QTL mapping of human retina DNA methylation identifies 87 gene-epigenome interactions in age-related macular degeneration.

DNA methylation provides a crucial epigenetic mark linking genetic variations to environmental influence. We have analyzed array-based DNA methylation profiles of 160 human retinas with co-measured RNA-seq and >8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 methylation quantitative trait loci and 12,505 expression quantitative trait loci) and 13,747 DNA methylation loci affecting gene expression, with over one-third specific to the retina. Methylation and expression quantitative trait loci show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration. Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of macular degeneration pathology by genotype-environment interaction in retina.

Quantitative Proteomics of Human Retinal Pigment Epithelium Reveals Key Regulators for the Pathogenesis of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people, with limited treatment options available for most patients. AMD involves the death of retinal pigment epithelium (RPE) and photoreceptor cells, with mitochondria dysfunction being a critical early event. In the current study, we utilized our unique resource of human donor RPE graded for AMD presence and severity to investigate proteome-wide dysregulation involved in early AMD. Organelle-enriched fractions of RPE were isolated from donors with early AMD (n = 45) and healthy age-matched controls (n = 32) and were analyzed by UHR-IonStar, an integrated proteomics platform enabling reliable and in-depth proteomic quantification in large cohorts. A total of 5941 proteins were quantified with excellent analytical reproducibility, and with further informatics analysis, many biological functions and pathways were found to be significantly dysregulated in donor RPE samples with early AMD. Several of these directly pinpointed changes in mitochondrial functions, e.g., translation, ATP metabolic process, lipid homeostasis, and oxidative stress. These novel findings highlighted the value of our proteomics investigation by allowing a better understanding of the molecular mechanisms underlying early AMD onset and facilitating both treatment development and biomarker discovery.

Ambra1 haploinsufficiency in CD1 mice results in metabolic alterations and exacerbates age-associated retinal degeneration.

Macroautophagy/autophagy is a key process in the maintenance of cellular homeostasis. The age-dependent decline in retinal autophagy has been associated with photoreceptor degeneration. Retinal dysfunction can also result from damage to the retinal pigment epithelium (RPE), as the RPE-retina constitutes an important metabolic ecosystem that must be finely tuned to preserve visual function. While studies of mice lacking essential autophagy genes have revealed a predisposition to retinal degeneration, the consequences of a moderate reduction in autophagy, similar to that which occurs during physiological aging, remain unclear. Here, we described a retinal phenotype consistent with accelerated aging in mice carrying a haploinsufficiency for Ambra1, a pro-autophagic gene. These mice showed protein aggregation in the retina and RPE, metabolic underperformance, and premature vision loss. Moreover, Ambra1+/gt mice were more prone to retinal degeneration after RPE stress. These findings indicate that autophagy provides crucial support to RPE-retinal metabolism and protects the retina against stress and physiological aging.Abbreviations : 4-HNE: 4-hydroxynonenal; AMBRA1: autophagy and beclin 1 regulator 1, AMD: age-related macular degeneration;; GCL: ganglion cell layer; GFAP: glial fibrillary acidic protein; GLUL: glutamine synthetase/glutamate-ammonia ligase; HCL: hierarchical clustering; INL: inner nuclear layer; IPL: inner plexiform layer; LC/GC-MS: liquid chromatography/gas chromatography-mass spectrometry; MA: middle-aged; MTDR: MitoTracker Deep Red; MFI: mean fluorescence intensity; NL: NH4Cl and leupeptin; Nqo: NAD(P)H quinone dehydrogenase; ONL: outer nuclear layer; OPL: outer plexiform layer; OP: oscillatory potentials; OXPHOS: oxidative phosphorylation; PCR: polymerase chain reaction; PRKC/PKCα: protein kinase C; POS: photoreceptor outer segment; RGC: retinal ganglion cells; RPE: retinal pigment epithelium; SI: sodium iodate; TCA: tricarboxylic acid.

