key: cord-0294724-23qojn9n authors: Liu, Shu; Heumüller, Stefanie-Elisabeth; Hossinger, André; Müller, Stephan A.; Buravlova, Oleksandra; Lichtenthaler, Stefan F.; Denner, Philip; Vorberg, Ina M. title: Endogenous retroviruses promote prion-like spreading of proteopathic seeds date: 2022-05-06 journal: bioRxiv DOI: 10.1101/2022.05.06.490866 sha: 1d92ce83c583a0a6c3a75916319946bb93452939 doc_id: 294724 cord_uid: 23qojn9n Endogenous retroviruses, remnants of viral germline infections, make up a substantial proportion of the mammalian genome. While usually epigenetically silenced, retroelements can become upregulated in neurodegenerative diseases associated with protein aggregation, such as amyotrophic lateral sclerosis and tauopathies. Here we demonstrate that spontaneous upregulation of endogenous retrovirus gene expression drastically affects the dissemination of protein aggregates between murine cells in culture. Viral glycoprotein Env mediates membrane association between donor and recipient cells and promotes the intercellular transfer of protein aggregates packaged into extracellular vesicles. Proteopathic seed spreading can be inhibited by neutralizing antibodies targeting Env as well as drugs inhibiting viral protein processing. Importantly, we show that also overexpression of a human endogenous retrovirus Env elevates intercellular spreading of pathological Tau. Our data highlight the potential influence of endogenous retroviral proteins on protein misfolding diseases and suggest that antiviral drugs could represent promising candidates for inhibiting protein aggregate spreading. Neurodegenerative diseases are associated with the aberrant folding of host-encoded proteins into insoluble, highly structured beta sheet-rich protein complexes, termed amyloid. Misfolding of proteins such as the microtubule-binding Tau is associated with highly prevalent Alzheimer's disease (AD) and other tauopathies. In AD, Tau deposition precedes grey matter atrophy, arguing that aberrant Tau is a major driver of pathogenesis (1) . Several different proteins such as TDP-43 or FUS accumulate in the central nervous systems of patients suffering from amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD) (2) . While mutations in aggregation-prone proteins account for few cases of familial neurodegenerative diseases, the etiologies of spontaneous disease are unknown (3) . Protein misfolding occurs through a process of templated conversion, in which small oligomers of misfolded proteins eventually fold into amyloid fibrils capable of templating their aberrant fold onto soluble homotypic proteins. Protein aggregation appears to proceed along neuroanatomical projections, suggesting that intercellular dissemination and propagation of protein misfolding underlies disease progression (4, 5) . This process resembles the spreading of prions, infectious protein aggregates composed of PrP that are the causative agents of transmissible spongiform encephalopathies (TSEs) (6) . Indeed, a growing number of studies, using cellular models or animals, provide substantial evidence for the spreading of protein aggregates between cells and within tissues (7, 8) . Small seeds of aggregated proteins can be either directly released by affected cells or transmitted to bystander cells via direct cell contact (9) . Proteopathic seeds capable of inducing protein aggregation in recipient cells can also be packaged into extracellular vesicles (EVs), which are normally secreted by cells for intercellular communication. We have recently shown that viral glycoproteins such as the vesicular stomatitis virus protein G or SARS Cov-2 spike S expressed by protein aggregate-bearing cells can mediate efficient intercellular contact with bystander cells, resulting in protein aggregate induction in the latter (10) . Moreover, viral glycoprotein decoration of EVs from donor cells harboring proteopathic seeds composed of Tau, the yeast Sup35 prion domain NM or prion protein PrP, strongly increased their aggregate inducing capacity in recipient cells. Thus, viral glycoproteins expressed during infection could act as "address codes" that enable delivery, receptor binding, efficient uptake and cytosolic release of proteopathic cargo in recipient cells. Viral genes are not only encoded by exogenous viruses invading mammalian cells, but are also remnants of mammalian germline infections that happened millions of years ago. Approximately 8-10 % of human and mouse genomes consists of retroviral elements (11) . Endogenous retroviruses (ERVs) share a common genome architecture with their exogenous counterparts, in which the coding regions for the capsid proteins (gag), reverse transcriptase, integrase and protease (pol) and envelope glycoprotein (env) are flanked by long terminal repeats. The majority of ERVs are reduced to proviral fragments, and only few are intact or at least contain full open reading frames that are transcribed and/or translated (12) . ERVs are subject to tight control by epigenetic