Advancing exRNA
Communication Research

The NIH Common Fund has announced new funding opportunities for advancing research into exRNA communication.
An informational webinar on the application process will be held August 24, 2018 from 2:00-3:30 pm EST. Join us!

This blog was adapted and simplified from a Spotlight in the Journal of Cell Biology.

Directed movement along a chemical gradient – chemotaxis – is a fundamental behavior of cells in the body during processes like inflammation, embryogenesis, and cancer metastasis. While the intracellular signaling mechanisms underlying chemotaxis have been investigated by many groups, it is not well known how stable gradients of chemoattractant chemicals are generated and maintained outside the cell, especially given the rapid diffusion of chemicals after secretion.

The amoeba Dictyostelium discoideum (fondly known as Dicty) is a model organism often studied since its biology can shed light on that of human cells. Recent work from Kriebel et al. (2018) showed that extracellular vesicles (EVs) are central to the biology of chemotaxis in Dicty by demonstrating that the main chemical Dicty follows during chemotaxis is synthesized within and released from EVs.

Dictyostelia are soil-living amoebas that undergo chemotaxis toward cyclic adenosine monophosphate (cAMP) under starvation conditions. As cAMP is released from the rear of migrating cells, chemotaxis toward cAMP leads to aggregation of the amoebas into multicellular structures.

Kriebel et al. previously showed (2008) that the enzyme that synthesizes cAMP, adenylyl cyclase (ACA), is present in multivesicular bodies (MVBs) located at the rear of migrating amoebas. They have now shown that leader cells release ACA-enzyme-containing vesicular trails and that follower cells stream onto them in a head-to-tail fashion. The vesicular trails are highly chemotactic and direct the migration of the follower cells.

Measurement of ATP, the precursor used by the enzyme to synthesize the product cAMP, revealed that it is also present inside EVs. Narrowing down from the 68 transporter proteins found in Dicty, the researchers identified the one protein – ABCC8 – that transports cAMP from the inside to the outside of EVs in order to promote chemotaxis. Dicty amoebas with that transporter knocked out exhibit much less chemotaxis and streaming behavior, and fluorescently tagged ABCC8 reintroduced into those amoebas is visible in the vesicular trails but not the plasma membrane.

Altogether, these data indicate that the entire machinery for generating and releasing chemoattractants is contained within EVs: the catalytically active enzyme (ACA), its substrate (ATP), the product (cAMP), and the transporter (ABCC8; see figure).

EVs secreted from Dictyostelia synthesize and release cAMP to promote chemotaxis. cAMP is converted from ATP by enzyme ACA in EVs and secreted through the ABCC8 transporter. Secreted cAMP makes a gradient and promotes chemotaxis of follower cells.

Overall, Kriebel et al. (2018) describe an elegant system by which chemotactic signals are generated and sustained to promote streaming in a complex multicellular system, and they elucidate a major mechanism by which cells leave a memory of themselves. EVs are left in a breadcrumb-like trail behind cells and continue sending chemotactic signals to surrounding cells. The finding that EVs act as cell-independent entities to not only carry but also generate bioactive products is important because it shows that they can amplify signals.

The same group made similar findings previously for neutrophils, one of the main sentry cells of the immune system (Majumdar et al., 2016). In both cases, the presence in EVs of enzymes that can generate a chemical product continuously amplifies and sustains a signal beyond that which would occur if the EVs carried just the chemical. The findings in amoeba and human cells together suggest that amplification of chemotactic signals is an important function of EVs that is conserved across organisms and necessary to promote effective directional sensing.

In a different context, it has been reported that precursor miRNAs can be processed into mature miRNAs in exosomes in a cell-independent manner due to the presence of the RNA interference–silencing complex (RISC) machinery in breast cancer–associated exosomes (Melo et al., 2014) and that this activity is important to promote tumor aggressiveness. Along with Melo et al. (2014), Kriebel et al. (2018) provide direct evidence that EVs can act as an independent machinery to regulate biological processes such as chemotaxis or tumorigenesis via enzymatic activities.

