Molecular Visualization of Neuronal TDP43 Pathology In Situ

Nuclear exclusion and cytoplasmic accumulation of the RNA-binding protein TDP43 are characteristic of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite this, the origin and ultrastructure of cytosolic TDP43 deposits remain unknown. Accumulating evidence suggests that abnormal RNA homeostasis can drive pathological TDP43 mislocalization, thereby enhancing RNA misprocessing due to the loss of nuclear TDP43, and engendering a cycle that ultimately leads to cell death. Here, we demonstrate that the addition of small monovalent oligonucleotides successfully recapitulates pathological TDP43 mislocalization and aggregation, aberrant splicing, and degeneration in iPSC-derived neurons (iNeurons). By employing a tailored multimodal in situ cryo-correlative light and electron microscopy pipeline, we examine the localization and aggregation of TDP43 in near-native conditions. We discover that mislocalized TDP43 accumulates and forms ordered fibrils within autophagosomes and lysosomes in iNeurons, as well as in ALS/FTLD patient tissue. We provide the first high-resolution snapshots of TDP43 aggregates in situ, delivering an unprecedented view of the earliest pathogenic events underlying ALS, FTLD, and related TDP43 proteinopathies.