High Frequency of Cognitive and Behavioral Impairment in Amyotrophic Lateral Sclerosis Patients with SOD1 Pathogenic Variants.

OBJECTIVE: While the cognitive-behavioral characteristics of amyotrophic lateral sclerosis (ALS) patients carrying C9orf72 pathological repeat expansion have been extensively studied, our understanding of those carrying SOD1 variants is mostly based on case reports. The aim of this paper is to extensively explore the cognitive-behavioral characteristics of a cohort of ALS patients carrying pathogenetic variants of SOD1 gene, comparing them to patients without pathogenetic variants of 46 ALS-related genes (wild-type [WT]-ALS) and healthy controls. METHODS: All ALS patients seen at the Turin ALS expert center in the 2009-2021 period who underwent both cognitive/behavioral and extensive genetic testing were eligible to be included in the study. Only patients with SOD1 pathogenetic variants (n = 28) (SOD1-ALS) and WT-ALS (n = 829) were enrolled in the study. A series of 129 controls was also included. RESULTS: Among the 28 SOD1-ALS patients, 16 (57.1%) had normal cognitive function, 5 (17.9%) isolated cognitive impairment (ALSci) (17.9%), 6 (21.4%) isolated behavioral impairment (ALSbi), 1 (3.6%) cognitive and behavioral impairment (ALScbi), and no one ALS-FTD. SOD1-ALS performed worse than controls in all explored domains, in particular Social Cognition and Language domains. SOD1-ALS patients had similar scores in all tests compared to WT-ALS, except the Story-based Empathy Task (SET), where they performed worse. INTERPRETATION: Cognitive-behavioral impairment is much more common in SOD1 patients than previously assumed. SOD1-ALS are characterized by a more frequent impairment of Social Cognition and, less markedly, of Language domains. These findings have relevant implication both in the clinical and in the research setting, also considering recently approved treatment for SOD1-ALS. ANN NEUROL 2024;96:150-158.

Giving Breath to Motor Neurons: Noninvasive Mechanical Ventilation Slows Disease Progression in Amyotrophic Lateral Sclerosis.

OBJECTIVE: Noninvasive mechanical ventilation (NIMV) improves amyotrophic lateral sclerosis (ALS) quality of life and survival. However, data about its effect on disease progression are still lacking. Here, we test whether NIMV use changed the rate of functional decline among ALS patients. METHODS: In this retrospective observational study, we included 448 ALS patients followed up at the ALS Center in Turin, Italy, who underwent NIMV during the disease course. The primary outcome was the change in functional decline after NIMV initiation adjusting for covariates. Functional decline was based on the nonrespiratory items of the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R). RESULTS: NIMV initiation resulted in a slower functional decline (mean improvement = 0.16 points per month, 95% confidence interval = 0.12-0.19, p < 0.001), with consistent effects observed across various demographic factors, including sex, age at diagnosis, and disease duration before NIMV initiation. This finding was replicated using the PRO-ACT (Pooled Resource Open-Access ALS Clinical Trials) dataset. The favorable impact of NIMV on ALSFRS-R progression was evident independently of disease stages. Notably, NIMV benefits were not dose-dependent but were particularly prominent for nighttime respiratory support. INTERPRETATION: NIMV significantly influences the rate of motor progression in ALS, and this effect is not determined by the nonlinearity of ALSFRS-R trajectory. The functional decline slowed following NIMV initiation, independently of the site of disease onset or disease severity at the time of NIMV initiation. Our findings underscore the importance of timely NIMV initiation for all ALS patients and highlight the need to consider NIMV-induced slowing of disease progression when evaluating clinical trial outcomes. ANN NEUROL 2024;95:817-822.

Sex Differences in Amyotrophic Lateral Sclerosis Survival and Progression: A Multidimensional Analysis.

