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 2

Apoptosis and extracellular matrix degradation in MAL iPSC-RPE cells.

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Apoptosis and extracellular matrix degradation in MAL iPSC-RPE cells. iP...
iPSC-RPE cells were cultured for 4 weeks, followed by an additional 4 weeks of incubation in the presence or absence of photoreceptor outer segments (POS). (A) Immunostaining of cross-sections of NOR and MAL iPSC-RPE, showing heat shock protein family A (Hsp70) member 5 (HSPA5, red), phalloidin (green), and DAPI (blue). (B and C) Western blot analysis of total and cleaved caspase-3 (t-Casp3 and c-Casp3) in NOR and MAL iPSC-RPE cells treated with POS. (D and E) TdT-mediated dUTP nick-end labeling (TUNEL) staining (green) of cross-sections of NOR and MAL iPSC-RPE cells. (F and G) The expression levels of growth arrest specific 6 (GAS6), tyrosine-protein kinase receptor (AXL), matrix metalloproteinase-2 (MMP2), and metalloproteinase inhibitors 1 and 3 (TIMP1 and TIMP3) in NOR and MAL iPSC-RPE cells were analyzed by RNA-seq. The y axis represents expression levels in log2 transcripts per million (TPM). Corresponding analyses, including both POS (–) and POS (+) conditions, are shown in Supplemental Figure 2. (H and I) Western blot analysis of MMP2, TIMP1, and TIMP3 expression in NOR and MAL iPSC-RPE cells. (J) Measurement of MMP2 activity in medium supernatants of NOR and MAL iPSC-RPE using gelatin zymography. Scale bar: 20 μm (A and D). *P < 0.05, ***P < 0.005. Data in C, E, I, and J were analyzed using a linear mixed-effects model followed by Tukey’s HSD test. Data in F and G were analyzed using Student’s t test; n = 3 (C, F, G, and J), n = 5 (I), n = 10 (E). Data are shown as mean ± SD.