Some people with amyotrophic lateral sclerosis or frontotemporal dementia accumulate deposits of the nuclear protein FUS in the cytoplasm. What drives this relocation? In the July 24 Cell Reports, researchers led by Magdalini Polymenidou at the University of Zurich blame osmotic pressure. When Polymenidou and colleagues subjected brain slices to hyperosmotic conditions, FUS piled up in cytoplasm of neurons. The protein localized to stress granules, but even when the authors prevented those from forming, FUS still loitered in the cell body. The authors traced the cause of this mislocalization to transportin 1 and 2, the nuclear import factors that ferry FUS. These factors became marooned in cytoplasm during osmotic stress, they found.
FUS mislocalization could be the first step toward disease, Polymenidou suggested. She believes osmotic stress acts as the trigger that sends FUS to the wrong place, where a second stressor could then cause it to aggregate. Supporting the disease connection, hyperosmolarity had no effect on FUS in astrocytes. These cells do not develop FUS deposits in ALS and FTD.
“It is a really exciting observation that the relocalization of FUS is independent of stress granule formation,” Brian Freibaum at St. Jude Children’s Research Hospital in Memphis, Tennessee, wrote to Alzforum (full comment below). Others were more cautious. “The potential contribution of this mechanism to ALS/FTD pathogenesis is unclear,” noted Sami Barmada of the University of Michigan Medical School in Ann Arbor.
FUS contains a nuclear localization signal that allows transportins 1 and 2 (collectively called TNPO) to carry it into the nucleus. Without TNPO, FUS that escapes the nucleus via passive diffusion gradually amasses in cytoplasm (see May 2018 news). Even during most types of cellular stress, including oxidative stress, ER stress, and heat shock, TNPO keeps FUS corralled in the nucleus. One previous report flagged osmotic stress as the exception. Researchers led by Daryl Bosco at the University of Massachusetts Medical School found that subjecting immortalized somatic cell lines to hyperosmolarity forced FUS into stress granules (Sama et al., 2013).
Polymenidou and colleagues wondered if the same thing would happen in an environment closer to that of living brain. First author Eva-Maria Hock applied sorbitol, a sugar alcohol, to mouse cortico-hippocampal slices to induce osmotic stress. The high concentration of solutes in the extracellular fluid pulls water out of cells in the slices, shrinking them and causing intracellular molecular crowding. After two hours of this treatment, FUS began to disappear from nuclei, showing up in cytoplasmic stress granules. This occurred in neurons and microglia, but not astrocytes. Cytoplasmic FUS peaked at four hours. Then, curiously, it began to shift back to its normal location. After eight hours of continuous osmotic stress, FUS was confined to the nucleus again, and stress granules had dissolved, suggesting cells somehow adapted to these conditions. As with the cell lines, FUS mislocalization occurred only under osmotic stress; treating slices with arsenite to induce oxidative stress did not budge the protein.
Did FUS movement depend on stress granule formation? To test this idea, the authors treated the slices with inhibitors of protein synthesis, which prevent stress granule formation. FUS still migrated to cytoplasm under osmotic stress, although it remained diffuse. Likewise, artificially stabilizing stress granules did not trap FUS in cytoplasm; it still escaped back to the nucleus after eight hours.
“It surprised me that translocation [of FUS] to cytoplasm and incorporation into stress granules were independent processes. That’s an important conceptual change in how we think about these steps,” Polymenidou told Alzforum. Some evidence suggests this uncoupling might be specific to osmotic stress. Researchers at Johns Hopkins University in Baltimore recently reported that stress granules formed by oxidative stress actively sequester nucleocytoplasmic transport factors like TNPO. In this case, inhibiting granule formation prevented mislocalization of these factors and blocked neurodegeneration (Zhang et al., 2018).
The authors explored several possible mechanisms for this FUS mislocalization. Signaling pathways and transcription factors activated by hyperosmotic stress did not affect FUS movement. Turning to nuclear import, the authors saw that other TNPO cargos accumulated in the cytoplasm during osmotic stress as well, but cargos of other types of importins did not. Immunostaining revealed that TNPO fled the nucleus and localized to stress granules during osmotic stress, in contrast to its normal diffuse distribution throughout the nucleus and cytoplasm. Transfecting cells with a TNPO construct that trapped it in the nucleus kept FUS in the nucleus during hyperosmolarity.
Commenters were particularly interested in the TNPO connection. Wilfried Rossoll at the Mayo Clinic in Jacksonville, Florida, noted that several recent studies have shown a role for transportins in preventing RNA-binding proteins such as FUS from sticking together (see Apr 2018 news). “Since hyperosmolarity causes cell shrinkage and macromolecular crowding, these new findings suggest that cytoplasmic recruitment of transportins may act as a protective mechanism to counteract increased levels of protein aggregation,” Rossoll wrote to Alzforum.
Were the findings on FUS specific to mouse cells? The authors repeated the experiments in human neurons derived from stem cells, and saw the same response to osmotic stress. However, human neurons did not adapt as quickly or as well to this type of stress as mouse neurons did, with FUS remaining in the cytoplasm of some cells under osmolar conditions. By contrast, FUS in human astrocytes was unaffected, just as in mouse astrocytes, although the cells showed other signs of osmotic stress, such as the activation of signaling cascades.
“I was very surprised to see that FUS in astrocytes resisted cytoplasmic relocalization,” Freibaum said. Polymenidou said she is investigating the reason for this. She thinks the mechanism might point toward potential interventions that could help neurons deal better with osmotic stress. Keeping FUS nuclear might prevent pathogenic deposits from forming, she suggested. She believes these findings are particularly pertinent for FTD. In ALS, it is mutant FUS that gets trapped in the cell body, but in FTD, it is the wild-type protein. The cytoplasmic mislocalization of normal FUS was a mystery.
It is unclear, however, how common hyperosmolarity is in the brain, and whether this condition could precipitate disease. Hyperosmolarity is sometimes induced in brain after traumatic injuries to bring down swelling; Polymenidou speculated that this could be one reason for the association between TBI and neurodegeneration. Severe dehydration or an imbalance of electrolytes might also cause osmotic stress in the brain, she said.
Bosco noted that the experimental conditions used in this study, like those in in her own, are not physiological. However, the molecular crowding caused by hyperosmotic stress might mimic other conditions that occur in the brain, such as impaired proteostasis and protein accumulation, she suggested. She also noted that hyperosmolarity can trigger the release of proinflammatory cytokines (Schwartz et al., 2009; Brocker et al., 2012). Inflammation is a known risk factor for many neurodegenerative diseases.
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