JC Virus infected choroid plexus epithelial cells produce extracellular vesicles that infect glial cells independently of the virus attachment receptor

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy (PML) in immunosuppressed and immunomodulated patients. Initial infection with JCPyV is common and the virus establishes a long-term persistent infection in the urogenital system of 50–70% of the human population worldwide. A major gap in the field is that we do not know how the virus traffics from the periphery to the brain to cause disease. Our recent discovery that human choroid plexus epithelial cells are fully susceptible to virus infection together with reports of JCPyV infection of choroid plexus in vivo has led us to hypothesize that the choroid plexus plays a fundamental role in this process. The choroid plexus is known to relay information between the blood and the brain by the release of extracellular vesicles. This is particularly important because human macroglia (oligodendrocytes and astrocytes), the major targets of virus infection in the central nervous system (CNS), do not express the known attachment receptors for the virus and do not bind virus in human tissue sections. In this report we show that JCPyV infected choroid plexus epithelial cells produce extracellular vesicles that contain JCPyV and readily transmit the infection to human glial cells. Transmission of the virus by extracellular vesicles is independent of the known virus attachment receptors and is not neutralized by antisera directed at the virus. We also show that extracellular vesicles containing virus are taken into target glial cells by both clathrin dependent endocytosis and macropinocytosis. Our data support the hypothesis that the choroid plexus plays a fundamental role in the dissemination of virus to brain parenchyma.

Author summary

JC polyomavirus (JCPyV) is a common human pathogen that causes a central nervous system demyelinating disease known as progressive multifocal leukoencephalopathy (PML). To cause PML, JCPyV has to traffic from peripheral tissues to the central nervous system (CNS) and invade glial cells. In previous work we found that choroid plexus epithelial cells express receptors for JCPyV in vivo and are fully susceptible to virus infection in vitro. In contrast, glial cells do not express the receptors for JCPyV and virus does not bind to these cells in human tissue sections. Because choroid plexus epithelial cells are known to relay information between the blood and the brain using extracellular vesicles we hypothesized that this could be important for JCPyV neuroinvasion. We found that JCPyV infected choroid plexus epithelial cells produce extracellular vesicles containing JCPyV virions and that these extracellular vesicles transmit the infection to human glial cells independently of the virus attachment receptor. These findings support our hypothesis that the choroid plexus is important in the dissemination of virus to the brain to initiate disease.

Related collections

Most cited references 24

Record: found
Abstract: found
Article: found

Is Open Access

Extracellular Vesicle-Mediated Transfer of Genetic Information between the Hematopoietic System and the Brain in Response to Inflammation

Kirsten Ridder, Sascha Keller, Maria Dams … (2014)