OBJECTIVE: To investigate sex-related differences in amyotrophic lateral sclerosis (ALS) prognosis and their contributing factors. METHODS: Our primary cohort was the Piemonte and Aosta Register for ALS (PARALS); the Pooled Resource Open-Access ALS Clinical Trials (PRO-ACT) and the Answer ALS databases were used for validation. Survival analyses were conducted accounting for age and onset site. The roles of forced vital capacity and weight decline were explored through a causal mediation analysis. Survival and disease progression rates were also evaluated after propensity score matching. RESULTS: The PARALS cohort included 1,890 individuals (44.8% women). Men showed shorter survival when stratified by onset site (spinal onset HR 1.20, 95% CI 1.00-1.44, p = 0.0439; bulbar onset HR 1.36, 95% CI 1.09-1.70, p = 0.006917), although women had a steeper functional decline (+0.10 ALSFRS-R points/month, 95% CI 0.07-0.15, p < 0.00001) regardless of onset site. Instead, men showed worse respiratory decline (-4.2 forced vital capacity%/month, 95% CI -6.3 to -2.2, p < 0.0001) and faster weight loss (-0.15 kg/month, 95% CI -0.25 to -0.05, p = 0.0030). Causal mediation analysis showed that respiratory function and weight loss were pivotal in sex-related survival differences. Analysis of patients from PRO-ACT (n = 1,394, 40.9% women) and Answer ALS (n = 849, 37.2% women) confirmed these trends. INTERPRETATION: The shorter survival in men is linked to worse respiratory function and weight loss rather than a faster disease progression. These findings emphasize the importance of considering sex-specific factors in understanding ALS pathophysiology and designing tailored therapeutic strategies. ANN NEUROL 2024;96:159-169.

A mother and her daughter carrying a pathogenic expansion of the HTT gene with a phenotype encompassing motor neuron disease and Huntington's disease.

Recently, pathogenic expansions (range 40-64 CAG repeats) in the HTT gene have been found in patients diagnosed with pure frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). We report a mother with Huntington's disease (HD) associated with motor neuron disease (MND) signs and her daughter suffering from ALS with subtle signs of HD, both carrying a pathogenic allele of the HTT gene (i.e., >39 repeats). The co-occurrence of MND and chorea has been reported in previous cases. Subjects showing both ALS and HD signs and carrying HTT pathogenic expansions in two generations of the same kindred have never been reported so far. The study of the overlap of disease mechanisms at the cellular level between TDP-43 and Huntingtin is relevant in an era offering promising strategies of targeted treatments in neurodegenerative disorders.

Disentangling the relationship between social cognition, executive functions and behaviour changes in amyotrophic lateral sclerosis.

BACKGROUND: Social cognition (SC) deficits are included in the amyotrophic lateral sclerosis-frontotemporal spectrum disorder (ALS-FTDS) revised diagnostic criteria. However, the impact of SC assessment on cognitive classification and the cognitive-behavioural correlates of SC remain unclear. This cross-sectional study aimed to assess the impact of SC assessment on ALS-FTDS categorisation and explore the relationship of SC with executive functions (EF) and behaviour changes in a cohort of ALS patients. METHODS: 121 patients and 56 healthy controls from the Turin ALS Centre underwent cognitive/behavioural testing, including the SC subdomains of facial emotion recognition, and cognitive and affective theory of mind (ToM). RESULTS: Patients performed significantly worse than controls in all SC explored domains, and 45% of patients exhibited a deficit in at least one SC test, dissociated from the presence of EF deficits. In 13% of cases, the SC deficit was isolated and subclinical. SC assessment contributed to the attribution of cognitive impairment in 10% of patients. Through a statistical clustering approach, we found that ToM only partially overlaps with EF while behaviour changes are associated with emotional disorders (anxiety and depression). CONCLUSIONS: SC is overall independent of EF in ALS, with ToM only partially associated with specific EF measures, and behaviour changes associated with emotional disorders. The influence of SC on cognitive categorisation and the frequent identification of a subclinical SC impairment have implications in a clinical setting, considering the substantial impact of cognitive impairment on disease burden and therapeutic choices.

Long-term modifications of the peripheral immune repertoire after switching from sequestering disease-modifying treatments in multiple sclerosis.

