Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly population, is of two types based on the presence of choroidal neovascularization, dry and wet AMD (Apte, 2021; Jager et al., 2008). Several factors, including oxidative stress, genetic, environmental, and inflammatory stimulation, affect the development of AMD (Shaw et al., 2016). Dry, non-neovascular AMD is characterized by the progressive degeneration of the retinal pigment epithelium (RPE) (Jager et al., 2008). Pathogenesis of dry AMD is associated with the formation of small acellular amorphous deposits called drusen under the RPE in the macula. Enlarged and confluent drusen increase the risk of AMD progression, and disrupt the interaction between Bruch’s membrane and RPE, thereby inhibiting RPE function. Anti-vascular endothelial growth factor agents are the mainstay treatment options for wet AMD that substantially improve the visual symptoms in affected patients. However, only a few therapeutic approaches are currently available for dry AMD.

Amyloid-β (Aβ) is a substructural constituent of drusen deposits that is closely implicated in the development of AMD (Isas et al., 2010; Mullins et al., 2000; Wang et al., 2008). Proteolytic cleavage of amyloid precursor protein by β- and γ-secretases instead of α-secretase drives the production and accumulation of abnormal pathogenic Aβ (Cheng et al., 2013; Zeng et al., 2019). Pathogenic Aβ forms diffusible oligomers and insoluble plaque leading to chronic inflammation, toxicity, and autophagic cell death that cause age-associated disorders, such as Alzheimer disease and AMD (Cao et al., 2013; Tanokashira et al., 2017; Ziegler-Waldkirch et al., 2018). Specifically, Aβ accumulation causes barrier dysfunction and structural disruption of RPE. Notably, intracellular Aβ disrupts the tight junction of RPE (Jo et al., 2020; Park et al., 2014; 2015). Therefore, effective removal of Aβ in drusen is a key strategy for AMD treatment.

Aβ clearance is mediated by enzymatic degradation, efflux of the blood-brain barrier, and autophagic degradation pathways (Gonzalez-Marrero et al., 2015; Nixon, 2007; Wani et al., 2019; Zuroff et al., 2017). Dysfunction of the autophagy-lysosome pathway (ALP) leads to Aβ deposition and AMD; therefore, clearance of Aβ by increasing ALP activity may be a promising therapeutic strategy for AMD. Protein aggregates and cellular organelles are usually degraded by ALP in a multistep process involving the maturation and degradation of autophagosomes (Ciechanover, 2005; Pan et al., 2008). Autophagosome formation is mediated by the autophagy adaptor SQSTM1/p62, autophagy-related genes, and microtubule-associated protein 1A/1B-light chain 3 (LC3). Cytosolic LC3 (LC3-I) hydrolysis followed by conjugation to phosphatidylethanolamine (PE) to generate PE-LC3 (LC3-II) are involved in autophagosome formation. Autophagosomes fuse with lysosomes to form autolysosomes and are subsequently degraded by lysosomal enzymes, including Cathepsin B, D, and L. Many proteins, such as the small GTPase Rab7 and lysosome membrane-associated protein 2 (LAMP2), regulate autolysosome formation (Ferrington et al., 2016; Hyttinen et al., 2013; 2014; Rogov et al., 2014). This process promotes the adequate degradation of protein aggregates and maintains cellular homeostasis.

Autophagy is regulated by multiple signaling molecules including the AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), and nuclear factor-kappa B (NF-κB). mTOR, a serine/threonine kinase, forms two functionally distinct multiprotein complexes, mTORC1 and mTORC2. In particular, activation of mTORC1 suppresses the translocation of transcription factor EB (TFEB), a crucial regulator of lysosome biogenesis and autophagy, resulting in the inhibition of autophagy (Kim et al., 2021; Settembre et al., 2011; Shen and Mizushima, 2014; Zhou et al., 2013). Several inhibitors that induce autophagy via blockade of mTOR and activation of TFEB can be putative drug candidates for cancer and metabolic diseases (Maiese, 2016; Park et al., 2022; Wen et al., 2018). However, efficacy of targeting mTOR in AMD therapy remains unkown.

In this study, we determined the efficacy of the mTOR inhibitors, Torin and PP242, in clearing Aβ aggregates by inducing ALP in mice after subretinal injection of Aβ. Additionally, we assessed the potential of Torin and PP242 in ameliorating tight junction disruption induced by Aβ. We found that Torin and PP242 improved the Aβ oligomer-induced ALP impairment via activation of LAMP2 expression. Our data reveal the mechanism by which Torin and PP242 recovered lysosomal dysfunction to promote Aβ clearance in vitro and in vivo. Our findings can aid in the development of new therapeutic strategies for AMD.

