Research ArticleCELL BIOLOGY

Excessive exosome release is the pathogenic pathway linking a lysosomal deficiency to generalized fibrosis

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Science Advances  17 Jul 2019:
Vol. 5, no. 7, eaav3270
DOI: 10.1126/sciadv.aav3270
  • Fig. 1 Neu1−/− fibroblasts have characteristics of myofibroblasts.

    (A) Coronal gastrocnemius muscle sections of WT and Neu1−/− mice were stained with Masson’s trichrome, demonstrating massive collagen deposition (blue) in the Neu1−/− muscle. Scale bars, 50 μm. (B and C) FACS analysis of muscle connective tissue fibroblasts (CD45/CD31/αSMA/TCF4+, n = 5) (B) and myofibroblasts (CD45/CD31/αSMA+/TCF4+, n = 3) (C) isolated from WT and Neu1−/− skeletal muscle at 3 to 4 months of age. (D and E) FACS analysis of MPs (CD45/CD31/Sca1/integrin-α7+, n = 3) (D), and FAPs (CD45/CD31/Sca1+/integrin-α7, n = 4) (E) isolated from WT and Neu1−/− skeletal muscle at 2 and 4.5 months of age. (F) Cell proliferation assayed in WT and Neu1−/− (myo)fibroblasts (n = 6). (G) Representative images of the invasion/migration assays performed with WT and Neu1−/− (myo)fibroblasts. Scale bar, 200 μm. (H) Quantification of (G) (n = 3). (I) Representative images of ex vivo invasion/migration of WT and Neu1−/− (myo)fibroblasts using ex vivo cultured peritoneal membranes. Scale bar, 100 μm. (J) Quantification of (I) (n = 4). Values are expressed as means ± SD. Statistical analysis was performed using the Student t test; *P < 0.05, ***P < 0.001, ****P < 0.0001.

  • Fig. 2 TGF-β1 signaling drives fibrosis in Neu1−/− muscle, which is rescued by AAV-mediated gene therapy.

    (A to C) Immunohistochemical (IHC) analysis of WT and Neu1−/− gastrocnemius (GA) muscle sections performed with anti–TGF-β1 (A), anti-SMAD2 (B), and anti-pSMAD2 (C) antibodies. Scale bars, 25 μm. (D) Immunoblot analysis of WT and Neu1−/− total GA muscle lysates probed with anti–TGF-β1, anti-SMAD2, and anti-pSMAD2 antibodies. (E and F) Transverse sections of the WT and Neu1−/− muscles were costained with anti-vimentin (green) and anti–collagen IV (red) (E) and anti-TCF4/TCF7 (red) and anti-αSMA (green) (F) antibodies. (G) Transverse sections of the WT and Neu1−/− muscles were costained with anti-TCF4 (green) and anti-TGF-β1 (red) antibodies. (H) Immunoblot analysis of WT and Neu1−/− (myo)fibroblast lysates probed with anti–TGF-β1, anti-SMAD2, and anti-pSMAD2 antibodies. (I) Immunoblots of WT and Neu1−/− (myo)fibroblasts were probed with and anti-vimentin, anti–collagen IV, anti-αSMA, and anti-TCF4 antibodies. (J) RT-qPCR of the indicated mRNAs in WT, Neu1−/−, and Neu1−/−-AAV muscles (n = 7). mRNA levels are normalized by 18S ribosomal RNA (rRNA) and relative to WT control. (K) Coronal gastrocnemius skeletal muscle sections of WT, Neu1−/−, and Neu1−/−-AAV (scAAV2/8-CMV-huNEU and scAAV2/8-CMV-huPPCA) treated mice stained with Masson’s trichrome, showing complete rescue of the muscle fibrosis. Scale bars, 200 μm. Values are expressed as means ± SD. Statistical analysis was performed using the Student t test; ****P < 0.0001.

  • Fig. 3 Neu1−/− muscle and myofibroblasts up-regulate canonical markers of EMT.

    (A to D) RT-qPCR of the indicated mRNAs in Neu1−/− muscle from 1-, 3.5-, and 5-month-old mice (n ≥ 3) (A to C) and in Neu1−/− myofibroblasts (n ≥ 3) (D). Levels of normalized mRNA expression (by 18S rRNA) were relative to WT control. Values are expressed as means ± SD. Statistical analysis was performed using the Student t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 4 Neu1−/− exosomes contain increased levels of components of the TGF-β and WNT signaling pathways.

    (A) Exosomes exocytosed by Neu1−/− myofibroblasts were verified on immunoblots probed with antibodies against established exosomal markers [Alix, CD81, CD9, flottilin-1 (Flot1), Syndecan-1 (SDC1), and syntenin 1 (SDCBP1)]. (B) Immunoblot analyses of WT and Neu1−/− exosomes performed with antibodies against canonical (β-catenin, GSK3β, LRP5, and WNT3a) and noncanonical (WNT5a/b) WNT signaling. (C and D) Immunoblots of WT and Neu1−/− exosomes probed with anti–TGF-β1 (C) and LAP (D) antibodies. (E) Exosomes treated with proteinase K were analyzed on immunoblots probed with anti–TGF-β1, anti-LAP, anti-WNT5a/b, and anti–β-catenin antibodies.