BACKGROUND: Scarce data are available on the long-term immunological effects of multiple sclerosis (MS) disease-modifying treatments (DMTs). OBJECTIVES: This study aimed to investigate the long-term modifications of the peripheral immune repertoire on interruption of a sequestering DMT (natalizumab, fingolimod) and switch to another high-efficacy DMT. METHODS: Lymphocyte subpopulations were assessed, every 6 months up to 48 months, in patients switched from fingolimod or natalizumab to ocrelizumab, and in patients switched from fingolimod to natalizumab, compared to patients switched to ocrelizumab or natalizumab from a moderate-efficacy DMT and to naive patients. RESULTS: We included 389 MS patients (200 ocrelizumab and 189 natalizumab). After adjusting for baseline variables, patients switched from fingolimod to ocrelizumab showed lower CD3 + and CD4 + lymphocytes up to 48 months after switch (with lower percentage of naive CD4 +), and increased odds of total, CD3+, CD4+ lymphopenia. Patients switched from natalizumab to ocrelizumab showed higher CD3 + lymphocytes up to 36 months after switch, and higher CD4+, CD8+ lymphocytes up to 24 months. The frequency of infections was not influenced by previous treatment. CONCLUSIONS: A long-term persistence of the residual effects of the exposure to sequestering DMTs (fingolimod and less natalizumab) on the peripheral immune repertoire was observed after switching to another high-efficacy DMT.

Use of the combination of spirometry, arterial blood gas analysis and overnight oximetry to predict the outcomes of patients affected by motor neuron disease: The Milan-Torin respiratory score (Mi-To-RS).

BACKGROUND AND PURPOSE: The use of multiple tests, including spirometry, arterial blood gas (ABG) analysis and overnight oximetry (OvOx), is highly recommended to monitor the respiratory function of patients with motor neuron disease (MND). In this study, we propose a composite score to simplify the respiratory management of MND patients and better stratify their prognosis. MATERIALS AND METHODS: We screened the clinical charts of 471 non-ventilated MND patients referred to the Neuro-rehabilitation Unit of the San Raffaele Scientific Institute of Milan (January 2001-December 2019), collecting spirometric, ABG and OvOx parameters. To evaluate the prognostic role of each measurement, univariate Cox regression for death/tracheostomy was performed, and the variables associated with survival were selected to design a scoring system. Univariate and multivariate Cox regression analyses were then carried out to evaluate the prognostic role of the score. Finally, results were replicated in an independent cohort from the Turin ALS Center. RESULTS: The study population included 450 patients. Six measurements were found to be significantly associated with survival and were selected to design a scoring system (maximum score = 8 points). Kaplan-Meier analysis showed significant stratification of survival and time to non-invasive mechanical ventilation adaptation according to score values, and multivariate analysis confirmed the independent effect of the respiratory score on survival of each cohort. CONCLUSION: Forced vital capacity, ABG and OvOx parameters provide complementary information for the respiratory management and prognosis of MND patients and the combination of these parameters into a single score might help neurologists predict prognosis and guide decisions on the timing of the implementation of different diagnostic or therapeutic approaches.

Distinct neural signatures of pulvinar in C9orf72 amyotrophic lateral sclerosis mutation carriers and noncarriers.