Reagents and antibodies

Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 media (LM002-04), penicillin-streptomycin streptomycin (LS202-02) and Dulbecco’s phosphate-buffered saline (LB001-02) were purchased from WELGENE (Korea). Fetal bovine serum (F0900-050), dimethylsulfoxide (DMSO; D2680-010) and ultrapure bovine serum albumin (BSA; A0100-010) were purchased from GenDEPOT (USA). 4, 6-diamidino-2-phenylindole (DAPI; 62248) and LysoTracker^TM Red DND-99 (L7528) were purchased from Thermo Fisher Scientific (USA). Anti-Aβ (2450S) and anti-p62 (5114) antibodies were purchased from Cell Signaling Technology (USA). Anti-LC3B (ab48394) and anti-GAPDH (ab8245) antibodies were obtained from Abcam (USA). Anti-β-actin (sc-4778) antibody was obtained from Santa Cruz Biotechnology (USA). OPTI-MEM medium (11058021) was purchased from Gibco (USA). Anti-LAMP2 (51-2200 and PA1-655) antibody, anti-zonula occludens (ZO)-1 (33-9100) antibody, goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (A-11008), goat anti-rabbit IgG Alexa Fluor 594 secondary antibody (A32740) and Lipofectamine 3000 (L3000015) were purchased from Invitrogen. HRP-goat anti-rabbit IgG (111-035-144), HRP-goat anti-mouse IgG (115-035-003) were purchased from Jackson ImmunoResearch (USA). Aβ1-42 peptide (AG968) was obtained from Millipore (USA). Chloroquine (CQ) (C6628; Sigma, USA), Torin (10997; Cayman Chemical, USA), PP242 (S2218; Selleck Chemicals, USA), and a protease inhibitor cocktail (11697498001; Roche, Switzerland) were also used in this study.

Preparation of Aβ oligomers

Aβ oligomers was prepared as described previously (Park et al., 2014). Briefly, Aβ peptide (AG968; Millipore) and fluorescein isothiocyanate (FITC)-Aβ peptide (4033502; Bachem, Switzerland) were dissolved in hexafluoro-2-propanol (105228; Sigma) at room temperature for 3 days. The solution was dried in vacuum concentrator. Then Aβ peptides were stored desiccated at –80°C until further processing. To form Aβ oligomers, the peptide was dissolved in DMSO to 2 mM. Then, the Aβ was diluted in Dulbecco’s modified Eagle medium: Nutrient Mixture F-12 media to the indicated concentration.

Cell culture

ARPE-19 cells were purchased from the American Type Culture Collection (CRL-2302) and maintained in Dulbecco’s modified Eagle medium: Nutrient Mixture F-12 media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were maintained at 37°C with 5% CO₂. The cells were treated with varying concentrations of Aβ for 48 h or cotreated Aβ with 50 μM of CQ, 200 nM of Torin and 1 μM of PP242.

Cell viability assay

For the cell viability assay, the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) (G3582; Promega, USA) was used according to the manufacturer’s instructions. ARPE-19 cells (2 × 10⁴ cells) were seeded in a 96-well plate. Cells were treated with Aβ oligomers for 24 h and 48 h and then 20 μl per well of CellTiter 96 AQueous One Solution reagent was added. After 2 h incubation at 37°C, the absorbance at 490 nm was measured using a VersaMax^TM microplate reader.

Western blot analysis

After various treatments, ARPE-19 cells were lysed with a radioimmunoprecipitation assay (RIPA) lysis buffer containing 1% protease inhibitor cocktail. Thirty micrograms of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (IPVH00010; Millipore). The membrane was subjected to blocking with 5% milk for 1 h at room temperature and then incubated with primary antibodies for overnight at 4°C. Then, the membrane was incubated with the appropriate secondary antibodies for 1 h at room temperature. Blots were developed using the West-Q Pico ECL Solution (W3652-020; GenDEPOT) and then chemiluminescence signal was detected using the Amersham ImageQuant 800 (29399481; Cytiva, USA). The band intensities were quantified using the ImageJ program.