modifications that repress transcription (13) . Failure to silence ERVs is associated with cancer as well as autoimmune, inflammatory and neurodegenerative disorders (14, 15) . Importantly, several human ERV members are upregulated in the brains of tauopathy and ALS patients (16) (17) (18) (19) (20) (21) (22) . No HERVderived infectious virions have so far been detected, but HERV-expressing human cell lines can produce viral-like particles (23) . Murine ERV of the Moloney leukemia virus (MLV) clade have integrated into the germline of ancestors millions of years ago. Some inbred mouse lines constitutively generate infectious MLV particles (24) . In most inbred mouse lines, however, the propensity of individual MLV loci to produce infectious virions is low. Still, restoration of ERV infectivity by recombination events between distinct loci in immunocompromised mice can result in ERV viremia (25, 26) . Here we uncover that de-repression of endogenous MLVs strongly affects spreading of proteopathic seeds between cells. By studying the spreading behavior of cytosolic protein aggregates composed of a yeast prion domain in cell culture, we demonstrate that the reactivation of MLVs strongly increases intercellular aggregate transmission. Reconstitution of HEK donor cells propagating cytosolic NM prions or Tau aggregates with MLV Env was sufficient to promote protein aggregate transfer between cells. Targeting of receptor binding or viral glycoprotein maturation drastically reduced intercellular proteopathic seed spreading. Further, expression of a human ERV glycoprotein also increased Tau aggregate dissemination in cocultures. These findings raise the possibility that de-repression of ERVs accelerates prion-like spreading of protein aggregates and suggests that ERVs represent potential therapeutic targets for disease intervention. For this study, we made use of our cell model which is based on the Sup35 prion domain NM. NM is the prion domain of the Saccharomyces cerevisiae translation termination factor Sup35 that can form self-templating protein aggregates. The prion domain exhibits compositional similarity to prion-like domains of RNA-binding proteins FUS and TDP-43, known to form protein aggregates in ALS and FTLD (27) . Soluble NM expressed in mammalian cells can be induced to aggregate by recombinant NM amyloid fibrils, resulting in cell populations that faithfully replicate NM aggregates over multiple passages (28) . Once induced, NM aggregates can also be transmitted to bystander cells by direct cell contact or via EVs, thereby inducing ongoing NM aggregation (29, 30) . Using our mouse neuroblastoma N2a Sup35 NM model system, we isolated subclone s2E with HA epitopetagged Sup35 NM prion aggregates (NM-HA agg ) (Suppl. Figure 1a) , which outcompetes other clones in its aggregate inducing capacity in recipient cells (30) . For simplicity, we here call this donor clone N2a NM-HA agg . Surprisingly, its aggregate-inducing activity in coculture experiments was strongly increased when cells were cultured over prolonged periods of time (Fig. 1a-c; Suppl. Figure 1b) . The increase in aggregate inducing capacity was reproducible, occurring approximately between 7 to 16 passages post thawing of the cryopreserved cells (approx. 32 to 72 d). Donor cells at high passage number retained their NM aggregate inducing activity even when cryopreserved at passage 21 and subsequently taken into culture (Suppl. Figure 1c) . For convenience, donor cells cryopreserved at P1 or P21 were subsequently used for experiments for up to 6 passages if not otherwise noted. We refer to cell populations as early and late passage donors (EP and LP, respectively). Increased donor passage number also increased aggregate induction in recipients cultured with conditioned medium from donors. This effect was abolished when conditioned medium was sonicated prior to addition to recipients, suggesting that aggregates might have been contained in EVs that were destroyed by sonication (Suppl. Figure 1d, e) . We previously demonstrated that NM aggregates are transmitted to bystander cells by EVs. To test if this was also the case at late passage, EVs from donors of different passages were purified by differential centrifugation. Strong aggregate induction was observed with EVs from late passage donors, demonstrating that EVs were involved in cell-free aggregate spreading (Fig. 1d, e) . Sonication abolished aggregate-inducing activity of EVs (Suppl. Fig. 1f, g) . Increased aggregate induction was not due to increased EV secretion, as particle numbers did not change significantly over prolonged culture (Fig. 1f) . EVs isolated from late passage donor cells also increased NM aggregate induction in primary cortical neurons, arguing that the effect was independent of recipient cells (Fig. 1g ). To identify changes in the proteome of donor cells that might contribute to protein aggregate spreading, we performed mass spectrometry analyses of total cell lysate ( Fig. 1h ) and donor EV fractions (Fig. 1i) at early and late passages. Among the proteins increased in donor cells and EVs upon prolonged culture, we identified mouse endogenous γ-retroviruses MLV proteins to be highly and significantly increased (Source data). Endogenous MLV are remnants of ancient germline infections that constitute approx. 