Abstract Background and aims Up to 30% of cases of acute type A aortic dissection (ATAAD) are associated with carotid artery dissection, a significant risk factor for acute ischemic stroke. Genetic testing is often performed to exclude the involvement of variants that can cause or predispose to this condition. We present the genetic study of a 58-year-old Italian woman affected by acute type A aortic dissection with right carotid artery dissection and subsequent acute ischemic stroke. Methods A Next Generation Sequence (NGS) panel of 25 genes involved in structural and functional mechanism of arterial wall was tested in the patient. Results Patient’s anamnesis included hypertension, active tobacco use and past alcohol abuse as vascular risk factors, while neither atraumatic fractures nor spontaneous dislocations except for congenital hip dislocation had ever occurred. Her 33-year-old daughter suffered from headache. No cases of arterial dissection were documented in other family members. Patient’s genetic study showed a heterozygous c.4649T>A p.(Ile1550Asn) missense variant in the protein kinase domain of MYLK gene, with a potential deleterious effect according to in silico models. This variant is rare, has never been previously described in literature and has therefore been classified of uncertain clinical significance (ACMG class 3). Conclusions This case highlights the possible pathogenic role of this novel missense MYLK variant in this case of ATAAD. We are currently monitoring first-grade relatives for any further clinical suggestion and screening them with aortic CT angiography potentially followed by genetic testing in order to better characterize the role of this variant. Conflict of interest Manuel Menabue: nothing to disclose Figure 1 - belongs to Results

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Abstract Background and aims Acute large vessel occlusions (LVOs) represent a severe form of acute ischemic stroke (AIS). In our university hospital, a collaborative approach between neuroradiologists and neurologists in performing endovascular thrombectomy (EVT) has been set up, shaping the role of "interventional neurologist". The study analyses the characteristics of patients treated in our center between December 2017 and May 2024, focusing on outcomes and treatment differences, particularly on a five-year period, during which all EVTs were performed by a team of neuroradiologists and neurologists. Methods This is a retrospective, single-center, observational cohort study that included patients with AIS, treated with EVT between December 2017 and May 2024, divided into two groups: (A) treated by a team composed of neuroradiologist and neurologist (2018-2023), and (B) treated only by the neuroradiologist (2023-2024). We collected data about clinical characteristics, times of treatment, recanalization (TICI), complications and outcomes. Results During the study period, 1091 patients received EVT. Group B showed significantly longer door-to-imaging (DTI) and door-to-groin (DTG) times, with a lower groin-to-recanization time (GTR), indicating delays in accessing neuroradiology and EVT, but shorter procedures. Reducing the sample of group A to patients treated between June 2021 and May 2023, only DTG differences remained significant. Logistic regression identified the presence of interventional neurologist, IV thrombolysis at the spoke hospital, and anterior circulation involvement as independent predictors of DTG <90 minutes, while procedures at night or on holidays predicted delays. Conclusions Interventional neurologist can improve access times to neuroimaging and endovascular treatment, contributing to the efficiency and duration of the angiographic procedure. Conflict of interest Dr. Zini received consulting and speaker fees from Angels Initiative, Boehringer-Ingelheim, Alexion, Daiichi Sankyo, Pfizer, Amgen, Aurora Biopharma, and fees for Advisory Board from Bayer, Daiichi Sankyo, Astra Zeneca, and Boehringer-Ingelheim, none relevant to this work. The other authors have nothing to disclose.

Tofersen is a gene-targeted therapy for superoxide dismutase 1 (SOD1)-associated amyotrophic lateral sclerosis (ALS), but neurofilament light chain (NfL) may not fully capture the biological response to treatment. We performed a multicentre retrospective longitudinal study including 24 patients with SOD1-ALS treated with intrathecal tofersen at four Italian referral centres between 2022 and 2025. Cerebrospinal fluid (CSF) and serum biomarkers were assessed at baseline, month 3, month 6, and last available administration using single-molecule array assays to quantify NfL, glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase L1 (UCHL-1), and total Tau. NfL decreased after treatment initiation in both CSF and serum, providing the clearest pharmacodynamic signal. In contrast, CSF GFAP increased progressively over follow-up, while CSF total Tau and UCHL-1 rose mainly at later timepoints; serum GFAP, total Tau, and UCHL-1 also showed increases during follow-up. ALS Functional Rating Scale-Revised trajectories were broadly stable, whereas disease progression rate was lower at last follow-up than at baseline. Greater reductions in CSF NfL were observed in pathogenic versus uncertain SOD1 variants, and early serum NfL and UCHL-1 changes were associated with longer-term changes in disease progression. These findings suggest that longitudinal multi-analyte profiling may refine biological response stratification beyond NfL alone in tofersen-treated SOD1-ALS.