Investigating mitochondrial fission, fusion, and autophagy in retinal pigment epithelium from donors with age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in developed countries, characterized by the death of retinal pigment epithelial (RPE) cells and photoreceptors. Previous studies report an accumulation of damaged and dysfunctional mitochondria in RPE of human donors with AMD. Understanding how damaged mitochondria accumulate in AMD is an important step in discovering disease mechanisms and identifying therapeutic targets. In this report, we assessed mitochondrial fission and fusion by quantifying proteins and measured mitochondrial autophagy (mitophagy) via protein analysis and advanced imaging techniques using mitochondrial targeted mKeima in primary human RPE from donors with or without AMD. We report disease-specific differences in mitochondrial proteins that regulate fission, fusion, and mitophagy that were present at baseline and with treatments to stimulate these pathways. Data suggest AMD RPE utilize receptor-mediated mitophagy as a compensatory mechanism for deficits in the ubiquitin-mediated mitophagy pathway. These changes in mitochondrial homeostasis could lead to the buildup of damaged and dysfunctional mitochondria observed in the RPE of AMD donors.

Dissecting Regulators of Aging and Age-Related Macular Degeneration in the Retinal Pigment Epithelium.

Age-related macular degeneration (AMD), the leading cause of blindness in elderly populations, involves the loss of central vision due to progressive dysfunction of the retinal pigment epithelium (RPE) and subsequent loss of light-sensing photoreceptors. While age is a key risk factor, not every aged individual develops AMD. Thus, the critical question is what specific cellular changes tip the balance from healthy aging to disease. To distinguish between changes associated with aging and AMD, we compared the RPE proteome in human eye bank tissue from nondiseased donors during aging (n = 50, 29-91 years) and in donors with AMD (n = 36) compared to age-matched donors without disease (n = 28). Proteins from RPE cells were separated on two-dimensional gels, analyzed for content, and identified using mass spectrometry. A total of 58 proteins displayed significantly altered content with either aging or AMD. Proteins involved in metabolism, protein turnover, stress response, and cell death were altered with both aging and AMD. However, the direction of change was predominantly opposite. With aging, we detected an overall decrease in metabolism and reductions in stress-associated proteins, proteases, and chaperones. With AMD, we observed upregulation of metabolic proteins involved in glycolysis, TCA, and fatty acid metabolism, with a concurrent decline in oxidative phosphorylation, suggesting a reprogramming of energy utilization. Additionally, we detected upregulation of proteins involved in the stress response and protein turnover. Predicted upstream regulators also showed divergent results, with inhibition of inflammation and immune response with aging and activation of these processes with AMD. Our results support the idea that AMD is not simply advanced aging but rather the culmination of perturbed protein homeostasis, defective bioenergetics, and increased oxidative stress within the aging RPE, exacerbated by environmental factors and the genetic background of an individual.

Airway epithelial immunoproteasome subunit LMP7 protects against rhinovirus infection.

Immunoproteasomes (IP) serve as an important modulator of immune responses to pathogens and other pathological factors. LMP7/ß5i, one of the IP subunits, plays a critical role in autoimmune diseases by downregulating inflammation. Rhinovirus (RV) infection is a major risk factor in the exacerbations of respiratory inflammatory diseases, but whether LMP7 regulates RV-mediated inflammation in the lung particularly in the airway epithelium, the first line of defense against RV infection, remains unclear. In this study, we determined whether airway epithelial LMP7 promotes the resolution of RV-mediated lung inflammation. Inducible airway epithelial-specific LMP7-deficient (conditional knockout, CKO) mice were generated to reveal the in vivo anti-inflammatory and antiviral functions of LMP7. By using LMP7-deficient primary human airway epithelial cells generated by CRISPR-Cas9, we confirmed that airway epithelial LMP7 decreased pro-inflammatory cytokines and viral load during RV infection. Additionally, airway epithelial LMP7 enhanced the expression of a negative immune regulator A20/TNFAIP3 during viral infection that may contribute to the anti-inflammatory function of LMP7. We also discovered that induction of LMP7 by a low dose of polyinosinic:polycytidylic acid (PI:C) reduced RV-mediated inflammation in our CKO mice infected with RV. Our findings suggest that airway epithelial LMP7 has anti-inflammatory and antiviral functions that is critical to the resolution of RV-mediated lung inflammation. Induction of airway epithelial LMP7 may open a novel avenue for therapeutic intervention against RV infection.