10 % of the mouse genome. Partially overlapping MLV open reading frames code for polyproteins Gag (comprising matrix, p12, capsid and nucelocapsid), Pol (reverse transcriptase, integrase and protease) and for the envelope glycoprotein Env, giving rise to full genome RNAs as well as mRNAs coding for gag/pol and env, respectively. Prolonged cell culture increased mRNA levels coding for env and gag in both cell lysates and EVs (Fig. 1j) . Western blot analyses confirmed increased expression of Env and Gag in cell lysates and EV fractions from donors upon prolonged culture (Fig. 1k) . Gag was processed into capsid (CA, p30), nucleocapsid core (MA) and nucleocapsid (NC) by the viral protease. Env was cleaved into a surface subunit (SU, gp70) and a transmembrane domain (TM, Pr15/p15E) (31) . The finding that MLV mRNA and proteins increased in donor cells upon prolonged culture suggested that donor cells secrete active retrovirus, as has been observed for few cell lines before (32) (33) (34) (35) . In line with this, increased reverse transcriptase activity was observed upon prolonged culture of donors (Fig. 2a) . MLVs are classified by their respective Env proteins as ecotropic, polytropic and xenotropic, depending on their receptor preferences (36, 37) . To test if expression of ERVs had been triggered by NM aggregation, we preformed quantitative real-time PCR on mRNA extracted from different N2a NM populations before and after exposure to recombinant NM fibrils. Aggregate induction had no influence on induction of endogenous MLV subgroups, arguing that upregulation of MLV is likely influenced by other means (Suppl. figure 2a-f ). To demonstrate that donor cells produced infectious virus, vesicle fractions derived from donors were added to murine melan-a cells, a cell line permissive for MLV. Detection of Gag and Env by Western blot demonstrated that cells produced infectious virus ( Fig. 2b ) (38) . We further tested if Vectofusin-1, a compound that increases viral interaction with cellular membranes, could enhance aggregate induction. Aggregate induction was also increased when conditioned medium was added to recipients in the presence of Vectofusin-1 (Suppl figure 2g, h) (39) . To test which vesicle fractions released by the donor cells were NM-seeding competent, we separated EVs from viral particles by an OptiPrep velocity gradient previously used to separate HIV-1 virions from microvesicles ( Fig. 2c) (40) . Fractions with highest particle concentrations (fractions 2-6) harbored Alix and NM-HA, arguing that they contained EVs (Fig. 2d, e) . Gag and Env were distributed throughout the gradient, with highest levels present in Alix-positive fractions (Fig. 2e) . Reverse transcriptase (RT) activity was almost exclusively present in fractions 8-11, arguing that these fractions contained active virus (Fig. 2f) . This was confirmed by electron microscopy, which revealed membranous 80-100 nm spherical particles with an electrondense core, characteristic of γ-retroviral particles in fractions 9 and 10 ( Fig. 2g) (41) . By contrast, vesicles in fractions 2 and 3 exhibited a cup-shaped morphology, characteristic of EVs in TEM (42) . Next, we tested the NM aggregate inducing activity of fractions by adding them to recipient N2a NM-GFP sol cells (Fig. 2h) . Highest aggregate induction was associated with RT-negative EV fractions 2-6. Interestingly, aggregate induction could also be inhibited by antibodies against MLV Env in both coculture and by EVs (Fig. 2i) . We conclude that NM aggregate seeding activity is mainly associated with the RT-negative EV fraction. Still, aggregate induction can be inhibited by antibodies directed against MLV Env. The foregoing experiments suggested that Env plays a prominent role in intercellular aggregate transmission and induction. We tested if silencing of MLV in donors affects aggregate induction in recipient cells. Transfection of donor cells with three individual siRNAs targeting MLV env or gag/pol only slightly decreased MLV gene products, likely due to multicopy ERVs (24, 43) (Fig. 3a-c) . Still, knock-down of either gag/pol or env significantly decreased NM aggregate induction in cocultured N2a NM-GFP cells (Fig. 3d ). Since Gag/Pol and Env are encoded by different mRNAs, these results argue that Env as well as Gag/Pol contribute to enhanced proteopathic seed spreading. Expression of MLV underlies epigenetic control by silencing of proviral promoter regions through DNA methylation early during development (44) . As DNA methyltransferase inhibitors can induce expression of ERVs (45), we tested if erasing epigenetic marks affects aggregate inducing activity of donor cells (Fig. 3e) . Treatment of donor cells at early