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Abstract Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motoneurons (MN) degeneration. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by aggregation of mutant proteins, among which the RNA binding protein FUS. Here we show that, in neuronal cells and in iPSC-derived MN expressing mutant FUS, such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished. Interestingly, stress granules formed in ALS conditions showed a distinctive transcriptome with respect to control cells, which reverted to similar to control after m6A downregulation. Notably, cells expressing mutant FUS were characterized by higher m6A levels suggesting a possible link between m6A homeostasis and pathological aggregates. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, an inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.

Abstract The Popeye domain containing (POPDC) genes encode sarcolemma-localized cAMP effector proteins. Mutations in blood vessel epicardial substance (BVES) also known as POPDC1 and POPDC2 have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms. Biopsy material of patients carrying mutations in BVES were immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein–protein interaction. Patients carrying a novel homozygous variant, BVES (c.547G > T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. In contrast, variants such as BVES p.Q153X or POPDC2 p.W188X were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1–POPDC2 complex. Mutations in POPDC proteins that cause an impairment in membrane localization affect POPDC complex formation while mutations which leave protein–protein interaction intact likely affect some other essential function of POPDC proteins.

Researchers studying Amyotrophic Lateral Sclerosis (ALS) have made significant efforts to find a unique mechanism to explain the etiopathology of the different forms of the disease. However, despite several mutations associated with ALS having been discovered in recent years, the link between the mutated genes and its onset has not yet been fully elucidated. Among the genes associated with ALS, superoxide dismutase 1 (SOD1) was the first to be identified, but its role in the etiopathogenesis of the disease is still unclear. In recent years, research has been focused on the non-coding part of the genome to fully understand the mechanisms underlying gene regulation. Non-coding RNAs are conserved molecules and are not usually translated in protein. A total of 98% of the human genome is composed of non-protein coding sequences with roles in the transcriptional and post-transcriptional regulation of gene expression. In this study, we characterized a divergent nuclear lncRNA (SOD1-DT) transcribed in the antisense direction from the 5′ region of the SOD1 coding gene in both the SH-SY5Y cell line and fibroblasts derived from ALS patients. Interestingly, this lncRNA seems to regulate gene expression, since its inhibition leads to the upregulation of surrounding genes including SOD1. SOD1-DT represents a very complex molecule, displaying allelic and transcriptional variability concerning transposable elements (TEs) included in its sequence, widening the scenario of gene expression regulation in ALS disease.

Background and aimsEdoxaban proved to be safe and effective also in fragile patients, but its administration through percutaneous endoscopic gastrostomy (PEG) has not been previously investigated. The purpose of this study was to evaluate the feasibility and the preliminary safety and efficacy profiles of edoxaban administered via PEG in patients with an indication for long-term oral anticoagulation.MethodsORIGAMI was a prospective, single-arm, observational study (NCT04271293). Patients with PEG and an indication for long-term anticoagulation were prospectively enrolled. Crushed edoxaban at approved doses was administered via PEG. The primary endpoint was the composite of cardio-embolic events consisting of ischemic stroke, systemic embolism, or symptomatic deep venous thrombosis/pulmonary embolism (DVT/PE). Secondary endpoints were the number of bleeding events and edoxaban plasma concentrations at steady state. We here report the 12-month results.ResultsA total of 12 patients were enrolled. The main indication for PEG implantation was amyotrophic lateral sclerosis (10/12). The primary endpoint of cardio-embolic events did not occur in any patients at 12 months. All patients were in the therapeutic range of steady-state edoxaban plasma levels. Three minor bleedings were observed, while no major bleedings occurred during the observational period. A total of five patients died. All deaths were from non-cardiovascular causes and were consistent with the natural history of the pre-existing severe disease.ConclusionOur study suggests that edoxaban administration via PEG is feasible and appears safe and effective in fragile, comorbid patients, resulting in therapeutic plasma concentrations of edoxaban.Clinical trial registration[ClinicalTrials.gov], identifier [NCT04271293].