Immunofluorescence analysis

At the end of treatment, cells were washed twice in phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min at room temperature and washed with PBS. Cells were incubated with 5% BSA for 1 h and stained using specific primary antibodies for overnight at 4°C. From the enucleated eyes at 3 days after the injection of Aβ, Torin, and PP242, RPE-choroid-scleral complexes were prepared for immunofluorescence analysis. The primary antibodies were used as follows: anti-Aβ and anti-LAMP2 for ARPE-19 cells; anti-LAMP2 and anti-ZO-1 for RPE-choroid-scleral complexes. Then, cells were incubated with corresponding secondary antibodies and DAPI. The immunostained cells were analyzed using a confocal laser-scanning microscope at 64× (LSM800; Zeiss, Germany).

Tandem monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP) LC3 analysis

For transfection of mRFP-GFP-LC3, ARPE-19 cells were seeded on coverslips at a density of 4 × 10⁵ cells. These cells were transfected with mRFP-GFP-LC3 plasmid using Lipofectamine 3000 into OPTI-MEM media. After 24 h of transfection, cells were treated with 10 μM of Aβ oligomers or cotreated Aβ oligomers with 200 nM of Torin and 1 μM of PP242 and samples were analyzed by immunofluorescence using confocal microscope at 64× (LSM800; Zeiss).

SA-β-gal senescence assay

To confirm the senescence of ARPE cells, the Senescence β-Galactosidase Staining Kit (#9860; Cell Signaling Technology) was employed, following the manufacturer’s instructions. After the treatment, the cells were rinsed with PBS and fixed with a fixation solution for 15 min at room temperature, followed by another PBS wash. The fixed cells were then incubated overnight with β-Galactosidase staining solution at 37°C. The β-Galactosidase staining was analyzed using a fluorescence microscope at 20× magnification (EVOS5000; Thermo Fisher Scientific).

Animal experiments

All animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research. The protocols were approved by the Institutional Animal Care and Use Committee of both Seoul National University and Seoul National University Hospital (No. SNU-230519-4). Five-week-old C57BL/6 male mice were purchased from KOATECH and maintained under a 12-h dark-light cycle. After 1 week of acclimatization, mice underwent subretinal injection of PBS (2 μl) or FITC-Aβ (10 μM in 2 μl PBS) after deep anesthesia. Then, mice received intravitreal injection of PBS (1 μl), Torin (200 nM in 1 μl PBS) or PP242 (1 μM in 1 μl PBS). At 3 days after the subretinal and intravitreal injection, the eyes were prepared for immunofluorescence analysis.

For the quantification of Aβ- or LAMP2-positive areas in the RPE, the areas with green or red fluorescence in the ×400 magnification confocal images were analyzed using the ImageJ program. For the quantification of ZO-1-positive cells, 30 cells near the injection sites were examined to check the integrity of ZO-1 expression along the cell borders by two independent observers (D.H.J. and C.S.C.).

Optomotor response measurement

The optomotor response was assessed employing a virtual optomotor system (OptoMotry HD; CerebralMechanics Inc.) according to the manufacturer’s instructions. The mice were positioned on a platform to see a small rotating cylinder displayed on a monitor. The system tracked the mice’s head movements as they attempted to follow the rotating cylinder. The system generated values indicating the maximum number of cycles per degree that the mouse could track the cylinder without response.

Electroretinography (ERG)

Before conducting the ERG, mice were subjected to overnight dark adaptation. Deep anesthesia was induced, and the pupils were dilated using tropherine ophthalmic solution containing phenylephrine hydrochloride and tropicamide. Electroretinogram measurements were performed using the Universal Testing and Electrophysiologic System 2000 (UTAS E-2000; LKC, USA). The response to a 0 dB xenon flash was recorded at 60 Hz with a notch filter and a gain of 2 k. The data were filtered between 0.1 and 1,500 Hz, and graphs were generated. Amplitudes were estimated using Prism 10 (GraphPad Software, USA). A-wave amplitude was determined by measuring from the baseline to the lowest negative voltage, while b-wave amplitude was measured from the trough of the a-wave to the highest peak of the positive b-wave.

Statistical analysis

All data are expressed as the mean ± SEM. Statistical analyses were performed using Prism 10. Each statistical method was indicated in figure legends. P values less than 0.05 were considered statistically significant.

