TDP-43-related cryptic splicing reflects pathological heterogeneity in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) involves TAR DNA-binding protein 43 (TDP-43) dysfunction, leading to cryptic splicing, including UNC13A cryptic exon inclusion and truncated STMN2 production. Pathologically, ALS is classified into three phosphorylated TDP-43 subtypes (Types 1, 2a, and 2b) based on the distribution of neuronal and glial cytoplasmic inclusions and the density of dystrophic neurites. However, the relationship between these pathological subtypes and TDP-43-associated cryptic splicing remains unclear. This retrospective postmortem study aimed to analyze motor cortex tissue from sporadic ALS cases (Type 1, n = 12; Type 2a, n = 12; Type 2b, n = 11) with previously described subtype-specific morphological features. UNC13A cryptic RNA and truncated STMN2 RNA levels were quantified using quantitative real-time PCR (qRT-PCR). Compared with Types 1 and 2a, Type 2b, characterized by abundant thread-, dot-, and grain-like dystrophic neurites, exhibited significantly higher levels of UNC13A cryptic RNA and truncated STMN2 RNA. Droplet digital PCR revealed a similar subtype-dependent trend and showed strong correlations with qRT-PCR measurements, providing partial support for the primary findings. UNC13A cryptic RNA and truncated STMN2 RNA levels were positively correlated (Spearman's ρ = 0.58), indicating coordinated dysregulation of multiple TDP-43-dependent splicing targets. Our results demonstrate that the burden of TDP-43-associated cryptic splicing differs across ALS pathological subtypes. Additional analyses indicated that these subtype-dependent differences were not readily accounted for by disease duration, RNA quality, normalization strategy, or semiquantitative neuronal loss scores. These findings suggest that molecular heterogeneity parallels pathological heterogeneity in ALS and provide additional insight into the biological diversity of TDP-43 proteinopathy.