here we investigated whether intracerebroventricular (Icv) administration of cladribine has effects on EAE. Icv infusion of cladribine reduced the clinical deficits of EAE mice and reversed EAE-induced enhancement of excitatory postsynaptic current (sEPSC) frequency, a neurophysiological measure of glutamatergic synaptopathy associated with central inflammation. Cladribine failed to interfere with EAE-induced microglial and astroglial activation, but blocked EAE synaptic alterations by interfering with interleukin-1β effects. Cladribine possesses neuroprotective properties in experimental MS that are independent of its peripheral immunosuppressant action.

Dimethyl fumarate (DMF), recently approved as an oral immunomodulatory treatment for relapsing-remitting multiple sclerosis (MS), metabolizes to monomethyl fumarate (MMF) which crosses the blood–brain barrier and has demonstrated neuroprotective effects in experimental studies. We postulated that MMF exerts neuroprotective effects through modulation of microglia activation, a critical component of the neuroinflammatory cascade that occurs in neurodegenerative diseases such as MS. To ascertain our hypothesis and define the mechanistic pathways involved in the modulating effect of fumarates, we used real-time PCR and biochemical assays to assess changes in the molecular and functional phenotype of microglia, quantitative Western blotting to monitor activation of postulated pathway components, and ex vivo whole-cell patch clamp recording of excitatory post-synaptic currents in corticostriatal slices from mice with experimental autoimmune encephalomyelitis (EAE), a model for MS, to study synaptic transmission. We show that exposure to MMF switches the molecular and functional phenotype of activated microglia from classically activated, pro-inflammatory type to alternatively activated, neuroprotective one, through activation of the hydroxycarboxylic acid receptor 2 (HCAR2). We validate a downstream pathway mediated through the AMPK–Sirt1 axis resulting in deacetylation, and thereby inhibition, of NF-κB and, consequently, of secretion of pro-inflammatory molecules. We demonstrate through ex vivo monitoring of spontaneous glutamate-mediated excitatory post-synaptic currents of single neurons in corticostriatal slices from EAE mice that the neuroprotective effect of DMF was exerted on neurons at pre-synaptic terminals by modulating glutamate release. By exposing control slices to untreated and MMF-treated activated microglia, we confirm the modulating effect of MMF on microglia function and, thereby, its indirect neuroprotective effect at post-synaptic level. These findings, whereby DMF-induced activation of a new HCAR2-dependent pathway on microglia leads to the modulation of neuroinflammation and restores synaptic alterations occurring in EAE, represent a possible novel mechanism of action for DMF in MS. Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1422-3) contains supplementary material, which is available to authorized users.

Our aim is to establish the predictive value of LTP-like cortical plasticity as a prognostic biomarker of disease progression in terms of cognitive decline. We applied different neurophysiological protocols in a sample of 60 AD patients and 30 age matched healthy controls. Results: We found that area under curve (AUC) was 0.90 for LTP, indicating an excellent diagnostic accuracy of this biomarker, but only 0.64 for SAI. We performed univariate regression analyses for LTP, SAI, CSF biomarkers, APOE4 genotype, neuropsychological evaluation, disease duration, education and demographic factors. Results showed that LTP was the only significant predictor of disease progression ( p = 0.02), while there was no effect for SAI ( p = 0.84), CSF total tau ( p = 0.10), CSF p-tau ( p = 0.21), CSF A β ( p = 0.54), APOE4 genotype ( p = 0.32), neuropsychological battery and other demographic factors. The probability of disease progression significantly decreased with every point increase of LTP ( p = 0.04). ROC curve was also performed to evaluate the sensitivity and specificity of LTP biomarker in predicting cognitive decline in AD patients. We found an AUC = 0.71 indicating a fair prognostic accuracy of this biomarker in discriminating among AD patients those with faster disease progression. LTP impairment can be considered as a biomarker of progression in AD patients.