High concentration Aβ oligomers induce dysfunction of autophagy and lysosome activity in RPE cells

To determine the effects of Aβ oligomers in the RPE, we treated an increasing concentration of Aβ oligomers in ARPE-19 cells. Aβ oligomers treatment decreased cell viability and led to cellular changes in a concentration and time-dependent manner (Figs. 1A and 1B). Especially, high concentration of Aβ oligomers aggravated the intact morphology of RPE cells and induced cell death. To investigate whether the effects of Aβ oligomers are associated with autophagic occurrence, we examined the commonly used indicator of autophagosome markers such as p62 and LC3-II using western blot analysis. Low concentration of Aβ oligomers (1 μM) increased the ratio of the lipidated LC3-II and decreased p62 expression at the same time, while high concentration of Aβ oligomers (10 μM) simultaneously accumulated the expression of p62 and LC3-II (Fig. 1C). The ubiquitin-binding protein, p62, also serves to assess autophagy flux. According to the autophagy activation, p62 degradation accompanies an increase of LC3-II. Therefore, these results suggest that high concentration Aβ oligomers induce loss of cell viability and suppress the formation of autophagosomes.

In addition, as the accumulated p62 and LC3-II reflect impairment of lysosome activity during autophagy progression (Yoshii and Mizushima, 2017), we next explored the role of Aβ oligomers on lysosomal function. We incubated Aβ oligomers in ARPE-19 cells with CQ, a lysosomotropic agent that inhibits the fusion of autophagosome and lysosome. CQ treatment attenuated cell viability in Aβ oligomer-treated ARPE-19 cells (Fig. 1D). When CQ was added to Aβ oligomer-treated ARPE-19 cells, the expression of p62 and LC3-II was significantly increased (Fig. 1E). Additionally, we evaluated the effect of Aβ oligomers on lysosome activity using the lysotracker. Immunofluorescence analysis showed that Aβ oligomers treatment significantly decreased the intensity of lysotracker staining in a concentration-dependent manner (Figs. 1F and 1G). Taken together, these data indicate that high concentration Aβ oligomers prevent autophagic occurrence by inhibiting lysosomal function.

Torin and PP242 enhance Aβ clearance by improving lysosomal dysfunction in RPE cells

Numerous studies show that mTOR signaling inhibits TFEB activation, which contributes to lysosome biogenesis and activity (Roczniak-Ferguson et al., 2012; Zhou et al., 2013). Therefore, we hypothesized that mTOR inhibitors might improve lysosomal dysfunction and facilitate Aβ clearance in ARPE-19 cells treated with high concentration of Aβ oligomers. To investigate the role of mTOR inhibitors on Aβ clearance, we utilized Torin and PP242 as mTOR inhibitors in Aβ oligomers-treated ARPE-19 cells. Immunofluorescence analysis showed that treatment of Torin and PP242 significantly reduced Aβ oligomers expression (Fig. 2A). Furthermore, Torin and PP242 also decreased Aβ aggregation, as assessed by western blot analysis (Fig. 2B). These data suggest that Torin and PP242 enhance Aβ clearance.

We further examined whether Torin and PP242 recover reduced lysosome activity in Aβ oligomers-treated ARPE-19 cells. Immunofluorescence analysis showed that treatment of Torin and PP242 significantly rescued the relative fluorescent intensity of lysotracker compared to control cells (Figs. 2C and 2D). Moreover, to assess whether Torin and PP242 restore the ALP activity, we confirmed the change of autophagic flux using a tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) fusion protein based on different pH stability. As autophagosome (mRFP+/GFP+, yellow puncta) and autolysosome (mRFP+/GFP–, red puncta) are distinguishable, we analyzed the effect of Torin and PP242 on the change from autophagosome to autolysosome using confocal microscopy. High concentration of Aβ oligomer treatment accumulated the number of yellow puncta, indicating an increased number of autophagosomes and impaired fusion of the autophagosome with the lysosome. However, Torin and PP242 treatment markedly decreased the number of yellow puncta and increased the number of red puncta in Aβ oligomers-treated ARPE-19 cells, suggesting an increased number of autolysosomes (Figs. 2E and 2F). Taken together, these data demonstrate that Torin and PP242 enhance lysosomal function to facilitate the clearance of Aβ.