Introduction The influence of the immune system on the brain in the context of inflammation is highly relevant for a number of diseases, yet mechanisms for this interaction are not fully understood. The stereotypical response is the secretion of pro-inflammatory cytokines by immune cells. These peripheral cytokines in turn can have a direct effect on neural cells or activate brain inflammatory cytokine signaling, usually via microglia, the principle innate immune cells of the brain [1]. Recently, heterotypic cell fusion of hematopoietic cells with Purkinje neurons in the brain has been suggested as a conceptually different mechanism of response to inflammation. When transplanted into lethally irradiated mice, genetically labeled hematopoietic donor cells have been found to contribute to a number of host tissues including skeletal and cardiac myofibers, hepatocytes in the liver, intestinal crypt cells, and Purkinje neurons in the brain. Initially seen as evidence for an unexpected differentiation potential of hematopoietic stem cells, it was eventually demonstrated that the experimentally observed plasticity was largely attributable to cell fusion, rather than transdifferentiation [2]–[4]. For the brain, fusion of hematopoietic cells has so far mainly been reported with Purkinje neurons. Although the number of fusion events is very low in the healthy animal, peripheral inflammation induces cell fusion events to increase by a factor of 10–100, giving the first indication that heterotypic fusion is regulated by a pathologic stimulus and may therefore be of biological significance [5]. We were interested in studying the contribution of hematopoietic cells to neural tissue without the accompanying confounding factors such as lethal irradiation, chemoablation, or parabiosis normally associated with replacing the host bone marrow. In contrast, to irreversibly label hematopoietic cells to follow their fate in vivo, we used transgenic mice expressing Cre recombinase under the hematopoietic-specific promoter vav [6] in a Cre reporter background. Although in our mouse model we could observe recombination events in the same tissues and at a frequency comparable to those observed in transplantation studies, we did not find any evidence for cell fusion in Purkinje neurons, marked by the absence of a second nucleus [7]. We now show that recombination in neural cells is caused by the intercellular transfer of Cre recombinase messenger RNA. More specifically, biochemical analysis demonstrates that Cre mRNA is contained in extracellular vesicles (EVs), including exosomes. These EVs are sufficient to induce recombination in neural cells after direct intracerebellar injection. Recombination events occur rarely and are restricted to very few Purkinje neurons in the healthy animal. However, when inducing a peripheral inflammation or an entorhinal cortex lesion (ECL), the number of recombined cells increases dramatically and extends to other neuronal cell populations. Importantly, recombined versus nonrecombined Purkinje neurons display differences in their miRNA profile several days after inflammation, indicating biologically significant changes. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation. Results Expression of Cre Recombinase Specifically in the Hematopoietic Lineage Leads to Recombination Events in Purkinje Neurons Without Cell Fusion To monitor the contribution of hematopoietic cells to other tissues without any of the confounding factors associated with host bone marrow replacement, we previously utilized transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage under the Vav1 promoter [7]. For this study, we additionally included a mouse model expressing Cre under the hematopoietic- and endothelial-specific promoter Tie2 [8] to minimize the possibility of false positive results due to a leaky expression of Cre. Thus, in hematopoietic cells, marker gene expression is irreversibly induced (Figure 1A), allowing the tracing of hematopoietic contribution to any other tissue. Confocal analysis of different tissue sections of both Vav-iCre- and Tie2-Cre-GFP/LacZ mice showed GFP- or LacZ-positive cells in organs such as the liver, lung, and small intestine (Figure 1B–D) similar to observations made in animals transplanted with bone marrow from constitutively marker-gene-expressing cells. In the cerebellum, only Purkinje neurons could be observed expressing the marker gene (Figure 1E). However, all marker-gene–positive Purkinje neurons from both mouse lines contained only a single nucleus in line with our previous findings [7], suggesting a contribution of hematopoietic-to-neural cells independent of cell fusion (Figure 1F). Thus, in both our transgenic models we could make observations similar to what had been reported for chimeric animals but with no evidence of cell fusion in neurons. 10.1371/journal.pbio.1001874.g001 Figure 1 Noninvasive tracing of hematopoietic Cre recombinase activity. (A) Expressing Cre recombinase specifically in the hematopoietic lineage reveals the contribution of hematopoietic cells to nonhematopoietic tissues by irreversibly switching on reporter gene expression after excision of a floxed stop codon. β-galactosidase expression after recombination can be observed in multiple tissues such as liver (B), lung epithelia (C), and small intestine (D). In the intestine, the labeling of an entire crypt indicates recombination of an intestinal stem cell with concomitant inheritance of the marker to all progeny. (E) Overview of a cerebellar section showing a single recombined Purkinje neuron expressing GFP (green). (F) High magnification of a Purkinje neuron in serial section confocal analysis confirming absence of a second nucleus, excluding cell fusion. Scale bar, 50 µm (B), 20 µm (C), 50 µm (D), 50 µm (E), and 10 µm (F). Hematopoietic Cell-Derived Exosomes Contain Cre mRNA The observation of Cre-dependent reporter gene expression without evidence of cell fusion events led us to consider alternative explanations for a transfer of Cre recombinase from hematopoietic cells to neurons. A growing body of evidence suggests a role for EVs in cell-to-cell communication [9]. Moreover, EVs can transfer functional mRNA between cells in vitro [10]. We therefore asked if reporter gene expression in nonhematopoietic cells was the result of an RNA-based transfer of Cre recombinase via EVs, leading to the