Chloroquine treatment of ARPE-19 cells leads to lysosome dilation and intracellular lipid accumulation: possible implications of lysosomal dysfunction in macular degeneration - PubMed (original) (raw)

Chloroquine treatment of ARPE-19 cells leads to lysosome dilation and intracellular lipid accumulation: possible implications of lysosomal dysfunction in macular degeneration

Patrick M Chen et al. Cell Biosci. 2011.

Abstract

Background: Age-related macular degeneration (AMD) is the leading cause of vision loss in elderly people over 60. The pathogenesis is still unclear. It has been suggested that lysosomal stress may lead to drusen formation, a biomarker of AMD. In this study, ARPE-19 cells were treated with chloroquine to inhibit lysosomal function.

Results: Chloroquine-treated ARPE-19 cells demonstrate a marked increase in vacuolation and dense intracellular debris. These are identified as chloroquine-dilated lysosomes and lipid bodies with LAMP-2 and LipidTOX co-localization, respectively. Dilation is an indicator of lysosomal dysfunction. Chloroquine disrupts uptake of exogenously applied rhodamine-labeled dextran by these cells. This suggests a disruption in the phagocytic pathway. The increase in LAMP protein levels, as assessed by Western blots, suggests the possible involvement in autophagy. Oxidative stress with H2O2 does not induce vacuolation or lipid accumulation.

Conclusion: These findings suggest a possible role for lysosomes in AMD. Chloroquine treatment of RPE cells may provide insights into the cellular mechanisms underlying AMD.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Cell Viability Assay. MTT assay shows chloroquine toxicity is both time and dose dependent. Chloroquine concentrations of 10-30 μg/ml (p < 0.05) do not significantly affect cell viability.

Figure 2

Figure 2

Cytoplasmic Changes with Chloroquine in ARPE-19. Phase contrast microscopy of ARPE-19 with 0 (A), 10 (B), 20 (C), 40 (D), 80 (E) μg/ml chloroquine. Chloroquine treatment causes membrane-enclosed vacuoles (red arrows) and membranous dense bodies (blue arrows). ARPE treated with high chloroquine dosage show signs of cell death. Bar size = 25 microns.

Figure 3

Figure 3

Co-localization of LAMP-2 and LipidTOX. (A-D) Control ARPE-19. (E-H) 20 μg/ml chloroquine-treated ARPE-19. Control phase contrast (A) shows no vacuolation and LAMP-2 (B, green), and LipidTOX(C, red) staining is observed throughout cell. D is fluorescent overlay. Chloroquine-treated cell in phase contrast (E) shows intense vacuolation (white arrows). These vacuoles co-localize with LAMP-2 staining (F, corresponding white arrows). Chloroquine treatment also induces dense body formation (E, yellow arrows). Dense formations co-localize with neutral lipid (G, corresponding yellow arrows). Overlay (H) shows no overlap between lipids and vacuoles. Bar size = 25 microns.

Figure 4

Figure 4

Co-localization of LAMP-1 in NIH/3T3. NIH/3T3 cells show a similar pattern of vacuole formation with treatment of chloroquine. Phase contrast and corresponding LAMP-1 co-localization of 25 μg/ml chloroquine-treated cells (C, D) show vacuolation of LAMP-1, when compared with control (A, B). The arrows in C and D note that the borders of the vacuoles correspond with LAMP-1 protein. Bar size = 25 microns.

Figure 5

Figure 5

Chloroquine Effect on ARPE-19 Golgi and Mitochondria. (A-D) Mitochondria co-localization in (A-B) control and (C-D) 20 μg/ml-treated ARPE-19. There is no difference in MitoTracker staining (red) intensity, shape or localization between control (B) and treatment (D). Similarly, there were no noticeable changes in Golgi apparatus (E-H). Golgi co-localization of control (E-F) and 20 μg/ml chloroquine treatments. Golgi is stained with Golgin-97 antibody staining (green). Bar size = 25 microns.

