MLST8 overexpression in RPE cells disrupts autophagy through novel mechanisms affecting AMD pathogenesis

ABSTRACT

Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly, with dysfunction of the retinal pigment epithelium (RPE) central to disease pathogenesis. Using our uniquely developed MLST8 (MTOR associated protein, LST8 homolog) knock-in animal model with RPE-specific overexpression, which drives MTOR (mechanistic target of rapamycin kinase) upregulation, we demonstrate that increased MTOR complexes 1 and 2 in the RPE disrupts macroautophagy/autophagy by suppressing autophagosome formation genes and impairing MAP1LC3/LC3 processing. This leads to autophagosome accumulation and defective autolysosome formation, driving RPE dysfunction and AMD-like pathology, including subretinal debris build up and photoreceptor degeneration. Notably, MTOR inhibition with torin1 treatment or CRYBA1 overexpression rescues these defects, restoring autophagy and RPE integrity. Our findings reveal that autophagy disruption mediated by both MTORC1 and MTORC2 drives AMD-like pathology in our mouse model, establishing autophagy regulation as a promising avenue for therapeutic intervention in this vision-threatening disease.

Age-related macular degeneration (AMD) is a leading cause of vision loss in the elderly, posing a substantial socio-economic burden on society, with projections indicating 288 million affected individuals in the next decade. AMD is characterized by progressive degeneration of the retinal pigment epithelium (RPE) and photoreceptors, potentially leading to complete vision loss. RPE cells are essential for retinal health, supporting photoreceptor outer segment phagocytosis, the visual cycle, immune regulation and oxidative stress defense. Autophagy plays a crucial role in RPE function, and its impairment leads to toxic waste buildup and release, thereby contributing to the accumulation of subretinal drusenoid deposits and drusen, which are hallmarks of atrophic AMD. While lysosomal dysfunction and the role of autophagy in AMD are well recognized, the molecular mechanisms governing autophagy in RPE cells remain incompletely understood.

MTOR (mechanistic target of rapamycin kinase) exists as two distinct complexes, MTORC1 and MTORC2, regulating diverse cellular processes including protein synthesis, metabolism, and autophagy. While previous studies have established definitive roles of MTORC1 in RPE homeostasis, the combined function of both MTOR complexes in regulating autophagy has remained poorly elucidated. MLST8 (MTOR associated protein, LST8 homolog) serves as a core component in MTORC1 and MTORC2, making it an ideal target for developing our animal model with activation of both these complexes specifically in the RPE cells. Using our MLST8 knock-in (KI) mouse model, we establish MLST8 as a critical regulator of RPE autophagy, essential for RPE and photoreceptor health as well as visual function.

Our findings reveal several novel aspects of MLST8’s function in autophagy regulation in RPE cells, particularly its disruptive impact on macroautophagy [1]. Transcriptomic analysis revealed downregulation of key autophagosome formation/maturation genes (Atg3, Atg5, Atg9b, Uvrag, Rb1cc1, Map1lc3a, and Gabarap) in MLST8 KI RPE. These abnormalities likely indicate that MTORC1 and MTORC2 inhibit transcription factors such as TFEB and FOXO, both essential regulators of autophagy-related genes, resulting in severely compromised autophagy flux and RPE homeostasis. Our results confirmed that MLST8-overexpressing ARPE19 cells demonstrate compromised LC3 processing and autophagy flux. Tandem fluorescent reporter assays further revealed disrupted autolysosome formation which is corroborated by SQSTM1/p62 accumulation and suggests impaired autophagosome initiation, maturation and degradation. Thus, activation of both MTORC1 and MTORC2 perturbs autophagy/lysosomal processes in RPE cells. Functionally, MLST8 upregulation-induced autophagy impairment directly correlates with downregulation of essential visual cycle proteins (RPE65, RDH5) and significant electrophysiological deficits. We observed reduced a-wave, b-wave, and c-wave amplitudes, indicative of compromised photoreceptor, inner retinal, and RPE function, respectively. These functional impairments establish a clear link between molecular defects in autophagy and visual dysfunction in our AMD model.