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that selectively affects motor neurons. In 20% of cases, ALS appears in comorbidity with frontotemporal dementia (FTD). We generated patient-derived-induced Pluripotent Stem Cells (iPSCs), from an ALS/FTD patient. The patient had a familial form of the disease and a missense variant in TARDBP gene. We used an established protocol based on Sendai virus to reprogram fibroblasts. We confirmed the stemness and the pluripotency of the iPSC clones, thus generating embryoid bodies. We believe that the iPSC line carrying a TARDBP mutation could be a valuable tool to investigate TDP-43 proteinopathy linked to ALS.

Abstract Background An increasing number of studies evidences that amyotrophic lateral sclerosis (ALS) is characterized by extensive alterations in different cell types and in different regions besides the CNS. We previously reported the upregulation in ALS models of a gene called fibroblast-specific protein-1 or S100A4, recognized as a pro-inflammatory and profibrotic factor. Since inflammation and fibrosis are often mutual-sustaining events that contribute to establish a hostile environment for organ functions, the comprehension of the elements responsible for these interconnected pathways is crucial to disclose novel aspects involved in ALS pathology. Methods Here, we employed fibroblasts derived from ALS patients harboring the C9orf72 hexanucleotide repeat expansion and ALS patients with no mutations in known ALS-associated genes and we downregulated S100A4 using siRNA or the S100A4 transcriptional inhibitor niclosamide. Mice overexpressing human FUS were adopted to assess the effects of niclosamide in vivo on ALS pathology. Results We demonstrated that S100A4 underlies impaired autophagy and a profibrotic phenotype, which characterize ALS fibroblasts. Indeed, its inhibition reduces inflammatory, autophagic, and profibrotic pathways in ALS fibroblasts, and interferes with different markers known as pathogenic in the disease, such as mTOR, SQSTM1/p62, STAT3, α-SMA, and NF-κB. Importantly, niclosamide in vivo treatment of ALS-FUS mice reduces the expression of S100A4, α-SMA, and PDGFRβ in the spinal cord, as well as gliosis in central and peripheral nervous tissues, together with axonal impairment and displays beneficial effects on muscle atrophy, by promoting muscle regeneration and reducing fibrosis. Conclusion Our findings show that S100A4 has a role in ALS-related mechanisms, and that drugs such as niclosamide which are able to target inflammatory and fibrotic pathways could represent promising pharmacological tools for ALS.

Abstract Background Hereditary transthyretin amyloidosis (hATTR), alias ATTR variant (ATTRv) is a severe and disabling disease causing sensory and motor neuropathy, autonomic dysfunction, and cardiomyopathy. The progressive decline of patient’s functional autonomy negatively affects the patient’s quality of life and requires increasing involvement of relatives in the patient’s daily life. Family caregiving may become particularly demanding when the patient is no longer able to move independently. This study is focused on the psychosocial aspects of ATTRv from the patient and relative perspectives. In particular, it explored: the practical and psychological burdens experienced by symptomatic patients with ATTRv and their key relatives and the professional and social network support they may rely on; whether burden varied in relation to patients’ and relatives’ socio-demographic variables, patients’ clinical variables, and perceived professional and social network support; and, any difference in burden and support between patients and their matched relatives. Methods The study was carried out on symptomatic patients included in the ATTRv Italian national registry and living with at least one adult relative not suffering from severe illness and being free from ATTRv symptoms. Patients and relatives’ assessments were performed using validated self-reported tools. Results Overall, 141 patients and 69 relatives were evaluated. Constraints of leisure activities, feelings of loss and worries for the future were the consequences of ATTRv most frequently reported by patients and relatives. Both in patients and their relatives, the burden increased with the duration of symptoms and the level of help in daily activities needed by the patient. In the 69 matched patient-relative pairs, the practical burden was significantly higher among the patients than among their relatives, while the psychological burden was similar in the two groups. Moreover, compared to their relatives, patients with ATTRv reported higher levels of professional and social network support. Conclusions These results show that ATTRv is a disease affecting quality of life of both patients and their families. Supporting interventions should be guaranteed to patients, to facilitate their adaptation to the disease, and to their families, to cope as best as possible with the difficulties that this pathology may involve.