BACKGROUND:The contribution of environmental determinants in the etiology of amyotrophic lateral sclerosis (ALS) is still unclear. Among the various environmental factors, exposure to green spaces, also known as greenness, is attracting considerable interest as many studies have reported its beneficial associations to health outcomes, particularly to neurodegenerative diseases. METHODS:To investigate the relation between greenness and ALS risk, we conducted a population-based case-control study in a Northern Italy population (from Modena, Reggio Emilia and Parma provinces), including 499 cases of ALS newly-diagnosed from 1998 to 2011 and 1,935 sex-, age-, and province-matched controls randomly selected from study provinces residents. We evaluated the association between greenness in the proximity of residence and ALS risk, assessing exposure through multiple satellite-based and land-use derived indices, both conventional and novel devised, for a total of six indices, each providing specific information, including annual and seasonal Normalized Difference Vegetation Index (NDVI), NDVI-weighted to green areas, green cover ratio, accessibility index, and their combined Green Exposure Index (GEI). We used conditional logistic regression models to evaluate disease risk for increasing exposure through both fixed-categories and non-linear restricted cubic splines. RESULTS:We observed a non-linear U-shaped association between greenness and ALS risk with increased odds ratios at both low and high levels. Results were more defined when using NDVI-based indices, while the associations were smoother when considering GEI. The higher risk at low levels may be related to lower accessibility to green spaces with lower physical activity and higher exposure to outdoor air pollutants, whilst elevated greenness may reflect higher exposure to neurotoxic pesticides. These results were confirmed also after adjustment for potential confounders, namely magnetic fields and light at night. Sex stratified analysis yielded similar results, except for more distinct associations in females for GEI. CONCLUSIONS:Despite the limitations due to possible unmeasured confounding and exposure misclassification related to the use of residential data, our results provide evidence of an inverse association between intermediate residential greenness and ALS risk, and may have public health implications including disease prevention and urban planning.

Growing evidence suggests that microplastics, particularly nanoplastics, may be neurotoxic. However, there are few studies of neurologic diseases, especially amyotrophic lateral sclerosis (ALS). In a hospital-based case-control study, we measured in vivo concentrations of nano- and microplastics (size 0.1-10 µm) in serum and CSF among 24 newly diagnosed cases of ALS and 20 controls. Adjusting for sex and age, we found a strong positive association between serum and CSF microplastic concentrations and higher microplastic concentrations in the CSF and serum of ALS cases compared with controls. In spline regression analyses, CSF microplastic concentrations showed a positive monotonic association with odds of ALS. Serum microplastic concentrations also showed a positive association with ALS, but only above a certain threshold. Among ALS cases, serum-but not CSF-microplastic concentrations were positively associated with neurofilament light chain, a biomarker of neuroaxonal damage. Given the case-control design, we cannot rule out reverse causation (i.e. that ALS-related factors caused greater bioaccumulation of microplastics) or the possibility that the association reflects changes in lifestyle or other chemical exposures. However, the observed pattern raises the possibility of an aetiologic role of nano- and microplastics in motor neuron degeneration and indicates higher CNS concentrations of these chemicals in ALS cases.

