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.

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.

The revised ALS Functional Rating Scale (ALSFRS-R) and the blood plasma accumulation of neurofilament light chain (NfL) are key markers for tracking amyotrophic lateral sclerosis (ALS) progression. Based on recent studies indicating that dietary supplementation with conjugated linoleic acid (CLA) may boost antioxidant enzyme activity in ALS patients, in this study, ALSFRS-R scores (and subscores), NfL and thiol-bound protein (P-SH) levels, and intracellular activity of antioxidant enzymes (G6PD and GSR) were measured in ALS patients after 6 months (T6) of treatment with riluzole (R) or riluzole + CLA (R + CLA); we also investigated the correlations between these markers. At baseline (T0), G6PD activity in the R group was positively correlated with ALSFRS-R score (p < 0.001), whereas plasma P-SH levels in the R + CLA group were inversely correlated with ALSFRS-R score (p = 0.033). No significant baseline association was found between NfL levels and total ALSFRS-R score in the R group. At six months (T6), GSR and G6PD activities in the R + CLA group were significantly higher than in the R group (p < 0.05). Furthermore, a significant inverse correlation between plasma NfL levels and ALSFRS-R total score emerged at T6 exclusively in the R group (p = 0.002). Subscore analysis at T6 showed that plasma NfL levels negatively correlated with bulbar function in both groups (R: p = 0.010; R + CLA: p = 0.020) and with fine motor function in the R group (p = 0.031); whereas no significant correlation was observed for the respiratory subscore in either treatment group. These findings are consistent with an enhanced antioxidant activity associated with CLA supplementation. Overall, these preliminary exploratory results suggest that systemic redox biomarkers and plasma NfL may provide complementary, non-redundant information on distinct aspects of ALS pathobiology, though their precise temporal and mechanistic interactions require confirmation by dedicated longitudinal studies. The need for the combined use of neurodegeneration and oxidative-stress biomarkers for monitoring the progression of ALS is highlighted.