Unlocking the
Mysteries of
Extracellular RNA

Once thought to exist only inside cells, RNA is
known to travel outside of cells and play a role in newly
discovered mechanisms of cell-to-cell communication.

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.

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.

This commentary originally appeared as an Editor’s Choice in Science Translational Medicine. Thanks to STM and Steven Jay for permission to reprint here.

The sci-fi thriller I, Robot tells the story of robots attempting to take over the world based on their interpretation of the three governing laws of their programming. This plan is thwarted with the help of Sonny, a unique robot who can ignore the three laws due to being programmed differently. This movie illustrates how selective programming can be a powerful tool that can be used to turn a subset of a population against the rest. This same concept underlies the strategy of gene-directed enzyme prodrug therapy (GDEPT) for cancer, which involves specific delivery of a gene to cancer cells that allows for subsequent activation of a systemically administered prodrug into a toxic form only in cells where an enzyme encoded by the delivered gene is present. Several GDEPT strategies have advanced to clinical trials; however, the specificity and fidelity of gene delivery are still limiting factors to successful translation.

Toward addressing these limitations, Wang et al. describe the use of modified extracellular vesicles (EVs) for targeted delivery of mRNA to cancer cells overexpressing the HER2 receptor. EVs are nanoscale vesicles secreted by many cell types that have been co-opted for a variety of therapeutic applications. However, targeted delivery using EVs has been challenging, as has encapsulation of large nucleic acid cargo. To address cargo encapsulation, the authors applied a transfection-based approach to successfully load exogenous mRNA encoding for the enzyme HChrR6 into EVs. To address targeting, the authors created a novel chimeric protein consisting of a HER2 antibody fragment to target the receptor on cancer cells and the C1C2 domain of lactadherin, which interacts with the EV membrane. By mixing mRNA-loaded EVs with purified chimeric protein, the EVs were endowed with targeting capability for HER2-overexpressing cancer cells. Delivery of these EVs followed by systemic administration of the prodrug 6-chloro-9-nitro-5-oxo-5H-benzo-(a)-phenoxazine (CNOB) resulted in near complete growth arrest of orthotopically implanted HER2-overexpressing breast tumors in mice.

This report establishes a new and versatile approach for improving GDEPT that could be applied to a wide variety of cancers and other diseases. Significant barriers to translation of this approach remain, most notably the problem of scalability of EV-based approaches. However, the methods and strategy described are likely to have broad utility in further developing both GDEPT and therapeutic EVs.

Highlighted Article
J.-H. Wang, A. V. Forterre, J. Zhao, D. O. Frimannsson, A. Delcayre, T. J. Antes, B. Efron, S. S. Jeffrey, M. D. Pegram, A. C. Matin, Anti-HER2 scFv-directed extracellular vesicle-mediated mRNA-based gene delivery inhibits growth of HER2-positive human breast tumor xenografts by prodrug activation. Mol. Cancer Ther. (2018) 17:1133-1142. PMID:29483213 doi:10.1158/1535-7163.MCT-17-0827