Introduction APOE E4 allele associates not only with AD risk and a lower age onset, but also with faster cognitive decline and greater cerebral atrophy, suggesting a key role of this polymorphism in modulating both disease risk and clinical outcome. Objectives In this study we investigated the correlation between cognitive decline, motor cortical plasticity and cerebrospinal fluid (CSF) biomarkers profile of AD patients divided by APOE polymorphism in E4 allele carriers (E4) and homozygous E3 carriers. Materials and methods A monophasic Magstim 200 device was used to deliver intermitted/continuous theta burst stimulation (iTBS/cTBS) protocols. ELISA was used for determination of CSF biomarkers level. Forty-one AD patients underwent lumbar puncture for CSF withdrawal, blood screening for APOE polymorphism, stimulation protocols applied over the primary motor cortex and mini mental state examination (MMSE) at baseline and at 6-, 12- and 18-months. Results No difference was found in CSF biomarkers profile within the APOE variants group. I-TBS after-effects were significantly reduced in E3 in comparison with E4 AD patients. Correlation analyses revealed that the individual amount of iTBS induced plasticity correlated with delta-MMSE and total Tau showing that a less pronounced LTP-like plasticity and higher total-Tau CSF levels were associated with a higher delta-MMSE. Only in apoE4 patients Tau pathology correlates with cortical plasticity impairment and cognitive decline. A multivariate analysis showed that APOE polymorphism and LTP-like plasticity, but not t-Tau levels, are independently able to predict delta-MMSE in AD patients. Conclusions APOE variants show different level of cortical plasticity and are independently associated with clinical progression in AD patients. Tau pathology is specific for ApoE4 group driving cortical plasticity impairment and cognitive decline. ApoE4 patients represent a pure model of TAU-driven AD pathology. ApoE3 patients are characterized by different mechanisms of cortical plasticity impairment and clinical symptoms. LTP impairment is a marker of pathophysiological dysfunction in AD and, as such, it should be taken in account also for the adoption of new pharmacological strategies, considering AD as a disorder of synaptic plasticity.

Background: Acute optic neuritis is often in association with multiple sclerosis (MS). Proinflammatory cytokines trigger neuronal damage in neuroinflammatory disorders but their role in optic neuritis is poorly investigated. Objective: The objective of this work is to investigate the associations of intrathecal contents of proinflammatory cytokines with transient and persistent dysfunctions after optic neuritis. Methods: In 50 MS patients followed for up to six months, cerebrospinal fluid (CSF) levels of IL-1β, TNF and IL-8 were determined, along with clinical, neurophysiological and morphological measures of optic neuritis severity. Results: Visual impairment, measured by high- and low-contrast visual acuity, and delayed visual-evoked potential (VEP) latencies were significantly correlated to IL-8 levels during optic neuritis. IL-8 at the time of optic neuritis was also associated with persistent demyelination and final axonal loss, inferred by VEP and optical coherence tomography measures, respectively. Contents of IL-8 were correlated to functional visual outcomes, being higher among patients with incomplete recovery. Multivariate analysis confirmed that IL-8 significantly predicted final visual acuity, at equal values of demographics and baseline visual scores. Conclusion: Our study points to IL-8 as the main inflammatory cytokine associated with demyelination and secondary neurodegeneration in the optic nerve after optic neuritis.

nized with PLP139-151, a clearly defined relapsing–remitting disease course similar to relapsing–remitting multiple sclerosis. OLC populations in the CNS were quantified by flow cytometry throughout an EAE disease course. To prevent the large infiltration of immune cells from skewing resident CNS cell analysis, we incorporated an antibody against the hematopoietic marker CD45 into the panel and gated out those cells in OLC analysis. Flow cytometry results demonstrated a robust loss of oligodendrocytes during onset of EAE and an early expansion of OPCs. By disease peak the early OPCs expressed more mature markers of OLCs but never fully differentiated into mature myelin-forming oligodendrocytes throughout the rest of the disease course. The early expansion of OPCs was further associated with an increase in the chemotactic protein CD44 in OPCs from the brain. In summary we present here quantification of OLCs and functional global analysis of the myelinproducing cells in EAE mice by flow cytometry. Characterization in mouse models of human CNS disease presents a rapid means to evaluate potential therapeutic interventions in vivo for direct and indirect effects on demyelination and myelin regeneration.

