Detection of TDP-43 Proteinopathies in Brain and Cerebrospinal Fluid Using Seed Amplification Assay

Misfolded TAR DNA-binding protein 43 (TDP-43) is the primary pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). While seed amplification assays (SAAs), such as real-time quaking-induced conversion (RT-QuIC), have shown promise in detecting misfolded TDP-43 in cerebrospinal fluid (CSF) and olfactory mucosa, technically accessible methodologies are urgently required for widespread clinical application. We developed a streamlined, non-immunoprecipitation-based TDP-43 RT-QuIC assay to assess seeding activity in brain tissue and CSF. We evaluated its diagnostic performance using CSF from patients with TDP-43 proteinopathies and control subjects, and further examined its association with neurofilament light chain (NfL) and tau-related biomarkers. In CSF analysis, the assay demonstrated positive seeding activity in 70% (21/30) of patients with ALS and dementia, 50% (5/10) of patients with FTLD, and 40% (8/20) of patients with ALS alone. The assay exhibited excellent specificity, yielding negative results in >99% (199/200) of control samples, including those with autoimmune or electrophysiological abnormalities. Furthermore, CSF analysis demonstrated significantly higher NfL levels in TDP-43 SAA-positive cases compared to SAA-negative cases (p < 0.0008). The highest NfL concentrations were observed in the SAA-positive ALS with dementia and ALS cohorts, contrasting with lower levels in FTLD. Tau-related biomarkers exhibited no significant differences between the groups. Our streamlined, non-immunoprecipitation TDP-43 RT-QuIC assay provides highly specific detection of pathological TDP-43 seeding activity. While the assay detects the underlying TDP-43 proteinopathy rather than distinguishing between ALS and FTLD clinical phenotypes, its technical simplicity and combined utility with NfL measurements offer a robust, scalable framework for biomarker development. This approach provides a practical foundation for future multi-center validation and international standardization efforts.