OBJECTIVE:The objective of this study was to evaluate the prognostic value of several muscle damage biomarkers. METHODS:Data from Piemonte and Valle d'Aosta Amyotrophic Lateral Sclerosis (PARALS) were considered for this study. Survival was defined as the time from diagnosis to death, tracheostomy, or the censoring date. Blood levels of potassium, creatinine, creatine kinase, phosphorus, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) diagnosis were evaluated as potential prognostic biomarkers. A Cox model was developed for each biomarker and adjusted for sex, onset age, onset site, and diagnostic delay. Significant findings from PARALS were evaluated in the Pooled Resource Open-Access Amyotrophic Lateral Sclerosis Clinical Trials (PRO-ACT) database. Additionally, a joint model was constructed to evaluate the prognostic role of phosphatemia slope over time using longitudinal data from PRO-ACT. RESULTS:A total of 1,444 and 1,023 patients were included in the PARALS and PRO-ACT cohorts, respectively. Only creatinine (hazard ratio [HR] = 0.65, 95% confidence interval [CI] = 0.50-0.85) and phosphorus (HR = 1.14, 95% CI = 1.04-1.24) showed a significant association with survival in the PARALS cohort. These findings were further validated in the PRO-ACT cohort (creatinine HR = 0.21, 95% CI = 0.13-0.35, p < 0.0001; phosphorus HR = 2.35, 95% CI = 1.13-4.88, p = 0.02). Longitudinal data from the PRO-ACT database showed that an increase of 0.1 mmol/l per month in phosphate levels was also associated with a HR of 8.26 (95% CI = 1.07-96.6, p = 0.044). INTERPRETATION:Creatininemia was confirmed as a prognostic marker in amyotrophic lateral sclerosis (ALS). Additionally, both phosphatemia levels at diagnosis and its rate of change over time were identified as a potential prognostic marker for ALS. As with other blood biomarkers, phosphate levels are cost-effective and minimally invasive to measure, supporting their potential use in clinical trials. ANN NEUROL 2025;98:286-293.

OBJECTIVE:Despite its clinical heterogeneity, amyotrophic lateral sclerosis is unified by early and prominent alterations in cortical excitability, increasingly recognized as contributors to disease progression. This study assessed whether the ratio between motor evoked potential (MEP) amplitude, reflecting upper motor neuron integrity, and compound muscle action potential (CMAP) amplitude, indexing lower motor neuron function, could provide an accessible marker of corticospinal excitability to stratify patients by phenotype, stage, and survival. METHODS:In this multicenter retrospective study, 743 amyotrophic lateral sclerosis patients from 16 tertiary centers in Italy were analyzed. The MEP:CMAP ratio, recorded from upper limb muscles, was categorized as hyperexcitable, normal, or hypoexcitable. Phenotypes included progressive muscular atrophy (or lower motor neuron), flail arm/leg, classic, bulbar, patient with predominant upper motor neuron signs (or pyramidal), and primary lateral sclerosis. Disease stage was assessed using King's staging. Survival was analyzed using Kaplan-Meier curves and Cox regression models. RESULTS:The MEP:CMAP ratio differed significantly across phenotypes (p < 0.0001), with hyperexcitability predominating in lower motor neuron, flail, classic, and bulbar forms, and hypoexcitability in pyramidal and primary lateral sclerosis. Hypoexcitability increased in advanced King's stages (p < 0.0001). Hyperexcitable patients had shorter survival (p = 0.004), including when tested within 1 year of onset (p = 0.006). Cox regression identified the MEP:CMAP ratio as an independent survival predictor (HR 1.84, 95% CI 1.12-3.03, p = 0.016). INTERPRETATION:This real-world study supports the clinical value of the MEP:CMAP ratio as a scalable biomarker of cortical excitability in amyotrophic lateral sclerosis, with prognostic relevance across phenotypes and disease stages. ANN NEUROL 2025;98:801-813.

