Molecular Mechanisms That Regulate Cellular Clearance Functions in the RPE

The goal of this project is to develop novel targets with significant therapeutic potential to enhance cellular clearance functions in the RPE in order to protect these cells against cell death. The goal of this project is to develop effective therapeutic strategies to induce cellular clearance in the RPE. Transcription factor EB (TFEB) is a master regulator of autophagy and lysosomal function. We hypothesize that induction of TFEB will alleviate substrate accumulation in the RPE. We propose in Specific Aim 1, to study the functional effects of gene transfer of constitutively active TFEB in enhancing lysosomal function and autophagy in the RPE. In Specific Aim 2, we will study the cellular effects of inhibiting micro-RNA (miRNA)-mediated suppression of TFEB expression.

Age-related macular degeneration (AMD) is a common degenerative disease of the eye and is the leading cause of vision loss among the elderly. One of the causative factors for the disease is build-up of cellular waste with advancing age due to defective cellular waste-disposal mechanisms. Lysosomes are cellular organelles responsible for clearance of cellular waste and these organelles decline in their ability to process cellular waste with advancing age. Our proposal addresses strategies by which the degradative ability of lysosomes can be enhanced or restored to augment clearance of cellular waste. These strategies help not only keep the intracellular environment of the cell clean but also promote the overall cellular health. All cells have the ability to activate expression of proteins that help with their ability to clear cellular waste. In the cells of the eye that are affected in AMD, these proteins are either non-functional or not expressed in optimal levels. In this study, we propose to develop therapeutics that allow these cells to express functional levels of proteins that help alleviate accumulation of pathological cellular material.

We used novel approaches to study the cellular impact of induction of TFEB, a master cellular regulator of autophagy and lysosomal function. These strategies are expected to enhance clearance of undigested cellular material in the RPE. We propose to 1). Use an adeno-associated viral vector (AAV)-mediated delivery of constitutively active TFEB to the RPE, and 2). Use of oligonucleotides to that inhibit microRNA-binding to TFEB. These studies are expected to establish a strategy for sustained activation of TFEB expression in the RPE.

Restoring lysosomal function and autophagy in RPE cells can be effective in preventing RPE cell dysfunction in the pathogenesis of Age-related Macular Degeneration (AMD). In order to achieve this, it is imperative to understand the mechanisms regulating autophagy and lysosomal function in the RPE and our knowledge in this regard is limited. The goal of this project is to identify molecular mechanisms regulating autophagy and lysosomal function and to use this knowledge to develop effective therapeutic strategies to inhibit the loss of RPE cells in AMD.