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with motor neuron degeneration, muscle atrophy and paralysis. To date, multiple panels of biomarkers have been described in ALS patients and murine models. Nevertheless, none of them has sufficient specificity and thus the molecular signature for ALS prognosis and progression remains to be elucidated. Here we overcome this limitation through a longitudinal study, analyzing serum levels of circulating miRNAs, stable molecules that are recently used as promising biomarkers for many types of human disorders, in ALS patients during the progression of the pathology. We performed next-generation sequencing (NGS) analysis and absolute RT quantification of serum samples of ALS patients and healthy controls. The expression levels of five selected miRNAs were quantitatively analyzed during disease progression in each patient and we demonstrated that high levels of miR-206, miR-133a and miR-151a-5p can predict a slower clinical decline of patient functionality. In particular, we found that miR-206 and miR-151a-5p serum levels were significantly up-regulated at the mild stage of ALS pathology, to decrease in the following moderate and severe stages, whereas the expression levels of miR-133a and miR-199a-5p remained low throughout the course of the disease, showing a diagnostic significance in moderate and severe stages for miR-133a and in mild and terminal ones for miR-199a-5p. Moreover, we found that miR-423–3p and 151a-5p were significantly downregulated respectively in mild and terminal stages of the disease. These data suggest that these miRNAs represent potential prognostic markers for ALS disease.

Background: Data from published studies about the effect of HFE polymorphisms on ALS risk, phenotype, and survival are still inconclusive. We aimed at evaluating whether the p.H63D polymorphism is a modifier of phenotype and survival in SOD1-mutated patients. Methods: We included 183 SOD1-mutated ALS patients. Mutations were classified as severe or mild according to the median survival of the study population. Patients were screened for the HFE p.H63D polymorphism. Survival was calculated using the Kaplan–Meier modeling, and differences were measured by the log-rank test. Multivariable analysis was performed with the Cox proportional hazards model (stepwise backward). Results: SOD1 severe mutation carriers show more frequent familial history for ALS and shorter survival compared to mild mutation carriers. Carriers and non-carriers of the p.H63D polymorphism did not differ in terms of sex ratio, frequency of positive familial history, age at onset, and bulbar/spinal ratio. In univariate and in Cox multivariable analysis using sex, age at onset, site of onset, family history, country of origin, and mutation severity as covariates, p.H63D carriers had a longer survival (p = 0.034 and p = 0.004). Conclusions: We found that SOD1-mutated ALS patients carrying the p.H63D HFE polymorphism have a longer survival compared to non-carriers, independently of sex, age and site of onset, family history, nation of origin, and severity of mutations, suggesting a possible role as disease progression modifier for the p.H63D HFE polymorphism in SOD1-ALS.

Abstract Objective Prevalence estimate of amyotrophic lateral sclerosis (ALS) ranged between 1.1/100,000 and 11.2/100,000 inhabitants with different design of the study (prospective or retrospective) and sample size. The aim of this study is to conduct for the first time an estimate of the ALS prevalence in the Latium region. Materials and methods The study was performed in Latium, a region located in the center of Italy, with a population, as of January 1, 2016, of 5888.472 inhabitants. In this region, a network of 15 clinical centers (of which 4 referral ALS centers are located in Rome) and 10 local health authorities involved in the diagnosis and treatment of ALS patients has been identified. Each patient was classified according to the El Escorial revised criteria. Results The prevalence study in 2016 identified 353 ALS cases (200 males). By considering population aged >=20 years, the total crude prevalence rate resulted 7.33 (CI95% 6.59–8.14) × 100,000 and 8.75 and 6.05 in males and females, respectively. Age‐specific prevalence rates did not differ among males and females in the population aged less than 49 years. The difference emerged in population aged > 50 years. This type of diagnosis was recorded for 343 patients (11 missing). 68% of these patients have a definite diagnosis, 14% likely, 11% possible, and 12% defined as suspect. Conclusions The estimate of prevalence rates observed in this study is probably in line with the values reported in the literature for prospective prevalence studies.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting upper and lower motor neurons. We generated patient-derived-induced Pluripotent Stem Cells (iPSCs), from an ALS patient affected by an early-onset and aggressive form of the disease, carrying a missense pathogenic variant in FUS gene. We reprogrammed somatic cells using an established Sendai virus protocol and we obtained clones of iPSC. We confirmed their stemness and further generated embryoid bodies, showing their potential of differentiating in all three germ layers. This iPSC line, carrying a pathogenic FUS variant, is a valuable tool to deeply investigate pathogenic mechanisms leading to ALS.