Inflammasome Activation in Retinal Pigment Epithelium from Human Donors with Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, is characterized by the death of retinal pigment epithelium (RPE) and photoreceptors. One of the risk factors associated with developing AMD is the single nucleotide polymorphism (SNP) found within the gene encoding complement factor H (CFH). Part of the innate immune system, CFH inhibits alternative complement pathway activation. Multi-protein complexes called inflammasomes also play a role in the innate immune response. Previous studies reported that inflammasome activation may contribute to AMD pathology. In this study, we used primary human adult RPE cell cultures from multiple donors, with and without AMD, that were genotyped for the Y402H CFH risk allele. We found complement and inflammasome-related genes and proteins at basal levels in RPE tissue and cell cultures. Additionally, treatment with rotenone, bafilomycin A, and ATP led to inflammasome activation. Overall, the response to priming and activation was similar, irrespective of disease state or CFH genotype. While these data show that the inflammasome is present and active in RPE, our results suggest that inflammasome activation may not contribute to early AMD pathology.

Inflammatory adipose activates a nutritional immunity pathway leading to retinal dysfunction.

Age-related macular degeneration (AMD), the leading cause of irreversible blindness among Americans over 50, is characterized by dysfunction and death of retinal pigment epithelial (RPE) cells. The RPE accumulates iron in AMD, and iron overload triggers RPE cell death in vitro and in vivo. However, the mechanism of RPE iron accumulation in AMD is unknown. We show that high-fat-diet-induced obesity, a risk factor for AMD, drives systemic and local inflammatory circuits upregulating interleukin-1ß (IL-1ß). IL-1ß upregulates RPE iron importers and downregulates iron exporters, causing iron accumulation, oxidative stress, and dysfunction. We term this maladaptive, chronic activation of a nutritional immunity pathway the cellular iron sequestration response (CISR). RNA sequencing (RNA-seq) analysis of choroid and retina from human donors revealed that hallmarks of this pathway are present in AMD microglia and macrophages. Together, these data suggest that inflamed adipose tissue, through the CISR, can lead to RPE pathology in AMD.

Human iPSC- and Primary-Retinal Pigment Epithelial Cells for Modeling Age-Related Macular Degeneration.

Primary cultures of retinal pigment epithelium (RPE) from human adult donors (haRPE) and induced pluripotent stem cell derived-RPE (iPSC-RPE) are valuable model systems for gaining mechanistic insight and for testing potential therapies for age-related macular degeneration (AMD). This study evaluated the treatment response of haRPE and iPSC-RPE to oxidative stress and potential therapeutics addressing mitochondrial defects. haRPE and iSPC-RPE were derived from donors with or without AMD. Mitochondrial function was measured after treatment with menadione, AICAR, or trehalose and the response to treatment was compared between cell models and by disease status. In a subset of samples, haRPE and iPSC-RPE were generated from the same human donor to make a side-by-side comparison of the two cell models' response to treatment. Disease-specific responses to all three treatments was observed in the haRPE. In contrast, iPSC-RPE had a similar response to all treatments irrespective of disease status. Analysis of haRPE and iPSC-RPE generated from the same human donor showed a similar response for donors without AMD, but there were significant differences in treatment response between cell models generated from AMD donors. These results support the use of iPSC-RPE and haRPE when investigating AMD mechanisms and new therapeutics but indicates that attention to experimental conditions is required.

Quantification of mitophagy using mKeima-mito in cultured human primary retinal pigment epithelial cells.

The retinal pigment epithelium is a pigmented monolayer of cells that help maintain a healthy retina. Loss of this essential cell layer is implicated in a number of visual disorders, including age-related macular degeneration (AMD). Utilizing primary RPE cultures to investigate disease is an important step in understanding disease mechanisms. However, the use of primary RPE cultures presents a number of challenges, including the limited number of cells available and the presence of auto-fluorescent pigment that interferes with quantifying fluorescent probes. Additionally, primary RPE are difficult to transfect with exogenous nucleic acids traditionally used for fluorescent imaging. To overcome these challenges, we used an adeno-associated viral (AAV) vector to express a pH sensitive fluorescent protein, mKeima, fused to the mitochondrial targeting sequence of cytochrome oxidase subunit 8A (mKeima-mito). mKeima-mito allows for quantification of mitochondrial autophagy (mitophagy) in live-cell time-lapse imaging experiments. We also developed an image analysis pipeline to selectively quantify mKeima-mito while removing the signal of auto-fluorescent pigment from the dataset by utilizing information from the mKeima fluorescent channels. These techniques are demonstrated in primary RPE cultures expressing mKeima-mito treated with 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP), an uncoupler that depolarizes the mitochondrial membrane and leads to mitochondrial fragmentation and mitophagy. The techniques outlined provide a roadmap for investigating disease mechanisms or the effect of treatments utilizing fluorescent probes in an important cell culture model.

