Calpain-2 Facilitates Autophagic/Lysosomal Defects and Apoptosis in ARPE-19 Cells and Rats Induced by Exosomes from RPE Cells under NaIO3 Stimulation

Abstract

Although accumulated evidence supports the notion that calpain contributes to eye disease, the mechanisms by which calpain promotes RPE injury are not defined. The present study is aimed at investigating whether the effect of NaIO₃-exos (exosomes derived from RPE cells under NaIO₃ stimulation) on the dysfunction of the autophagy-lysosomal pathway (ALP) and apoptosis is based on its regulation of calpain activation in ARPE-19 cells and rats. The results showed that calpain-2 activation, ALP dysfunction, and apoptosis were induced by NaIO₃-exos in ARPE-19 cells. NaIO₃-exo significantly increased autophagic substrates by activating lysosomal dysfunction. ALP dysfunction and apoptosis in vitro could be eliminated by knocking down calpain-2 (si-C2) or the inhibitor calpain-2-IN-1. Further studies indicated that NaIO₃-exo enhanced calpain-2 expression, ALP dysfunction, apoptosis, and retinal damage in rats. In summary, these results demonstrate for the first time that calpain-2 is one of the key players in the NaIO₃-exo-mediated ALP dysfunction, apoptosis, and retinal damage and identify calpain-2 as a promising target for therapies aimed at age-related macular degeneration (AMD).

Figure 1

The characterization of exosomes secreted by ARPE-19 cells or NaIO₃-stimulated ARPE-19 cells. (a) Exosome morphology detected by TEM. (b) The expression levels of the specific exosomal biomarker TSG101 and CD63 proteins were examined by western blotting. (c) Diameter distribution of the particles detected by NTA (nm). (d) Flow cytometry results indicated that the exosomes in the control group were CD63 (99.0%) and TSG101 (99.6%) positive. (e) Flow cytometry results indicated that the exosomes were CD63 (99.0%) and TSG101 (94.7%) positive in the NaIO₃ group.

Figure 2

The effects of exosomes (NaIO₃) on calcium fluxes, calpain activation, autophagic flux defects, and apoptosis in ARPE-19 cells. (a, b) The cytosolic-free calcium was detected using a flow cytometer. (c–e) Western blot analysis was performed to detect the expression levels of calpain-1, calpain-2, and autophagy-related proteins in ARPE-19 cells. (f, g) The apoptosis rate of ARPE-19 cells was evaluated by flow cytometry. ^#P < 0.05, ^##P < 0.01 vs. Con (control group).

Figure 3

Exosome (NaIO₃) induced lysosome dysfunction in ARPE-19 cells. (a–d) The levels of LAMP1, CTSB, ATP6V0A1, and VAMP7 were detected by western blotting. (e, f) Confocal microscopy images of ARPE-19 cells labeled with anti-LAMP1 (green) and nuclear-stained labeled with DAPI (blue). ^#P < 0.05, ^##P < 0.01 vs. Con (control group).

Figure 4

The effects of CQ or 3-MA on the ALP dysfunction and calpain-2 in ARPE-19 cells. (a–c) Calpain-2 and ALP-related proteins were examined by western blotting. (d, e) Representative immunofluorescence images and quantitative analysis of autophagosomes (LC3-positive cells shown in red). ^#P < 0.05, ^##P < 0.01 vs. Con (control group). ^∗P < 0.05, ^∗∗P < 0.01 vs. NaIO₃-exo group. ^&P < 0.05 vs. CQ group.

Figure 5

Knockdown of the calpain-2 enhanced ALP function and attenuated apoptosis in ARPE-19 cells. (a–c) Western blotting showed the expression of proteins related to ALP and calpain-2. (d, e) Knockdown of calpain-2 reduced apoptotic cells (green). ^##P < 0.01 vs. Con (control). ^∗P < 0.05, ^∗∗P < 0.01 vs. si-NC group.

Figure 6

Inhibition of calpain-2 by calpain-2-IN-1 attenuated lysosomal membrane permeabilization, autophagosome formation, and apoptosis in ARPE-19 cells. (a–c) The expression of calpain-2 and ALP-related proteins was examined by western blotting. (d, e) The apoptotic cells were measured by TUNEL. ^#P < 0.05, ^##P < 0.01 vs. Con (control). ^∗∗P < 0.01 vs. NaIO₃-exo group.

Figure 7

Exosome (NaIO₃) induced calpain-2 activation, ALP dysfunction, and apoptosis in rat retina. (a) Exosome tracking analysis was used to assess the location of exosomes in the retina on day 0, day 7, and day 14 after injection. The white arrows are pointing toward the RPE cell layer. (b) HE staining and light micrographs were performed on the retinal cross-sections. The black arrows are pointing toward the RPE cells. (c, d) The TUNEL assay examined the retinal cell apoptosis in rats. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; POS: photoreceptor outer segments; RPE: retinal pigment epithelium; CH: choroid. (e–g) Calpain-2 and ALP-related proteins were examined by western blotting. ^#P < 0.05, ^##P < 0.01 vs. Con (control group).

Figure 8

Schematic description of NaIO₃-exo induced autophagic/lysosomal defects and apoptosis by activating calpain-2 in ARPE-19 cells and rats.