References

Kriebel PW, Barr VA, Rericha EC, Zhang G & Parent CA . (2008) Collective cell migration requires vesicular trafficking for chemoattractant delivery at the trailing edge. J. Cell Biol. 183:949–961. doi: 10.1083/jcb.200808105

Kriebel PW, et al. (2018) Extracellular vesicles direct migration by synthesizing and releasing chemotactic signals. J. Cell Biol. doi: 10.1083/jcb.201710170

Majumdar R, Tavakoli Tameh A & Parent CA. (2016) Exosomes mediate LTB4 release during neutrophil chemotaxis. PLoS Biol. 14:e1002336. doi: 10.1371/journal.pbio.1002336

Melo, et al. 2014. Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer Cell 26:707–721. doi: 10.1016/j.ccell.2014.09.005

Sung BH & Weaver AM. (2018) Directed migration: Cells navigate by extracellular vesicles. J. Cell Biol. doi: 10.1083/jcb.201806018

When someone finds out that I work at the National Institute on Aging, they usually ask me “How can I stop aging?”. Although aging is inevitable, it is well-known that individuals age at different rates and that certain groups age more rapidly than others. Hence, one of our laboratory’s objectives is to identify why certain population groups age differently, with the goal to find biomarkers that can tell us an individual’s biological age (how old you seem) versus chronological age (the actual time you have been alive).

Several years ago, we began this journey by examining whether small non-coding RNAs called microRNAs (miRNAs) change with age. We focused first on these regulatory RNAs because data has shown that these RNAs are particularly stable in biofluids, such as serum and plasma, and can be identified readily using small RNA sequencing. We identified several serum miRNAs that change significantly with age in both humans and rhesus monkeys (Noren Hooten, Fitzpatrick, et al. 2013). In most cases, their abundance in circulation decreases as we get older. Interestingly, some of these miRNAs target and negatively regulate the expression of various inflammatory markers, suggesting that decreased expression of these circulating miRNAs may contribute to higher levels of inflammatory markers that have already been observed in the elderly.

More recently we have focused on establishing a more complete extracellular RNA (exRNA) profile of human aging. To do so, we developed a sequencing pipeline that enables us to sequence both small and long RNAs in one sequencing reaction, which lowers both the cost and labor required (Dluzen, Noren Hooten, et al. 2018). Cataloging what is the “normal” distribution of exRNA in young and old individuals and identifying age-dependent differences will aid in establishing important references for the study of age-related disease. The Ensembl database classifies RNA into various categories, termed biotypes. We found that most RNA biotypes were similar in distribution between young and old, but several biotypes, including mitochondrial tRNAs (Mt_tRNA), mitochondrial ribosomal RNAs (Mt_rRNA), and unprocessed pseudogenes, were significantly higher in older individuals.


Figure 1. Changes in circulating factors with age. A decrease in circulating levels of specific miRNAs occurs with age. On the other hand, unprocessed pseudogenes, Mt_tRNAs, and Mt_rRNAs increase in abundance with age.

Pathway analysis revealed that RNAs related to mitochondria, response to oxidative stress, and chromatin remodeling were all enriched in the circulation of older individuals, providing potential clues as to what pathways may be deregulated as humans age. We also further validated our sequencing results in a larger cohort of individuals and found age-related changes in a messenger RNA, a small nucleolar RNA, a pseudogene transcript, a small nuclear pseudogene transcript, and several additional miRNAs.

What was very interesting was that we identified many circular RNAs (circRNAs) in serum, which we have named ex-circRNAs. Recent attention has been focused on circRNAs, as this class of ncRNAs may be important modulators of gene expression. CircRNAs have long half-lives (i.e. are stable) compared to mRNAs, making them an attractive new serum biomarker. However, little is known about ex-circRNAs, and I anticipate that this will be an active area of interest in the coming years.

As we have begun to establish an exRNA profile of human aging, there remain important unanswered questions in the field. Currently, we do not fully understand how exRNA in the circulation reflects the health status of our cells and tissues. It has also proven difficult to ascertain which cell type is contributing exRNA into the circulation. Further research is needed to better understand these questions.

