Abstract
Exosomes are secreted membrane vesicles of endosomal origin present in biological fluids. Exosomes may serve as shuttles for amyloidogenic proteins, notably infectious prions, and may participate in their spreading in vivo. To explore the significance of the exosome pathway on prion infectivity and release, we investigated the role of the endosomal sorting complex required for transport (ESCRT) machinery and the need for ceramide, both involved in exosome biogenesis. Silencing of HRS-ESCRT-0 subunit drastically impairs the formation of cellular infectious prion due to an altered trafficking of cholesterol. Depletion of Tsg101-ESCRT-I subunit or impairment of the production of ceramide significantly strongly decreases infectious prion release. Together, our data reveal that ESCRT-dependent and -independent pathways can concomitantly regulate the exosomal secretion of infectious prion, showing that both pathways operate for the exosomal trafficking of a particular cargo. These data open up a new avenue to regulate prion release and propagation.
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Acknowledgments
This work was supported by CNRS, INSERM, INRA and the ANR program (ExoPrion AO2008). KL obtained a one-year FINOVI fellowship. We thank Jennifer T. Miller (NCI-Frederick) for carefully reading the manuscript. We acknowledge the PLATIM microscope platform at ENS-Lyon (SFR BioSciences Gerland–Lyon Sud UMS3444/US8, France).
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18_2015_1945_MOESM1_ESM.eps
Supplementary material Figure S1: HRS depletion inhibits PrP res formation. (a) Mov 127S cells were transduced with lentivectors encoding ShRNAs Sh-CT (lane 1) or three different Sh-HRS (1, 2 and 3; lanes 2-4). Cell lysates (20 μg) were immunoblotted using anti-HRS (upper, left panel) or anti-PrP antibodies (bottom, left panel). Cell lysates (300 μg) were PK digested and immunoblotted using anti-PrP antibodies (right panel). Note the strong decrease of PrPres in Sh-HRS1, 2 and 3 (lanes 2-4) compared to Sh-CT (lane 1). Non-PK digested Sh-CT lysate was used as a negative control of PK digestion (lane 5). (b) Mov 127S cells transduced with lentivectors encoding Sh-CT or Sh-HRS1 were fixed and analyzed by confocal microscopy using anti-PrP (red) and anti-Caveolin1 (green) antibodies. Images are single sections through the middle of the cells. Abnormal PrP was detected after incubating the cells 5 min with 3 M guanidine thiocyanate. Note the strong decrease of abnormal PrP signal in Sh-HRS1 cells. Nuclei were stained with DAPI. Scale bars represent 10 μm. (c) Infected scN2a#22L cells were transduced with ShRNAs Sh-CT (non-specific target negative control; lanes 5 and 7) or Sh-HRS1 (lanes 6 and 8). After puromycin selection, cells were harvested and HRS depletion was assessed by immunoblotting (upper panel). Note the strong decrease of HRS signal (lane 6) compared to CT ShRNA (lane 5). Loading control was assessed using coomassie staining (bottom panel). Cell lysates from Sh-CT (lane 7) and Sh-HRS KD (lane 8) cells were PK digested and PrPres was detected by Western blotting. Note the strong decrease of PrPres signal in HRS-depleted cells (lane 8) compared to control cells (lane 7). The normal N2a#58 (lanes 1 and 3) and infected N2a#22L cells (lanes 2 and 4) were used as negative and positive controls for PK digestion and PrPres detection. (EPS 13072 kb)
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Supplementary material Figure S2: HRS depletion does affect exosomal release. (a) Mov 127S cells were transduced with lentivectors encoding ShRNAs Sh-CT (lane 1) or Sh-HRS1 (lane 2). Cell lysates were immunoblotted using anti-HRS (upper panel) or anti-GAPDH antibodies (bottom panel) for loading control. (b) Cell lysates (lanes 1&2) and the 100 K pellets (lanes 3&4) from supernatants of Mov 127S cells transduced with ShRNA-CT (lanes 1 and 3) and ShRNA-HRS1 (lanes 2 and 4) lentivectors were analyzed by Western blotting using antibodies against Tsg101 or Flotillin-1 as exosomal proteins and against Calnexin as negative exosomal control or anti-GAPDH for cell lysate loading control. Note that Tsg101 and Flotillin-1 signals were similar in 100 K pellets from ShRNA-HRS1 and shRNA-CT. The data are representative of two experiments carried out with independent transduced cells. (EPS 2230 kb)
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Supplementary material Figure S3: HRS depletion causes accumulation of cholesterol in infected Mov cells. Mov 127S cells transduced with lentivectors encoding Sh-CT or Sh-HRS1 were stained with Filipin (green) to visualize the distribution of free cholesterol. Note the punctuate signals of free cholesterol in Sh-HRS1 cells (see white arrows) compared to the diffuse signal observed in Sh-CT cells. Representative images are shown. Scale bar represents 10 μm. (EPS 9726 kb)