Mitochondrial dysfunction is a key pathological hallmark in amyotrophic lateral sclerosis (ALS), yet the role of circulating cell-free mitochondrial DNA (Cf-mtDNA) as a biomarker remains unclear. This study aimed to investigate serum Cf-mtDNA levels in ALS patients compared to healthy controls and explore its associations with disease biomarkers, clinical progression, and survival. We conducted a case-control study measuring Cf-mtDNA levels in serum samples from 54 ALS patients and 36 age- and sex-matched healthy controls using quantitative droplet digital PCR. Correlations between Cf-mtDNA levels and clinical features, neurofilament concentrations, inflammatory indices, and survival were assessed. The average Cf-mtDNA level in ALS patients was 2,426,315 copies/mL of serum (IQR: 865000-2475000), compared to 1,885,667 copies/mL of serum (IQR: 394250-2492500) in controls (p = 0.308). ROC analysis yielded an AUC of 0.595 (95% CI: 0.468-0.721), indicating very limited discriminant ability. Cf-mtDNA levels were inversely correlated with serum creatinine concentrations (r = -0.335, p = 0.018), but showed no significant associations with ALS phenotype, disease staging, neurofilaments, inflammatory indices, or survival. These findings suggest that, in a predominantly sporadic ALS cohort, serum Cf-mtDNA may not serve as a standalone diagnostic or prognostic biomarker, in contrast to previous reports. Methodological differences, cohort composition, and genetic heterogeneity may account for these discrepancies. Our results underscore the importance of further large-scale, longitudinal studies incorporating genetic stratification and multi-biomarker approaches to better elucidate the role of Cf-mtDNA in ALS pathophysiology.

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two neurodegenerative disorders that share common genes and pathomechanisms and are referred to as the ALS-FTD spectrum. A hallmark of ALS-FTD pathology is the abnormal aggregation of proteins, including Cu/Zn superoxide dismutase (SOD1), transactive response DNA-binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS/TLS), and dipeptide repeat proteins resulting from C9orf72 hexanucleotide expansions. Genetic mutations linked to ALS-FTD disrupt protein stability, phase separation, and interaction networks, promoting misfolding and insolubility. This review explores the molecular mechanisms underlying protein aggregation in ALS-FTD, with a particular focus on TDP-43, as it represents the main aggregated species inside pathological inclusions and can also aggregate in its wild-type form. Moreover, this review describes the protective mechanisms activated by the cells to prevent protein aggregation, including molecular chaperones and post-translational modifications (PTMs). Understanding these regulatory pathways could offer new insights into targeted interventions aimed at mitigating cell toxicity and restoring cellular function.

Type 2 diabetes mellitus (T2DM) as a comorbidity in amyotrophic lateral sclerosis (ALS) has sparked interest for its potential impact on disease expression and prognosis. In this retrospective cohort study, we investigated the prevalence and clinical correlates of T2DM in a large cohort of patients from the ALS registry of a Northern Italy region, Emilia Romagna, established in 2009. Out of 1756 ALS patients enrolled up to 2021, 145 were affected by T2DM (diALS). Patients with diALS were older than those without T2DM (ndALS) (71.56 vs. 65.76 years, p < 0.001), had a higher body mass index (25.63 vs. 24.23, p < 0.001), but experienced greater weight loss at diagnosis (6.87% vs. 5.44%, p < 0.007). Respiratory onset (6.2% vs. 2.6%, p = 0.013) and respiratory phenotype (4.2% vs. 1.4%, p = 0.04) were more frequent among diALS. Coherently, diALS presented a lower forced vital capacity (74.9% vs. 87.9%, p ≤ 0.001) and more frequently adopted Non-Invasive Ventilation (NIV) (50.35% vs. 37.61%, p = 0.003), with significant influence on time to NIV (HR 1.71, 95% CI 1.07-2.74, p = 0.024). Exploring genetic background, among all the genes examined C9ORF72 emerged as underrepresented among diALS (7.64% in ndALS vs. 0% in diALS, p = 0.039). In conclusion, we confirmed a more severe respiratory dysfunction in diALS, suggesting a specific frailty in respiratory muscles, together with some peculiar clinical features consistent with the previous literature data, such as a later onset. The lower prevalence of C9ORF72 expansion in this population may hint towards a specific role of the gene in metabolism and inflammation, granting more space to non-genetic causes, warranting further studies for confirmation.