BACKGROUND AND PURPOSE: Thalamic alterations have been reported as a major feature in presymptomatic and symptomatic patients carrying the C9orf72 mutation across the frontotemporal dementia-amyotrophic lateral sclerosis (ALS) spectrum. Specifically, the pulvinar, a high-order thalamic nucleus and timekeeper for large-scale cortical networks, has been hypothesized to be involved in C9orf72-related neurodegenerative diseases. We investigated whether pulvinar volume can be useful for differential diagnosis in ALS C9orf72 mutation carriers and noncarriers and how underlying functional connectivity changes affect this region. METHODS: We studied 19 ALS C9orf72 mutation carriers (ALSC9+) accurately matched with wild-type ALS (ALSC9-) and ALS mimic (ALSmimic) patients using structural and resting-state functional magnetic resonance imaging data. Pulvinar volume was computed using automatic segmentation. Seed-to-voxel functional connectivity analyses were performed using seeds from a pulvinar functional parcellation. RESULTS: Pulvinar structural integrity had high discriminative values for ALSC9+ patients compared to ALSmimic (area under the curve [AUC] = 0.86) and ALSC9- (AUC = 0.77) patients, yielding a volume cutpoint of approximately 0.23%. Compared to ALSmimic, ALSC9- showed increased anterior, inferior, and lateral pulvinar connections with bilateral occipital-temporal-parietal regions, whereas ALSC9+ showed no differences. ALSC9+ patients when compared to ALSC9- patients showed reduced pulvinar-occipital connectivity for anterior and inferior pulvinar seeds. CONCLUSIONS: Pulvinar volume could be a differential biomarker closely related to the C9orf72 mutation. A pulvinar-cortical circuit dysfunction might play a critical role in disease progression and development, in both the genetic phenotype and ALS wild-type patients.

Prognostic communication in amyotrophic lateral sclerosis: findings from a Nationwide Italian survey.

BACKGROUND: Amyotrophic Lateral Sclerosis (ALS) is a fatal motor neuron disease with a highly variable prognosis. Among the proposed prognostic models, the European Network for the cure of ALS (ENCALS) survival model has demonstrated good predictive performance. However, few studies have examined prognostic communication and the diffusion of prognostic algorithms in ALS care. OBJECTIVE: To investigate neurologists' attitudes toward prognostic communication and their knowledge and utilization of the ENCALS survival model in clinical practice. METHODS: A web-based survey was administered between May 2021 and March 2022 to the 40 Italian ALS Centers members of the Motor Neuron Disease Study Group of the Italian Society of Neurology. RESULTS: Twenty-two out of 40 (55.0%) Italian ALS Centers responded to the survey, totaling 37 responses. The model was known by 27 (73.0%) respondents. However, it was predominantly utilized for research (81.1%) rather than for clinical prognostic communication (7.4%). Major obstacles to prognostic communication included the unpredictability of disease course, fear of a negative impact on patients or caregivers, dysfunctional reaction to diagnosis, and cognitive impairment. Nonetheless, the model was viewed as potentially useful for improving clinical management, increasing disease awareness, and facilitating care planning, especially end-of-life planning. CONCLUSIONS: Despite the widespread recognition and positive perceptions of the ENCALS survival model among Italian neurologists with expertise in ALS, its implementation in clinical practice remains limited. Addressing this disparity may require systematic investigations and targeted training to integrate tailored prognostic communication into ALS care protocols, aligning with the growing availability of prognostic tools for ALS.

Impact of Universal Use of the McGrath Videolaryngoscope as a Device for All Intubations in the Cardiac Operating Room. A Prospective Before-After VIDEOLAR-CAR Study.

OBJECTIVE: Tracheal intubation in cardiac surgery patients has a higher incidence of difficult laryngoscopic views compared with patients undergoing other types of surgery. The authors hypothesized that using the McGrath Mac videolaryngoscope as the first intubation option for cardiac surgery patients improves the percentage of patients with "easy intubation" compared with using a direct Macintosh laryngoscope. DESIGN: A prospective, observational, before-after study. SETTING: At a tertiary-care hospital. PARTICIPANTS: One thousand one hundred nine patients undergoing cardiac surgery. INTERVENTION: Consecutive patients undergoing cardiac surgery were intubated using, as the first option, a Macintosh laryngoscope (preinterventional phase) or a McGrath Mac videolaryngoscope (interventional phase). MEASUREMENTS AND MAIN RESULTS: The main objective was to assess whether the use of the McGrath videolaryngoscope, as the first intubation option, improves the percentage of patients with "easy intubation," defined as successful intubation on the first attempt, modified Cormack-Lehane grades of I or IIa, and the absence of the need for adjuvant airway devices. A total of 1,109 patients were included, 801 in the noninterventional phase and 308 in the interventional phase. The incidence of "easy intubation" was 93% in the interventional phase versus 78% in the noninterventional phase (p < 0.001). First-success-rate intubation was higher in the interventional phase (304/308; 98.7%) compared with the noninterventional phase (754/801, 94.1%; p = 0.005). Intubation in the interventional phase showed decreases in the incidence of difficult laryngoscopy (12/308 [3.9%] v 157/801 [19.6%]; p < 0.001), as well as moderate or difficult intubation (5/308 [1.6%] v 57/801 [7.1%]; p < 0.001). CONCLUSIONS: The use of the McGrath videolaryngoscope as the first intubation option for tracheal intubation in cardiac surgery improves the percentage of patients with "easy" intubation," increasing glottic view and first-success-rate intubation and decreasing the incidence of moderate or difficult intubation.