The objective of the study is to provide age-related normative values for dorsal sural nerve (DSN) and to analyse its application during follow-up of hereditary transthyretin amyloidosis (ATTRv) pre-symptomatic subjects. We consecutively recruited ATTRv pre-symptomatic carriers in which clinical examination, cardiological evaluation, and nerve conduction studies of the sural nerve and DSN were performed. To provide normative data of DSN, neurophysiologic parameters from healthy controls referred to our service were entered into linear regression analyses to check the relative influence of age and height. A correction grid was then derived. We collected 231 healthy subjects: the mean DSN sensory nerve action potential (SNAP) amplitude was 9.99 ± 5.48 μV; the mean conduction velocity was 49.01 ± 5.31 m/s. Significant correlations were found between age and height with DSN SNAP amplitude. Fifteen ATTRv pre-symptomatic carriers were examined. Sural nerve NCS were normal in 12/15 and revealed low/borderline values in three subjects. Considering our correction grid, we found an abnormal DNS amplitude in 9/15 subjects and low/borderline values in 2/15. In ATTRv, early detection of peripheral nerve damage is crucial to start a disease-modifying treatment. DSN may be easily and reliably included in the routine neurophysiological follow-up of ATTRv pre-symptomatic subjects.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative condition with phenotypic and genetic heterogeneity. It is characterized by the selective vulnerability and the progressive loss of the neural population. Here, an induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of an individual carrying the p.G376D mutation in the TDP-43 protein. Fibroblasts were reprogrammed using non-integrating episomal plasmids. There were no karyotype abnormalities, and iPSCs successfully differentiated into all three germ layers. This cell line may prove useful in the study of the pathogenic mechanisms that underpin ALS syndrome.

Abstract Extensive data support the safety of direct oral anticoagulants compared with vitamin K antagonists in patients with non‐valvular atrial fibrillation, leading to a significantly increase in the use of these compounds in clinical practice. However, there is no compelling evidence supporting the use of direct oral anticoagulant in individuals who are intubated or have a percutaneous endoscopic gastrostomy (PEG): patients with several co‐morbidities are underrepresented in clinical trials, so the best long‐term strategy for anticoagulation is difficult to ascertain. The aim of the present report was to evaluate the safety and efficacy of edoxaban administered via PEG in a patient with heart failure and a history of atrial fibrillation affected by amyotrophic lateral sclerosis (ALS). A 71‐year‐old man with atrial fibrillation, advanced ALS, type II diabetes mellitus, and hypertension presented to the emergency department with dyspnoea and tachycardia. Because vitamin K antagonist and rivaroxaban 15 mg were dropped because of difficult international normalized ratio control (time in therapeutic range

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of the upper and lower motor neurons (MNs). About 10% of patients have a family history (familial, fALS); however, most patients seem to develop the sporadic form of the disease (sALS). SOD1 (Cu/Zn superoxide dismutase-1) is the first studied gene among the ones related to ALS. Mutant SOD1 can adopt multiple misfolded conformation, lose the correct coordination of metal binding, decrease structural stability, and form aggregates. For all these reasons, it is complicated to characterize the conformational alterations of the ALS-associated mutant SOD1, and how they relate to toxicity. In this work, we performed a multilayered study on fibroblasts derived from two ALS patients, namely SOD1L145F and SOD1S135N, carrying the p.L145F and the p.S135N missense variants, respectively. The patients showed diverse symptoms and disease progression in accordance with our bioinformatic analysis, which predicted the different effects of the two mutations in terms of protein structure. Interestingly, both mutations had an effect on the fibroblast energy metabolisms. However, while the SOD1L145F fibroblasts still relied more on oxidative phosphorylation, the SOD1S135N fibroblasts showed a metabolic shift toward glycolysis. Our study suggests that SOD1 mutations might lead to alterations in the energy metabolism.