Testing Mitochondrial-Targeted Drugs in iPSC-RPE from Patients with Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. No universally effective treatments exist for atrophic or "dry" AMD, which results from loss of the retinal pigment epithelium (RPE) and photoreceptors and accounts for ≈80% of all AMD patients. Prior studies provide evidence for the involvement of mitochondrial dysfunction in AMD pathology. This study used induced pluripotent stem cell (iPSC) RPE derived from five AMD patients to test the efficacy of three drugs (AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), Metformin, trehalose) that target key processes in maintaining optimal mitochondrial function. The patient iPSC-RPE lines were used in a proof-of-concept drug screen, utilizing an analysis of RPE mitochondrial function following acute and extended drug exposure. Results show considerable variability in drug response across patient cell lines, supporting the need for a personalized medicine approach for treating AMD. Furthermore, our results demonstrate the feasibility of using iPSC-RPE from AMD patients to develop a personalized drug treatment regime and provide a roadmap for the future clinical management of AMD.

No association between cataract surgery and mitochondrial DNA damage with age-related macular degeneration in human donor eyes.

PURPOSE: To determine whether age-related macular degeneration (AMD) severity or the frequency of retinal pigment epithelium mitochondrial DNA lesions differ in human donor eyes that have undergone cataract surgery compared to phakic eyes. METHODS: Eyes from human donors aged ≥ 55 years were obtained from the Minnesota Lions Eye Bank. Cataract surgery status was obtained from history provided to Eye Bank personnel by family members at the time of tissue procurement. Donor eyes were graded for AMD severity using the Minnesota Grading System. Quantitative PCR was performed on DNA isolated from macular punches of retinal pigment epithelium to quantitate the frequency of mitochondrial DNA lesions in the donor tissue. Univariable and multivariable analyses were performed to evaluate for associations between (1) cataract surgery and AMD severity and (2) cataract surgery and mitochondrial DNA lesion frequency. RESULTS: A total of 157 subjects qualified for study inclusion. Multivariable analysis with age, sex, smoking status, and cataract surgery status showed that only age was associated with AMD grade. Multivariable analysis with age, sex, smoking status, and cataract surgery status showed that none of these factors were associated with retinal pigment epithelium mitochondrial DNA lesion frequency. CONCLUSIONS: In this study of human donor eyes, neither retinal pigment epithelium mitochondrial DNA damage nor the stage of AMD severity are independently associated with cataract surgery after adjusting for other AMD risk factors. These new pathologic and molecular findings provide evidence against a relationship between cataract surgery and AMD progression and support the idea that cataract surgery is safe in the setting of AMD.

OGT Regulates Mitochondrial Biogenesis and Function via Diabetes Susceptibility Gene Pdx1.

O-GlcNAc transferase (OGT), a nutrient sensor sensitive to glucose flux, is highly expressed in the pancreas. However, the role of OGT in the mitochondria of ß-cells is unexplored. In this study, we identified the role of OGT in mitochondrial function in ß-cells. Constitutive deletion of OGT (ßOGTKO) or inducible ablation in mature ß-cells (ißOGTKO) causes distinct effects on mitochondrial morphology and function. Islets from ßOGTKO, but not ißOGTKO, mice display swollen mitochondria, reduced glucose-stimulated oxygen consumption rate, ATP production, and glycolysis. Alleviating endoplasmic reticulum stress by genetic deletion of Chop did not rescue the mitochondrial dysfunction in ßOGTKO mice. We identified altered islet proteome between ßOGTKO and ißOGTKO mice. Pancreatic and duodenal homeobox 1 (Pdx1) was reduced in in ßOGTKO islets. Pdx1 overexpression increased insulin content and improved mitochondrial morphology and function in ßOGTKO islets. These data underscore the essential role of OGT in regulating ß-cell mitochondrial morphology and bioenergetics. In conclusion, OGT couples nutrient signal and mitochondrial function to promote normal ß-cell physiology.

