Restoration of impaired lysosomal function mitigates drusen-like deposit formation and cell death in Malattia Leventinese
Yumi Inoue, Hanako O. Ikeda, Masayuki Hata, Yuto Iida, Keiko Okamoto-Furuta, Isao Asaka, Makoto Arita, Akitaka Tsujikawa
Yumi Inoue, Hanako O. Ikeda, Masayuki Hata, Yuto Iida, Keiko Okamoto-Furuta, Isao Asaka, Makoto Arita, Akitaka Tsujikawa
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Research Article Cell biology Ophthalmology

Restoration of impaired lysosomal function mitigates drusen-like deposit formation and cell death in Malattia Leventinese

Abstract

Malattia Leventinese (MAL) is an inherited macular degeneration disorder characterized by retinal drusen formation in adolescence, leading to vision loss. A mutation in the fibulin-3 gene (EFEMP1) causes MAL; however, the mechanisms underlying disease onset and drusen formation remain unclear. In this study, we generated induced pluripotent stem cell–derived retinal pigment epithelial (iPSC-RPE) cells from a patient with MAL to investigate disease mechanisms and potential therapies. MAL iPSC-RPE exhibited fibulin-3 and apolipoprotein E (ApoE) aggregation, increased endoplasmic reticulum stress, and enhanced apoptosis. Long-term culture with photoreceptor outer segments led to drusen-like deposits containing ApoE, complement components, and collagen IV accumulation, and it showed activation of matrix metalloproteinase-2 (MMP2). Untargeted lipid analysis revealed increased hexosylceramide and bis-monoacylglycerophosphate levels in MAL iPSC-RPE cells. A key pathological feature was lysosomal dysfunction associated with altered regulation of lysosomal gene programs, including reduced transcription factor EB transcript levels. Treatment with trehalose, a lysosome-modulating compound, increased lysosomal content and function, reducing drusen-like deposit formation, inhibiting MMP2 activation, and suppressing apoptosis. This study highlighted lysosomal dysfunction as a contributor to RPE damage, drusen-like deposit accumulation, and extracellular matrix degradation. Pharmacological restoration of lysosomal function alleviated these defects, suggesting therapeutic potential for MAL and other drusen-related diseases, including age-related macular degeneration.

Authors

Yumi Inoue, Hanako O. Ikeda, Masayuki Hata, Yuto Iida, Keiko Okamoto-Furuta, Isao Asaka, Makoto Arita, Akitaka Tsujikawa

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Figure 8

Improvement of lysosomal function by trehalose in MAL iPSC-RPE cells.

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Improvement of lysosomal function by trehalose in MAL iPSC-RPE cells. iP...
iPSC-RPE cells were cultured for 4 weeks, followed by the addition of POS and treatment with or without 100 mM trehalose (tre) for an additional 4 weeks. (A and B) Immunostaining of MAL iPSC-RPE cells treated with trehalose showing z stack reconstructions stained for transcription factor EB (TFEB, red), phalloidin (green), and DAPI (blue). (B) Quantification of the nuclear-to-total TFEB fluorescence intensity ratio in NOR, NOR + trehalose, MAL, and MAL + trehalose iPSC-RPE cells. Sample sizes: NOR, n = 43 cells; NOR + trehalose, n = 41; MAL, n = 38; MAL + trehalose, n = 20. (C and D) Western blot analysis and statistical analyses of LAMP2, CTSD, Rab7, Rab9, and LC3 expression in NOR and MAL iPSC-RPE cells. (E and H) Immunostaining of NOR and MAL iPSC-RPE cross-sections for LAMP2 (red), phalloidin (green), and DAPI (blue, E), as well as separate cross-sectional images stained for mannose-6-phosphate receptor (M6PR) (red), phalloidin (green), and DAPI (blue, H). (F) The quantification of LAMP2 expression is presented as fluorescence intensity per cell. (G) En face image and z stack reconstructions of MAL iPSC-RPE cells treated with trehalose stained for LAMP2 (red), phalloidin (green), and DAPI (blue). (I and J) Western blot analysis of pEGFR in MAL iPSC-RPE cells treated with EGF and assessed at 0, 15, 30, and 60 minutes after EGF removal. *P < 0.05, ***P < 0.005, 1-way ANOVA followed by the Tukey-Kramer test (B), a linear mixed-effects model followed by Tukey’s HSD test (D, F, and J). In J, comparisons were made versus 0 min, n = 5 (D), n = 10 (F), n = 3 (J). Data are shown as mean ± SD. (-), not treated with trehalose; tre, treated with trehalose. Scale bar: 20 μm (A, E, G, and H).