translation of Cre RNA in target cells and nuclear recombination followed by irreversible reporter gene expression. Because low levels of recombination events could be detected in uninjured animals, we hypothesized that Cre messenger RNA should be detectable in the blood of mice with hematopoietic Cre recombinase expression. Peripheral blood was drawn from six Vav-iCre mice and processed according to a protocol for the isolation of secreted membrane vesicles [11]. In all vesicle isolation experiments, the pellet was treated with RNaseA to remove RNA that was not contained in vesicles. RNA was extracted from vesicle preparations and reverse transcribed, followed by nested primer RT-PCR. Cre recombinase cDNA could be detected in four out of six analyzed blood samples (Figure 2A), showing that Cre mRNA is present at low levels in blood plasma. To obtain larger amounts of secreted membrane vesicles for a more detailed analysis, we prepared in vitro cultures from pooled peripheral blood and bone marrow of Vav-iCre mice (n = 6 separate preparations from one to two mice each) and added lipopolysaccharide (LPS) at 200 ng/ml to stimulate an inflammatory reaction. Cell culture supernatants were collected after 3 d of stimulation, and membrane vesicle fractions enriched for exosomes were prepared via differential ultracentrifugation. The resulting supernatants and pellets were used for RNA extraction, reverse transcription, and subsequent detection of cDNA using PCR. Consistently, Cre recombinase cDNA could be detected in the vesicle pellet but not in the supernatant (Figure 2B) in all experiments, suggesting that Cre mRNA was not part of the soluble cell culture medium but was contained in physical structures amenable to separation by centrifugation. To directly show that Cre mRNA is contained in vesicles and does not sediment because of an association with protein complexes, we took vesicle pellets prepared as above and treated them with RNaseA alone or in combination with detergent. In this way, vesicles would be lysed and the RNA exposed to RNase digestion. Indeed, in the samples treated with detergent and RNAse, we could no longer detect Cre mRNA in contrast to vesicle pellets that were treated with RNAse alone (n = 2 independent sample preparations) (Figure 2C). Electron microscopic images from our vesicle preparations showed predominantly structures with sizes between 50 and 100 nm and a round or cup-shaped morphology typically associated with exosomes (Figure 2D) [12]. EVs are a heterogeneous population and their typology is not established with regard to their possible biological functions. However, the most prominent subclass are exosomes as defined by their size, protein marker load, and enrichment profile by differential density ultracentrifugation [9]. To specifically determine whether exosomes contain Cre mRNA, we fractionated the vesicle preparations using sucrose density gradient ultracentrifugation [13]. Exosomal identity was confirmed by Western blot analysis for the specific surface markers ADAM10 and CD9 (Figure 2E,F). Using antibodies against Cre recombinase, we could not detect Cre recombinase protein in any of the fractions (Figure 2G). We could also not detect any Cre protein by ELISA with a sensitivity of 0.1 ng/ml in total EV preparations of bone marrow and peripheral blood of a single mouse (unpublished data). Thus, our EV preparations do not contain Cre protein or only at exceedingly low concentrations. Cre recombinase RNA could always be detected by RT-PCR in all exosomal fractions (Figure 2H). Nonexosomal fractions varied with regard to the presence of Cre mRNA, ranging from its complete absence (Figure S1A) to individual subfractions being positive for Cre-derived cDNA (n = 5 preparations) (Figure 2H). The latter observation indicated either trace amounts of exosomes in fractions negative for exosomal markers or Cre mRNA containing nonexosomal vesicles. Quantitative analysis of vesicle number and size revealed an increase in the share of larger membrane blebs or apoptotic vesicles in the in vitro preparations compared to blood plasma (Figure S1B). Our results demonstrate that Cre mRNA but not Cre recombinase protein is contained predominantly in exosomes and suggests that functional Cre recombinase protein was generated from Cre RNA contained in vesicles rather than recombination activity being a result of the direct transfer of Cre protein. 10.1371/journal.pbio.1001874.g002 Figure 2 Cre mRNA is present in the blood plasma of Vav-iCre mice and contained in EVs including exosomes. (A) Cre mRNA can be detected in vesicle preparations enriched for exosomes from the blood plasma of Vav-iCre mice by RT-PCR. Each lane represents a result from an individual animal. For detection of Cre mRNA, nested primer PCR was used. The PCR product at 100 bp represents the signal after the second round of amplification. (B) Cre mRNA is localized in the pellet but not in the supernatant after ultracentrifugation of conditioned medium from primary Vav-iCre–positive hematopoietic cells after stimulation by LPS in vitro. Cre mRNA was resistant to RNaseA treatment in all experiments. (C) After treatment with Triton-X to lyse EVs in combination with RNaseA digestion, Cre mRNA is no longer detectable in contrast to RNaseA treatment alone. (D) Vesicular structures between 50 and 100 nm in size were visualized in electron micrographs from Vav-iCre hematopoietic-cell-derived vesicle preparations (scale bar, 50 nm). (E and F) Secreted membrane vesicle subspecies can be separated by density by sucrose gradient ultracentrifugation. Exosomal identity was confirmed by blotting against the specific protein markers ADAM10 and CD9 for all subfractions. (G) Cre protein could not be detected in any of the fractions. Positive controls for all antibodies are shown in boxes to the right. (H) Cre mRNA is present in the exosomal fractions 2–7. The nonexosomal vesicles fractions or apoptotic bodies are characterized by their variability of positive subfractions to complete absence of Cre mRNA. In this experiment, subfractions 9 and 10 are positive, whereas 8, 11, and 12 do not contain any Cre RNA. Injection of Cre mRNA-Containing EV Preparations Is Sufficient to Induce Recombination in the Cerebellum We wanted to test whether secreted Cre recombinase RNA-containing EVs are sufficient to induce recombination in Purkinje neurons in