  • Fig. 5 NEU1-deficient exosomes convert fibroblasts into myofibroblast.

    (A and B) Cell proliferation was measured in murine (A) WT fibroblasts (WT Fs) cocultured with murine WT or Neu1−/−-derived exosomes (exo) (n = 3) and in human (B) skin fibroblasts (Hu-Fs) cocultured with exosomes isolated from cultured medium of patients with type I or II sialidosis (n = 3). (C) Invasive/migratory capacity of murine WT fibroblasts after coculturing them with WT or Neu1−/− exosomes (n = 8). (D to K) Increased mRNA expression of markers of the TGF-β and WNT signaling pathways was detected in WT murine fibroblasts (WT) cocultured with Neu1−/− exosomes but not with exosomes isolated from pCL20c-NEU1–transduced cells (Neu1−/−/NEU1) or from Neu1−/−/Lamp1−/− cells (dKO) (n ≥ 5). (L) Increased mRNA expression of markers of the TGF-β and WNT signaling pathways in human skin fibroblasts cocultured with human control (cHu), sialidosis type I, or sialidosis type II exosomes (n = 3). Normalized (by 18S rRNA or HPRT1) mRNA expression relative to phosphate-buffered saline (PBS) control. Values are expressed as average ± SD. Statistical analysis was performed using the Student t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 6 Down-regulation of NEU1 correlates with activation of profibrotic signals in IPF fibroblasts.

    (A) RT-qPCR of NEU1 mRNA in control (n = 13) and IPF (n = 14) patients’ fibroblasts. (B) Representative IF images of control (NL) and IPF fibroblasts labeled with anti-NEU1 antibody. Scale bar, 100 μm. (C) RT-qPCR of Postn, Ctnnb1, Tgfb1, and Smad2 mRNAs in mouse WT and Neu1−/− lung (myo)fibroblasts (n = 5). (D) RT-qPCR of POSTN, CTNNB1, TGFB1, and SMAD2 mRNAs in human control and IPF lung fibroblasts (n = 12). Levels of mRNA expression were normalized by 18S rRNA or GAPDH. Values are expressed as means ± SD. Statistical analysis was performed using the Student t test; *P < 0.05, **P < 0.01, ***P < 0.001. (E) Proposed model of fibrosis induced by NEU1 deficiency. (1) Schematic representation of Neu1−/− myofibroblasts that have exacerbated lysosomal exocytosis of soluble lysosomal enzymes and exosomes. (2) Magnification of a Neu1−/− exosome containing, among others, increased amounts of activated components of the TGF-β and WNT signaling pathways, responsible for propagating fibrotic signals to neighboring or distant cells. (3) Consequences of excessive lysosomal exocytosis of lysosomal hydrolases and exosomes by Neu1−/− myofibroblasts that result in massive ECM degradation/remodeling and full-blown fibrosis leading to myofiber degeneration.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaav3270/DC1

    Fig. S1. Neu1−/− muscles have increased levels of TGF-β and WNT signaling components.

    Fig. S2. Neu1−/− muscles show increased levels of periostin and osteopontin.

    Fig. S3. WB analyses of WT and Neu1−/− (myo)fibroblast lysates.

    Fig. S4. Neu1−/− muscle and myofibroblasts up-regulate canonical markers that drive an EMT process.

    Fig. S5. Characterization of WT and Neu1−/− exosomes.

    Fig. S6. Neu1−/− exosomes contain increased levels of components of the TGF-β and WNT signaling pathways.

    Fig. S7. NEU1-deficient exosomes induce a fibroblast to myofibroblast program in recipient normal fibroblasts.

    Fig. S8. General fibrosis in the Neu1−/− mouse model and decreased NEU1 protein levels in IPF fibroblasts.

    Table S1. Murine and human RT-qPCR primers.

    Table S2. Comparative high-throughput proteomic analyses of Neu1−/− and WT exosomes.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Neu1−/− muscles have increased levels of TGF-β and WNT signaling components.
    • Fig. S2. Neu1−/− muscles show increased levels of periostin and osteopontin.
    • Fig. S3. WB analyses of WT and Neu1−/− (myo)fibroblast lysates.
    • Fig. S4. Neu1−/− muscle and myofibroblasts up-regulate canonical markers that drive an EMT process.
    • Fig. S5. Characterization of WT and Neu1−/− exosomes.
    • Fig. S6. Neu1−/− exosomes contain increased levels of components of the TGF-β and WNT signaling pathways.
    • Fig. S7. NEU1-deficient exosomes induce a fibroblast to myofibroblast program in recipient normal fibroblasts.
    • Fig. S8. General fibrosis in the Neu1−/− mouse model and decreased NEU1 protein levels in IPF fibroblasts.
    • Table S1. Murine and human RT-qPCR primers.
    • Table S2. Comparative high-throughput proteomic analyses of Neu1−/− and WT exosomes.

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