B cells and/or the enhanced inflammatory milieu in the subarachnoid space are supposed to have a role in cortical pathology of progressive multiple sclerosis (PMS). The efficacy of intravenous rituximab to deplete circulating B cells is remarkable in MS, and its intrathecal delivery could target compartmentalized inflammation in PMS. We describe the central and peripheral effects of repeated intrathecal rituximab administrations in a patient with severe PMS. Peripheral CD20+ B cells were reduced, while oligoclonal bands were unaffected. Several central proinflammatory cytokines, and markers of neurodegeneration were markedly reduced. Central B cells modulation should be investigated in PMS.

Background Understanding how inflammation causes neuronal damage is of paramount importance in multiple sclerosis (MS) and in other neurodegenerative diseases. Here we addressed the role of the apoptotic cascade in the synaptic abnormalities and neuronal loss caused by the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor (TNF-α) in brain tissues, and disease progression caused by inflammation in relapsing-remitting MS (RRMS) patients. Results The effect of IL-1β, but not of TNF-α, on glutamate-mediated excitatory postsynaptic currents was blocked by pifithrin-α (PFT), inhibitor of p53. The protein kinase C (PKC)/transient receptor potential vanilloid 1 (TRPV1) pathway was involved in IL-1β-p53 interaction at glutamatergic synapses, as pharmacological modulation of this inflammation-relevant molecular pathway affected PFT effects on the synaptic action of IL-1β. IL-1β-induced neuronal swelling was also blocked by PFT, and IL-1β increased the expression of p21, a canonical downstream target of activated p53. Consistent with these in vitro results, the Pro/Pro genotype of p53, associated with low efficiency of transcription of p53-regulated genes, abrogated the association between IL-1β cerebrospinal fluid (CSF) levels and disability progression in RRMS patients. The interaction between p53 and CSF IL-1β was also evaluated at the optical coherence tomography (OCT), showing that IL-1β-driven neurodegenerative damage, causing alterations of macular volume and of retinal nerve fibre layer thickness, was modulated by the p53 genotype. Conclusions Inflammatory synaptopathy and neurodegeneration caused by IL-1β in RRMS patients involve the apoptotic cascade. Targeting IL-1β-p53 interaction might result in significant neuroprotection in MS.

Background and purpose Multiple sclerosis (MS) patients discontinuing natalizumab are at risk of rebound of disease activity. Methods In the present multi-center, open-label, non-randomized, prospective, pilot study, we tested whether treatment with glatiramer acetate (GA) is safe and effective after natalizumab in MS patients. The study was performed at academic tertiary medical centers. Forty active relapsing–remitting MS patients who never failed GA therapy and who discontinued natalizumab after 12–18 months of therapy were enrolled. GA was initiated 4 weeks after the last dose of natalizumab. Results 62.5% of patients were relapse-free 12 months after GA initiation. Annualized relapse rate and time to relapse were significantly lower than before natalizumab. Notably, the frequency of relapses was significantly lower amongst those patients who had experienced ≤2 relapses the year before initiation of natalizumab therapy, compared with patients who had had three or more relapses. No evidence of rebound was observed in magnetic resonance imaging scans. Furthermore, Expanded Disability Status Scale and Multiple Sclerosis Functional Composite were stable in our patients, again suggesting that 12 months of post-natalizumab–GA therapy is not associated with clinical deterioration. Conclusions Following discontinuation of natalizumab, 12 months of therapy with GA is safe and well tolerated in MS patients. GA can reduce the risk of early reactivation/rebound of disease activity in this setting.