Role of brain 2-[18F]fluoro-2-deoxy-D-glucose-positron-emission tomography as survival predictor in amyotrophic lateral sclerosis.

PURPOSE: The identification of prognostic tools in amyotrophic lateral sclerosis (ALS) would improve the design of clinical trials, the management of patients, and life planning. We aimed to evaluate the accuracy of brain 2-[18F]fluoro-2-deoxy-D-glucose-positron-emission tomography (2-[18F]FDG-PET) as an independent predictor of survival in ALS. METHODS: A prospective cohort study enrolled 418 ALS patients, who underwent brain 2-[18F]FDG-PET at diagnosis and whose survival time was available. We discretized the survival time in a finite number of classes in a data-driven fashion by employing a k-means-like strategy. We identified "hot brain regions" with maximal power in discriminating survival classes, by evaluating the Laplacian scores in a class-aware fashion. We retained the top-m features for each class to train the classification systems (i.e., a support vector machine, SVM), using 10% of the ALS cohort as test set. RESULTS: Data were discretized in three survival profiles: 0-2 years, 2-5 years, and > 5 years. SVM resulted in an error rate < 20% for two out of three classes separately. As for class one, the discriminant clusters included left caudate body and anterior cingulate cortex. The most discriminant regions were bilateral cerebellar pyramid in class two, and right cerebellar dentate nucleus, and left cerebellar nodule in class three. CONCLUSION: Brain 2-[18F]FDG-PET along with artificial intelligence was able to predict with high accuracy the survival time range in our ALS cohort. Healthcare professionals can benefit from this prognostic tool for planning patients' management and follow-up. 2-[18F]FDG-PET represents a promising biomarker for individual patients' stratification in clinical trials. The lack of a multicentre external validation of the model warrants further studies to evaluate its generalization capability.

Epidemiology of Spinocerebellar Ataxias in Europe.

Spinocerebellar ataxias (SCAs) are a heterogenous group of rare neurodegenerative conditions sharing an autosomal dominant pattern of inheritance. More than 40 SCAs have been genetically determined. However, a systematic review of SCA epidemiology in Europe is still missing. Here we performed a narrative review of the literature on the epidemiology of the most common SCAs in Europe. PubMed, Embase, and MEDLINE were searched from inception until 1 April 2023. All English peer-reviewed articles published were considered and then filtered by abstract examination and subsequently by full text reading. A total of 917 original articles were retrieved. According to the inclusion criteria and after reviewing references for useful papers, a total of 35 articles were included in the review. Overall, SCA3 is the most frequent spinocerebellar ataxia in Europe. Its frequency is strikingly higher in Portugal, followed by Germany, France, and Netherlands. None or few cases were described in Italy, Russia, Poland, Serbia, Finland, and Norway. SCA1 and SCA2 globally displayed similar frequencies, and are more prevalent in Italy, United Kingdom, Poland, Serbia, and France.

Association of MRI leptomeningeal enhancement with disability worsening in progressive multiple sclerosis: A clinical and post-mortem study.