Mitochondria: The Retina's Achilles' Heel in AMD.

Strong experimental evidence from studies in human donor retinas and animal models supports the idea that the retinal pathology associated with age-related macular degeneration (AMD) involves mitochondrial dysfunction and consequent altered retinal metabolism. This chapter provides a brief overview of mitochondrial structure and function, summarizes evidence for mitochondrial defects in AMD, and highlights the potential ramifications of these defects on retinal health and function. Discussion of mitochondrial haplogroups and their association with AMD brings to light how mitochondrial genetics can influence disease outcome. As one of the most metabolically active tissues in the human body, there is strong evidence that disruption in key metabolic pathways contributes to AMD pathology. The section on retinal metabolism reviews cell-specific metabolic differences and how the metabolic interdependence of each retinal cell type creates a unique ecosystem that is disrupted in the diseased retina. The final discussion includes strategies for therapeutic interventions that target key mitochondrial pathways as a treatment for AMD.

Impaired Mitochondrial Function in iPSC-Retinal Pigment Epithelium with the Complement Factor H Polymorphism for Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is characterized by loss of the retinal pigment epithelium (RPE). While the disease mechanism remains unclear, prior studies have linked AMD with RPE mitochondrial defects and genetic polymorphisms in the complement pathway. This study used RPE generated from induced pluripotent stem cells (iPSC-RPE), which were derived from human donors with or without AMD and genotyped for the complement factor H (CFH) AMD high-risk allele (rs1061170, Y402H) to investigate whether donor disease state or genotype had a detrimental effect on mitochondrial function and inflammation. Results show that cells derived from donors with AMD display decreased mitochondrial function under conditions of stress and elevated expression of inflammatory markers compared to iPSC-RPE from individuals without AMD. A more pronounced reduction in mitochondrial function and increased inflammatory markers was observed in CFH high-risk cells, irrespective of disease state. These results provide evidence for a previously unrecognized link between CFH and mitochondrial function that could contribute to RPE loss in AMD patients harboring the CFH high-risk genotype.

Glutathione Metabolism and the Novel Role of Mitochondrial GSH in Retinal Degeneration.

Glutathione (GSH) is present ubiquitously, and its role as a crucial cellular antioxidant in tissues, including the retina, is well established. GSH's antioxidant function arises from its ability to scavenge reactive oxygen species or to serve as an essential cofactor for GSH S-transferases and peroxidases. This review summarizes the general functions, retinal distribution, disorders linked to GSH deficiency, and the emerging role for mitochondrial GSH (mGSH) in retinal function. Though synthesized only in the cytosol, the presence of GSH in multiple cell organelles suggests the requirement for its active transport across organellar membranes. The localization and distribution of 2-oxoglutarate carrier (OGC) and dicarboxylate carrier (DIC), two recently characterized mitochondrial carrier proteins in RPE and retina, show that these transporters are highly expressed in human retinal pigment epithelium (RPE) cells and retinal layers, and their expression increases with RPE polarity in cultured cells. Depletion of mGSH levels via inhibition of the two transporters resulted in reduced mitochondrial bioenergetic parameters (basal respiration, ATP production, maximal respiration, and spare respiratory capacity) and increased RPE cell death. These results begin to reveal a critical role for mGSH in maintaining RPE bioenergetics and cell health. Thus, augmentation of mGSH pool under GSH-deficient conditions may be a valuable tool in treating retinal disorders, such as age-related macular degeneration and optic neuropathies, whose pathologies have been associated with mitochondrial dysfunction.

Automating Human Induced Pluripotent Stem Cell Culture and Differentiation of iPSC-Derived Retinal Pigment Epithelium for Personalized Drug Testing.

