The Vollrath lab studies mechanisms of homeostasis, aging, and neurodegeneration of the mammalian outer retina. Much of our work centers on understanding interactions of glial-like retinal pigment epithelial (RPE) cells with light-sensing photoreceptor neurons. We are particularly interested in how changes in cellular energy metabolism affect RPE and photoreceptor function, and in identifying genes and pathways that counteract photoreceptor degeneration.
The RPE is critical for retinal homeostasis.
The RPE is a highly differentiated, post-mitotic cell layer interposed between the choroidal blood supply and photoreceptors in the outer retina. The RPE plays a crucial role in vision by performing a host of important functions. The RPE transports carbon dioxide, water, lactate and ions from the subretinal space to the choroidal circulation, thus maintaining the chemical composition of the RPE/PR interface. A majority of glucose utilized by the entire retina is transported from the choroid by the RPE. The RPE vectorally secretes growth factors such as VEGF and PEDF, and performs multiple steps in retinoid (vitamin A) metabolism including regeneration of the rhodopsin chromophore, 11-cis retinal. Melanin in RPE melanosomes absorbs stray photons providing directionality to the light stimulus and enhancing retinal image resolution. The circadian phagocytosis of photoreceptor outer segment (OS) tips by the RPE acts in concert with new disc synthesis by the photoreceptors to ensure the continuous renewal of OS proteins and lipids, which are subject to oxidative and photic damage. Genetic disorders and animal models demonstrate the essential nature of many of these functions to outer retinal health.
Altered RPE energy metabolism is implicated in retinal pathogenesis.
The RPE requires ample energy to fuel membrane pumps and drive the biochemical reactions necessary to degrade and synthesize cellular molecules, consistent with an abundance of mitochondria in the tissue. Compromised RPE mitochondrial function has been posited as a contributing cause of age-related macular degeneration (AMD). Individuals with diminished OXPHOS due to mitochondrial DNA mutations frequently exhibit macular pigmentary retinopathy, and specific mitochondrial haplogroups have been associated with AMD risk. Importantly, RPE mitochondrial DNA damage is more extensive in AMD, and the degree of damage correlates with disease severity.
RPE energy metabolism has a profound influence on cell phenotype and photoreceptor viability.
Work from our lab has shown that enforced changes in cellular energy metabolism in vivo can drive dedifferentiation and transdifferentiation of the RPE. Our study in mice demonstrated that selective ablation of RPE OXPHOS capability triggers increased aerobic glycolysis, activation of the mTOR pathway, RPE dedifferentiation and a series of gradual morphological alterations, some of which are reminiscent of those documented in dry AMD. Photoreceptors suffer from these RPE changes and ultimately degenerate, but inhibition of mTOR can blunt both RPE and photoreceptor dysfunction. These findings emphasize the causal links between RPE cellular energy metabolism, RPE cell phenotype, and photoreceptor vitality.
Current metabolism studies
Our goal is to obtain a detailed understanding of the relationship between RPE cellular energy metabolism and RPE and photoreceptor cell phenotypes in health, aging, and disease. We are genetically manipulating our OXPHOS-deficient RPE mouse model to determine which aspects of the altered phenotype result from lack of ATP production via OXPHOS versus those arising from loss of electron transfer to oxygen. We have also created a new mouse model to determine whether increased aerobic glycolysis alone, in the presence of intact OXPHOS, can activate cell growth pathways and cause RPE dedifferentiation/transdifferentiation, and to tell us which features of the RPE glycolytic phenotype are reversible through rebalancing of metabolism. To better understand the human RPE, we are studying a collection of primary cell lines from different donors to correlate specific genetic variants with changes in gene expression [i.e., expression quantitative trait loci (eQTL)] and with variation in cellular metabolic and phagocytic functions. We are also developing a new human RPE cell culture model of induced mitochondrial dysfunction.
Saving photoreceptors from degeneration
The daily ‘big breakfast’ of OS material ingested by the post-mitotic RPE, summed over the life of an animal, distinguishes it as likely the most phagocytic cell type in the body. Defective RPE phagocytosis in Royal College of Surgeons (RCS) rats causes photoreceptor degeneration and demonstrates that OS phagocytosis is essential for homeostasis of the mammalian retina. By positional cloning of the mutant RCS gene, we identified MERTK as a critical part of the phagocytic mechanism. We also identified mutations in the human MERTK gene in individuals with a retinal degenerative disease known as retinitis pigmentosa, thereby defining the RP38 locus. TYRO3, AXL and MERTK constitute the TAM family of receptor tyrosine kinases. It is now appreciated that TAM receptors function in a diverse array of phagocytic processes. Our identification of Mertk as the gene mutated in RCS rats was the first connection of TAM receptors to the process of phagocytosis. Mice homozygous for a targeted null allele of Mertk exhibit a rapid loss of photoreceptors, just like RCS rats. While crossing the Mertk knockout allele to a new genetic background, we discovered a modifier gene that almost completely suppresses photoreceptor degeneration in MERTK-deficient mice. Our new work indicates that Tyro3 is the putative modifier gene, and that the likely mechanism of the modifier effect is variation in the level of TYRO3 expression in the RPE due to a cis (or local) eQTL. Our findings highlight the potential for eQTL to impact the phenotypic variability of human inherited photoreceptor degenerations, and motivate our global search for eQTL in the human RPE.
Our modifier and preclinical gene therapy studies (see Publications), address the need for therapy for MERTK-associated photoreceptor degeneration. However, inherited photoreceptor degenerations are incredibly genetically heterogeneous; more than 200 genes are currently associated with various types and new genes are regularly identified. Development of specialized therapies for each genetic form of photoreceptor degeneration would take many years and may ultimately be impractical. Therapies that counteract photoreceptor degeneration, regardless of genetic cause, could improve the quality of life of those suffering from these blinding diseases, and provide increased time for discovery of curative therapies. We have devised an unbiased screen to identify gene mutations that slow photoreceptor degeneration in mouse models. The genes/proteins and pathways we identify will be excellent targets for the development of more general human therapies. Our approach can be extended to other forms of neurodegeneration. Comparison of survival-promoting genes among different types of nerves dying due to a variety of underlying causes will be revelatory.