vivo. To this end, EVs enriched for the exosomal fraction were prepared from peripheral blood and bone marrow cultures from Vav-iCre mice as described above. These preparations were brought into the circulation of ROSA26-lacZ reporter mice intravenously (Figure 3A). Four days after injection, animals were transcardially perfused and analyzed for recombination events by X-Gal staining and immunohistochemistry in serial sections of the cerebellum. We could not observe recombination events in any of the brains of the animals analyzed (n = 4, unpublished data). These results suggested either that the amount of exosomes in our preparations was too low, leading to the quantitative absorption of vesicles en route to the brain, or that EVs could not cross the blood–brain barrier (BBB). To circumvent these problems and to test whether induction of reporter gene expression in Purkinje neurons by Cre mRNA-containing EVs is formally possible, we directly injected 1 µl vesicle preparations into the cerebella of ROSA-LacZ reporter mice (n = 3, two injections in a single hemisphere of each mouse; Figure 3A). Serial sections of the complete cerebellar hemisphere were analyzed for reporter gene expression 4 d after injection. This analysis identified Purkinje neurons expressing the reporter gene in all three injected animals with one to seven recombined Purkinje neurons in a single hemisphere (Figure 3B). Interestingly, we also observed other reporter gene-positive cell types with glial (Figure 3C) or microglial (Figure 3D) morphology in addition to Purkinje neurons in all injected animals. Hence, Cre mRNA-containing EV preparations are sufficient to induce recombination events in the brain, and the transfer of functional Cre mRNA by EVs appears not to be necessarily restricted to Purkinje neurons. Control brains from ROSA-LacZ mice that were injected with purified recombinant Cre recombinase protein (1 µl of 1 U/µl) (n = 4) or lysate prepared from Vav-iCre bone marrow cells (BMCs) (n = 2) did not display reporter gene expression in Purkinje neurons or any other cell types (Figure 3E). 10.1371/journal.pbio.1001874.g003 Figure 3 EVs containing Cre mRNA are sufficient to induce recombination in Purkinje neurons after intracerebellar injection in vivo. (A) EV preparations enriched for exosomes prepared from the peripheral blood and bone marrow of Vav-iCre mice were brought into the circulation by tail vein injection or were directly injected into the cerebellum. Injection of Cre RNA-containing EVs into tail veins did not lead to recombination events in the brain (n = 4). (B) β-galactosidase–positive Purkinje neuron in the cerebellum of a reporter mouse 4 d after intracerebellar injection of EVs. (C) Other reporter-gene–positive cells with a shape and size reminiscent of glial cells in proximity to the Purkinje cell layer. (D) Reporter-gene–positive cells displaying a microglia-like morphology. (E) Quantification of reporter-gene–expressing Purkinje neurons after intracerebellar injection of vesicle preparations from Vav-iCre–positive peripheral blood. Control mice (shaded part) were injected with 1 µl purified Cre-recombinase protein at 1 U/µl (light grey) or lysate prepared from Vav-iCre bone marrow (dark grey) and never showed any recombined cells. Scale bar, 50 µm (B and C) and 25 µm (D). Recombination Events in Purkinje Neurons Are Not the Result of an Endogenous Misexpression of Cre Recombinase To formally rule out the possibility that the recombination events we observe were resulting from an unspecific expression of Cre, we performed additional control experiments. First, we established cerebellar slice cultures from Vav-iCre- or Tie2-Cre-ROSA-LacZ/GFP mice (n = 4). After 2 d in culture, we induced an injury by transversally cutting into the tissue with a blade, and 3 d later slices were analyzed. We could only detect occasional recombination events in the Purkinje cell layer and no recombination in the injured areas, indicating a lack of Cre activity in the absence of a hematopoietic system. Next, we generated blood chimeras by transplanting Vav-iCre-GFP bone marrow into lethally irradiated ROSA-LacZ reporter mice (n = 6). In this way, we could identify cells that received Cre recombinase from the donor population while controlling for the possibility of cell fusion through the constitutive expression of GFP in the donor cells. After 5 wk or 2 mo, respectively, we induced peritonitis and 4 d later the chimeric animals were killed (for an experimental scheme, see Figure 4A). We could observe LacZ-positive, GFP-negative cells in the livers of all animals (n = 6; Figure 4B). In the cerebellum, recombined cells became apparent only in the longer surviving animals (2 mo, n = 3). We could identify β-galactosidase–positive, GFP-negative cells in the Purkinje layer (3.3±1.2 SD in one hemisphere) but also in the granular layer (5±1.7 SD in one hemisphere) as well as in cells associated with blood vessels (Figure 4C–E). In the latter two locations, we never observed recombined cells in the Vav-iCre/Tie2-Cre-GFP/LacZ mice, indicating physiological changes caused by the irradiation. To test whether we could still observe recombination events in chimeras without the confounding influence of lethal irradiation, we performed adoptive transfer experiments whereby 20 million spleen/lymph node cells from Vav-iCre-GFP mice were i.v. injected into ROSA-LacZ reporter mice (n = 8). Ten days after the transfer, animals were killed and their livers and brains analyzed. Although we could detect recombined, GFP-negative cells in the livers of four mice, we did not observe any recombination in the brain, probably due to the low and short-lived cell number present in the recipients (Figure 4A). Control animals for both types of experiments included ROSA-LacZ reporter mice as recipients injected with wild-type donor cells (n = 4). In sum, these experiments present formal evidence of a lateral transfer of Cre mRNA originating from blood cells, excluding fusion or endogenous misexpression. 