passage with DNA methyltransferase inhibitors 5-Azacytidine (Aza) or Decitabine (Dec) (46, 47) resulted in increased expression of total MLV env and gag mRNA ( Fig. 3f) and increased Env and Gag protein (Fig. 3g) . Both drugs also significantly increased NM aggregate induction in recipient cells when these were cocultured with pretreated donors (Fig. 3h) . In a reverse experiment, increased methylation with treatment of late passage donors with l-methionine, betaine or choline chloride resulted in decreased env and gag/pol mRNA (Fig. 3i) , protein levels ( Fig. 3j) and reduced aggregate induction in recipients (Fig. 3k) . The findings that siRNA against MLV mRNAs and epigenetic drugs that modulate MLV expression affect intercellular aggregate spreading suggest that MLV expression in involved intercellular aggregate spreading in our N2a cell culture system. MLV proteins Env and Gag require the MLV viral protease for processing into mature proteins. (48) (49) (50) . To investigate if EV-mediated NM aggregate induction depends on proper maturation of viral proteins, we tested anti-HIV-1 drugs for their effects on NM aggregate induction in coculture. Among the four tested HIV protease inhibitors, Amprenavir and Atazanavir have previously been shown to inhibit MLV protease (51) . Treatment of cocultures with these compounds (Fig. 4a ) had no effect on the percentage of donor cells with pre-existing NM-HA aggregates (Suppl. figure 3a) . However, the two MLV protease inhibitors Atazanavir and Amprenavir reduced the percentage of recipient cells with induced NM-GFP aggregates (Fig. 4b) . Other HIV protease inhibitors, reverse transcriptase inhibitors or Hepatitis C virus (HCV) protease inhibitors had no effect on donor aggregates or aggregate induction in recipients in coculture (Suppl. figure 3b). We hypothesized that Amprenavir inhibited MLV protease-driven viral protein maturation in the donor cells or during EV formation and thus had no effect on recipients. Thus, we tested the effect of the HIV protease inhibitor Amprenavir on NM-GFP aggregate induction by treating recipient cells 1 h prior to addition of EVs isolated from donor cells (LP) and incubating cells in the presence of drug for further 12 h. As a control, donor cells (LP) were also cocultured with recipients in the presence of the drug (Fig. 4c) . As expected, Amprenavir only inhibited aggregate induction in cocultures, but not in drug-treated recipients exposed to EVs (Fig. 4d) . In a reverse experiment, Amprenavir pretreated donor cells (LP) were cocultured with recipient cells in the absence of Amprenavir. Additionally, EVs isolated from Amprenavir-treated donors (LP) were tested for their aggregate inducing activity in recipient cells (Fig. 4e) . Amprenavir pre-treatment of donor cells significantly inhibited intercellular aggregate induction during coculture (Fig. 4f) and also basically abolished EV-mediated aggregate induction in recipient cells (Fig. 4g) , without affecting secreted particle numbers (Fig. 4h) . These experiments suggest that maturation of endogenous MLV encoded gene products in donor cells or donor-derived EVs is required for efficient aggregate induction in recipient cells. MLV envelope proteins mediate specific contact of virions with their receptors on target cells and induce cargo release into the cytosol by enforcing fusion of lipid bilayers. According to our proteomic analysis, polytropic env was upregulated in cells of late passage, suggesting that contact between polytropic Env and its receptor XPR1 was involved in NM aggregate spreading. Silencing of recipient MLV receptor XPR1 but not mCat-1, the receptor for ecotropic MLV (Fig. 5a, b) , strongly reduced NM aggregate induction in cocultures (Fig. 5c ) and by EV exposure (Fig. 5d) , confirming that upregulated MLVs belong to the group X/P-MLVs using this receptor. Silencing of both receptors in recipient cells had no effect on NM aggregate induction by recombinant NM fibrils, arguing that NM aggregate uptake was mediated by EV-receptor contact and not the direct contact of an NM seed with the receptor (Suppl figure 4a ). XPR1 is a receptor with eight putative transmembrane domains and four extracellular loops (ECL) (Fig. 5e) (52) . At least six polymorphic variants of XPR1 restrict infection by specific X/P-MLVs, with polymorphisms in ECL 3 and 4 affecting entry of certain X/P-MLV subtypes (53) . Analysis of XPR1 of susceptible N2a NM-GFP sol cells demonstrated that its Env recognition domain differed at 9 residues within ECL 3 and 4 from XPR1 expressed by HEK NM-GFP sol cells, a cell line refractory to NM aggregate induction by N2a NM-HA agg -derived EVs (Fig. 5f) . Expression of the N2a XPR1 variant ( Fig. 5g ) conferred susceptibility to HEK cells, both in coculture or upon exposure to donor-derived EVs (Fig. 5h, i) . By contrast, expression of the N2a polymorphic XPR1 variant had no effect on aggregate induction by recombinant NM fibrils (Suppl fig. 4b ). We conclude that efficient NM aggregate