BACKGROUND: Leptomeningeal enhancement (LME) has been described as a biomarker of meningeal inflammation in multiple sclerosis (MS). OBJECTIVE: The aim of this study was to (1) assess if LME is predictive of disability worsening in progressive MS (pMS) patients and (2) investigate the pathological substrates of LME in an independent post-mortem MS series. METHODS: In total, 115 pMS patients were imaged yearly with 1.5T MRI, using post-contrast CUBE 3D FLAIR for LME detection. Endpoint: to identify the baseline variables predictive of confirmed disability worsening (CDW) at 24 months follow-up. Post-mortem, inflammation, and structural changes of the leptomeninges were assessed in 12 MS/8 control brains. RESULTS: LME (27% of patients at baseline) was associated with higher EDSS and lower brain volume (nBV). LME was unchanged in most patients over follow-up. LME at baseline MRI was independently associated with higher risk of 24 months CDW (HR 3.05, 95% CI 1.36-6.84, p = 0.007) in a Cox regression, including age, nBV, T2 lesion volume, high-efficacy treatments, and MRI disease activity. Post-mortem, focal structural changes (fibrosis) of the leptomeninges were observed in MS, usually associated with inflammation (Kendall's Tau 0.315, p < 0.0001). CONCLUSIONS: LME is frequently detected in pMS patients using 1.5T MRI and is independently predictive of disability progression. LME could result from both focal leptomeningeal post-inflammatory fibrosis and inflammation.

Amyotrophic lateral sclerosis regional progression intervals change according to time of involvement of different body regions.

BACKGROUND AND PURPOSE: The prediction of disease course is one of the main targets of amyotrophic lateral sclerosis (ALS) research, particularly considering its wide phenotypic heterogeneity. Despite many attempts to classify patients into prognostic categories according to the different spreading patterns at diagnosis, a precise regional progression rate and the time of involvement of each region has yet to be clarified. The aim of our study was to evaluate the functional decline in different body regions according to their time of involvement during disease course. METHODS: In a population-based dataset of ALS patients, we analysed the functional decline in different body regions according to time and order of regional involvement. We calculated the regional progression intervals (RPIs) between initial involvement and severe functional impairment using the ALS Functional Rating Scale revised (ALSFRS-r) subscores for the bulbar, upper limb, lower limb and respiratory/thoracic regions. Time-to-event analyses, adjusted for age, sex, ALSFRS-r pre-slope (ΔALSFRS-R), cognitive status, and mutational status were performed. RESULTS: The duration of RPI differed significantly among ALS phenotypes, with the RPI of the first region involved being significantly longer than the RPIs of regions involved later. Cox proportional hazard models showed that in fact a longer time between disease onset and initial regional involvement was related to a reduced duration of the RPI duration in each different body region (bulbar region: hazard ratio [HR] 1.11, 95% confidence interval [CI] 1.06-1.16, p < 0.001; upper limb region: HR 1.16, 95% CI 1.06-1.28, p = 0.002; lower limb region: HR 1.11, 95% CI 1.03-1.19, p = 0.009; respiratory/thoracic region: HR 1.10, 95% CI 1.06-1.14, p = 0.005). CONCLUSIONS: We found that the progression of functional decline accelerates in regions involved later during disease course. Our findings can be useful in patient management and prognosis prediction.

Effect of RNS60 in amyotrophic lateral sclerosis: a phase II multicentre, randomized, double-blind, placebo-controlled trial.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with limited treatment options. RNS60 is an immunomodulatory and neuroprotective investigational product that has shown efficacy in animal models of ALS and other neurodegenerative diseases. Its administration has been safe and well tolerated in ALS subjects in previous early phase trials. METHODS: This was a phase II, multicentre, randomized, double-blind, placebo-controlled, parallel-group trial. Participants diagnosed with definite, probable or probable laboratory-supported ALS were assigned to receive RNS60 or placebo administered for 24 weeks intravenously (375 ml) once a week and via nebulization (4 ml/day) on non-infusion days, followed by an additional 24 weeks off-treatment. The primary objective was to measure the effects of RNS60 treatment on selected biomarkers of inflammation and neurodegeneration in peripheral blood. Secondary objectives were to measure the effect of RNS60 on functional impairment (ALS Functional Rating Scale-Revised), a measure of self-sufficiency, respiratory function (forced vital capacity, FVC), quality of life (ALS Assessment Questionnaire-40, ALSAQ-40) and survival. Tolerability and safety were assessed. RESULTS: Seventy-four participants were assigned to RNS60 and 73 to placebo. Assessed biomarkers did not differ between arms. The mean rate of decline in FVC and the eating and drinking domain of ALSAQ-40 was slower in the RNS60 arm (FVC, difference 0.41 per week, standard error 0.16, p = 0.0101; ALSAQ-40, difference -0.19 per week, standard error 0.10, p = 0.0319). Adverse events were similar in the two arms. In a post hoc analysis, neurofilament light chain increased over time in bulbar onset placebo participants whilst remaining stable in those treated with RNS60. CONCLUSIONS: The positive effects of RNS60 on selected measures of respiratory and bulbar function warrant further investigation.

