TDP-43 pathology is a hallmark of fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and limbic-predominant age-related TDP-43-encephalopathy (LATE). In affected patients, cytoplasmic TDP-43 aggregates are accompanied by disruption of its normal nuclear localization and function. Because TDP-43 is an RNA binding protein that controls transcript processing, including repression of cryptic exon splicing, its loss leads to dysregulation of gene expression. Despite its central significance in disease, the connection between TDP-43 aggregation and dysfunction remains poorly understood, and models to study the underlying mechanisms are limited. Here, we characterize a robust and quantitative cell-based reporter that captures both aggregation and the resulting loss of function. Using this human biosensor cell line, we show that aggregation initiated by prion-like seeding drives progressive depletion of nuclear TDP-43 and induces signature features of diminished TDP-43 activity, such as increased DNA damage and activation of cryptic exon splicing. We find that aggregate seeding also induces cryptic exon splicing in human neurons implying that this pathological link extends to disease-relevant models. The seeding model provides a platform for dissecting mechanisms that underlie TDP-43 pathology and for identifying factors that modulate the aggregation-to-dysfunction transition. Our data shows that aggregate seeding impacts TDP-43 autoregulation, initiating a toxic feed-forward mechanism that disrupts TDP-43 homeostasis. Furthermore, reducing ataxin-2 levels decreases aggregation and restores TDP-43 activity. Together, these findings reveal a molecularly guided strategy to directly impact TDP-43 activity by decreasing its misfolding and aggregation, highlighting approaches to prevent TDP-43 dysfunction and mitigate toxicity under pathological conditions.

Pathological inclusions of the C-terminal domain (CTD) of TAR DNA binding protein-43 (TDP-43) are neurodegenerative hallmarks in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, yet CTD’s aggregation propensity complicates structural characterization of native TDP-43. Here we propose structural models for the physiological multimerization of TDP-43 CTD’s conserved region (CR) essential for TDP-43 RNA processing. Using NMR spectroscopy, we establish that the native state of TDP-43 CR at physiological conditions is α-helical. Hydrophobic residues drive CR helix-helix assembly, phase separation, and TDP-43 nuclear retention, while polar residues down regulate these processes. An integrative approach combining analytical ultracentrifugation, NMR-derived contacts, AlphaFold2-Multimer modeling, and all-atom molecular dynamics simulations together suggest that TDP-43 CR forms dynamic, multimeric helical assemblies stabilized by a methionine-rich core with specific contributions from a tryptophan/leucine pair. These structures show how ALS-associated mutations disrupt TDP-43 function and provide pharmacologically targetable structures to prevent its conversion into pathogenic β-sheet aggregates.

Cytosolic aggregation of the nuclear protein TAR DNA-binding protein 43 (TDP-43) is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43-enriched phase within stress granules, which subsequently transition into pathological aggregates. Intra-condensate demixing of TDP-43 is observed in iPS-motor neurons, a disease mouse model, and patient samples. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We suggest that up-concentration inside condensates followed by intra-condensate demixing could be a general pathway for protein aggregation.

TAR DNA binding protein 43 (TDP-43) is an RNA binding protein that accumulates as aggregates in the central nervous systems of some patients with neurodegenerative diseases. However, TDP-43 aggregation is also a sensitive and specific pathologic feature found in a family of degenerative muscle diseases termed inclusion body myopathy. TDP-43 aggregates from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia brain lysates may serve as self-templating aggregate seeds in vitro and in vivo, supporting a prion-like spread from cell to cell. Whether a similar process occurs in patient muscle is not clear. We developed a mouse model of inducible, muscle-specific cytoplasmic localized TDP-43. These mice develop muscle weakness with robust accumulation of insoluble and phosphorylated sarcoplasmic TDP-43, leading to eosinophilic inclusions, altered proteostasis, and changes in TDP-43-related RNA processing that resolve with the removal of doxycycline. Skeletal muscle lysates from these mice also have seeding-competent TDP-43, as determined by a FRET-based biosensor, that persists for weeks upon resolution of TDP-43 aggregate pathology. Human muscle biopsies with TDP-43 pathology also contain TDP-43 aggregate seeds. Using lysates from muscle biopsies of patients with sporadic inclusion body myositis (IBM), immune-mediated necrotizing myopathy (IMNM), and ALS, we found that TDP-43 seeding capacity was specific to IBM. TDP-43 seeding capacity anticorrelated with TDP-43 aggregate and vacuole abundance. These data support that TDP-43 aggregate seeds are present in IBM skeletal muscle and represent a unique TDP-43 pathogenic species not previously appreciated in human muscle disease.

Motor neuron (MN) demise is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Post-transcriptional gene regulation can control RNA’s fate, and defects in RNA processing are critical determinants of MN degeneration. N-methyladenosine (mA) is a post-transcriptional RNA modification that controls diverse aspects of RNA metabolism. To assess the mA requirement in MNs, we depleted the mA methyltransferase-like 3 (METTL3) in cells and mice. METTL3 depletion in embryonic stem cell-derived MNs has profound and selective effects on survival and neurite outgrowth. Mice with cholinergic neuron-specific METTL3 depletion display a progressive decline in motor behavior, accompanied by MN loss and muscle denervation, culminating in paralysis and death. Reader proteins convey mA effects, and their silencing phenocopies METTL3 depletion. Among the mA targets, we identified transactive response DNA-binding protein 43 (TDP-43) and discovered that its expression is under epitranscriptomic control. Thus, impaired mA signaling disrupts MN homeostasis and triggers neurodegeneration conceivably through TDP-43 deregulation.