induction via coculture or EVs depends on specific interaction of Env with its receptor. The foregoing experiments demonstrated that epigenetic upregulation of MLV strongly affected intercellular aggregate spreading. So far it was unclear if generation of active viral particles was required for this process. To test this, HEK NM-GFP agg cells not coding for MLV were transfected with combinations of plasmids coding for MLV gag/pol, amphotropic MLV env 10A1 and MLV transfer vector (coding for mCherry) for virus production (Fig. 6a) . Vero cells stably expressing NM-GFP sol (10) were chosen as recipients due to their high expression level of amphotropic Env receptor Pit-2 ( Fig. 6b) . Ectopic expression of viral proteins Env and Gag/Pol resulted in significantly increased aggregate induction rates in cocultured recipients, with higher induction rates when env, and gag/pol were expressed simultaneously (Fig. 6c, d) . Interestingly, highest induction rates were observed when donor cells also produced virus (Fig. 6e, f ). Next, we tested if retroviral proteins also promoted intercellular transmission of protein aggregates associated with neurodegenerative diseases. To this end, we made use of our recently developed cell culture model propagating aggregates composed of the repeat domain of a mutant human Tau protein ( Fig. 6g) (54) . HEK cells stably expressing a soluble GFP-tagged repeat domain variant of human Tau (hereafter termed Tau-GFP sol ) stably produce and maintain Tau-GFP aggregates upon exposure to Alzheimer's disease brain homogenate (Tau-GFP AD ) (10, 54) . Upon donor transfection with retroviral plasmids (Fig. 6h) , induction rates in cocultured Vero cells expressing the same Tau variant fused to FusionRed (Tau-FR sol ) (10) were significantly increased ( Fig. i-k) . Again, highest induction rates were observed when donors were transfected with plasmids coding for gag/pol, env and the transfer vector (Fig. 6k) . Transfection of combinations of gag/pol and env vectors also strongly increased vesicle release (Fig. 6l) . When conditioned medium was adjusted for comparable particle numbers, highest induction rates were observed when donors were transfected with both gag/pol and env constructs (Fig. 6m) . We conclude that expression of retroviral proteins Env, Gag and Pol is sufficient to promote intercellular proteopathic seed spreading, but that proteopathic seed spreading is most efficient when donor cells produce active viral particles. In contrast to murine endogenous retroviruses of the MLV clade, HERV have so far not been shown to produce infectious virions in vivo. However, under certain circumstances, HERVs become reactivated, resulting in transcript and even protein expression. We thus tested if Env proteins encoded by HERVs could affect spreading of Tau misfolding. To this end, donor HEK Tau-GFP AD cells were transfected with a plasmid coding for the HERV-W Env Syncytin-1 or empty vector (Fig. 7a, b) . Coculture experiments with both HEK and Vero cells expressing Tau-FR sol revealed that Syncytin-1 resulted in a significant increase in recipient cells with Tau-FR aggregates ( Fig. 7c-f ). We conclude that gene products of both murine and human ERVs can facilitate intercellular protein aggregate spreading. Accumulating evidence argues that ERVs, resulting from retroviral germline invasions throughout evolution, are upregulated in NDs. While some HERV gene products can be directly neurotoxic, such as HERV-W Env (55) and HERV-K Env (56) , also inflammatory responses due to viral transcripts have been implicated in ND development (19) . Our data suggest an additional mechanism of how ERV proteins could contribute to neurodegeneration, namely by accelerating intercellular dissemination of protein particles. Activation of polytropic endogenous MLV proviruses resulted in the production of infectious virions as well as the secretion of protein aggregate-loaded EVs decorated with viral Env. As a result, MLV upregulation drastically increased the intercellular transmission of proteopathic seeds by EVs to bystander cells or to cells in direct contact and induced protein aggregation in the latter. Which cellular mechanism accounts for the consistent epigenetic switch upon continuous cell culture remains to be explored. The effect of epigenetic drugs on MLV expression argues that demethylation of MLV promoter regions at least partially explains this phenomenon. The activation of ERV was independent of transgene expression or the induction of protein aggregates. Activation and generation of ERV-derived retroviral particles has been reported for several cell lines in culture, including N2a cells (32-35, 57, 58) . Interestingly, receptor usage of viral particles produced in our N2a cell model clearly differed from the ecotropic MLV identified by others, arguing that within a given cell population, different MLVs can become de-repressed (33, 57) . Our detailed analysis reveals that the effect of reactivated endogenous MLV on protein aggregate