14 Apr 2023

Epidemiological evidence has linked infection with viruses, such as influenza and herpes, to higher odds of AD and other neurodegenerative diseases (Feb 2023 news). How might these microbes contribute to neurodegeneration? Perhaps by stealthily slipping tau aggregates into healthy cells, proposes Ina Vorberg of the German Center for Neurodegenerative Diseases in Bonn. At the International Conference on Alzheimer’s and Parkinson’s Diseases held from March 28 to April 1 in Gothenburg, Sweden, she reported that envelope proteins from endogenous retroviruses hidden within mouse and human genomes enabled tau seeds to spread in cell cultures. Inhibiting expression of these envelope proteins may be a target for tauopathies, Vorberg believes.

Vorberg previously reported that viral envelope proteins, namely a glycoprotein from the vesicular stomatitis virus (VSV) and the SARS-COV-2 spike protein, drive the spread of tau aggregates by promoting the release of extracellular vesicles (EVs) containing the toxic cargo (Oct 2021 news). The viral proteins decorate EVs containing tau aggregates, enabling their fusion onto nearby cell membranes and release of the aggregates into the cell.

In Gothenburg, Vorberg reported that proteins from endogenous retroviruses also speed the spread of tau seeds in mouse and human cells. While studying yeast prion aggregates in N2a mouse neuroblastoma cells, she and colleagues noticed that the longer they grew the cells, the more cells were infected with prion aggregates. Compared to cultures passaged seven times, those passaged 16 times contained fivefold more aggregate-filled cells, and they made EVs that spread aggregates more efficiently. Why? Proteomics showed that the highly passaged cells expressed more murine leukemia virus (MLV) proteins than their counterparts.

MLVs are endogenous retroviruses that are usually epigenetically silenced but can be activated to make leukemia virus particles (Kozak, 2014). When Vorberg separated the N2a culture medium via centrifugation, she found MLV envelope proteins enriched in the EV fractions, and the reverse transcriptase enzyme made by active viruses in other fractions. Only the EV fractions facilitated prion spreading.

Is the MLV envelope protein, not the active virus, responsible for prion seeding? Indeed, when Vorberg added small interfering RNA against the envelope protein to the N2a cells, neither EVs shuttling yeast prion nor prion-infected cells transmitted their aggregates to healthy cells.

Could the same be true for tau aggregates in human cells? Vorberg's team turned to HEK kidney cells expressing fluorescently labeled soluble tau, which aggregates upon adding AD brain tissue homogenate. While the aggregates barely spread between cells, transfecting these donor cells with plasmids encoding the MLV envelope protein enabled a bit more tau spread.

Seeding jumped fourfold upon expression of both the envelope and gag/pol genes. Gag encodes structural proteins; pol encodes viral enzymes, such as the reverse transcriptase, integrase, and protease. Vorberg attributed the better tau seeding to the structural proteins’ ability to increase virus-like particle secretion and the protease’s ability to activate the envelope protein to induce membrane fusion following receptor engagement. Adding EVs isolated from MLV protein-expressing cells similarly boosted tau seeding.

Intriguingly, cell-to-cell tau aggregate spread doubled in cells expressing all three MLV genes and a viral transfer vector, which enabled the assembly of active viral particles that were able to integrate into the genome but not replicate. Vorberg was not sure why this was, but speculated that perhaps the formation of active viral particles resulted in the formation of EVs with increased aggregate-inducing activity, or that virus production altered the ratio of viral proteins or their arrangement at cell membranes.

MLV is an animal virus. However, other retroviruses do hide in the genome of people. Human endogenous retroviruses (HERVs) are usually silent. They can become derepressed during aging, producing viral proteins though not active viruses. Proteins from one such virus, HERV-K, have been detected in postmortem brain tissue from people who had amyotrophic lateral sclerosis or frontotemporal dementia but not in healthy adults (Dec 2010 news; Phan et al., 2021).

Retroviruses Spread Tau. Compared to human kidney cells containing tau aggregates (red, left), those expressing the HEVR-W envelope protein spread aggregates to more cells (right). [Courtesy of Liu et al., 2022, bioRxiv.]

Vorberg expressed HERV-K and HERV-W envelope proteins in human kidney cells containing tau aggregates. These cells spread tau aggregates among themselves 1.5-fold better than did cells without the viral proteins (see image above). “You don't need an active virus [to increase tau seed spreading]; it's sufficient to express the HERV protein,” Vorberg said.

Vorberg found that certain antiviral drugs stop aggregate spreading in these cells. The HIV protease inhibitor amprenavir also inhibits the MLV protease and prevents cleavage of the envelope protein into its mature form. Adding amprenavir to N2a cells expressing the yeast prion prevented aggregates from spreading. However, Vorberg noted that HIV inhibitors may not work for HERVs because their envelope proteins do not need to be cleaved to be fully functional. Still, another type of HIV drug—the reverse transcriptase inhibitor TPN-101 —is being tested in Phase 2 trials in people with ALS, FTD, and progressive supranuclear palsy.