Torin and PP242 rescue autophagic flux blockade via activation of LAMP2 in RPE cells

To find a functional mechanism for how Torin and PP242 restore the lysosome activity in Aβ oligomer-treated ARPE-19 cells, we were interested in whether Torin and PP242 regulate the expression of a protein involved in lysosomal function. Since LAMP2 is a representative marker of lysosomes and it has been reported that LAMP2-deficient mice are much more susceptible to the pathogenesis of AMD (Notomi et al., 2019), we speculated that LAMP2 is associated with Aβ oligomer-induced lysosome impairment. Aβ oligomer treatment significantly decreased the expression of LAMP2 in a concentration-dependent manner, as assessed by immunofluorescence (Figs. 3A and 3B) and western blot analysis (Fig. 3C), suggesting that lysosomal dysfunction by Aβ oligomers is associated with a decrease of LAMP2 expression. Then, we further examined whether Torin and PP242 modulate the expression of LAMP2. Interestingly, Torin and PP242 restored LAMP2 expression in Aβ oligomers-treated ARPE-19 cells (Figs. 3D-3F). These results indicate that Torin and PP242 increase LAMP2 expression, thereby improving lysosomal function and autophagic flux. To investigate whether these effects were related to cell scenescence, we performed SA-β-gal staining, demonstrating that Aβ oligomers induced cell senescence, which was ameliorated by activating the ALP (Fig. 3G).

Torin and PP242 restores LAMP2 expression in RPE of Aβ-treated mice

Then, we further investigated in vivo effects of Aβ oligomers on senescence-like alterations. Subretinally administered Aβ oligomers were accumulated in the cytoplasm of RPE in the treated mice (Figs. 4A and 4B), as previously reported (Jo et al., 2020; Park et al., 2015). In lines with in vitro studies with ARPE-19 cells (Figs. 3A and 3B), Aβ oligomer treatment significantly decreased the expression of LAMP2 in the RPE (Fig. 4C). To verify that Torin and PP242 rescued the expression of LAMP2 in vivo, we subsequently injected them into the vitreous cavity of the mice after subretinal injection of Aβ oligomers. At 3 days after subretinal and intravitreal injection procedures, RPE-choroid-scleral complexes were examined for the LAMP2 expression. As Torin and PP242 increased LAMP2 expression in Aβ oligomers-treated ARPE-19 cells (Figs. 3D-3F), they increased the expression of LAMP2 in the RPE of mice which underwent co-treatment of them with subretinally administered Aβ oligomers (Figs. 4D and 4E).

Torin and PP242 decreases the accumulation of Aβ and ameliorates the tight junction disruption of RPE in Aβ-treated mice

Then, we examined the accumulation of Aβ oligomers in mice that underwent co-treatment of Torin and PP242 with subretinally administered Aβ oligomers. Confocal microscopy demonstrated that the burden of Aβ oligomers in the RPE of the mice treated with Torin and PP242 was dramatically decreased compared to the mice which only received subretinal injection of Aβ oligomers and intravitreal injection of PBS (Figs. 5A and 5B). Intracellular Aβ disrupts the tight junction of RPE in 5XFAD mice with Aβ accumulation, a representative animal model of Alzheimer disease, and mice treated with subretinal Aβ injection (Jo et al., 2020; Park et al., 2014; 2015; 2017). NF-κB activation by Aβ after intracellular uptake via the receptor for advanced glycation end products might be the mechanism of the tight junction disruption of the RPE (Jo et al., 2020; Park et al., 2015). As expected, the integrity of the expression of ZO-1, a tight junction marker, was significantly restored with the treatment of Torin and PP242 after Aβ (Figs. 5C and 5D). We also observed a definite increase in ZO-1 at the protein expression levels in Aβ oligomers-treated ARPE-19 cells (Fig. 5E). Taken together, these results suggest that Torin and PP242 recovered Aβ-induced tight junction disruption by LAMP2-mediated clearance of Aβ. To investigate whether these effects were linked to functional consequences, we measured oculomotor responses (Fig. 6A) and ERG (Figs. 6B-6D). Aβ oligomers decreaed the level of oculomotr responses (Fig. 6A) and amplitudes of a- and b-waves of ERG (Figs. 6B and 6C), which were rescued by the treatment with Torin and PP242.

Our study revealed the role of mTOR inhibitors such as Torin and PP242 in the clearance of abnormal accumulated Aβ oligomers in the RPE cells in vitro and in vivo. Aβ oligomers treatment suppressed lysosome activity and induced dysfunction of ALP, which resulted in aggravation of cell viability. Interestingly, Torin and PP242 as mTOR inhibitors recovered Aβ oligomer-induced lysosome impairment by increasing LAMP2 expression. These results implied that Torin and PP242 might be considered as potential drugs for treating AMD.