Background: Multiple sclerosis (MS) patients discontinuing natalizumab treatment are at risk of disease reactivation. No clinical or surrogate parameters exist to identify patients at risk of post-natalizumab MS reactivation. Objective: To determine the role of natalizumab-induced lymphocytosis and of Akt polymorphisms in disease reactivation after natalizumab discontinuation. Methods: Peripheral leukocyte count and composition were monitored in 93 MS patients during natalizumab treatment, and in 56 of these subjects who discontinued the treatment. Genetic variants of the anti-apoptotic protein Akt were determined in all subjects because natalizumab modulates the apoptotic pathway and lymphocyte survival is regulated by the apoptotic cascade. Results: Natalizumab-induced peripheral lymphocytosis protected from post-natalizumab MS reactivation. Subjects who relapsed or had magnetic resonance imaging (MRI) worsening after treatment cessation, in fact, had milder peripheral lymphocyte increases during the treatment, largely caused by less marked T cell increase. Furthermore, subjects carrying a variant of the gene coding for Akt associated with reduced anti-apoptotic efficiency (rs2498804T) had lower lymphocytosis and higher risk of disease reactivation. Conclusion: This study identified one functionally meaningful genetic variant within the Akt signaling pathway that is associated with both lymphocyte count and composition alterations during natalizumab treatment, and with the risk of disease reactivation after natalizumab discontinuation.

Abnormal glutamate-dependent synaptic excitation contributes to neuronal damage in multiple sclerosis (MS). Little is known about the involvement of the GABA system in this disorder. Here we found that cerebrospinal fluid (CSF) from MS patients with enhanced brain lesions on magnetic resonance imaging inhibited GABA transmission in mouse brain slices. Enhanced IL-1β neuronal action was responsible for this effect, because IL-1β receptor antagonist blocked, and exogenous IL-1β mimicked the synaptic effect of inflamed CSF. Our results provide evidence that focal inflammation in MS perturbs the cytokine milieu within the circulating CSF, resulting in diffuse GABAergic alteration in neurons.

Background There are two generally accepted strategies for treating multiple sclerosis (MS), preventing central nervous system (CNS) damage indirectly through immunomodulatory interventions and/or repairing CNS damage by promoting remyelination. Both approaches also provide neuroprotection since they can prevent, indirectly or directly, axonal damage. Objective Recent experimental and clinical evidence indicates that the novel immunomodulatory drug laquinimod can exert a neuroprotective role in MS. Whether laquinimod-mediated neuroprotection is exerted directly on neuronal cells or indirectly via peripheral immunomodulation is still unclear. Methods C57Bl/6 experimental autoimmune encephalomyelitis (EAE) mice, immunised with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, were treated for 26 days with subcutaneous daily injections of laquinimod (from 1 to 25 mg/kg). Patch clamp electrophysiology was performed on acute brain striatal slices from EAE mice treated with daily (25 mg/kg) laquinimod and on acute brain striatal slices from control mice bathed with laquinimod (1–30 µM). Results Both preventive and therapeutic laquinimod treatment fully prevented the alterations of GABAergic synapses induced by EAE, the first limiting also glutamatergic synaptic alterations. This dual effect might, in turn, have limited glutamatergic excitotoxicity, a phenomenon previously observed early during EAE and possibly correlated with later axonal damage. Furthermore, laquinimod treatment also preserved cannabinoid CB1 receptor sensitivity, normally lost during EAE. Finally, laquinimod per se was able to regulate synaptic transmission by increasing inhibitory post-synaptic currents and, at the same time, reducing excitatory post-synaptic currents. Conclusions Our data suggest a novel neuroprotective mechanism by which laquinimod might in vivo protect from neuronal damage occurring as a consequence of inflammatory immune-mediated demyelination.