Like a woolly mammoth in a tar pit, proteasomes can be doomed if they get stuck in gobs of the DNA-binding protein TARDP, better known as TDP-43. That’s according to new research from the labs of Dieter Edbauer at the German Center for Neurodegenerative Diseases, Munich, and Rubén Fernández-Busnadiego at the University of Göttingen, Germany. Scientists used cryo-electron tomography, proteomics and functional assays to show that fragments of TDP-43 form amorphous gel-like drops in the cytosol, and these are enriched with blocked proteasomes in the middle of catalysis. The authors believe this may be a common disease mechanism in amyotrophic lateral sclerosis and frontotemporal dementia, which may be caused by mutations in TDP-43.

  • The C-terminal fragments of TDP-43 form gel-like inclusions.
  • The latter trap blocked proteasomes.
  • This could be a common disease mechanism in ALS/FTD.

Insoluble TDP-43 inclusions are found in the brains of people with not only ALS/FTD, but also other neurodegenerative diseases, including Alzheimer’s disease and TARD, aka TDP-43 encephalopathy related to limbic predominant age (May 2019 news). Recently, scientists used cryo-electron microscopy to determine the fibril structure of TDP-43, but the protein also forms liquid droplets through a process called liquid-liquid phase separation (News December 2021; News September 2016) . Although rare, LLPS accounts for amorphous lumps formed by proteins that have limited secondary structure, including TDP43, FUS, tau, and other neurodegeneration-related proteins. Comprising a large domain of low complexity, the C-terminus of TDP-43 drives LLPS, and it is precisely the C-terminus of this predominantly nuclear protein that accumulates in the cytosol in ALS/FTD .

Have mercy on the proteasome. In primary neurons, 25 kDa C-terminal stubs of TDP-43 form gel-like drops (red), which contain ring-like structures (yellow) formed by proteasomes (blue). These are linked to an unknown structure (pink) which could be a substrate or an inhibitor. [Image courtesy Riemenschneider et al., EMBO Reports.]

To study these fragments and their role in disease, co-first authors Henrick Riemenschneider of DZNE and Qiang Guo of the Max Planck Institute for Biochemistry, Martinsried, also in Germany, turned to cryo-electron tomography, a type of cryo-EM which can restore high resolution images of complex structures in situ. It can discover the structures of membrane proteins in the cellulo, and Guo has already used it to discover that polyGA poly dipeptide ribbons trap proteasomes (February 2018 news).

Looking into primary rat cortical neurons, the scientists found that TDP-25, a 25 kDa C-terminal fragment of TDP43, forms spots that do the same thing. Wild-type TDP-25 and fragments carrying one of eight known ALS-linked TDP-43 mutations collapsed in this way. Cryo-ET also revealed ring structures in these drops which were proteasomes. Based on the structure, the researchers concluded that all of the trapped proteasomes were in the midst of degrading substrates. It’s unusual. In a normal neuron, only about 20% of the proteasomes would be in a substrate processing state, the majority passing through a “ground” state. The authors believe that the proteasomes in the TDP-25 gels have, in fact, stalled.

To test this, they expressed TDP43 variants in HEK293 cells that express a proteasome reporter, namely a mutant ubiquitin/green fluorescent protein chimera. Proteasomes readily degrade UbG76V-GFP, as they did in cells expressing full-length TDP-43; alas, in cells expressing even a small fraction of wild-type TDP-25, substrate began to accumulate. ALS mutations in TDP-25 further altered proteasome activity.

TARDP pit? Cryo-ET rendering neuronal cytosol shows proteasomes (purple) and some chaperonins (green) mired in a pit of TDP-25 (red). The ribosomes (yellow) are banished to the periphery. [Movie courtesy Riemenschneider et al., EMBO Reports.]

Interactome analysis supports the idea that cytosolic fragments of TDP-43 interact with the proteasome. Scientists have discovered a huge difference between the partners of full TDP-43 and TDP-25. The latter ignored 72 normal partners, mainly those involved in RNA splicing, but linked 400 new ones. These included proteasome subunits and other proteins related to the ubiquitin-proteasome system (UPS), such as the ubiquitin ligases ubiqulin 2 and E3. The authors speculate that these might contribute to an unknown density in the proteasome spotted by cryo-ET (see above).

Altogether, the authors concluded that “C-terminal fragments of TDP-43 adopt a gel-like conformation and impair proteostasis by sequestering blocked proteasomes in situ.” The similarity to their previous findings on polyGA suggests that this may be a common problem in ALS/FTD. ALS-causing mutations have also been found in other UPS-related genes, including ubiquilin 2, VCP, OPTN, C9orf72, and TBK1.—Tom Fagan

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