TDP-43 Seeding Induces Cytoplasmic Aggregation Heterogeneity and Nuclear Loss of Function of TDP-43

Neurology Basic Medical Sciences Life Sciences Medicine
TDP-43 cytoplasmic aggregation nuclear loss of function neurodegenerative disorders amyloid-like fibrils

Academic Background

TDP-43 (TAR DNA-binding protein 43) is an RNA-binding protein primarily located in the nucleus, involved in multiple processes of RNA metabolism, including transcription, splicing, RNA transport, and translation. However, in various neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), TDP-43 disappears from the nucleus and forms insoluble aggregates in the cytoplasm. This cytoplasmic aggregation and nuclear loss of function are considered key mechanisms in disease pathogenesis. Although the importance of TDP-43 in neurodegenerative diseases is widely recognized, its pathological mechanisms remain incompletely understood, particularly the challenge of simultaneously recapitulating cytoplasmic aggregation and nuclear loss of TDP-43 in cellular models.

To address this issue, this study aimed to trigger cytoplasmic aggregation and nuclear loss of function of TDP-43 by using amyloid fibrils formed from the TDP-43 low-complexity domain (LCD), thereby modeling pathological features in human neurons. Through this model, the researchers hope to uncover the molecular mechanisms of TDP-43 aggregation and provide a valuable tool for studying sporadic TDP-43 proteinopathies.

Source of the Paper

This paper was co-authored by Jens Rummens, Bilal Khalil, Günseli Yıldırım, and others, from multiple research institutions including the VIB-KU Leuven Center for Brain & Disease Research and Hasselt University. The paper was published on May 21, 2025, in the journal Neuron, titled “TDP-43 Seeding Induces Cytoplasmic Aggregation Heterogeneity and Nuclear Loss of Function of TDP-43.”

Research Process

1. Preparation and Characterization of TDP-43 LCD Amyloid Fibrils

The researchers first purified the low-complexity domain (LCD) of TDP-43 from E. coli and induced its spontaneous formation of fibrillar aggregates through shaking and time. Transmission electron microscopy (TEM) revealed that these fibrils exhibited typical amyloid structures, with a width of 16±3 nm, and most fibrils displayed a helical twist. Additionally, these fibrils showed green birefringence under Congo red staining, indicating their amyloid nature. Fourier-transform infrared spectroscopy (FTIR) further confirmed the amyloid properties of the fibrils.

To investigate whether these fibrils could act as seeds to trigger TDP-43 aggregation, the researchers sonicated the fibrils to generate small fragments (seeds) with an average length of 38±13 nm. In a cell-free environment, these seeds accelerated the aggregation of monomeric TDP-43 LCD, eliminating the lag phase of aggregation.

2. Fibril-Induced Cytoplasmic Aggregation of TDP-43

The researchers introduced the sonicated fibril seeds into human cells expressing TDP-43 via liposome-based transfection. The results showed that the fibril seeds triggered cytoplasmic aggregation of TDP-43, and these aggregates exhibited pathological features such as phosphorylation, ubiquitination, and p62 accumulation. Furthermore, the fibril seeds also induced the disappearance of endogenous TDP-43 from the nucleus and the formation of cytoplasmic aggregates.

Through fluorescence recovery after photobleaching (FRAP) and fluorescence lifetime imaging microscopy (FLIM) analyses, the researchers found that fibril-induced TDP-43 aggregates exhibited solid-like properties, distinct from the liquid-like TDP-43 droplets. Additionally, over time, TDP-43 aggregates displayed morphological heterogeneity, including compact, filamentous, and fragmented aggregates.

3. Fibril-Induced Nuclear Loss of Function of TDP-43

The researchers further assessed whether the fibril seeds could induce nuclear loss of function of TDP-43. The results showed that fibril seed treatment led to a significant reduction in nuclear TDP-43 levels and triggered RNA splicing defects, including the detection of disease-specific cryptic splicing in the ALS/FTD-related gene UNC13A. Through RNA sequencing (RNA-seq), the researchers found that TDP-43 aggregation and nuclear loss of function were associated with widespread transcriptomic changes, including upregulation of neurodegenerative disease-related genes and downregulation of TDP-43 binding targets.

4. Time-Dependent Degradation of TDP-43 Aggregates

The researchers also investigated the degradation process of TDP-43 aggregates in cells. The results showed that over time, the morphology of TDP-43 aggregates changed, transitioning from compact aggregates to filamentous and fragmented aggregates. This morphological change was associated with the activation of the proteasomal pathway, and proteasome inhibitors prevented this time-dependent morphological change. Additionally, by restricting TDP-43 expression, the researchers observed the gradual clearance of TDP-43 aggregates, further supporting the hypothesis of aggregate degradation.

5. Fibril-Induced TDP-43 Pathology in Human iPSC-Derived Neurons

Finally, the researchers validated the effects of fibril seeds in human induced pluripotent stem cell (iPSC)-derived neurons. The results showed that fibril seeds induced cytoplasmic aggregation and nuclear loss of function of TDP-43 in neurons, and triggered neuron-specific toxicity. Unlike in cell lines, fibril-induced TDP-43 pathology in neurons exhibited stronger time-dependent toxicity, indicating that neurons are more sensitive to TDP-43 pathology.

Research Conclusions

This study successfully recapitulated cytoplasmic aggregation and nuclear loss of function of TDP-43 in cellular and neuronal models by using TDP-43 LCD amyloid fibrils, uncovering the molecular mechanisms of TDP-43 aggregation. The results demonstrated that the morphological heterogeneity of TDP-43 aggregates is associated with a time-dependent degradation process, and fibril-induced TDP-43 pathology exhibited stronger toxicity in neurons. These findings provide a valuable model for studying sporadic TDP-43 proteinopathies and lay the foundation for future drug screening and disease mechanism research.

Research Highlights

  1. First use of TDP-43 LCD amyloid fibrils to trigger cytoplasmic aggregation and nuclear loss of function of TDP-43, successfully modeling pathological features in neurodegenerative diseases.
  2. Revealed the morphological heterogeneity and time-dependent degradation process of TDP-43 aggregates, providing new insights into the progression of TDP-43 pathology.
  3. Validated fibril-induced TDP-43 pathology in human iPSC-derived neurons, demonstrating that neurons are more sensitive to TDP-43 pathology.
  4. Provided extensive transcriptomic data, revealing the relationship between TDP-43 aggregation, nuclear loss of function, and expression changes in neurodegenerative disease-related genes.

Research Value

The scientific value of this study lies in uncovering the molecular mechanisms of TDP-43 aggregation and providing a new experimental model for studying sporadic TDP-43 proteinopathies. Through this model, researchers can further explore the molecular mechanisms of TDP-43 pathology and develop therapeutic strategies targeting TDP-43 aggregation. Additionally, this study offers new insights into the processes of protein aggregation and degradation in neurodegenerative diseases.