Author ORCID Identifier
Semester
Spring
Date of Graduation
2025
Document Type
Dissertation (Campus Access)
Degree Type
PhD
College
School of Medicine
Department
Exercise Physiology
Committee Chair
Jianhai Du
Committee Co-Chair
Paul D. Chantler
Committee Member
Paul D. Chantler
Committee Member
Dharendra Thapa
Committee Member
Emidio E. Pistilli
Committee Member
Saravanan Kolandaivelu
Abstract
Purpose: The retinal pigment epithelium (RPE) plays a critical role in maintaining vision by performing essential functions including visual cycle processing, nutrient transport, protein synthesis, protection against oxidative stress, cytokine secretion, and phagocytosis of outer segments. RPE dysfunction can cause photoreceptor cell death in inherited retinal degeneration and age-related retinal degeneration (AMD), resulting in blindness. RPE relies on its robust mitochondrial metabolism to support its multifaceted functions. However, it remains unclear how healthy RPE mitochondria fuel their metabolism and how substrate utilization is impaired in diseased RPE.
Nicotinamide N-methyl transferase, an enzyme that methylates nicotinamide into 1-methyl nicotinamide (1-MNAM) is a master regulator of mitochondrial metabolism. Transcriptomics data show that NNMT is highly expressed in RPE, and its expression is further upregulated in RPE from patients with age-related macular degeneration (AMD). A gap in knowledge remains regarding how NNMT regulates RPE substrate utilization and mitochondrial metabolism in RPE. The purpose of this work is to comprehensively evaluate nutrient utilization in healthy and diseased RPE and determine the role of NNMT in RPE metabolism. The central hypothesis is that healthy human RPE will have a high metabolic flexibility in nutrient utilization which will be disrupted in diseased RPE. NNMT is expressed in RPE to regulate its unique metabolism, but its excessive activity will contribute to metabolic changes in aging and AMD.
Methods and Results: A novel approach was developed using Biolog Phenotype Microarray Assays to screen substrate utilization in human RPE cells. Five human RPE cells were used for this substrate screening, including dedifferentiated fetal RPE (fRPE), induced pluripotent stem cell-derived RPE (iPSC RPE), Sorsby Fundus dystrophy (SFD) patient-derived iPSC RPE, CRISPR-corrected isogenic SFD (cSFD) iPSC RPE, and ARPE-19 cell lines. Some changes were further validated with stable isotope tracing using mass spectrometry. Differentiated fRPE and healthy iPSC RPE cells could use up to 51 nutrients. However, when dedifferentiated, fRPE used far fewer nutrients, primarily sugar and glutamine-related amino acids. SFD RPE can use 37 nutrients; however, compared to cSFD RPE and healthy iPSC RPE, they were unable to use lactate, some TCA cycle intermediates, and short-chain fatty acids. Nonetheless, they showed increased use of branched-chain amino acids (BCAAs) and BCAA-containing dipeptides. Dedifferentiated ARPE-19 cells grown in traditional culture media were incapable of utilizing lactate and ketone bodies. In contrast, nicotinamide supplementation promotes differentiation toward an epithelial phenotype, restoring the ability to use these nutrients.
NNMT expression was quantified by quantitative PCR and immunoblot. Human RPE cells, isolated mouse retina, and mouse RPE were incubated with deuterium nicotinamide to trace NAD synthesis and degradation. Mitochondrial oxygen consumption was measured using the Agilent Seahorse assay. Pharmacological inhibitors and constructs packaged in lentivirus or adeno-associated virus (AAV) were used for loss or gain of function studies. NNMT expression and activity were limited to the RPE but almost absent in the retina. RPE contained more 1-MNAM, the product of NNMT, than any other tissues. 1-MNAM was actively exported by the RPE to the retina. The inhibition of NNMT inhibited mitochondrial respiration and disrupted RPE nutrient utilization. Finally, NNMT was upregulated in aged RPE and RPE from AMD patients, and excessive NNMT activity impaired RPE morphology.
Conclusion: Healthy RPE cells have high flexibility in using different nutrients because of their epithelial phenotype. SFD RPE cells have reduced metabolic flexibility, relying on the oxidation of BCAAs. Our findings highlight the important roles of nutrient availability and use in RPE differentiation and diseases. NNMT is enriched in RPE, and its optimal expression is crucial for RPE nutrient utilization, metabolism, and morphology. Excessive NNMT activity in RPE may underlie pathology in aging and AMD.
Recommended Citation
Rizwan, Saira, "Nutrient Utilization and Regulation in Retinal Pigment Epithelium" (2025). Graduate Theses, Dissertations, and Problem Reports. 12829.
https://researchrepository.wvu.edu/etd/12829