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Supplementary material Figure S4: Decrease of PrP res in HRS-depleted cells is not inhibited by NH4Cl treatment. (a) Mov 127S cells transduced with Sh-CT (lanes 1 and 3) or Sh-HRS1 (lanes 2 and 4) were treated for 16 h with ammonium chloride (NH4Cl) (20 mM) (lanes 3 and 4) or with PBS (lanes 1 and 2). Cell lysates (20 μg) were analyzed by immunoblotting with anti-PrP, anti-p62 and anti-LC3 antibodies, as indicated. Cyclophilin A (Cyp A) was used as a loading control. Note the accumulation of PrP, p62 and LC3-II as evidence of successful inhibition of the degradation processes in NH4Cl-treated cells. (b) Mov 127S cells transduced with Sh-CT (lanes 1, 2, 5, 6) or Sh-HRS1 (lanes 3, 4, 7, 8) were treated for 16 h with ammonium chloride (NH4Cl) (20 mM) (lanes 5 to 8) or not (lanes 1 to 4) as in a). Cell lysates were analyzed by immunoblotting for PrP before (-) or after (+) PK digestion. Note that PrPres did not raise in NH4Cl-treated Mov 127S cells. Undigested lysates were also analyzed for p62 and for GAPDH (as a loading control). (EPS 3144 kb)
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Supplementary material Figure S5: Total PrP distribution in Mov 127S cells is not affected by HRS depletion. (a) DRMs isolation from Mov 127S cells transduced with lentivectors encoding Sh-CT and Sh-HRS1. Sh-CT and Sh-HRS1 cells were lysed in buffer containing 1 % triton X100 at 4 °C. Equivalent amount of cell lysates were fractionated by flotation on a 5-30-40 % sucrose step gradient. Twelve fractions were collected from the top of the gradient and were analyzed by Western blotting using anti-PrP and anti-Flotillin-1 (as a DRM-associated protein) antibodies. DRMs are in fractions 3-4 while fractions 9-12 correspond to soluble proteins. (b) Mov 127S cells transduced with lentivectors encoding Sh-CT or Sh-HRS1 were fixed and analyzed by confocal microscopy using anti-PrP (red) and anti-Caveolin1 (green) antibodies. Images are single sections through the middle of the cells. Nuclei were stained with DAPI. Scale bars represent 10 μm. (EPS 12567 kb)
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Supplementary material Figure S6: Release of the exosomal markers Alix and Flotillin-1 were marginally decreased in GW4869 treated cells. Quantifications of band signals for Alix (left panel) and Flotillin-1 (right panel) exosomal markers from 4 independent experiments. Values are given as mean ± SD. *P value < 0.05. Statistics and calculation of the P value were done by the GraphPad PRISM software with the Mann–Whitney test. (EPS 373 kb)
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Supplementary material Figure S7: The neutral Sphingomyelinase inhibitor GW4869 strongly reduces prion infectivity release in ovRK13 127S (Rov 127S) cellular model. Rov 127S cells were incubated with the diluent (DMSO, negative control) or with the neutral Sphingomyelinase inhibitor GW4869 (10 μM) for 26 h. Cells were collected, homogenized in PBS and 100 K pellets were harvested from the corresponding culture supernatants. a) Analysis of prion infectivity in DMSO- and GW4869-treated Rov 127S cells and in their corresponding 100 K pellets. For SCA experiments, recipient ovRK13 target cells were inoculated with 1/1, 1/3 and 1/9 inoculum dilutions (corresponding to 30, 10 and 3.3 μg of cellular proteins, respectively), of DMSO- and GW4869-treated Rov cells (lanes 1-7) and with the corresponding 100 K pellets (equivalent to 450 μl of conditioned medium) (lanes 8 to 10). No PrPres was detected when recipient ovRK13 cells that did not express the PrPC protein (-dox) were inoculated with cell homogenate and 100 K pellet (lanes 1 and 9). Note that GW4869 treatment did not affect cell infectivity but strongly inhibited the infectivity in the 100 K pellet. (b) Biochemical analysis of 100 K pellets from DMSO- (lane 1) and GW4869-treated (lane 2) Rov127S cells. 100 K pellets corresponding to 20 ml of culture medium were analyzed by immunoblotting for Alix, TSG101 and Flotillin-1 exosomal proteins. Note that Alix, Tsg101 and Flotillin-1 signals are not or marginally affected by the GW4869 treatment as observed in the Mov 127S cellular model. (EPS 1937 kb)
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Vilette, D., Laulagnier, K., Huor, A. et al. Efficient inhibition of infectious prions multiplication and release by targeting the exosomal pathway. Cell. Mol. Life Sci. 72, 4409–4427 (2015). https://doi.org/10.1007/s00018-015-1945-8
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DOI: https://doi.org/10.1007/s00018-015-1945-8