Mutations in NEK1, encoding for a serine/threonine kinase which regulates several biological processes, are associated with amyotrophic lateral sclerosis (ALS). NEK1 was analysed by amplicon deep sequencing in a cohort of 1016 Italian sporadic and familial ALS patients previously screened for C9orf72, SOD1, TARDBP and FUS mutations. We identified 28 rare NEK1 variants in 29 patients (2.85

IntroductionT regulatory cells (Tregs) inversely correlate with disease progression in Amyotrophic Lateral Sclerosis (ALS) and fast-progressing ALS patients have been reported to exhibit dysfunctional, as well as reduced, levels of Tregs. This study aimed to evaluate the longitudinal changes in Tregs among ALS patients, considering potential clinical and biological modifiers of their percentages and concentrations. Additionally, we explored whether measures of ALS progression, such as the decline over time in the revised ALS Functional Rating Scale (ALSFRS-r) or forced vital capacity (FVC) correlated Treg levels and whether Treg phenotype varied during the course of ALS.MethodsTotal Tregs (detected by CD3, CD4, FoxP3, CD25, and CD127) were quantified at five time points over 54 weeks in 21 patients in the placebo arm of the RAP-ALS trial; next they were characterized for the expression of surface markers including CD38, CD39, CXCR3, and PD1. Repeated measures mixed models were used to analyze the longitudinal course of Tregs, considering potential associations with other clinical and laboratory characteristics. Correlations between ALSFRS-r or FVC and Tregs over time were similarly investigated.ResultsOur study showed that Treg levels did not change significantly on average during the observation period in our ALS cohort. However, PD1+Tregs decreased and CD39+Tregs increased over time. Male sex and cholesterol levels were associated with increasing Tregs (%) over time, while monocytes positively affected Treg concentrations. Treg concentrations showed a modesty association with FVC decline but were not associated with ALSFRS-r decline.DiscussionTreg levels remained stable during the ALS observation period and were not significantly associated with ALSFRS-r variations, suggesting that Treg numbers alone may have limited utility as a pharmaco-dynamic biomarker for ALS trials. However the observed changes in Treg phenotypes, such as the decrease in PD1+Tregs, indicate that phenotypic variations may warrant further investigation for their potential role in ALS progression and therapeutic targeting.

The initiation of tofersen, a new specific antisense oligonucleotide (ASO) for SOD1 pathology, marked a significant turning point for SOD1-ALS patients. While clinical trials and early access program studies reported a significant reduction in plasma and cerebrospinal fluid (CSF) neurofilament levels, neuroinflammation following prolonged treatment was never assessed. In this multicenter study, we evaluated a cohort of 18 SOD1-ALS patients treated with tofersen, analyzing correlations between biomarkers of neurodegeneration/neuroinflammation and clinical variables indicative of disease progression. NfL, NfH, CHI3L1, and Serpina1 levels in serum and CSF were determined by semi-automated immunoassays (Ella™ technology). Generalized linear mixed models were employed to investigate longitudinal trends of these biomarkers. Our data highlighted a progressive decrease in CSF neurofilament levels during tofersen treatment (MR = 0.97, 95% CI 0.94–0.99, p = 0.006 and MR = 0.98, 95% CI 0.95–1.00, p = 0.076 for NfL and NfH in CSF, respectively). Conversely, CSF levels of SerpinA1 and CHI3L1 increased over time (MR = 1.12, 95% CI 1.08–1.16, p < 0.0001 and MR = 1.039, 95% CI 1.015–1.062, p = 0.001 for SerpinA1 and CHI3L1 in CSF, respectively), but these modifications were most apparent after six and twelve months of therapy, respectively. Disease progression rate did not correlate with these biomarker trends. We observed a significant decrease in neurofilament levels during Tofersen treatment, alongside an increase in neuroinflammatory markers, potentially linked to an immune response triggered by ASO treatment. Given the limited data on tofersen’s long-term efficacy in ALS due to its recent introduction, identifying biomarkers that predict clinical outcomes such as diminished therapeutic response or adverse effects is crucial. These biomarkers may help to better understand the underlying pathomechanisms of ALS and tofersen’s role in modulating disease progression.

