Spinal cord extracts of amyotrophic lateral sclerosis spread TDP-43 pathology in cerebral organoids

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by progressive loss of motor neurons and there is currently no effective therapy. Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein 43 kDa (TDP-43) within the CNS is a pathological hallmark in sporadic ALS and prion-like propagation of pathogenic TDP-43 is thought to be implicated in disease progression. However, cell-to-cell transmission of pathogenic TDP-43 in the human CNS has not been confirmed experimentally. Here we used induced pluripotent stem cells (iPSCs)-derived cerebral organoids as recipient CNS tissue model that are anatomically relevant human brain. We injected postmortem spinal cord protein extracts individually from three non-ALS or five sporadic ALS patients containing pathogenic TDP-43 into the cerebral organoids to validate the templated propagation and spreading of TDP-43 pathology in human CNS tissue. We first demonstrated that the administration of spinal cord extracts from an ALS patient induced the formation of TDP-43 pathology that progressively spread in a time-dependent manner in cerebral organoids, suggesting that pathogenic TDP-43 from ALS functioned as seeds and propagated cell-to-cell to form de novo TDP-43 pathology. We also reported that the administration of ALS patient-derived protein extracts caused astrocyte proliferation to form astrogliosis in cerebral organoids, reproducing the pathological feature seen in ALS. Moreover, we showed pathogenic TDP-43 induced cellular apoptosis and that TDP-43 pathology correlated with genomic damage due to DNA double-strand breaks. Thus, our results provide evidence that patient-derived pathogenic TDP-43 can mimic the prion-like propagation of TDP-43 pathology in human CNS tissue. Our findings indicate that our assays with human cerebral organoids that replicate ALS pathophysiology have a promising strategy for creating readouts that could be used in future drug discovery efforts against ALS. Author summaryAmyotrophic lateral sclerosis (ALS) is a disease characterized by the degeneration of selected groups of neuronal cells. Mislocalization of the TAR DNA-binding protein 43 kDa (TDP-43) from the nucleus of neuronal cells toward the cytoplasm is a pathological hallmark of most cases of ALS. Owing to its structure TDP-43 has a high aggregation propensity when outside the nucleus. Here we used a a form of self-organizing three-dimensional cell culture model (aka cerebral organoid) to test if TDP-43 could contribute the progression of the disease that occurs across the central nervous system of patients. TDP-43 is hypothesized to contribute to the progression of the disease by inducing the propagation of its aggregated form across cells. Protein extracts prepared from postmortem spinal cord of ALS patients showing aggregated TDP-43 were separately injected into cerebral organoids derived from an ALS case and from a control individual. This approach demonstrated the spreading of aggregated TDP-43 across the cells of the injected organoids derived from an ALS case; by comparison, the same spreading was very limited within organoids from the control individual. Critically, protein extracts prepared from non-diseased individuals did not result in the propagation of aggregated TDP-43. Our finding could be used to evaluate treatments aiming to prevent the propagation of aggregated TDP-43 established to be pathogenic.