Induction of Lysosomal Dilatation, Arrested Autophagy, and Cell Death by Chloroquine in Cultured ARPE-19 Cells

 

Therefore, a reduction of p62 indicates increased autophagic degradation, whereas an increase of p62 indicates inhibited autophagic degradation. 20 With CQ, the level of p62 significantly increased 6 to 12 hours after exposure (Fig. 2D), indicating that autophagic degradation was inhibited. After exposure to CQ, ARPE-19 cells developed cytosolic vacuoles within 1 hour and underwent the full details cell lysis within 24 hours. The levels of LC3-II, beclin-1 and, p62, as well as the number GFP-LC3 and RPF-LC3 positive autophagic vacuoles (AVs), increased after CQ treatment, indicating that autophagy was activated. However, lysosomal staining revealed that almost all AVs were separate from lysosomes; thus, fusion between AVs and lysosomes was completely blocked. Because the levels of some autophagic markers were increased, an autophagic mechanism of cell death might be plausible. However, our demonstration that 3-MA, an inhibitor of the PI3K involved in the early steps of autophagy, failed to attenuate cell death seem to exclude this possibility.

MARCH2 is upregulated in HIV-1 infection and inhibits HIV-1 production through envelope protein translocation or degradation.

In addition, the antioxidants (NAC and Trolox; Hoffman-LaRoche) failed to reduce cell death induced by CQ. This negative result lessens the possibility that oxidative stress plays a large role in CQ-induced RPE cell death. However, because we used only two kinds of antioxidants, we cannot completely rule out this possibility. Although our results do not precisely define the mechanisms of CQ-induced cell death, we speculate that marked increases in ubiquitinated proteins, which can be cytotoxic through as yet unidentified mechanisms, may play a role in the cytotoxicity of CQ. The final stage of autophagy is the fusion of autophagosomes with lysosomes 23. In addition, CQ-induced autophagy does not proceed to the level of the lysosomal stage because of this fusion failure.

Another mechanism by which CQ might induce cell death is caspase-mediated apoptosis. However, we failed to detect increases in levels of the active form of caspase3 in CQ-treated cells, and zVAD-FMK did not attenuate CQ-induced cell death. Hence, it seems unlikely that caspase-mediated apoptosis is the major mechanism underlying CQ-induced RPE cell death.

CQ increased the levels of the autophagic markers LC3-II and beclin-1 in ARPE-19 cells.

If acidity is a prerequisite for CQ accumulation in lysosomes, the inhibition of lysosomal acidification should block the toxic effects of

CQ.

In rapamycin-treated cells, in which autophagy was expected to be functional, LC3 and lysosomal signals significantly overlapped (Figs. 5A, 5B).

Fluorescent detection of lysosomes and autophagy

To examine this step, we stained RFP-LC3 transfected ARPE-19 cells with solution in dimethyl sulfoxide (LysoTracker Green DND-26; Invitrogen) and observed them with live-cell confocal microscopy. At the same time, we performed double immunostaining with anti LC3 and anti–LAMP-1 antibodies because the intensity of the dye (LysoTracker Green DND-26; Invitrogen) is changed by pH difference. As a positive control, ARPE-19 cells were treated with 0. 5 μM rapamycin for 6 hours, which induced normal autophagy, including fusion between autophagosomes and lysosomes. In rapamycin-treated cells, in which autophagy was expected to be functional, LC3 and lysosomal signals significantly overlapped (Figs. 5A, 5B). In contrast, ARPE-19 cells treated with 120 μM CQ for 2 and 6 hours exhibited almost complete separation between the two signals, indicating that autophagosome-lysosome fusion was severely inhibited (Figs. 5A, 5B). In addition, the levels of ubiquitinated proteins and GFP-mHttp aggregates in ARPE-19 cells were increased by CQ, providing further evidence that autophagic degradation was inhibited. CQ induced not only vacuolar changes but also cell death in the ARPE-19 cell line. Because RPE cells undergo degeneration and cell death in CQ-induced retinopathy, 22, 42 understanding the mechanism of cell death may be key to understanding the pathogenesis.

 

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