Our study also demonstrated how MLST8 overexpression disrupts melanosome homeostasis. As a key regulator of oxidative stress defense, melanosomes are essential for RPE health. However, MLST8 overexpression impairs autophagy-lysosomal function, causing abnormal basolateral melanosome accumulation (ultrastructural analysis) and defective melanosome-lysosome interactions, evidenced by reduced TCPN2/TPC2-LAMP1 colocalization. Proteomic analysis confirms downregulation of key melanosome regulators (TYRP1, MLANA, BLOC1S3), along with decreased melanin content. These anomalies compromise RPE function, promoting oxidative damage, thereby demonstrating how MLST8 overexpression mediates melanosome dysfunction in AMD pathogenesis.

Furthermore, impaired autophagy flux inhibits photoreceptor outer segment degradation, leading to lipofuscin and drusen accumulation that directly drives photoreceptor degeneration and AMD progression. This toxic accumulation creates a pathological feedback loop where oxidative stress disrupts AMPK-MTOR signaling, further exacerbating proteotoxic stress, and mitochondrial damage. This triggers inflammation, complement activation and epithelial-mesenchymal transition/EMT that ultimately accelerate RPE cell death and the progression from early to advanced stages of AMD. Importantly, pharmacological inhibition of MTOR kinase activity with the chemical inhibitor torin1 restores the pathway in vitro and overexpression of the biological regulatory protein CRYBA1, rescues autophagy in our KI mouse through modulation of MTORC1 and MTORC2 targets leading to subsequent normalization of autophagy-related protein levels and rescue of retinal structure and function.

In conclusion, this study establishes MLST8 as a critical negative regulator of autophagy in RPE cells, representing the first comprehensive characterization of this mechanism in ocular tissues. By demonstrating that MLST8 overexpression disrupts macroautophagy through downregulation of autophagosome formation genes and impaired LC3 processing, we have uncovered a novel regulatory pathway that directly links MTOR complex components to autophagy dysfunction in retinal degeneration. The elucidation of how MLST8-induced autophagy impairment leads to melanosome distribution abnormalities and compromised visual function provides critical insight into AMD pathogenesis. Importantly, our finding that MTOR inhibition with torin1 and CRYBA1 rescues autophagy regulator expression establishes a mechanistic foundation for developing targeted therapeutic interventions for AMD and related retinal disorders characterized by defective autophagy (Figure 1).

Figure 1.

Upregulation of MLST8 enhances MTORC1 and MTORC2 activity, suppressing the expression of key autophagosome maturation genes and reducing autophagic flux. This impairs LC3 processing, leading to SQSTM1/p62 accumulation and defective autophagosome-lysosome fusion, ultimately diminishing autolysosome formation and debris clearance. As a result, photoreceptor outer segment phagocytosis is disrupted, leading to photoreceptor degeneration. In addition, there is abnormal accumulation and altered function of melanosomes. Accumulated autophagosomes form large debris-filled vacuoles, contributing to drusen formation, a hallmark of atrophic AMD. Additionally, chronic autophagy dysfunction triggers oxidative stress, which further disrupts AMPK-MTOR signaling, reinforcing a pathological feedback loop that exacerbates disease progression. Treatment with the MTOR inhibitor torin1 or CRYBA1 overexpression restores autophagic function, reduces oxidative stress, and rescues the degenerative phenotype. These findings establish MLST8 as a critical regulator of MTOR-driven autophagy dysfunction in atrophic AMD, highlighting its potential as a therapeutic target. Created with Biorender.com.

Funding Statement

This work was supported by NIH R01EY031594 and R01EY032516 (to DS) and K99EY033421 (to SG), the Edward N. and Della L. Thome Memorial Foundation Awards Program in Age-Related Macular Degeneration Research (to DS), P30 core award (EY001765) from the National Eye Institute, NIH to the Wilmer Eye Institute, Johns Hopkins University School of Medicine, and unrestricted funds from The Research to Prevent Blindness Inc., NY to the Wilmer Eye Institute, Johns Hopkins University School of Medicine. DS is the Frieda Derdeyn Bambas Professor of Ophthalmology.

Disclosure statement

No potential conflict of interest was reported by the author(s).