Astrocyte-derived extracellular vesicle phosphorylated TDP-43 as a blood-based biomarker for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by central nervous system (CNS) neuroglial TAR DNA-binding protein 43 (TDP-43) pathology in ∼90-95% of cases. Non-invasive biomarkers are critically needed to reduce diagnostic delay, enable patient stratification, and assess treatment efficacy in clinical trials. Astrocyte-derived extracellular vesicles (ADEVs) represent promising CNS liquid biopsy biomarkers, given astrocyte involvement in ALS pathogenesis and the ability of ADEVs to cross the blood-brain barrier and carry TDP-43. Here, we assessed the diagnostic performance of TDP-43 and its hyperphosphorylated pathological form, phospho-Ser409 (pTDP-43), quantified in ADEVs, for distinguishing ALS cases from matched healthy controls. ADEVs were immunoisolated from whole blood (n = 76) and plasma (n = 86) across three ALS biorepositories/cohorts. ADEV quality and enrichment were validated in accordance with minimal information for studies of extracellular vesicles guidelines, and protein levels were normalized by ratio to tetraspanin cluster of differentiation 81 (CD81). Diagnostic accuracy was evaluated by logistic regression and random forest models with cross-validation, adjusting for age and sex across 100 random seeds. Plasma ADEV pTDP-43/CD81 ratio best predicted ALS case status, with a mean AUC of 0.89 (95% CI: 0.75-0.99) from logistic models, corresponding to a mean sensitivity and specificity of 87% and 89%, respectively, and a mean AUC of 0.86 (95% CI: 0.82-0.90) from random forest models. Although the specificity of ADEV pTDP-43 relative to disease mimics and associations with disease progression require further investigation, these findings support its potential as a biomarker candidate for ALS and for patient stratification in TDP-43-targeted clinical trials.