Our identification of changes in circulating miRNAs and exRNAs establishes baseline references for how these biomarkers change with human age. As the risk for many diseases including cancer, heart disease, and neurological diseases increase with age, it is important to consider age when examining these factors in relation to a specific disease. Although we have not identified the “fountain of youth” or the “magic elixir” for aging, we hope that establishing these profiles with normal aging will soon help to identify circulating biomarkers that can distinguish individuals with faster biological aging that may result in shortened health span and life span.

References
Dluzen DF, Noren Hooten N, De S, Wood H, Zhang Y, Becker KG, Zonderman AB, Tanaka T, Ferrucci L & Evans MK. Extracellular RNA profiles with human age. Aging Cell 2018;e12785. PMID 29797538.

Noren Hooten N, Fitzpatrick M, Wood WH, De S, Ejiogu N, Zhang Y, Mattison JA, Becker JG, Zonderman AB & Evans MK. Age-related changes in microRNA levels in serum. Aging (2013) 5: 725-740. PMID 24088671.

This post originated as a press release from Linköping University.

The waste-management system of the cell appears to play an important role in the spread of Alzheimer’s disease in the brain. A new study, published in the prestigious scientific journal Acta Neuropathologica, has focused on small membrane-covered droplets known as exosomes. It was long believed that the main task of exosomes was to help the cell to get rid of waste products. In simple terms, they were thought of as the cell’s rubbish bags. However, our understanding of exosomes has increased, and we now know that cells throughout the body use exosomes to transmit information. It’s now known that the exosomes can contain both proteins and genetic material, which other cells can absorb.

The Linköping researchers have shown in the new study that exosomes can also transport toxic aggregates of the protein amyloid beta, and in this way spread the disease to new neurons. Aggregated amyloid beta is one of the main findings in the brains of patients with Alzheimer’s disease, the other being aggregates of the protein tau. As time passes, they form ever-increasing deposits in the brain, which coincides with the death of nerve cells. The cognitive functions of a person with Alzheimer’s disease gradually deteriorate as new parts of the brain are affected.

“The spread of the disease follows the way in which parts of the brain are anatomically connected. It seems reasonable to assume that the disease is spread through the connections in the brain, and there has long been speculation about how this spread takes place at the cellular level,” says Martin Hallbeck, associate professor in the Department of Clinical and Experimental Medicine at Linköping University and senior consultant of clinical pathology at Linköping University Hospital.

Cells became diseased
In a collaboration with researchers at Uppsala University, he and his co-workers have investigated exosomes in brain tissue from deceased persons. The research team at Linköping University found more amyloid beta in exosomes from brains affected by Alzheimer’s disease than in healthy controls. Furthermore, the researchers purified exosomes from the brains from people with Alzheimer’s disease, and investigated whether they could be absorbed by cells cultured in the laboratory.

“Interestingly, exosomes from patients were absorbed by cultured neurons, and subsequently passed on to new cells. The cells that absorbed exosomes that contained amyloid beta became diseased,” says Dr. Hallbeck.

The researchers treated the cultured neurons with various substances that prevent exosomes from being formed, released, or absorbed by other cells. They were able to reduce the spread of the aggregated amyloid beta between cells by disrupting the mechanism in these ways. The methods used in these laboratory experiments are not yet suitable for treating patients, but the discovery is important in principle.

“Our study demonstrates that it is possible to influence this pathway, and possibly develop drugs that could prevent the spreading. The findings also open up the possibility of diagnosing Alzheimer’s disease in new ways, by measuring the exosomes,” says Martin Hallbeck.

The research has received financial support from donors that include the Swedish Research Council, the Swedish Alzheimer’s Foundation, and the Swedish Brain Foundation.

Sinha MS, Ansell-Schultz A, Civitelli L, Hildesjö C, Larsson M, Lannfelt L, Ingelsson M & Hallbeck M. Alzheimer disease pathology propagation by exosomes containing toxic amyloid-beta oligomers. Acta Neuropathologica AOP 13 June 2018. doi: 10.1007/s00401-018-1868-1

Translation by George Farrants.