Derivation and differentiation of human induced pluripotent stem cells (hiPSCs) provide the opportunity to generate medically important cell types from individual patients and patient populations for research and the development of potential cell therapies. This technology allows disease modeling and drug screening to be carried out using diverse population cohorts and with more relevant cell phenotypes than can be accommodated using traditional immortalized cell lines. However, technical complexities in the culture and differentiation of hiPSCs, including lack of scale and standardization and prolonged experimental timelines, limit the adoption of this technology for many large-scale studies, including personalized drug screening. The entry of reproducible end-to-end automated workflows for hiPSC culture and differentiation, demonstrated on commercially available platforms, provides enhanced accessibility of this technology for both research laboratories and commercial pharmaceutical testing. Here we have utilized TECAN Fluent automated cell culture workstations to perform hiPSC culture and differentiation in a reproducible and scalable process to generate patient-derived retinal pigment epithelial cells for downstream use, including drug testing. hiPSCs derived from multiple donors with age-related macular degeneration (AMD) were introduced into our automated workflow, and cell lines were cultured and differentiated into retinal pigment epithelium (RPE). Donor hiPSC-RPE lines were subsequently entered in an automated drug testing workflow to measure mitochondrial function after exposure to "mitoactive" compounds. This work demonstrates scalable, reproducible culture and differentiation of hiPSC lines from individuals on the TECAN Fluent platform and illustrates the potential for end-to-end automation of hiPSC-based personalized drug testing.

Improving retinal mitochondrial function as a treatment for age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Currently, there are no treatments for dry AMD, which is characterized by the death of retinal pigment epithelium (RPE) and photoreceptors. Reports from human donors with AMD suggest that RPE mitochondrial defects are a key event in AMD pathology. Thus, the most effective strategy for treating dry AMD is to identify compounds that enhance mitochondrial function and subsequently, preserve the RPE. In this study, primary cultures of RPE from human donors with (n = 20) or without (n = 8) AMD were used to evaluate compounds that are designed to protect mitochondria from oxidative damage (N-acetyl-l-cysteine; NAC), remove damaged mitochondria (Rapamycin), increase mitochondrial biogenesis (Pyrroloquinoline quinone; PQQ), and improve oxidative phosphorylation (Nicotinamide mononucleotide, NMN). Mitochondrial function measured after drug treatments showed an AMD-dependent response; only RPE from donors with AMD showed improvements. All four drugs caused a significant increase in maximal respiration (p < 0.05) compared to untreated controls. Treatment with Rapamycin, PQQ, or NMN significantly increased ATP production (p < 0.05). Only Rapamycin increased basal respiration (p < 0.05). Notably, robust responses were observed in only about 50% of AMD donors, with attenuated responses observed in the remaining AMD donors. Further, within the responders, individual donors exhibited a distinct reaction to each drug. Our results suggest drugs targeting pathways involved in maintaining healthy mitochondria can improve mitochondrial function in a select population of RPE from AMD donors. The unique response of individual donors to specific drugs supports the need for personalized medicine when treating AMD.

Investigating AKT activation and autophagy in immunoproteasome-deficient retinal cells.

Two major proteolytic systems, the proteasome and the autophagy pathway, are key components of the proteostasis network. The immunoproteasome, a proteasome subtype, and autophagy are upregulated under stress conditions, forming a coordinated unit designed to minimize the effect of cell stress. We investigated how genetic ablation of the LMP2 immunoproteasome subunit affects autophagy in retinal pigment epithelium (RPE) from WT and LMP2 knockout mice. We monitored autophagy regulation by measuring LC3, phosphorylation of AKT (S473), and phosphorylation of S6, a downstream readout of AKT (mTOR) pathway activation. We also evaluated transcription factor EB (TFEB) nuclear translocation, a transcription factor that controls expression of autophagy and lysosome genes. WT and LMP2 KO cells were monitored after treatment with EBSS to stimulate autophagy, insulin to stimulate AKT, or an AKT inhibitor (trehalose or MK-2206). Under basal conditions, we observed hyper-phosphorylation of AKT and S6, as well as lower nuclear-TFEB content in LMP2 KO RPE compared with WT. AKT inhibitors MK-2206 and trehalose significantly inhibited AKT phosphorylation and stimulated nuclear translocation of TFEB. Starvation and AKT inhibition upregulated autophagy, albeit to a lesser extent in LMP2 KO RPE. These data support the idea that AKT hyper-activation is an underlying cause of defective autophagy regulation in LMP2 KO RPE, revealing a unique link between two proteolytic systems and a previously unknown function in autophagy regulation by the immunoproteasome.