10.1371/journal.pbio.1001874.g004 Figure 4 Transfer of Cre mRNA in blood chimeras. (A) Schematic drawings of the experimental strategies to test for recombination events in blood chimeras. Lethally irradiated ROSA26-LacZ mice receive BM cells from Vav-iCre-ROSA-GFP mice. The bone marrow of recipient mice was tested for engraftment by flow cytometry analysis of GFP expression (representative analysis in right panel; wild-type bone marrow, red line; Vav-iCre-ROSA-GFP donor bone marrow, green line; bone marrow of ROSA26-LacZ recipient mouse after engraftment, blue line). For adoptive transfer experiments, the same combination of transgenes was used with spleen and lymph node cells as donor organs. Representative flow cytometry analysis of GFP expression of donor (green line) compared to wild-type cells (red line). At the time of analysis, GFP-positive cells were undetectable in recipient spleens (blue line). Two months after bone marrow transplantation, recombined cells can be detected in the liver (B), granular cell layer (C), and Purkinje cell layer (D), as well as associated with blood vessels (E). None of the recombined LacZ-/X-Gal–positive cells were positive for GFP, excluding cell fusion. Scale bar, 10 µm (B–E). Systemic and Central Nervous System (CNS) Inflammation Increases Recombination in Purkinje Neurons Hematopoietic cells of the myeloid and lymphoid lineage have been shown to contribute to nonhematopoietic tissues by heterotypic cell fusion [14]. These fusion events are very rare, but their number increases substantially after peripheral inflammation [5], leading to speculation that heterotypic fusion and subsequent reprogramming of the hematopoietic nucleus represent a sort of rescue mechanism for damaged tissues [15]. Likewise, inflammatory stimuli may affect EVs signaling [16],[17]. We induced a chronic inflammation by injecting Lewis lung carcinoma cells (LLC2) into the flanks of double transgenic mice, leading to the formation of a peripheral tumor after 12 d. For an acute inflammation, we induced peritonitis by intraperitoneal injection of thioglycolate broth and killed the animals 4 d after injection. In both models, we observed a significant increase in the number of recombined Purkinje neurons of up to three orders of magnitude (Figure 5A–C). To complement these models of a systemic inflammation with a pathology that directly affects neural tissue, we induced an ECL [18] in Vav-iCre-LacZ mice (n = 3), and 4 d postlesion, we observed a significant increase in the number of recombined Purkinje neurons (Figure 5C). Altogether, on the basis of a total population of approximately 150,000 Purkinje neurons per cerebellum, the relative quantity of recombined Purkinje neurons after inflammation thus averages 5.8% but goes as high as 26% in one individual case. Importantly, none of the recombined cells that we had analyzed in more detail were binucleated (Figure 5D,E), indicating that in our model systems injury is not sufficient to induce heterotypic cell fusion. The numbers of recombined Purkinje neurons for both Cre mouse lines were comparable to increases observed in heterologous transplantation models [5],[14]. Of note, when screening a large number of sections of postmortem human cerebellar tissue from patients (n = 12) that suffered from severe inflammatory injuries, we could very rarely detect Purkinje neurons containing two nuclei (Figure S2, Table S1), consistent with observations made in our transgenic mouse models. 10.1371/journal.pbio.1001874.g005 Figure 5 Peripheral inflammation increases the number of recombined Purkinje neurons. The number of recombined Purkinje neurons is low in healthy animals (A) but increases dramatically after peripheral inflammatory conditions (B). Inflammatory injuries were induced by subcutaneous injection of LLC2s or peritonitis. Animals were analyzed 12 d after injection when tumors were formed. Peritonitis was induced by a single i.p. injection of thioglycolate broth (1 ml in 3% PBS). Mice with peritonitis and ECL were analyzed 4 d after injection. (C) Filled bars represent results from Vav-iCre and empty bars from Tie2-Cre reporter mice. The p values were calculated by two-tailed t test for groups with unequal variance. (D and E) We did not observe any recombined Purkinje neurons that were binucleated in either transgenic mouse line after induction of an inflammation. (F and G) Microglia (white arrows) were always negative for the marker gene in healthy animals as well as after an inflammation. (H and I) Transendothelial electrical resistance (TEER, top panel) decreases and the corresponding capacitance (Ccl, bottom panel) of the bEnd5 endothelial monolayers increases significantly 24 h and 48 h after addition of bone-marrow-derived EVs compared to conditioned medium supernatant after ultracentrifugation. Vertical line at 0 h indicates media exchange. Scale bar, 100 µm (A and B), 50 µm (D and F), 10 µm (E), and 5 µm (G). Microglia are the main immune cells of the brain and are able to secrete EVs [19]. Therefore, we examined whether this cell type could be a possible source of Cre RNA-containing EVs in our model. We first analyzed brains from early postnatal (P4–P8) double transgenic Vav-iCre/Tie2-Cre-LacZ mice—the peak time of microglia invasion to the brain. In line with recent findings that the origin of microglia precedes definitive hematopoiesis [20], none of the microglia were positive for the reporter gene (unpublished data). Equally, we did not observe microglia expressing the reporter gene either in the healthy adult brain or after inflammatory injuries (Figure 5F,G). Thus, in our model, Cre-mediated recombination activity in neurons is not induced by EVs of microglial origin. EVs Targeting Neurons May Enter the Brain Via the Circulation To gain insight into whether blood-derived EVs reach Purkinje neurons directly via the circulation or by entry of leukocytes into the brain and subsequent local release of EVs, we screened serial cerebellar sections from mice with peritonitis for CD45-LacZ double-positive cells (n = 6). We did not detect a single double-positive cell in the brain parenchyma, arguing against a transfer of EVs from leukocytes to neurons over a short distance. Next, we wanted to test whether EVs themselves may influence BBB properties in order to facilitate their passage into the brain. To this end, we measured changes in the electric resistance and capacitance over a monolayer of bEnd5 brain endothelium cells, a system that has been previously described as an adequate approximation of BBB properties in vitro [21]. EVs from three separate preparations from the supernatant of BMCs were added to the transwells (three separate measurements in quadruplicate). The supernatants of these