ARPP21 defines a TDP-43-independent aggregation pathway in amyotrophic lateral sclerosis

Currently, pathological inclusions of ubiquitinated TDP-43 are considered central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, this view has yielded sparse attention to covert alternative pathways of aggregation in the disease. Here, we identified a pathological axis independent of TDP-43 led by ARPP21, a SUZ domain-containing RNA-binding protein encoded by a gene recently described in strong association with ALS. ARPP21 showed large fibrillar non-ubiquitinated aggregates in all the non-SOD1 ALS cases studied, consistently segregating from TDP-43 pathology. Biophysically, ARPP21 undergoes spontaneous phase separation, with a higher propensity for condensate formation than TDP-43 but without co-aggregating with it. ARPP21 also exhibited very slow soluble behavior and lower condensate dynamics compared to TDP-43, an effect that was more pronounced for the ARPP21-P529L disease-related variant. In human iPSC-derived motor neurons, expression of ARPP21 variants was sufficient to drive condensate formation and reduce cell viability. Mechanistically, ARPP21 promoted intercellular propagation by inducing tunnelling nanotube formation, enabling efficient cell-to-cell spreading of aggregates. Genetic analyses further identified multiple ARPP21 variants in ALS, supporting its clinical relevance. Together, our findings uncover ARPP21 as a previously unrecognized TDP-43-independent aggregation pathway in ALS, with implications for disease heterogeneity and therapeutic targeting.