When we tested the ALP activity and morphological changes of RPE cells following different concentrations of Aβ oligomers, low concentration of Aβ oligomers decreased p62 expression, maintained lysosome activity, and did not induce specific morphological changes. Moreover, the turnover of autophagosome-associated proteins, including p62 and LC3, showed a general autophagic flux (Fig. 1). However, high concentration of Aβ oligomers disrupted the regular lysosomal degradation system and was lethal to cell survival. Although further studies are needed to analyze the detailed kinetics of Aβ oligomers, we speculate that it may be necessary to consider Aβ concentration that affects the correlation between Aβ aggregation and lysosome activity in AMD development.

mTOR is well-known as the master negative regulator of autophagy and plays a crucial role in the autophagic flux (Kim and Guan, 2015; Yu et al., 2010). Thus, identifying mTOR targeting drugs that can be used to recover autophagy activity may offer a new way of treating age-related disorders. Furthermore, there might be further effects of mTOR inhibition by affecting autophagy and further signaling pathways related to Aβ-induced RPE alterations, such as NF-κB (Jo et al., 2020). NF-κB p65, which mediate Aβ-induced tight junction disruption (Jo el al., 2020), is targeted by autophagic degradation (Brischetto et al, 2022). In our study, mTOR inhibition by Torin and PP242 recovered Aβ oligomer-induced lysosome impairment leading to tight junction disruption. Many studies have shown that rapamycin or rapamycin derivate drugs delay the development of neurodegenerative disorder via inhibition of mTOR, and they have already been approved for clinical approach by European Medicines Agency and the U.S. Food and Drug Administration (Gensler et al., 2018; Petrou et al., 2014). Therefore, our results also suggest that Torin and PP242 can be considered promising new therapeutic strategies for AMD, providing a basis for more sophisticated and effective drug development.

Recently, a study showed that genetic LAMP2 deficiency in neurons and retina disrupted autophagy and lysosomal structure, leading to the pathogenesis of AMD (Eskelinen et al., 2002; Notomi et al., 2019; Tanaka et al., 2000). LAMP2 is highly expressed by RPE cells and continuously regulates retinal homeostasis (Schorderet et al., 2007; Thiadens et al., 2012). Also, we confirmed that high concentration of Aβ oligomers decreased LAMP2 expression in RPE cells and decreased lysosomal activity. However, Torin and PP242 restored decreased LAMP2 expression and autophagic flux by Aβ oligomer treatment, suggesting that Torin and PP242-mediated alterations of LAMP2 expression may enhance the lysosome biogenesis and function. In the future, further studies are needed to determine the detailed molecular mechanisms underlying these effects.

In conclusion, our results demonstrated that high concentration of Aβ oligomers induced dysregulation of ALP, while Torin and PP242 facilitated Aβ clearance by recovering the ALP and upregulation of LAMP2 expression. Therefore, a strategy to restore lysosome activity will be efficient for Aβ clearance and prevention of tight junction disruption associated with Aβ. In particular, the effects of Torin and PP242 have shown that they can potentiate the efficacy of therapy and emerge as promising therapeutic targets. Our findings will provide a basis for the new therapeutic approach of AMD and serve as essential guide to overcome the limitations of conventional therapy.

This study was supported by the Basic Science Research Program (NRF-2022R1A2C2010940 to H.K. and NRF-2022R1A2C1003768, NRF-2023M3A9I4009901 to D.H.J.), the Creative Materials Discovery Program (NRF-2018M3D1A1058826 to J.H.K.) from the National Research Foundation (NRF) of Korea funded by the Korean government, the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5362111 to J.H.K.), the Ministry of Trade, Industry, and Energy, and the Ministry of Health and Welfare (HN21C0917 to J.H.K.), Kun-hee Lee Child Cancer & Rare Disease Project, Republic of Korea (202200004004 to J.H.K.), Seoul National University Hospital Research Grant (18-2023-0010 to J.H.K.), and the Bio & Medical Technology Program of the NRF funded by the Korean government, MSIP (NRF-2022M3A9E4017127 to J.H.K.).

D.H.J. and S.H.L. conducted the experiments, analyzed data, and wrote the manuscript. M.J., C.S.C., and D.E.K. conducted the experiments. H.K. and J.H.K. were involved in study design, data interpretation, and writing and editing of the manuscript.

The authors have no potential conflicts of interest to disclose.