A paper published March 5 in Neuron takes a subcellular look at the biology of TDP-43 aggregation, a feature of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer’s disease. The authors, led by Sandrine Da Cruz and Don Cleveland, both at the University of California at San Diego, found that the protein forms droplets in the nucleus as part of its normal function. However, as described in another recent Neuron paper, Da Cruz and colleagues found that TDP43 condenses independently of stress granules in the cytoplasm of stressed cells. This steals away other proteins needed for effective transport in and out of the nucleus. The results suggest how wayward TDP-43 may disrupt nucleocytoplasmic transport and kill neurons.
- In the nucleus, TDP-43 normally forms liquid droplets.
- When cells are stressed, TDP-43 forms cytoplasmic droplets independently of stress granules.
- These cytoplasmic droplets deplete proteins needed for nucleocytoplasmic transport.
First author Fatima Gasset-Rosa examined endogenous TDP-43 using high-resolution microscopy in various kinds of somatic cells, including mouse and human neurons. In each, she found that normal TDP-43 formed droplets in the nucleus that dynamically fused and broke apart, rapidly exchanging with TDP-43 in the nucleoplasm. This suggested that dynamic liquid-liquid phase separation (LLPS) is a physiological part of TDP-43 function in the nucleus.
However, if the researchers treated human SH-SY5Y neuroblastoma cells with fibrils of either TDP-43 or FUS, TDP-43 drained out of the nucleus and accumulated in liquid droplets in the cytoplasm. The same droplets formed if the researchers overexpressed TDP-43 in the cytoplasm. These membraneless organelles were also dynamic, readily exchanging their TDP-43 with that in their surroundings. Like the TDP-43 droplets identified by Christopher Donnelly and colleagues at the University of Pittsburgh, they were distinct from stress granules (Mar 2019 news). These are liquid droplets of RNA and RNA-binding proteins, including TDP43, that temporarily form when cells are under duress. They are believed to protect cells by putting a brake on translation. One theory suggests these droplets provide an incubator for the aggregation of TDP43 and other proteins to eventually form solid aggregates (Jul 2010 conference news; April 2018 news). These new papers indicated that can happen sans stress granules.
Gasset-Rosa found that the cytoplasmic TDP-43 droplets appeared to be toxic. Initially, they didn’t kill cells, but after about two weeks, 60 percent of neurons had died, and by six weeks, all had. In dying cells, nuclear proteins RanGAP1 and Ran-GTP, both required for nucleocytoplasmic transport, had mislocalized and accumulated in the cytoplasm. Similarly, Nup62, a component of nuclear pores, hnRNPA1, which shuttles between nucleus and cytoplasm, and importin-α, which reads the TDP-43 nuclear localization signal, all aggregated with TDP43.
When the cells were exposed to additional stress, such as sodium arsenite, the cytoplasmic droplets congealed into gels or solids containing phosphorylated TDP-43. Most of it assumed amyloid conformations, as detected by binding to the A11 antibody, which recognizes prefibrillar oligomers of Aβ and other proteins.
Taken together, the results demonstrate that independently of stress granules, TDP-43 phase-separates in the cytoplasm in response to various stressors, and that this can both interfere with nucleocytoplasmic transport and siphon away nuclear TDP-43, resulting in cell death.
“It suggests that cytoplasmic liquid TDP-43 droplets can be toxic, without the need to mature into solid aggregates, as was commonly believed,” wrote Steven Boeynaems, Stanford University, to Alzforum (full comment below). That could mean the TDP-43 pathology observed in postmortem patient material may not necessarily represent aggregates, but could constitute an aberrant liquid/gel phase, he added.
The results bolster the idea that liquid TDP-43 droplets form in early stages of disease, then yield gels or solids as pathology worsens, suggested Gasset-Rosa. “This process may be central to understanding the emergence of TDP-43 pathology,” wrote Yuna Ayala, St. Louis University, who was not involved in the research (see full comment below). The result jibes with findings from Ayala’s own lab published last week, indicating that soluble, detergent-resistant oligomers of TDP-43 precede formation of large aggregates or fibrils (French et al., 2019).—Gwyneth Dickey Zakaib
- Stress Granules: No Incubator for Inclusions, After All?
- Honolulu: TDP-43 Gets a Place in the Sun
- Liquid Phase Transition: A Deluge of Data Points to Multiple Regulators
- French RL, Grese ZR, Aligireddy H, Dhavale DD, Reeb AN, Kedia N, Kotzbauer PT, Bieschke J, Ayala YM. Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation. J Biol Chem. 2019 Mar 1; PubMed.
- Prasad A, Bharathi V, Sivalingam V, Girdhar A, Patel BK. Molecular Mechanisms of TDP-43 Misfolding and Pathology in Amyotrophic Lateral Sclerosis. Front Mol Neurosci. 2019;12:25. Epub 2019 Feb 14 PubMed.
- Babinchak WM, Haider R, Dumm BK, Sarkar P, Surewicz K, Choi JK, Surewicz WK. The role of liquid-liquid phase separation in aggregation of the TDP-43 low complexity domain. J Biol Chem. 2019 Feb 27; PubMed.
- No, TDP-43 and FUS Are Not Actively Exported From the Nucleus
- TDP-43 Joins Cell-To-Cell Propagation Gang
- Structural Biology Sheds Light on Regulation of Liquid-Liquid Phase Transition
- How Does a Neuron Avoid Aggregation of Liquid Protein Droplets?
- Out of Chaos, Order: Reversible Amyloid Structure Seen in Phase Separation
- Tau Droplets Sprout Microtubules
- Gasset-Rosa F, Lu S, Yu H, Chen C, Melamed Z, Guo L, Shorter J, Da Cruz S, Cleveland DW. Cytoplasmic TDP-43 De-mixing Independent of Stress Granules Drives Inhibition of Nuclear Import, Loss of Nuclear TDP-43, and Cell Death. Neuron. 2019 Mar 5; PubMed.