23 Oct 2021

When it comes to spreading from cell to cell, viruses are the masters. To disseminate their progeny, many viruses encode glycoproteins that fuse virions to plasma membranes. According to a paper in the October 19 Nature Communications, those glycoproteins may unwittingly facilitate the intercellular transmission of their host’s cellular contents, including misfolded, prion-like proteins. Researchers led by Ina Vorberg, of the German Center for Neurodegenerative Diseases in Bonn, reported that two viral glycoproteins—VSV-G and the SARS-CoV2 S1 spike—dramatically boost the transfer of prions and misfolded tau between cells in culture. The viral ligands promote the release of extracellular vesicles, including any carrying proteopathic seeds, and their fusion to nearby cells. Once inside the recipient cell, the misfolded proteins co-opt their properly folded counterparts into aggregates.

“These results suggest a conceivable means whereby infections of various types might augment the probability of developing a neurodegenerative disease later in life,” commented Lary Walker of Emory University in Atlanta. “They also might help to explain why some infectious agents are stronger risk factors for Alzheimer's disease than are others.”

Prions seed aggregation by way of templated misfolding. Other proteins that cause neurodegeneration, including Aβ, tau, and α-synuclein, can propagate through the brain in a prion-like fashion. But how do the misfolded seeds spread from one cell to another? Numerous mechanisms have been proposed, ranging from the spewing of aggregates into the extracellular space, to tiny transcellular conduits, aka tunneling nanotubes, to gap junctions (Feb 2012 news; Tardivel et al., 2016; Sep 2021 news). Extracellular vesicles, including exosomes, are another potential vector. These tiny membrane packages ferry cellular material, including misfolded proteins Aβ and tau, between cells (Oct 2015 news; Dec 2020 news). Notably, viral infection instigates the formation and release of extracellular vesicles containing viral protein, and viral glycoproteins facilitate membrane fusion.

Might viral glycoproteins amplify proteopathic transfer via exosomes then? First author Shu Liu and colleagues addressed this with a series of cell culture models. They started by pairing a characterized prion—the NM prion domain from the yeast Sup35 protein—with the vesicular stomatitis virus glycoprotein. Made by rhabdoviruses, which include rabies, VSV-Gs strengthen the docking of EVs with cells by latching onto LDL receptors. Sup35-NM prions are known to spread between cells via direct contact as well as via EVs. Would adding VSV-G enhance prion dissemination?

Indeed, the scientists found that the glycoprotein ramped up prion transmission. HEK-293 or N2a neuroblastoma cells harboring aggregates of Sup35-NM poorly induced aggregation in co-cultured recipient cells containing only the soluble form. However, when the donor cells were transfected with VSV-G, aggregation in recipient cells ramped up dramatically. The researchers ultimately tied this enhanced transmission to both an increase in EV release from donor cells and more efficient fusion of EVs to recipient cells. VSV-G also cranked up prion transmission in primary human astrocytes.

This viral glycoprotein also helped transmit PrP^SC, the misfolded form of cellular prion protein, between cells. Unlike the intracellular Sup35 protein, PrPc is membrane-bound, thus extending the viral glycoprotein’s sphere of influence.

How about tau? To investigate, the researchers used HEK293 cell lines that express the repeat domains of tau—complete with pathogenic mutations—that are highly prone to aggregation. They induced aggregation in these cells by exposing them to homogenates extracted from postmortem brain samples of people who had died with different tauopathies, including AD, cortical basal degeneration, progressive supranuclear palsy, or frontotemporal lobar degeneration. They then co-cultured these aggregate-bearing cells with recipient cells expressing fluorescently labeled tau. Remarkably, recipient cells only lit up with tau aggregates when co-cultured with donor cells expressing VSV-G. This suggested that the glycoprotein was crucial for transfer of misfolded tau between cells.

Finally, the researchers asked whether another viral glycoprotein—the S1 spike protein from the infamous SARS-CoV2—would also amp up the intercellular transfer of protein miscreants. Studding the coronavirus capsid, the spike facilitates the entry of the virus into cells by binding to ACE2 receptors on their surface. The researchers transfected donor HEK cells with S1 and co-cultured them with HEK cells expressing ACE2, or Vero monkey kidney cells that are highly susceptible to coronavirus infection. They found S1 behaved similarly to VSV-G. Although the spike protein did not increase the release of EVs from donor cells, it enhanced the transmission of Sup35-NM as well as misfolded tau, leading to aggregation in recipient cells. S1-bearing EVs isolated from donor cell cultures could directly induce misfolding in recipient cells.