Phosphorylated TDP-43 aggregates in peripheral motor nerves of patients with amyotrophic lateral sclerosis.

Phosphorylated TDP-43 (pTDP-43) aggregates in the cytoplasm of motor neurons and neuroglia in the brain are one of the pathological hallmarks of amyotrophic lateral sclerosis. Although the axons exceed the total volume of motor neuron soma by several orders of magnitude, systematic studies investigating the presence and distribution of pTDP-43 aggregates within motor nerves are still lacking. The aim of this study is to define the TDP-43/pTDP-43 pathology in diagnostic motor nerve biopsies performed on a large cohort of patients presenting with a lower motor neuron syndrome and to assess whether this might be a discriminating tissue biomarker for amyotrophic lateral sclerosis and non-amyotrophic lateral sclerosis cases. We retrospectively evaluated 102 lower motor neuron syndrome patients referred to our centre for a diagnostic motor nerve biopsy. Histopathological criteria of motor neuron disease and motor neuropathy were applied by two independent evaluators, who were blind to clinical data. TDP-43 and pTDP-43 were evaluated by immunohistochemistry, and results compared to final clinical diagnosis. We detected significant differences between amyotrophic lateral sclerosis and non-amyotrophic lateral sclerosis cases in pTDP-43 expression in myelinated fibres: axonal accumulation was detected in 98.2% of patients with amyotrophic lateral sclerosis versus 30.4% of non-amyotrophic lateral sclerosis samples (P < 0.0001), while concomitant positive staining in Schwan cell cytoplasm was found in 70.2% of patients with amyotrophic lateral sclerosis versus 17.4% of patients who did not have amyotrophic lateral sclerosis (P < 0.001). Importantly, we were also able to detect pTDP-43 aggregates in amyotrophic lateral sclerosis cases displaying normal features at standard histopathological analysis. Our findings demonstrated that a specific pTDP-43 signature is present in the peripheral nervous system of patients with amyotrophic lateral sclerosis, and could be exploited as a specific, accessible tissue biomarker. The detection of pTDP-43 aggregates within motor nerves of living patients with amyotrophic lateral sclerosis, occurring before axonal degeneration, suggests that this is an early event that may contribute to amyotrophic lateral sclerosis pathogenesis.

Withdrawal of mechanical ventilation in amyotrophic lateral sclerosis patients: a multicenter Italian survey.

BACKGROUND: Law 219/2017 was approved in Italy in December 2017, after a years-long debate on the autonomy of healthcare choices. This Law, for the first time in Italian legislation, guarantees the patient's right to request for withdrawal of life-sustaining treatments, including mechanical ventilation (MV). OBJECTIVE: To investigate the current status of MV withdrawal in amyotrophic lateral sclerosis (ALS) patients in Italy and to assess the impact of Law 219/2017 on this practice. METHODS: We conducted a Web-based survey, addressed to Italian neurologists with expertise in ALS care, and members of the Motor Neuron Disease Study Group of the Italian Society of Neurology. RESULTS: Out of 40 ALS Italian centers, 34 (85.0%) responded to the survey. Law 219/2017 was followed by an increasing trend in MV withdrawals, and a significant increase of neurologists involved in this procedure (p 0.004). However, variations across Italian ALS centers were observed, regarding the inconsistent involvement of community health services and palliative care (PC) services, and the intervention and composition of the multidisciplinary team. CONCLUSIONS: Law 219/2017 has had a positive impact on the practice of MV withdrawal in ALS patients in Italy. The recent growing public attention on end-of-life care choices, along with the cultural and social changes in Italy, requires further regulatory frameworks that strengthen tools for self-determination, increased investment of resources in community and PC health services, and practical recommendations and guidelines for health workers involved.