spreading can be attributed to the expression of Env glycoprotein and retroviral Gag/Pol polyproteins. Upon increased expression, Env on the cell surface or on EVs mediates the binding to specific receptors on the cell surface of recipient cells. Cleavage of the MLV Env R-peptide by MLV protease then mediates fusion of cell membranes or EVs with the recipient cell or its endo-lysosomal membranes (48) , resulting in the release of proteopathic seeds into the cytosol of the recipient cell and subsequent aggregate induction. Surprisingly, reconstitution experiments in HEK cells demonstrate that MLV Env alone is sufficient to increase intercellular aggregate spreading, in analogy with our findings that vesicular stomatitis virus G and SARS-CoV2 spike S glycoproteins can elevate intercellular aggregate induction (10) . The additional positive effect of cotransfecting a plasmid coding for Gag and Pol polyproteins on aggregate induction is likely due to more efficient R peptide cleavage as well as concentration of Env and Gag polyprotein within rafts (48, 59) . However, highest induction rates were achieved when all plasmids for MLV virus production were transfected into donors. Thus, cells harboring protein aggregates and also actively producing infectious MLV virus most efficiently induce proteopathic seeds in bystanders. The reason for this is unclear, but might be related to the fact that MLV RNA increases the efficiency of proper Gag-Gag interactions, which in turn also affect proper Env positioning within rafts (60) . Most experiments in this study were performed using a model protein for cytosolic protein aggregates, which is based on the prion domain of the S. cerevisiae translation termination factor Sup35. The prion domain of Sup35 shares striking compositional similarity with so-called prion-like domains of a growing number of proteins associated with ALS and FTLD, such as FUS and TDP-43 (27) . In analogy to our study, experiments with transgenic mice have recently demonstrated that misfolded aggregated TDP-43 is secreted within EVs, suggesting that protein aggregates with similar domains are secreted by the same mechanism (61). Further, EVs isolated from plasma of ALS patients are enriched for TDP-43 and FUS (62) . Thus, it is feasible to assume that other protein aggregates with prion-like domains might also be affected by ERV de-repression. This hypothesis is supported by our findings that retroviral gene expression in donor cells harboring Tau aggregates also increased aggregate dissemination, demonstrating that this effect was independent of the type of cytosolic protein aggregate. Our results are consistent with a previous study, showing that simultaneous infection of cells with scrapie agent and friend retrovirus strongly enhanced intercellular spreading of pathologic prion protein and scrapie infectivity (63) . Viral Env and Gag association with prion-containing EV fractions has previously been observed for a prion-infected, endogenous ecotropic MLV producing N2a subpopulation. However, the role of Env expression in intercellular prion spreading has not been studied (33) . Interestingly, independent in vivo co-infections with MLV and scrapie showed no effect on scrapie incubation times, potentially because target cells for exogenous MLV and scrapie differ (64, 65) . Unfortunately, the effect of de-repressed MLVs on spreading of proteopathic seeds cannot easily be tested in ND mouse models, as endogenous MLV proviruses in common mouse lines such as C57BL/6 used in ND research are transcribed at low or undetectable levels (43, 66) . Endogenous polytropic MLV proviruses are highly polymorphic, but stable DNA elements, that are widespread in the murine genome (24) . Under certain circumstances, endogenous MLV can produce infectious virions, a characteristic that differs from HERVs which are generally considered non-infectious (67) . Still, also HERVs have been shown to produce viral-like particles in cell culture, cancer and autoimmune diseases (67) (68) (69) . For example, RNA and protein transcribed from LINE-1 retroelements are packaged into EVs (70) . Syncytin-1 is present on EVs derived from cutaneous T cell lymphoma, BeWo cells and placenta (71, 72) . Accumulating evidence suggests an association of HERV expression with NDs. Some but not all studies demonstrate that HERV-K transcripts are more abundant in ALS patients compared to unaffected individuals (22, 73) . Expression appears to be restricted to neurons rather than glia (56) . Elevated levels of HERV-K Env peptides in sera and CSF of ALS patients correlated with poor functional performance, suggesting that HERV-K Env contributes to disease progression (74) . Impaired ERV repression has also been correlated with Tau pathology (16, 17) . Elevated ERV transcripts have been reported for AD (18, 19) , PSP (20) , behavioral variant frontotemporal dementia (21) and sporadic CJD (75, 76) . Brains of bovine spongiform encephalopathy-infected macaques displayed increased ERV transcripts (77) . ERV Gag protein and RNA of the retroelement group IAP were also found to co-fractionate with CJD infectivity (78) (79) (80) . Importantly, HERVs are also upregulated during infection with exogenous pathogens, during inflammation und aging, processes which have been implicated in the progression of NDs (81, 82) . While directly being neurotoxic or indirectly activating microglia, our data suggest that ERV derepression could also contribute to prion-like spreading events. Murine XPR1 or human XPR1 were amplified from cDNA of N2a Preparation of cortical neurons was performed from the cortices of p13 SWISS pups as described previously (29) . Cortical neurons were transduced with lentivirus after 2 d cultivation on 96 well plates or Sarstedt 8 slice chambers. 2 d later, EVs were added to the cortical neurons and incubated for 2 d. The neurons were fixed for microscopy and imaging analysis. HEK293T cells were cotransfected with plasmids pRSV-Rev, pMD2.VSV-G, pMDl.g/pRRE, and pRRl.sin.PPT.hCMV.Wpre coding for Tau-FR or NM-GFP. Supernatants were harvested and concentrated with PEG according to published protocols (85) . Vero cells and primary neurons were transduced with lentivirus. Stable Vero cell clones with homogenous Tau-GFP/FR expression were produced by limiting dilution cloning (28) . To prepare EV-depleted medium, fetal calf serum was ultracentrifuged at 100,000 x g, 4° C for 20 h. Medium supplemented with EV-depleted FCS and antibiotics was subsequently and proteins was adjusted to less than 1 %. Label free quantification (LFQ) of proteins required at least two ratio counts of razor peptides. Only unique and razor peptides were used for quantification. The LFQ values were log2^ transformed and a two-sided Student's t-test was used to evaluate statistically significant changed abundance of proteins between cell lysates from passages 16 and 7 as well as EV lysates from passages 15 and 6. A pvalue less than 5 % was set as significance threshold. Additionally, a permutation based false discovery rate estimation was used to account for multiple hypotheses (89) . For separating EVs and viruses, the discontinuous iodixanol gradient in 1.2 % increments ranging from 6 to 18% were prepared as previously described (40) . The 100,000 x g pellet from 1050 ml culture supernatant (30 x T175 flasks) was resuspended in 1 ml PBS and overlaid onto the gradient. The gradient was subjected to high-speed centrifugation at 100,000 x g for 2 h at 4° C using a SW41Ti rotor (Beckman Coulter). 12 fractions of 1ml each were collected from the top of the gradient, diluted with PBS in 5 ml, and centrifuged at 100,000 x g for 1 h at 4° C. The pelleted fractions were resuspended in 100 µl PBS, and then used for further experiments. The reverse transcriptase activity of the viruses was measured by using the colorimetric Reverse Transcriptase Assay (Roche). EM imaging of EV and virus preparations were performed as previously described (42) . Briefly, the 100,000 x g pellets were fixed in 2 % paraformaldehyde, loaded on glow discharged Formvar / carbon-coated EM grids (Plano GmbH), contrasted in uranyl-oxalat (pH 7) for 5 min and embedded in uranyl-methylcellulose for 5 min. Samples were examined using a JEOL JEM-2200FS transmission electron microscope at 200 kV (JEOL). The infectivity assay was performed as previously described (57) . Briefly, melan-a cells were exposed to conditioned medium from different cell clones at either low or high passages in the presence of 4 µg Polybrene/ ml for 24 h. The medium was then replaced with normal culture medium. After 6 d, cells were lysed for Western blot analysis for the existence of retroviral Env and Gag proteins. The To block MLV Env on the surface of the donor cell clone and on EVs, mAb83A25, which reacts with almost all members of MLVs (90) was incubated with either EVs or donor cells in serial dilutions for 1 h at 37°C with rotation at 20 rpm. Afterwards, the donor cells were mixed with recipient cells and EVs were added to the pre-seeded recipient cells for 1 d. To transiently knock-down the upregulated specific MLV Env and Gag genes in N2a NM-HA agg donors, custom-designed Silencer select siRNAs (Thermo Fisher) against AAO37244.2 (env) and AID54952 (gag) were used. Pre-designed siRNAs against murine XPR1 and mCat-1 were used to knock-down both genes. For transfection, cells were pre- The image analysis was performed using the CellVoyager Analysis support software Alternatively, recipients were cultured with donor EVs that had been pre-incubated with anti-env antibodies for 1 h. Anti-env antibodies were present throughout the experiment. with two siRNAs against XPR1, one mCat-1 siRNA or a non-silencing siRNA control (mock). 48 h later, recipient cells were cocultured with donor cells (LP) . Alternatively, recipients were exposed to EVs isolated from conditioned medium of N2a NM-HA agg cells similar results. P-values calculated by two-tailed unpaired Student´s t-test. Source data are provided as a Source Data file. Percentage of recipient cells with Tau-FR agg exposed to conditioned medium of donors adjusted for comparable particle numbers. 