Background/Objectives: C9ORF72 expansion is associated with significant phenotypic heterogeneity. This study aimed to characterize the clinical features of C9ALS patients from the Emilia Romagna ALS registry (ERRALS) and compare them with non-mutated ALS (nmALS) patients matched for sex, age at onset, and diagnostic delay, sourced from the same register. Methods: In total, 67 C9ALS patients were compared to 201 nmALS. Clinical data, phenotype, and prognostic factors were analyzed in the two groups and within the C9ALS group after stratification by sex. Results: C9ALS patients displayed a higher disease progression rate and shorter times to gastrostomy and invasive ventilation, despite no differences in overall survival. Female C9ALS had a more severe bulbar and upper motor neuron involvement compared to males. Cognitive and behavioral symptoms were more common in the C9ALS group, and the former was an independent prognostic factor. Prevalences of, autoimmune diseases, and dyslipidemia were significantly higher among C9ALS patients. Conclusions: In our dataset, we show an overall increased disease progression rate in C9ALS patients and hint at sex-specific discrepancies in some phenotypical characteristics. We also suggest a possible clinically relevant involvement of C9ORF72 expansion in metabolism and autoimmunity.

While the role of systemic and neuroinflammatory players is actually consolidated in amyotrophic lateral sclerosis (ALS) pathogenesis, a representative and reliable panel of inflammatory prognostic biomarkers is still lacking. This retrospective study on 182 ALS patients from the Modena ALS Center, investigates biomarkers of axonal injury (neurofilaments) and microglial and astrocytic activation (SerpinA1, TREM2, CHI3L1, OPN and S100B, respectively). In a subpopulation of patients, we examined blood count-derived parameters, including the Systemic-Immune-Inflammation index (SII), Systemic Inflammation Response Index (SIRI) and Aggregate Systemic Inflammation Index (AISI). All variables were analyzed for association with clinical features in the entire cohort and then in sex- and age-based subgroups. SerpinA1CSF was significantly lower in females [4.43 μg/ml (IQR 2.76-6.3) vs 5.61 μg/ml (IQR 3.39-9.16),p = 0.032], while SII indexes was oppositely distributed [higher in females, 540.67 (IQR 363.05-807.24) vs 384.89 (IQR 307.5-578.52),p = 0.015]. In univariate survival analysis, without overcoming neurofilaments, SIRI (HR 1.43, 95 %CI 1.03-1.966,p = 0.03), AISI (HR 1.002, 95 %CI 1.001-1.003, p = 0.002) and SII (HR 1.001, 95 %CI 1.0003-1.001,p = 0.003) impact negatively on survival, with AISI retaining its power at multivariate analysis (HR 1.002, 95 %CI 1.0004-1.001,p = 0.012). SerpinA1CSF levels influenced survival only for females (HR 1.042, 95 %CI 1.0065-1.078,p = 0.02), in a similar manner of SIRI (HR 1.483, 95 %CI 1.082-2.0334,p = 0.014) and SII (HR 1.00099, 95 %CI 1.0008-1.003,p = 0.001). Our data revealed the influence of sex on survival by SerpinA1CSF, CHI3L1CSF and systemic biomarkers in females. However, both neurofilaments and CHI3L1CSF outperform the other neuroinflammatory biomarkers at predicting rate of disease progression, so the prognostic meaning of these measure alone remains unconclusive, requiring larger collaborative studies.

Amyotrophic Lateral Sclerosis (ALS) is a complex neurodegenerative disorder characterized by wide phenotypic heterogeneity. Despite efforts to carefully define and stratify ALS patients according to their clinical and genetic features, prognosis prediction still remains unreliable. Biomarkers that reflect changes in the central nervous system would be useful, but the physical impossibility of direct sampling and analysis of the nervous system makes them challenging to validate. A highly explored option is the identification of neuronal-specific markers that could be analyzed in peripheral biofluids. This review focuses on the description of the physical and biological barriers to the central nervous system and of the composition of biofluids in which ALS disease biomarkers are actively searched. Finally, we comment on already validated biomarkers, such as the neurofilament light chain, and show the potential of extracellular vesicles (EVs) and cell-free DNA as additional biomarkers for disease prediction.