Serum exosomes can restore cellular function in vitro and be used for diagnosis in dysferlinopathy

Abstract

Purpose: It is challenging to deliver the full-length dysferlin gene or protein to restore cellular functions of dysferlin-deficient (DYSF^-/-) myofibres in dysferlinopathy, a disease caused by the absence of dysferlin, which is currently without effective treatment. Exosomes, efficient membranous nanoscale carriers of biological cargoes, could be useful. Experimental design: Myotube- and human serum-derived exosomes were investigated for their capabilities of restoring dysferlin protein and cellular functions in murine and human DYSF^-/- cells. Moreover, dysferlinopathic patient serum- and urine-derived exosomes were assessed for their abilities as diagnostic tools for dysferlinopathy. Results: Here we show that exosomes from dysferlin-expressing myotubes carry abundant dysferlin and enable transfer of full-length dysferlin protein to DYSF^-/- myotubes. Exogenous dysferlin correctly localizes on DYSF^-/- myotube membranes, enabling membrane resealing in response to injury. Human serum exosomes also carry dysferlin protein and improve membrane repair capabilities of human DYSF^-/- myotubes irrespective of mutations. Lack of dysferlin in dysferlinopathic patient serum and urine exosomes enables differentiation between healthy controls and dysferlinopathic patients. Conclusions: Our findings provide evidence that exosomes are efficient carriers of dysferlin and can be employed for the treatment and non-invasive diagnosis of dysferlinopathy.

Keywords: Dysferlinopathy; diagnostics; exosome; therapeutics.

Figure 1

Characterization of exosomes derived from murine myotubes (EXOdysf). (A) Western blot to examine the level of dysferlin expression in EXOdysf. 20 μgor 50 μg total protein from cell lysates or exosomes was loaded and Cytochrome C was used as an organelle marker. (B) Representative transmission electron microscopy (TEM) image of EXOdysf (scale bar = 200 nm (left) or 100 nm (right)).Arrowheads point to the typical sauce-cup shape of exosomes. (C) Analysis of the size distribution of EXOdysf with a nanoparticle tracking analysis (NTA) system. (D) Western blot to evaluate the presence and localization of dysferlin protein in EXOdysf. 25 μg total protein from exosomes derived from myotubes was loaded and Alix was used as an exosomal marker. Tryp, sap, or tryp+sap refer to trypsin-, saponin- or both trypsin- and saponin-treated EXOdysf.

Figure 2

Examination of the restoration and localization of dysferlin protein in EXOdysf-treated murine dysferlin-deficient (DYSF^-/-) myotubes. The restoration of dysferlin protein was examined 48 h after the addition of 100 μg/mL EXOdysf in DYSF^-/- myotubes. (A) Western blot to measure the level of dysferlin protein in EXOdysf-treated DYSF^-/- myotubes. GAPDH was used as a loading control and 20 μgor 200 μg total protein from cell lysates or exosomes was loaded, respectively. (B) Confocal fluorescence microscopy images to reflect the localization of restored dysferlin protein in EXOdysf-treated DYSF^-/- myotubes. Nuclei were counterstained with DAPI (blue) (scale bar = 30μm; for boxed areas, scale bar= 15 μm). Arrowheads refer to the co-localization of caveolin-3 (red) and exosome (green).

Figure 3

Functional restoration of EXOdysf-treated murine DYSF^-/- myotubes. (A) Two-photon confocal microscopy images showing the membrane repair capacity of EXOdysf in DYSF^-/- myotubes. Arrowheads point to the laser injury sites. The FM4-64 dye (red) was used to show the integrity of the membrane (scale bar = 10 μm). WT: wild-type; BF: bright field. (B) Quantitative analysis of fluorescence intensity at different time-points in WT (circles), DYSF^-/- (squares) or EXOdysf-treated DYSF^-/- (triangles) myotubes. Significant reductions in fluorescence intensity were achieved in EXOdysf-treated DYSF^-/- myotubes compared to untreated myotubes (n=15, two-tailed test, *P<0.05). (C) Immunostaining of dysferlin and lysosome-associated membrane protein-1 (LAMP-1) in EXOdysf-treated DYSF^-/- myotubes. Nuclei were counterstained with DAPI (blue). Arrowheads point to the scrape wounding sites (scale bar =100 μm). (D) Two-photon confocal microscopy images showing the Ca²⁺ influx in EXOdysf-treated DYSF^-/- myotubes. Arrowheads point to the laser injury sites (scale bar = 10 μm). (E)Quantitative analysis of fluorescence intensity at different time-points in WT (circles), DYSF^-/- (squares) or EXOdysf-treated DYSF^-/- (triangles) myotubes. Significant reductions in fluorescence intensity were achieved in EXOdysf-treated DYSF^-/- myotubes compared to untreated myotubes at earlier time-points (n=15, two-tailed test, *P<0.05).

Figure 4

Restoration of dysferlin protein and membrane repair capacities in human DYSF^-/- myotubes treated with human serum exosomes (EXOser). (A) Western blot to examine the level of dysferlin protein in human serum exosomes. 20 μgor 50 μg total protein from cell lysates or exosomes was loaded and Cytochrome C was used as an organelle marker. EXOdysf refers to exosomes derived from normal human myotubes. (B) Western blot to examine the level of dysferlin restoration in human DYSF^-/- myotubes at 48 h after the addition of 100 μg/mL human EXOser. GAPDH was used as a loading control and 100 μg or 200 μg total protein from cell lysates or exosomes was loaded, respectively. (C) Two-photon confocal microscopy image showing the membrane repair capacity of EXOser-treated DYSF^-/- myotubes. Arrowheads point to the laser injury sites (scale bar =20 μm). (D) Quantitative analysis of fluorescence intensity at different time-points in WT (circles), DYSF Exon22^-/- or Exon55^-/- (squares) or EXOser-treated DYSF^-/- (triangles) myotubes. Significant reductions in fluorescence intensity were achieved in EXOser-treated DYSF Exon22^-/- or Exon55^-/- myotubes compared to untreated myotubes (n=15, two-tailed test, *P<0.05).

Figure 5

Examination of dysferlin proteinin exosomes from normal controls' and dysferlinopathic patients' serum and urine. (A) Western blot to examine levels of dysferlin protein in exosomes from normal controls' and dysferlinopathic patients' serum. 30 μg total protein from exosomes was loaded. Pn stands for the patient No. EXOcon refers to exosomes from normal controls' serum. (B) Representative gene sequencing results for dysferlinopathic patients (patient no. 20 and her parents). (C) Western blot to analyze the expression of exosomes derived from a spectrum of dysferlinopathic patients' serum. P20-F and P20-M mean patient no.20's father and mother. (D) Western blot to examine levels of dysferlin protein in exosomes from normal controls' and dysferlinopathic patients' urine. 50 μg total protein from exosomes was loaded. EXOcon refers to exosomes derived from normal human urine. (E) Western blot to analyze the expression of dysferlin in urine exosomes derived from a spectrum of dysferlinopathic patients.