EV preparations after the ultracentrifugation step served as negative controls. Addition of EVs, but not the supernatant, led to a decrease in the resistance and a concomitant increase in the capacitance at 24 h and 48 h but not any more at 72 h (Figure 5H,I). This suggests that EVs from the bone marrow are sufficient to make the BBB more permeable without lasting toxic effects. Reporter Gene-Positive Neurons Are Present in Various Areas of the Brain Heterotypic cell fusion in the brain induced by peripheral inflammatory injury has only been reported in Purkinje neurons [5],[22],[23]. However, because inflammation contributes to various neural pathologies such as epilepsy [24], neurodegenerative diseases [25],[26], and sickness behavior [27], we tested whether other neuronal populations apart from Purkinje neurons could display Cre-mediated marker gene expression. Analyzing mice with an inflammation revealed different brain areas with recombined neurons. These were tyrosine hydroxylase (TH)-immunoreactive dopaminergic neurons in the substantia nigra/ventral tegmental area (SN/VTA) (Figure 6A,B). For this area, we could also observe recombined cells with a neuronal morphology that were negative for TH (Figure 6C), indicating loss of TH expression due to inflammation. Additionally we detected recombination in cortical neurons (Figure 6D–F) and in the granular cell layer of the hippocampus (Figure 6G–I). In the animals with an ECL, neurons in the hippocampal areas CA1 and CA3 were recombined as well as cells at the lesion site that were neither of a neuronal nor astrocytic lineage (Figure 6J–L). All neurons displaying marker gene expression contained only one nucleus, consistent with our observations in the cerebellum that cell fusion between hematopoietic cells and neurons is not the reason for the observed recombination events. 10.1371/journal.pbio.1001874.g006 Figure 6 Reporter-gene–positive neurons can be found in multiple areas of the brain. (A and B) β-galactosidase–positive cells in the SN/VTA that can be TH-positive dopaminergic neurons as well as TH-negative cells with neuronal morphology (C). (D–F) β-galactosidase–positive cells in the cortex are also positive for NeuN. (G–I) Overview of the dentate gyrus in the hippocampus with recombined neurons in the granular cell layer that are positive for the neuronal marker NeuN. (J and K) Recombined neurons after ECL in hippocampal areas CA2 and CA3 and nonneuronal GFAP-negative recombined cells at the lesion site (L). Scale bar, 100 µm (D, G, H, and J), 50 µm (A, E, K, and L), and 10 µm (B, C, F, and I). Next we wanted to test if an increase in recombination levels in Purkinje neurons is restricted to our injury model or constitutes a general response to any type of injury. Thus, we analyzed if a lesion that is specific for a selected group of neurons and that does not induce a systemic inflammation is capable of increasing recombination events in the Purkinje neuron population. To this end, we injected 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into Vav-iCre-LacZ mice (n = 3). MPTP is converted to the toxin MPP+ (1-methyl-4-phenylpyridinium) in glial cells that is then released and taken up specifically via the dopamine transporter of dopaminergic neurons, leading to cell death by inhibiting mitochondrial complex I. Animals were intraperitoneally injected with MPTP (30 mg/Kg body weight per day) on 5 consecutive days. Two weeks after the last injection, animals were killed and their brains analyzed. MPTP treatment leads to a massive reduction in the number of dopaminergic neurons in the SN, more than in the VTA (Figure S3 A,B). As a result, we did not observe an increase in the number of recombined Purkinje neurons in the cerebellum comparable to levels seen after peripheral inflammation (53.3±14.7 SD labeled Purkinje neurons per cerebellar hemisphere, n = 3). Therefore, an increase in recombined Purkinje neurons seems to be more injury-specific, particularly for a peripheral inflammation and not to limited mechanical damage, similar to findings in the context of cell fusion where mechanical trauma did not lead to an increase in heterokaryon formation [5]. Interestingly, we could detect numerous TH-negative LacZ-positive cells with neuronal morphology in the SN/VTA (Figure S3C–E). This could indicate an effect of a cell-type-specific lesion on a heightened signaling of blood-derived EVs, but we did not formally test this possibility. Purkinje Neurons Display a Different miRNA Profile After Recombination Finally, we wanted to test whether the EV transfer of Cre mRNA to Purkinje neurons is accompanied by other cellular changes that may indicate a more than transient physiological response. EVs and exosomes contain short noncoding RNAs including microRNAs (miRNAs), and it has been shown in vitro that functional miRNAs can be transferred by exosomes to target cells where they exert their effects on gene regulation [10],[28]. Furthermore, miRNAs are abundant in the nervous system, and there is compelling evidence for a role of miRNAs in all aspects of neuronal function from development to plasticity in the adult brain [29]. Hence we asked whether we could observe differences in the miRNA profile in recombined Purkinje neurons compared to their nonrecombined counterparts. To this end, we induced peritonitis in two Vav-iCre-LacZ mice. Four days later, these animals were killed and their cerebella snap-frozen and serially cut. After brief fixation, sections were stained for X-Gal and screened for marker-positive Purkinje neurons on a microscope fitted for laser capture microdissection (LCM). Cells that were X-Gal–positive and clearly identifiable as Purkinje neurons based on morphology and location were cut out and collected. To obtain a reference population that was as similar as possible, for each recombined cell a corresponding neighboring X-Gal–negative Purkinje neuron ( 1.2 and 35 were considered to be not expressed. (PDF) Click here for additional data file. Table S5 Comparison of microdissected Purkinje neuron miRNAs with published miRNA profiles. Comparison of miRNAs detected in microdissected Purkinje neurons with miRNAs in three libraries (PKN 1–3, >10 pm reads) obtained from healthy animals in He et al. [31]. The total number of identified miRNAs contained in each library is given in brackets. (DOCX) Click here for additional data file.