The findings suggest a mechanism through which viral infections might egg on the spread of protein pathology in the brain. “Although the study is limited to overexpression systems using immortalized cell lines, the results are striking and may have translational relevance to virus infection-mediated enhancement of tau propagation via EVs, including by COVID-19, HSV, or HIV infection,” wrote Tsuneya Ikezu of the Mayo Clinic in Jacksonville, Florida. However, as Ikezu and other commentators pointed out, in vivo experiments will be crucial to address if, and how, this occurs within the brain.

The idea that viruses instigate or exacerbate neurodegenerative diseases by encouraging release and transmission of toxic proteins is not far-fetched. Previous studies reported that retroviral glycoproteins boosted the infectivity of prions, and also triggered the formation of Aβ plaques (Leblanc et al., 2006; Kodidela et al., 2019). In regard to SARS-CoV2, studies have found that not only can the virus infect the brain, but the spike protein itself can go rogue and cross the blood-brain barrier (Heneka et al., 2020; Jan 2021 news; Rhea et al., 2020). 

Herpesviruses, such as HSV-1, also infect the CNS, and have been implicated in AD. Vorberg noted that, like many viruses, HSV-1 encodes its own fusion protein—glycoprotein B—which forms a complex with other viral proteins to fuse membranes together. “Importantly, herpesvirus glycoproteins and HIV envelope proteins are also sorted onto EV,” she wrote to Alzforum.

Vorberg’s group is analyzing patient brain samples to get an idea of which viruses, and which types of protein aggregates, might sway neurodegenerative disease. They also plan to study mouse models. “Our work unveils an important aspect of proteopathic seed transmission. Uptake, and especially release, of proteopathic seeds in the cytosol are bottlenecks that might represent targets for drug development,” she added.—Jessica Shugart

Comments

1. • Lary Walker
     Emory University
   • Posted: 23 Oct 2021

   A link between infectious pathogens and neurodegenerative disorders later in life is plausible but mechanistically enigmatic. It is fair to say that no single infectious agent is the cause of Alzheimer's disease (Walker, 2020), as has been proposed by advocates of diverse microbes and viruses (see, e.g., Itzhaki et al., 2020; Itzhaki et al., 2016; Sochocka et al., 2017). However, there is good evidence that certain infections are risk factors for the disease (Walker, 2020). 

   In this compelling investigation in cultured cells, Liu and colleagues present evidence that two viral glycoproteins—VSV-G and CoV-2 spike S—each promote the spread of prions and tau proteopathic seeds among cells. The findings indicate that specific receptor-ligand interactions expedite the fusion of extracellular, seed-laden donor vesicles with recipient cells. Interestingly, while glycoproteins from different types of viruses can facilitate the spread of proteopathic seeds, they do so with different efficiencies.

   These results suggest a conceivable means whereby infections of various types might augment the probability of developing a neurodegenerative disease later in life. They also might help explain why some infectious agents are stronger risk factors for Alzheimer's disease than are others. The analysis includes a nice experimental progression from yeast prions to pathogenic agents in humans, though it will be important to extend the cell culture findings to animal models. One question, for example, is whether the age at which an infection occurs influences the risk of later neurodegenerative disease—is infection at older or younger ages more problematic? Is there a cumulative effect of multiple infections? Might different pathogens interact to further increase disease risk?

   It also will be interesting to determine whether this process of intercellular spread applies to other neurodegenerative proteopathies, such as the synucleinopathies. From a translational standpoint, the study suggests that blocking the receptor-ligand interactions that enable the transfer of cargo from one cell to another might reduce both the pathogenicity of an infectious agent and its downstream impact on the risk of neurodegenerative disease.   

   References:

   Itzhaki RF, Golde TE, Heneka MT, Readhead B. Do infections have a role in the pathogenesis of Alzheimer disease?. Nat Rev Neurol. 2020 Apr;16(4):193-197. Epub 2020 Mar 9 PubMed.

   Itzhaki RF, Lathe R, Balin BJ, Ball MJ, Bearer EL, Braak H, Bullido MJ, Carter C, Clerici M, Cosby SL, Del Tredici K, Field H, Fulop T, Grassi C, Griffin WS, Haas J, Hudson AP, Kamer AR, Kell DB, Licastro F, Letenneur L, Lövheim H, Mancuso R, Miklossy J, Otth C, Palamara AT, Perry G, Preston C, Pretorius E, Strandberg T, Tabet N, Taylor-Robinson SD, Whittum-Hudson JA. Microbes and Alzheimer's Disease. J Alzheimers Dis. 2016;51(4):979-84. PubMed.