Figure 6

Figure 6

Phagocytic Activity and LAMP Protein. (A) Comparisons of LAMP-1 and LAMP-2 protein levels at different concentrations of chloroquine. Protein levels measured in ARPE-19 by Western blot with beta-actin as loading control. Western shows marked qualitative increase in LAMP 1-2 band size between control and 10 μg/ml chloroquine treatment, while loaded control is constant. (B) There is a significant increase in LAMP-1 and 2 (p < 0.05) at 10 μg/ml with overall upregulation trend response to chloroquine treatment. Immunoblots (n = 6) were scanned, and densitometry was performed on bands with ImageJ (NIH, Bethesda MD). Relative band density measurement was repeated several times to ensure maximum and minimum values.

Figure 7

Figure 7

Phagocytic Activity Measured by Dextran Uptake. (A-B) Immunofluorescence of exogenous rhodamine-labeled dextran (red) uptake with ImageJ markings (crosshairs) of counted dextran maximas. 20 μg/ml chloroquine-treated cells at 24 hours (B) showed striking decrease in fluorescent dextran uptake when compared to control (A). (C) Quantization of dextran uptake between control and chloroquine-treated ARPE-19. There is a significant (p < 0.05) decrease in relative dextran uptake in individual sampled ARPE-19 cells between control and chloroquine-treated cells. Quantitation was performed by using ImageJ, isolating cells of relative same size (~20-25 microns), and then calculating florescent maximas (tolerance = 20, based on control). Maximas were averaged and standard t-test was performed (n = 10).

Figure 8

Figure 8

Oxidative Stress Test. (A) phase contrast, (B) LAMP-2 and (C) LipidTOX staining of 10 mM hydrogen peroxide-treated ARPE-19. (D) Overlay showing co-localization of neutral lipid and LAMP-2. There are no differences in cell cytoplasm (vacuoles, lipids) in phase or fluorescence between control (Fig 2 A-D) and hydrogen peroxide treatment.

Figure 9

Figure 9

Possible LAMP-2/Lysosomal Inhibition Model of Pathogenesis of AMD. An unknown cause results in loss of the protective glycocalyx of LAMP-2. Proteolysis of LAMP-2 occurs. Loss of LAMP-2 results in either 1) A pH shift and loss of acidity of lysosome, 2) dynein no longer moving late phagosome to microtubule sorting center near Golgi for fusion with lysosome or 3) perturbation of M6PR/Rab recycling such that M6PR/Rab7 does not tag late endosomes. The loss of functionality of M6PR/Rab7 results in a lack of phagosome lysosome-fusion. Any of these results in loss of the lysosome's ability to degrade intra- and extracellular material. Subsequently, undegraded material is oxidized, turning to lipofuscin. Ultimately, the combination of oxidative stress, Fe+ accumulation, senile mitochondria and decrease in ATP results in inefficient turnover of organelles and increased inhibition of lysosomes. RPE death triggers photoreceptor and macula death.

Similar articles

Cited by

References

    1. Buch H. Fourteen-year incidence of age-related maculopathy and cause-specific prevalence of visual impairment and blindness in a Caucasian population: The Copenhagen city eye study. Acta Opthalmol. 2005;83:400–401. doi: 10.1111/j.1600-0420.2005.00474.x. - DOI - PubMed
    1. la Cour M, Kiilgaard JF, Nissen M. Age-related macular degeneration: epidemiology and optimal treatment. Drugs Aging. 2002;19:101–133. doi: 10.2165/00002512-200219020-00003. - DOI - PubMed
    1. Klein R. Overview of progress in the epidemiology of age-related macular degeneration. Opthalmic Epidemiol. 2007;14:184–187. doi: 10.1080/09286580701344381. - DOI - PubMed
    1. Jong P. Mechanisms of disease: age-related macular degeneration. N Engl J Med. 2006;355:1474–1485. doi: 10.1056/NEJMra062326. - DOI - PubMed
    1. Young RW, Bok D. Participation of retinal pigment epithelium in rod outer segment renewal process. J Cell Biol. 1969;42:392–403. doi: 10.1083/jcb.42.2.392. - DOI - PMC - PubMed

LinkOut - more resources