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Preparation and use Universal sample preparation method for proteome analysis Stop and go extraction tips for matrixassisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ Significance analysis of microarrays applied to the ionizing radiation response A neutralizable epitope common to the envelope glycoproteins of ecotropic, polytropic, xenotropic, and amphotropic murine leukemia viruses Donor N2a NM-HA agg cells (LP) were treated with Amprenavir for 3 d. Cells were subsequently cocultured with recipient N2a NM-GFP sol . Alternatively, EVs from treated donors (LP) were added to recipient cells. Aggregate formation in recipients was assessed 16 h later. f. Amprenavir inhibits NM-GFP aggregation in cocultured recipient cells. g. Effect of Amprenavir-treatment of donor cells (LP) on the induction of NM-GFP aggregates in recipient cells upon exposure to donor-derived NM-HA agg bearing EVs. h. Effect of Amprenavir on particle release by donor cells (LP) NM-GFP aggregate induction was determined 16 h post exposure or coculture Knock-down of receptor mRNA in N2a NM-GFP sol cells was assessed 48 h post siRNA transfection by qRT-PCR. Shown is the fold change of mRNA expression normalized to mock control. c., d. Recipients with NM-GFP aggregates following receptor knock-down Shown are results of coculture (c) recipients exposed to donor derived EVs (LP) (d) Transmembrane structure of XPR1. The receptor contains 4 extracellular loops MLV receptor XPR1 in mouse N2a and human HEK cells. Shown are mismatches in the surface-exposed loops ECL 3 and 4. ECL3 and 4 are required for binding of X/P-MLV (37). g. Ectopic expression of the N2a XPR1 receptor variant in poorly permissive HEK NM-GFP sol cells. Ectopically expressed XPR1-HA was detected using anti-HA antibodies. GAPDH served as a loading control. h. HEK NM-GFP sol cells were transfected with mouse XPR1-HA or mock Alternatively, transfected HEK NM-GFP sol cells were exposed to EVs from N2a NM-HA agg cells (LP) Three (b-d, h-i) independent experiments were carried out with Supplementary figures Increased aggregate induction by donors that have been in culture for prolonged time. a. N2a NM cell culture model upper left image) or soluble NM-GFP (NM-GFP sol , upper right) (28) Shown are early (P7) and late passages of the clone (P16). b. Cocultures of N2a NM-GFP sol with N2a NM-HA agg cells of early and late passage. As controls, recipients were also cocultured with N2a NM-HA sol cells. c. Cryopreserved N2a NM-HA agg cells of late passage (P21) retain NM aggregate high inducing activity. Donor cells were frozen at passage 1. Donors of different passaging history were subsequently defrosted and cultured for less than 6 passages Donors were cocultured with N2a NM-GFP sol cells and recipients with NM-GFP agg were detected 16 h later. As control, recipients were cultured with N2a NM-HA sol cells Confocal images of recipient cells exposed to isolated EVs from donors of early or late passage. g. Quantitative analysis of aggregate induction by EVs. To destroy vesicles, EVs were sonicated. Insets show higher magnifications of cells. All data are shown as the means ± SD from three (g) or six (c, e) replicate cell cultures. Three (c, e, g) independent experiments were carried out with preexisting NM-HA aggregates in donor cells. Shown are the percentages of donor cells with aggregates upon exposure to different concentrations of drugs. b. Experimental scheme. N2a NM-HA agg and N2a NM-GFP sol cells were cocultured in the presence of HIV or HCV inhibitors. Effect of different concentrations of inhibitors on the percentage of donor and recipient cells harboring NM-HA agg or NM-GFP agg , respectively. % cells with aggregates were normalized to control cocultures only treated with DMSO. All data are shown as the means ± SD from three (a-b) replicate cell cultures Recipients with NM-GFP agg were quantified 16 h post exposure. b. Recipient HEK NM-GFP sol cells were transfected with a plasmid coding for the N2a XPR1 receptor or empty plasmid for 2 d. Afterwards, pretreated recipients were exposed to 10 µm recombinant NM fibril (monomer equivalent). As controls All data are shown as the means ± SD from three (a) or six (b) replicate cell cultures. Three (a-b) independent experiments were carried out with similar results. P-values calculated by two-tailed unpaired Student´s t-test (b) or one-way ANOVA (a). ns: non-significant We thank Leonard Henry "Pug" Evans for generously sharing anti-MLV antibodies. We are grateful to Paolo Salomoni and Dan Ehninger for critical reading of the manuscript.The light microcopy (LMF) and laboratory automation facilities (LAT) of the DZNE Bonn were used for image acquisition. This work was funded by the Helmholtz Portfolio "Wirkstoffforschung", the "Deutsche The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.