   Sochocka M, Zwolińska K, Leszek J. The infectious etiology of Alzheimer's Disease. Curr Neuropharmacol. 2017 Mar 13; PubMed.

   Walker LC. Aβ Plaques. Free Neuropathol. 2020;1 Epub 2020 Oct 30 PubMed.

   View all comments by Lary Walker
2. • Tsuneya Ikezu
     Mayo Clinic Florida
   • Posted: 23 Oct 2021

   This is an interesting study showing the potential of virus-infected cells for spreading of extracellular vesicles (EVs) containing protein aggregates. It earmarks viral glycoprotein for enhanced binding and endocytosis of EVs to recipient cells for subsequent spread of protein aggregates.

   The study tested VSV-G and SARS-CoV-2 spike S viral proteins and the prion domain NM of Saccharomyces cerevisiae Sup35 fused with GFP, the mutated 4R domain of Tau fused with GFP, and the scrapie strain 22 L (N2a22L) as transiently expressed prion seeds. Although the study is limited to overexpression systems using immortalized cell lines, the results are striking and may have translational relevance for virus infection-mediated enhancement of tau propagation via EVs, including via COVID-19, HSV, or HIV infection. The study would be more convincing if the key findings were reproduced in more physiological conditions, such as primary cultured neurons or animal models using physiological protein aggregates as seeds.

   We have originally reported the role of EVs for tau propagation (Asai et al., 2015), which was further enhanced in the presence of amyloid plaque in the brain (Clayton et al., 2021). We have also recently shown that AD-brain-derived EV-mediated tau propagation is more efficient than the same amount of tau in fibril or oligomer forms (Ruan et al., 2021). 

   This study could be evaluated using the tau-propagation mouse model or human brain-derived EV from virus-infected cases in a similar manner. The study also shows that VSV-G loaded EVs endocytose into recipient cells via clathrin-dependent endocytosis, presumably via receptor-mediated endocytosis. This is different from conventional EV uptake, which is by macropinocytosis, and the endocytosis may lead to endolysosomal degradation of the EVs. Indeed, suppression of this pathway is known to enhance tau propagation in recipient cells, which is different from this report. It is unclear how protein seeds can escape this degradation pathway and be efficiently propagated in recipient cells.

   View all comments by Tsuneya Ikezu

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References

News Citations

1. Mice Tell Tale of Tau Transmission, Alzheimer’s Progression 3 Feb 2012
2. Microglia Share Synuclein Aggregates with Each Other Via Nanotubes 30 Sep 2021
3. Deadly Delivery: Microglia May Traffic Tau Via Exosomes 9 Oct 2015
4. Fatal Gift Wrap: Neurons Fall for Packaged Tau Oligomers 22 Dec 2020
5. How Does COVID-19 Affect the Brain? 29 Jan 2021

Paper Citations

1. Tardivel M, Bégard S, Bousset L, Dujardin S, Coens A, Melki R, Buée L, Colin M. Tunneling nanotube (TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies. Acta Neuropathol Commun. 2016 Nov 4;4(1):117. PubMed.
2. Leblanc P, Alais S, Porto-Carreiro I, Lehmann S, Grassi J, Raposo G, Darlix JL. Retrovirus infection strongly enhances scrapie infectivity release in cell culture. EMBO J. 2006 Jun 21;25(12):2674-85. Epub 2006 May 25 PubMed.
3. Kodidela S, Gerth K, Haque S, Gong Y, Ismael S, Singh A, Tauheed I, Kumar S. Extracellular Vesicles: A Possible Link between HIV and Alzheimer's Disease-Like Pathology in HIV Subjects?. Cells. 2019 Aug 24;8(9) PubMed.
4. Heneka MT, Golenbock D, Latz E, Morgan D, Brown R. Immediate and long-term consequences of COVID-19 infections for the development of neurological disease. Alzheimers Res Ther. 2020 Jun 4;12(1):69. PubMed.
5. Rhea EM, Logsdon AF, Hansen KM, Williams LM, Reed MJ, Baumann KK, Holden SJ, Raber J, Banks WA, Erickson MA. The S1 protein of SARS-CoV-2 crosses the blood-brain barrier in mice. Nat Neurosci. 2020 Dec 16; PubMed.

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