Fibroblasts isolated from an Amyotrophic Lateral Sclerosis (ALS)-patient carrying a mutation in Matrin-3 (p.Q66K -MATR3) gene were reprogrammed to the pluripotency stage by using non-integrating episomal plasmids. We generated the Q66K#44DRM induced pluripotent stem cell (iPSC) line that showed regular karyotype, expressed pluripotency-associated markers and were able to properly differentiate into the three germ layers. The heterozygous missense mutation in the MATR3 gene (p.Q66K), which is associated to ALS disease, was present in the generated iPSC line.Resource tableUnlabelled TableUnique stem cell line identifierCIBIOi001-AAlternative name(s) of stem cell lineQ66K#44DRMInstitutionCIBIO, University of Trento, ItalyContact information of distributorAlessandro Provenzani alessandro.provenzani@unitn.itType of cell lineiPSCs (induced pluripotent stem cells)OriginHumanAdditional origin infoAge:51Sex: MaleEthnicity if known: European, Italian.Cell sourcePatient derived fibroblastsClonalityClonal, clone number 44Method of reprogrammingEpisomal iPSC Reprogramming PlasmidGenetic modificationNOType of modificationSpontaneous mutation (missense)Associated diseaseAmyotrophic Lateral Sclerosis (ALS)Gene/locusMATR3/5q31.2Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock date16/01/2018Cell line repository/bankN/AEthical approvalPatient signed informed consent, number of protocol P/740/CE/2012 released by the ethical committee of the Catholic University School of Medicine

Objective: Hereditary transthyretin amyloidosis (ATTRv) represents a diagnostic challenge considering the great variability of clinical presentation and multiorgan involvement. In the present study, we report the prevalence of kidney involvement and kidney function over time in a cohort of ATTRv patients with different transthyretin gene mutations. Patients and Methods: For this study, we systematically collected data from all patients with a diagnosis of ATTRv followed at the Neurology Unit of Fondazione Policlinico Universitario A. Gemelli IRCCS. Kidney involvement was defined as presence of estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2 obtained with CKD-EPI equation, abnormal urinary protein excretion (UPE) (>150 mg/24 h) and/or albuminuria > 30 mg/24 h (or mg/g creatinine). The analysis included data from 46 patients with 122 measurements of serum creatinine. Results: Among the 46 patients included in the analysis, kidney involvement was present in 37%, with 15% showing reduced eGFR and 22% abnormal UPE (63% of patients with available UPE data). No single predictor was associated with either eGFR values or its slope over time. Conclusions: Kidney involvement is quite common in patients with ATTRv regardless of the underlying genetic variant. In particular, abnormal UPE appears to be a common feature of the disease.

Hereditary transthyretin amyloidosis (hATTR) is a rare disease caused by a point mutation in the transthyretin (TTR) gene and inherited in an autosomal dominant fashion. TTR is a plasma protein that functions as a carrier for thyroxine (T4) and retinol (vitamin A). Ophthalmological manifestations are due to both the hepatic and ocular production of mutated TTR. In this case series, we report the ocular manifestations of hATTR in eighteen eyes of nine consecutive patients. Corneal nerve abnormalities as well as morphological and functional changes in the retina were investigated. The study was a single-center, retrospective, observational, clinical case series. In all patients, corneal confocal microscopy (CCM), multimodal imaging of the retina, including fundus photography and Optical Coherence Tomography (OCT), as well as rod and cone electroretinography (ERG) were performed. Eight patients had active disease and one was an unaffected carrier. In all study eyes, corneal nerve plexa examined with CCM were poorly represented or absent. Mixed rod-cone and cone ERG b-wave amplitudes were reduced, and photopic b-wave responses were significantly delayed. Photopic Negative Response (PhNR) amplitude was significantly reduced, while PhNR latency was significantly augmented. In 13/18 eyes, vitreous opacities and abnormalities of vitreo-retinal interface were found. The current results highlight the presence of corneal nerve damage. Functional retinal abnormalities, detected by ERG, can be found even in the presence of minimal or absent structural retinal damage. These findings support the use of CCM and ERGs to detect early biomarkers for primary hATTR.