0 comments Cited 169 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Choroid plexus transcytosis and exosome shuttling deliver folate into brain parenchyma.

Jutta Gärtner, Nicolas Snaidero, Arne Wrede … (2012)

Loss of folate receptor-α function is associated with cerebral folate transport deficiency and childhood-onset neurodegeneration. To clarify the mechanism of cerebral folate transport at the blood-cerebrospinal fluid barrier, we investigate the transport of 5-methyltetrahydrofolate in polarized cells. Here we identify folate receptor-α-positive intralumenal vesicles within multivesicular bodies and demonstrate the directional cotransport of human folate receptor-α, and labelled folate from the basolateral to the apical membrane in rat choroid plexus cells. Both the apical medium of folate receptor-α-transfected rat choroid plexus cells and human cerebrospinal fluid contain folate receptor-α-positive exosomes. Loss of folate receptor-α-expressing cerebrospinal fluid exosomes correlates with severely reduced 5-methyltetrahydrofolate concentration, corroborating the importance of the folate receptor-α-mediated folate transport in the cerebrospinal fluid. Intraventricular injections of folate receptor-α-positive and -negative exosomes into mouse brains demonstrate folate receptor-α-dependent delivery of exosomes into the brain parenchyma. Our results unravel a new pathway of folate receptor-α-dependent exosome-mediated folate delivery into the brain parenchyma and opens new avenues for cerebral drug targeting.