Vorberg and colleagues are now focusing on finding drugs that prevent HERV protein expression in hopes they might treat tauopathies. The scientists are partnering with clinicians to isolate and clone antibodies against HERV proteins from people with tauopathies, as they likely have higher levels of these antibodies. Another approach might be resilencing HERVs, though it's still unknown why they become derepressed in the first place.

“We know very little, so researchers should be encouraged to look at these HERVs much more closely to understand what they really do and how they contribute to disease,” Vorberg told Alzforum.—Chelsea Weidman Burke

Comments

1. • Tsuneya Ikezu
     Mayo Clinic Florida
   • Posted: 14 Apr 2023

   This is an extension of this group's previous paper published in 2021 and is further supporting the in vitro evidence that retrovirus activation potentially enhances intracellular aggregation of proteins. XP1 as the viral receptor for the potential target of intervention and the use of amprenavir to suppress retrovirus protein maturation is intriguing.

   The characterization of the involvement of EVs in the transmission process is not rigorously tested, as most of the experiments are not conducted with isolated EVs. This applies to their examination for tau aggregation, which is conducted by co-cultures of donor and recipient cells. The lack of validation in primary cultured cells also remains as a concern.

   View all comments by Tsuneya Ikezu
2. • Benjamin Wolozin
     Boston University School of Medicine
   • Posted: 18 Apr 2023

   This work presents a new twist on factors that might promote seeding and propagation of protein aggregates. The authors find that production of endogenous retroviruses can promote formation of extracellular vesicles (EVs) that contain and propagate protein aggregates such as the Sup35 prion protein or tau aggregates. The mechanism is plausible because groups such as Josh Dubnau’s, Bess Frost’s, and Josh Shulman’s have shown increased production of viral genes in models of neurodegenerative disease.

   The Vorberg group shows that induction of aggregation can reactivate latent endogenous retroviruses, which then stimulate the generation of EVs containing aggregates. Importantly, the group shows that inhibiting viral production of EV production inhibits propagation/seeding.

   Although interesting, the work also has important limitations. It is entirely done with dividing cells, such as murine N2a neuroblastoma or human HEK cells. These are cell lines, which are by definition dividing cells and have very different biologies than nondividing neurons or other cell types, such as astrocytes or microglia.

   In addition, even if proven, a classic quantitative question remains: While this can be shown in an experimental cell line, to what extent does the process accelerate the disease process in humans? This last question is a huge challenge for all of us working in preclinical models in that our systems might overestimate actual effects in patients. Nevertheless, this work adds to our knowledge of the pathophysiology of disease and could prove to be very important in particular subsets of patients.

   View all comments by Benjamin Wolozin
3. • Lary Walker
     Emory University
   • Posted: 18 Apr 2023

   This intriguing research from the Vorberg lab makes a strong case for the potential involvement of endogenous retroviruses (ERVs) in the spread of proteopathic seeds. The integration of work on yeast prions with mammalian cell culture models is a nice example of how basic biological investigations can illuminate disease mechanisms. ERVs are a salient component of the human genome, and they have been implicated in other aspects of neurodegenerative pathobiology such as neurotoxicity and inflammation. This study further indicates that de-repression of ERVs increases the intercellular transfer of pathogenic proteins such as aberrant tau.

   The experiments suggest that the spread of other proteopathic seeds might be similarly amplified by ERVs; in this regard, it would be interesting to know if the spread of Aβ—the probable prime mover of Alzheimer's disease—also is promoted by ERVs. As a next step, it will be important to extend the insights gained from in vitro work to animal models.

   For instance, advancing age is the most prevalent risk factor for most neurodegenerative diseases; if aging increases the risk of neurodegenerative disease via the de-repression of ERVs, how and when does it do so? And how might this be mitigated pharmacologically? If the strategy can be validated in vivo, these elegant cell culture experiments suggest a new approach to delaying or preventing neurodegenerative diseases.

   View all comments by Lary Walker

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References

News Citations

1. Nothing to Sneeze At: Viruses Raise Risk of Neurodegenerative Disease 2 Feb 2023
2. Viral Proteins Help Shuttle Tau Aggregates Among Cells 23 Oct 2021
3. Does an Ancient Retrovirus Come Out of Hiding in ALS? 23 Dec 2010

Therapeutics Citations

1. TPN-101

Paper Citations

1. Kozak CA. Origins of the endogenous and infectious laboratory mouse gammaretroviruses. Viruses. 2014 Dec 26;7(1):1-26. PubMed.
2. Phan K, He Y, Fu Y, Dzamko N, Bhatia S, Gold J, Rowe D, Ke YD, Ittner LM, Hodges JR, Piguet O, Kiernan MC, Halliday GM, Kim WS. Pathological manifestation of human endogenous retrovirus K in frontotemporal dementia. Commun Med (Lond). 2021;1 Epub 2021 Dec 9 PubMed.

Further Reading

Papers

1. Liu S, Heumüller SE, Hossinger A, Müller SA, Buravlova O, Lichtenthaler SF, Denner P, Vorberg IM. Reactivated endogenous retroviruses promote protein aggregate spreading. Nat Commun. 2023 Aug 18;14(1):5034. PubMed.