Hereditary transthyretin amyloidosis (ATTRv, v for variant) prevalence in Italy, a non-endemic region, has been established by ATTRv amyloidosis Italian Registry. However, values of prevalence were extremely heterogeneous, considering different regions. To properly establish the prevalence of the disease in the Lazio region, a survey was sent to university regional hospitals and to main regional hospitals, in order to collect all affected patients regularly followed. We identified 100 ATTRv patients and, considering a Lazio population of 5.8/million, we estimated a ATTRv prevalence of 17.2/million. The ATTRv amyloidosis Italian Registry reported a prevalence of 8.0/million in Lazio, while our survey showed a value of double this. Our survey documented a high-prevalence for a non-endemic country. The increased awareness of the disease among general practitioners and medical specialists is a fundamental step to reduce the diagnostic delay and start an effective treatment of this disease.

Hereditary amyloidosis associated with mutations in the transthyretin gene (hATTR) is a progressive devastating disease, with a fatal outcome occurring within 10years after onset. In recent years, TTR gene silencing therapy appeared as a promising therapeutic strategy, showing evidence that disease progression can be slowed and perhaps reversed. We report here 18 subjects affected by hATTR amyloidosis treated with patisiran, a small interfering RNA acting as TTR silencer, and evaluated with a PND score, the NIS and NIS-LL scale, and a Norfolk QOL-DN questionnaire at baseline and then every 6 months. A global clinical stabilizationwas observed for the majority of the patients, with mild-moderate improvements in some cases, even in advanced disease stage (PND score > 2). Analysis of NIS, NIS-LL and Norfolk QOL-DN results, and PND score variation suggest the possible presence of a 6-month latency period prior to benefit of treatment.

We investigated whether the C9orf72 repeat expansion is associated with specific clinical features, comorbidities, and prognosis in patients with amyotrophic lateral sclerosis (ALS). A cohort of 1417 ALS patients, diagnosed between January 1, 2009 and December 31, 2013 by 13 Italian ALS Referral Centers, was screened for the C9orf72 repeat expansion, and the analyses were performed comparing patients carrying this expansion (ALS-C9Pos) to those negative for this and other explored ALS-related mutations (ALS without genetic mutations, ALSwoGM). Compared to the ALSwoGM group, ALS-C9Pos patients (n = 84) were younger at disease onset, at the first clinical observation and at diagnosis (p < 0.001). After correcting for these differences, we found that ALS-C9Pos patients had higher odds of bulbar onset, diagnosis of frontotemporal dementia (FTD) and family history of ALS, FTD, and Alzheimer's disease and had lower odds of spinal onset, non-invasive ventilation, hypertension and psychiatric diseases than ALSwoGM patients. Among these variables, those related to shorter survival time were: bulbar onset, presence of FTD, hypertension, psychiatric disease, and family history of ALS (p < 0.05). Cox proportional hazards regression multivariate analysis suggested that carrying the C9orf72 repeat expansion was an independent factor negatively impacting on survival time in men (HR 1.58, 95% CI 1.07–2.33, p = 0.021), but not in women (p > 0.05) as well as in the whole sample (p > 0.05). When compared to ALSwoGM, ALS-C9Pos showed an earlier disease onset, no significant diagnostic delay and a higher odds of bulbar onset, FTD and family history of ALS and dementia. Moreover, male sex drove the negative effect of expanded variant on survival, confirming the hypothesis that sex is likely to be a crucial factor in the biology of C9orf72-related disease.

Chronic exposure to low levels of mercury is involved in the development of motor neuron diseases (MND). Genetic alterations may have a crucial role in the onset and progression. We presented a case of a TANK-binding kinase 1 (TBK1)-mutated 54-year-old male worker who developed a MND due to chronic mercury exposure at work. He was employed in a chlor-alkali plant in Central Italy. After two years of employment he had acute mercury intoxication with suggestive neurological symptoms and a high urinary level of the metal. Through years, many episodes of intoxication occurred, but he continued to perform the same job and be exposed to mercury. After yet another episode of intoxication in 2013, he showed fasciculations of the upper limbs and trunk, and electromyographic activity patterns were consistent with amyotrophic lateral sclerosis (ALS). In 2016, a genetic test revealed a mutation of TBK1, an ALS-related gene. This case highlights the important role of genetics in personalized occupational medicine. Occupational physicians should use genetic tests to identify conditions of individual susceptibility in workers with documented frequent episodes of mercury intoxication recorded during health surveillance programs to customize prevention measures in the workplace and act before damage appears.

