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Cellular Hitchhiking—a New Way to Understand ALS Pathways—and Perhaps Provide a Potential Target to Stop It

Article In Brief

A new study suggests that annexin A11 mutations in ALS patients may impair movement of RNA granules, providing a novel way to think about a critical function that is disrupted in ALS. It may also provide a potential avenue for therapeutics, experts said.

Figure

RNA granules (blue) move around cells by attaching to lysosomes (red).

A recently discovered gene involved in amyotrophic lateral sclerosis (ALS) plays a critical role in a heretofore unknown transport process in neurons, one in which messenger RNA granules “hitchhike” a ride on lysosomes to carry them down the axon for translation.

RNA granules are composed of messenger RNA and RNA-binding proteins that coalesce to form coherent, membrane-less structures that are thought to sequester, protect, and promote transport of mRNAs. For many cell organelles, proteins embedded in their membranes allow them to link to the cytoskeletal motor network for long-range transport. RNA granules, however, have no membrane, and so how they are moved around the cell has not been clear. The new findings suggest that this “transit” or hitchhiking may go wrong in people with ALS.

“This [hitchhiking] is a new concept in cell biology,” said Jennifer Lippincott-Schwartz, PhD, a group leader at the Howard Hughes Medical Institute Janelia Farm Research Campus in Ashburn, VA, and the principal investigator of the study published September 19 in the journal Cell.

The protein encoded by the gene is called annexin A11. Mutations in annexin A11 were shown in 2017 to be responsible for cases of both familial and sporadic ALS in individuals of European descent and have been reported to be a common cause of ALS in the Chinese population, she said.

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“The discovery that annexin A11 mutations impairs movement of RNA granules provides a new way to think about a critical function that is disrupted in ALS, and that may provide new ideas for therapeutics.”—DR. GENE YEO

Commenting on the study, Erika Holzbaur, PhD, professor of physiology at the University of Pennsylvania Perelman School of Medicine in Philadelphia, pointed out that intracellular transport defects have been previously implicated in ALS, as have RNA granule dynamics. But, she said, “This paper is really interesting because it links RNA granule biology to [the] cytoskeletal motor biology in a single pathway that is involved in locally distributing RNAs in neurons.”

“That connection is interesting and may be important for understanding ALS disease pathways,” added Dr. Holzbaur, who researches axonal transport in health and disease, and who was not involved in the new study.

That connection also may have therapeutic implications for future research. “The discovery that annexin A11 mutations impairs movement of RNA granules provides a new way to think about a critical function that is disrupted in ALS, and that may provide new ideas for therapeutics,” said Gene Yeo, PhD, professor of cellular and molecular medicine at the University of California, San Diego, who was not involved in the study.

Study Design, Findings

Dr. Lippincott-Schwartz has been a leader in live cell imaging, using a variety of techniques to tag and record the movements of cell organelles and other structures. In the current study, her team promoted the formation of RNA granules by heat shocking neurons in culture—a standard technique for granule formation, which is thought to occur in response to stress. They then visualized the movements of the granules and lysosomes with fluorescent tags. The researchers found that the granules and lysosomes moved together along microtubule networks and that inhibiting lysosomal movement blocked granule transport. Because the granules appeared to be dependent on the motility of the lysosomes for their transport, the association appeared to be the first reported instance in mammalian cells of “RNA granule hitchhiking,” a phenomenon recently described for the first time in fungi.

Microscopy revealed that the granules were very closely associated with the lysosomes, but were not engulfed in them, “and that suggested the presence of some type of molecular tether between them,” Dr. Lippincott-Schwartz said. In collaboration with Michael E. Ward, MD, PhD, at the National Institutes of Neurological Disorders and Stroke, the team captured a large number of proteins at the granule-lysosome interface, and used mass spectrometry and proteomics, along with a list of RNA granule-interacting proteins from another lab, to identify six possible tethering proteins. One of these was annexin A11, which they confirmed was the actual tethering protein.

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“We are speculating that the decision to release an RNA granule at a particular site is very much dependent on the lysosome. If the lysosome stops releasing calcium, it is no longer going to bind the RNA granule. That granule would then be able to start releasing messenger RNA that could be locally translated.”—DR. JENNIFER LIPPINCOTT-SCHWARTZ

Structurally, annexin A11 appeared to be well-suited to play a linking role between a membrane-bound organelle such as the lysosome and an RNA granule. At its C-terminal end, it has four calcium-dependent membrane-binding domains suitable for lysosome association, while the N-terminal end has a “low-complexity region,” composed of relatively few types of amino acids, often seen in RNA granule-associated proteins. “It is that dual structure that allows annexin A11 to link lysosomes and RNA granules together,” Dr. Lippincott-Schwartz said.

ALS-causing mutations are found at both ends of the protein, and Dr. Lippincott-Schwartz showed that two different mutations, one C-terminal and one N-terminal, each interfered with the dynamic ability of RNA granules to form and disperse, and that C-terminal mutations disrupted its ability to link RNA granules with lysosomes. That prevented RNA granules from hitchhiking on the lysosome for delivery along the axon.

There is much more to learn about this system, Dr. Lippincott-Schwartz stressed, including how the transport and delivery of the RNA granules is regulated. “If you are hitchhiking, you have to know where to get off,” she said, adding that a likely key step is a change in calcium release by the lysosome, acting on the calcium-binding sites at the C-terminal end.

“We are speculating that the decision to release an RNA granule at a particular site is very much dependent on the lysosome. If the lysosome stops releasing calcium, it is no longer going to bind the RNA granule. That granule would then be able to start releasing messenger RNA that could be locally translated.”

Expert Commentary

“There has been an extraordinary increase in the discovery of new genetic causes of ALS,” said Dr. Holzbaur, “and they are starting to organize into a few common cellular pathways,” including trafficking, RNA dynamics, and lysosome biology.

“This paper is now highlighting a new mechanism in cell biology—hitchhiking—that brings some of those pathways together. That is important,” she said, noting that it will also be important going forward to keep these convergences in mind when interpreting new findings. “We have to be careful that a lysosomal transport or localization defect could also have a knock-on effect of altering RNA localization.”

Figure

“This paper is really interesting because it links RNA granule biology to [the] cytoskeletal motor biology in a single pathway that is involved in locally distributing RNAs in neurons. That connection is interesting and may be important for understanding ALS disease pathways.”—DR. ERIKA HOLZBAUR

“This finding is very exciting,” said Dr. Yeo, who studies RNA granule components and dynamics and led the team that created the list of RNA granule-interacting proteins, including annexin A11, which Dr. Lippincott-Schwartz used. “I expect there will be other adapter proteins like annexin A11 that bind RNA granules and may serve to facilitate transport as well. RNA transport is maybe something we should pay more attention to.”

“The field of basic biology in ALS has been really exploding in the last decade or two,” Dr. Holzbaur noted, “with new mechanisms that are revealing new cell biology. That has given us new insights into the disease, and hopefully will begin to tell us where therapy has to be aimed.”

Disclosures

Drs. Lippincott-Schwartz, Yeo, and Holzbaur had no disclosures.

Link Up for More Information

• Liao YC, Fernandopulle MS, Wang G, et al. RNA granules hitchhike on lysosomes for long-distance transport, using annexin A11 as a molecular tether https://www.cell.com/cell/pdf/S0092-8674(19)30969-9.pdf. Cell 2019;179(1):147–164.
• Smith BN, Topp SD, Fallini C, et al. Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis https://stm.sciencemag.org/content/9/388/eaad9157.short. Sci Transl Med 2017;9(388).