0 comments Cited 105 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics.

Berthold Streubel, Claus Riedl, Paul Jennings … (2008)

Telomere-dependent replicative senescence is one of the mechanisms that limit the number of population doublings of normal human cells. By overexpression of telomerase, cells of various origins have been successfully immortalized without changing the phenotype. While a limited number of telomerase-immortalized cells of epithelial origin are available, none of renal origin has been reported so far. Here we have established simple and safe conditions that allow serial passaging of renal proximal tubule epithelial cells (RPTECs) until entry into telomere-dependent replicative senescence. As reported for other cells, senescence of RPTECs is characterized by arrest in G1 phase, shortened telomeres, staining for senescence-associated beta-galactosidase, and accumulation of gamma-H2AX foci. Furthermore, ectopic expression of the catalytic subunit of telomerase (TERT) was sufficient to immortalize these cells. Characterization of immortalized RPTEC/TERT1 cells shows characteristic morphological and functional properties like formation of tight junctions and domes, expression of aminopeptidase N, cAMP induction by parathyroid hormone, sodium-dependent phosphate uptake, and the megalin/cubilin transport system. No genomic instability within up to 90 population doublings has been observed. Therefore, these cells are proposed as a valuable model system not only for cell biology but also for toxicology, drug screening, biogerontology, as well as tissue engineering approaches.

0 comments Cited 92 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Contributors

Bethany A. O’Hara:

ORCID: http://orcid.org/0000-0002-6158-5159

Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing

Jenna Morris-Love: Role: ConceptualizationRole: InvestigationRole: MethodologyRole: Writing – review & editing

Gretchen V. Gee:

ORCID: http://orcid.org/0000-0003-0403-3240

Role: ConceptualizationRole: InvestigationRole: Writing – review & editing

Sheila A. Haley:

ORCID: http://orcid.org/0000-0003-2855-5668

Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SupervisionRole: ValidationRole: Writing – review & editing

Walter J. Atwood:

ORCID: http://orcid.org/0000-0002-3763-9073

Role: ConceptualizationRole: Funding acquisitionRole: SupervisionRole: Writing – review & editing

David Ornelles: Role: Editor

Journal

Journal ID (nlm-ta): PLoS Pathog

Journal ID (iso-abbrev): PLoS Pathog

Journal ID (publisher-id): plos

Journal ID (pmc): plospath

Title: PLoS Pathogens

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Print): 1553-7366

ISSN (Electronic): 1553-7374

Publication date (Electronic): 4 March 2020

Publication date Collection: March 2020

Volume: 16

Issue: 3

Electronic Location Identifier: e1008371

Affiliations

[1 ] Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, United States of America

[2 ] Graduate Program in Pathobiology, Brown University, Providence, Rhode Island, United States of America

[3 ] MassBiologics, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America

Wake Forest University, UNITED STATES

Author notes

The authors have declared that no competing interests exist.

* E-mail: sheila_haley@ 123456brown.edu (SAH); walter_atwood@ 123456brown.edu (WJA)

Author information

Bethany A. O’Hara http://orcid.org/0000-0002-6158-5159

Gretchen V. Gee http://orcid.org/0000-0003-0403-3240

Sheila A. Haley http://orcid.org/0000-0003-2855-5668

Walter J. Atwood http://orcid.org/0000-0002-3763-9073

Article

Publisher ID: PPATHOGENS-D-19-01946

DOI: 10.1371/journal.ppat.1008371

PMC ID: 7075641

PubMed ID: 32130281

SO-VID: 163fa2d1-047d-4a90-8e36-b6de00afac8e

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 24 October 2019

Date accepted : 31 January 2020

Page count

Figures: 8, Tables: 0, Pages: 20

Funding

Funded by: funder-id http://dx.doi.org/10.13039/100000065, National Institute of Neurological Disorders and Stroke;

Award ID: R01NS043097

Award Recipient :

ORCID: http://orcid.org/0000-0002-3763-9073

Walter J. Atwood

Funded by: funder-id http://dx.doi.org/10.13039/100000065, National Institute of Neurological Disorders and Stroke;

Award ID: P01NS065719

Award Recipient :

ORCID: http://orcid.org/0000-0002-3763-9073

Walter J. Atwood

These studies were supported by grants from the National Institute of Neurological Disease and Stroke, R01NS043097 and P01NS065719 to WJA. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Custom metadata

PLOS Publication Stage vor-update-to-uncorrected-proof

Publication Update 2020-03-16

Data Availability All relevant data are within the manuscript and its Supporting Information files.

JC Virus infected choroid plexus epithelial cells produce extracellular vesicles that infect glial cells independently of the virus attachment receptor

Read this article at

Abstract

Author summary

Related collections

Bacterial extracellular vesicles

Most cited references 24

Extracellular Vesicle-Mediated Transfer of Genetic Information between the Hematopoietic System and the Brain in Response to Inflammation

Choroid plexus transcytosis and exosome shuttling deliver folate into brain parenchyma.

hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics.

Author and article information

Contributors

Journal

Affiliations

Author notes

Author information

Article

History

Page count

Funding

Categories

Custom metadata

Comments

Comment on this article

Similar content 231

Cited by 33

Most referenced authors 619