Pathological evidence of amyloid on nerve biopsy has been the gold standard for diagnosis in hereditary transthyretin amyloidosis polyneuropathy (hATTR-PN) for a long time. In this article, we reviewed the pathological findings of a large series of sural nerve biopsies from a cohort of hATTR-PN patients, collected by different Italian referral centers. Patients and Methods: We reviewed clinical and pathological data from hATTR-PN patients, diagnosed and followed in five Italian referral centers for peripheral neuropathies. Diagnosis was formulated after a positive genetic test for transthyretin (TTR) mutations. Sural nerve biopsy was performed according to standard protocols. Results: Sixty-nine sural nerve biopsies from hATTR-PN patients were examined. Congo red positive deposits were found in 73% of cases. Only the Phe64Leu mutation failed to show amyloid deposits in a high percentage of biopsies (54%), as already described. Unusual pathological findings, such as myelin abnormalities or inflammatory infiltrates, were detected in occasional cases. Conclusions: Even if no longer indicated to confirm hATTR-PN clinical suspicion, nerve biopsy remains, in expert hands, a rapid and inexpensive tool to detect amyloid deposition. In Italy, clinicians should be aware that a negative biopsy does not exclude hATTR-PN, particularly for Phe64Leu, one of the most frequent mutations in this country.

The development of high-throughput sequencing technologies and screening of big patient cohorts with familial and sporadic amyotrophic lateral sclerosis (ALS) led to the identification of a significant number of genetic variants, which are sometimes difficult to interpret. The American College of Medical Genetics and Genomics (ACMG) provided guidelines to help molecular geneticists and pathologists to interpret variants found in laboratory testing. We assessed the application of the ACMG criteria to ALS-related variants, combining data from literature with our experience. We analyzed a cohort of 498 ALS patients using massive parallel sequencing of ALS-associated genes and identified 280 variants with a minor allele frequency < 1%. Examining all variants using the ACMG criteria, thus considering the type of variant, inheritance, familial segregation, and possible functional studies, we classified 20 variants as “pathogenic”. In conclusion, ALS’s genetic complexity, such as oligogenic inheritance, presence of genes acting as risk factors, and reduced penetrance, needs to be considered when interpreting variants. The goal of this work is to provide helpful suggestions to geneticists and clinicians dealing with ALS.

Objective: Multifocal motor neuropathy (MMN) is a rare inflammatory neuropathy, clinically characterized by exclusive motor involvement. We wished to evaluate the possible presence of sensory dysfunction, including the evaluation of small fibres, after a long-term disease course. Patients and methods: seven MMN patients, regularly followed in our Neurology Department, underwent clinical evaluation, neurophysiological examination by nerve conduction studies (NCSs), and Sudoscan. We compared neurophysiological data with a group of patients with other disorders of the peripheral nervous system. Results: NCSs showed a reduction of sensory nerve action potential amplitude in 2/7 MMN patients. Sudoscan showed borderline electrochemical skin conductance (ESC) values in 3/7 MMN patients (two of them with abnormal sensory NCSs). Conclusions: Our results confirm that sensory involvement may be found in some MMN after a long-term disease course, and it could also involve the small fibres.

Objective: Segmental demyelination is the pathological hallmark of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), but other elementary lesions are frequently observed, configuring a series of different pathological pictures. In this article, we review the pathological findings of a large series of sural nerve biopsies from our cohort of CIDP patients. Patients and Methods: Patients with CIDP who underwent nerve biopsy were retrospectively selected from those referred to the Institute of Neurology of the “Università Cattolica del Sacro Cuore” in Rome, Italy, from 1982 to February 2020. Sural nerve biopsy was performed according to standard protocols. Results: Sural nerve biopsy was performed in 43/130 CIDP patients. Demyelinating abnormalities and axonal loss were found in 67.4% and 83.7% of biopsies, respectively. Conversely, onion bulbs and inflammatory infiltrates were rare (18.6% and 4.7%, respectively). In three cases, we observed normal pathological findings. Conclusions: A pathognomonic pathological finding of CIDP cannot be established, but we confirm the utility of nerve biopsy in this setting to confirm the diagnosis (also in atypical phenotypes) and to elucidate pathogenic mechanisms.