SOMAscan Proteomics Identifies Novel Plasma Proteins in Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex disease characterized by the interplay of genetic and environmental factors for which, despite decades of intense research, diagnosis remains rather delayed, and most therapeutic options fail. Therefore, unravelling other potential pathogenetic mechanisms and searching for reliable markers are high priorities. In the present study, we employ the SOMAscan assay, an aptamer-based proteomic technology, to determine the circulating proteomic profile of ALS patients. The expression levels of ~1300 proteins were assessed in plasma, and 42 proteins with statistically significant differential expression between ALS patients and healthy controls were identified. Among these, four were upregulated proteins, Thymus- and activation-regulated chemokine, metalloproteinase inhibitor 3 and nidogen 1 and 2 were selected and validated by enzyme-linked immunosorbent assays in an overlapping cohort of patients. Following statistical analyses, different expression patterns of these proteins were observed in the familial and sporadic ALS patients. The proteins identified in this study might provide insight into ALS pathogenesis and represent potential candidates to develop novel targeted therapies.

Identification of BiP as a CB1 Receptor-Interacting Protein That Fine-Tunes Cannabinoid Signaling in the Mouse Brain.

Cannabinoids, the bioactive constituents of cannabis, exert a wide array of effects on the brain by engaging Type 1 cannabinoid receptor (CB1R). Accruing evidence supports that cannabinoid action relies on context-dependent factors, such as the biological characteristics of the target cell, suggesting that cell population-intrinsic molecular cues modulate CB1R-dependent signaling. Here, by using a yeast two-hybrid-based high-throughput screening, we identified BiP as a potential CB1R-interacting protein. We next found that CB1R and BiP interact specifically in vitro, and mapped the interaction site within the CB1R C-terminal (intracellular) domain and the BiP C-terminal (substrate-binding) domain-α. BiP selectively shaped agonist-evoked CB1R signaling by blocking an "alternative" Gq/11 protein-dependent signaling module while leaving the "classical" Gi/o protein-dependent inhibition of the cAMP pathway unaffected. In situ proximity ligation assays conducted on brain samples from various genetic mouse models of conditional loss or gain of CB1R expression allowed to map CB1R-BiP complexes selectively on terminals of GABAergic neurons. Behavioral studies using cannabinoid-treated male BiP+/- mice supported that CB1R-BiP complexes modulate cannabinoid-evoked anxiety, one of the most frequent undesired effects of cannabis. Together, by identifying BiP as a CB1R-interacting protein that controls receptor function in a signaling pathway- and neuron population-selective manner, our findings may help to understand the striking context-dependent actions of cannabis in the brain.SIGNIFICANCE STATEMENT Cannabis use is increasing worldwide, so innovative studies aimed to understand its complex mechanism of neurobiological action are warranted. Here, we found that cannabinoid CB1 receptor (CB1R), the primary molecular target of the bioactive constituents of cannabis, interacts specifically with an intracellular protein called BiP. The interaction between CB1R and BiP occurs selectively on terminals of GABAergic (inhibitory) neurons, and induces a remarkable shift in the CB1R-associated signaling profile. Behavioral studies conducted in mice support that CB1R-BiP complexes act as fine-tuners of anxiety, one of the most frequent undesired effects of cannabis use. Our findings open a new conceptual framework to understand the striking context-dependent pharmacological actions of cannabis in the brain.

Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis.

TDP-43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP-43 function at physiological levels both in vitro and in vivo Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain-of-function mutation in endogenous Tardbp causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.