Peripheral neuropathies are a vast group of diseases with heterogeneous aetiologies, including genetic and acquired causes. Several haematological disorders may cause an impairment of the peripheral nervous system, with diverse mechanisms and variable clinical, electrophysiological and pathological manifestations. In this practical review we considered the main phenotypes of peripheral nervous diseases and examined the haematological disorders which may associate with each of them. The area of intersection of neurological and haematological fields is of particular complexity and raises several problems in clinical practice. The personal crosstalk between neurologists and haematologists remains a fundamental tool for a proper diagnostic process which may lead to successful treatments in most cases.

S100A4, belonging to a large multifunctional S100 protein family, is a Ca2+-binding protein with a significant role in stimulating the motility of cancer and immune cells, as well as in promoting pro-inflammatory properties in different cell types. In the CNS, there is limited information concerning S100A4 presence and function. In this study, we analyzed the expression of S100A4 and the effect of the S100A4 transcriptional inhibitor niclosamide in murine activated primary microglia. We found that S100A4 was strongly up-regulated in reactive microglia and that niclosamide prevented NADPH oxidase 2, mTOR (mammalian target of rapamycin), and NF-κB (nuclear factor-kappa B) increase, cytoskeletal rearrangements, migration, and phagocytosis. Furthermore, we found that S100A4 was significantly up-regulated in astrocytes and microglia in the spinal cord of a transgenic rat SOD1-G93A model of amyotrophic lateral sclerosis. Finally, we demonstrated the increased expression of S100A4 also in fibroblasts derived from amyotrophic lateral sclerosis (ALS) patients carrying SOD1 pathogenic variants. These results ascribe S100A4 as a marker of microglial reactivity, suggesting the contribution of S100A4-regulated pathways to neuroinflammation, and identify niclosamide as a possible drug in the control and attenuation of reactive phenotypes of microglia, thus opening the way to further investigation for a new application in neurodegenerative conditions.

Myasthenia gravis (MG) is an autoimmune disease in which 90% of patients have autoantibodies against the muscle nicotinic acetylcholine receptor (AChR), while autoantibodies to muscle-specific tyrosine kinase (MuSK) have been detected in half (5%) of the remaining 10%. Recently, the low-density lipoprotein receptor-related protein 4 (LRP4), identified as the agrin receptor, has been recognized as a third autoimmune target in a significant portion of the double sero-negative (dSN) myasthenic individuals, with variable frequency depending on different methods and origin countries of the tested population. There is also convincing experimental evidence that anti-LRP4 autoantibodies may cause MG.The aim of this study was to test the presence and diagnostic significance of anti-LRP4 autoantibodies in an Italian population of 101 myasthenic patients (55 dSN, 23 AChR positive and 23 MuSK positive), 45 healthy blood donors and 40 patients with other neurological diseases as controls. All sera were analyzed by a cell-based antigen assay employing LRP4-transfected HEK293T cells, along with a flow cytofluorimetric detection system.We found a 14.5% (8/55) frequency of positivity in the dSN-MG group and a 13% frequency of co-occurrence (3/23) in both AChR and MuSK positive patients; moreover, we report a younger female prevalence with a mild form of disease in LRP4-positive dSN-MG individuals.Our data confirm LRP4 as a new autoimmune target, supporting the value of including anti-LRP4 antibodies in further studies on Myasthenia gravis.

Recent evidence suggested that muscle degeneration might lead and/or contribute to neurodegeneration, thus it possibly play a key role in the etiopathogenesis and progression of amyotrophic lateral sclerosis (ALS). To test this hypothesis, this study attempted to categorize functionally relevant genes within the genome-wide expression profile of human ALS skeletal muscle, using microarray technology and gene regulatory network analysis. The correlation network structures significantly change between patients and controls, indicating an increased inter-gene connection in patients compared to controls. The gene network observed in the ALS group seems to reflect the perturbation of muscle homeostasis and metabolic balance occurring in affected individuals. In particular, the network observed in the ALS muscles includes genes (PRKR1A, FOXO1, TRIM32, ACTN3, among others), whose functions connect the sarcomere integrity to mitochondrial oxidative metabolism. Overall, the analytical approach used in this study offer the possibility to observe higher levels of correlation (i.e. common expression trends) among genes, whose function seems to be aberrantly activated during the progression of muscle atrophy.