Restoration of Rod Bipolar Cells Following Retinitis Pigmentosa: Changes in Dendritic Complexity after Retinal Degeneration, Retinal Prostheses Design, and AMIGO2-Knockdown Gene Therapy Assessment - PROJECT SUMMARY/ABSTRACT Vision begins when rod and cone photoreceptors in the retina capture light and send signals through interneurons to ganglion cells, which relay the information to the brain. In retinitis pigmentosa (RP), an inherited retinal disease affecting over two million people, photoreceptors progressively degenerate, triggering degeneration of nearby neurons. This disrupts signal transmission, leading to night blindness, peripheral vision loss, and eventually total blindness. As photoreceptors degenerate, transplanted photoreceptors are a promising therapy for restoring vision. However, recent trials highlight challenges in re-establishing retinal signal flow. Transplanted photoreceptors must connect with remaining interneurons, which are about 80 percent intact, but often fail to integrate effectively. Although progress has been made in therapies targeting photoreceptor restoration, these are limited by the degeneration of interneurons, such as rod bipolar cells (RBCs). RBCs are critical interneurons that relay signals from photoreceptors to ganglion cells. During RP progression, RBC dendritic arbors deteriorate. This deterioration disrupts their ability to form new connections with transplanted rods or retinal prosthetics, creating a major obstacle to restoring the retinal signal pathway and achieving full visual recovery. This project focuses on understanding and reversing RBC degeneration to enhance retinal therapies. Aim 1 seeks to delineate the sequence of RBC dendritic changes during RP progression using the RhoP23H mouse model, representing the most common genetic mutation causing RP in humans. Sparse labeling of RBCs, combined with confocal imaging, will quantify dendritic complexity. Fractal dimension (D) will assess arbor complexity, while convex hull volume (V) will measure arbor volume across disease stages. This approach will establish a timeline of RBC morphological decline, identify windows for therapeutic intervention, and generate data on RBC arbors to inform future retinal prosthesis design. Aim 2 investigates the potential of AMIGO2 knockdown using AAV2-CRISPR to restore RBC dendritic architecture. AMIGO2, a membrane protein implicated in dendritic growth, has shown promise in promoting structural recovery in preliminary studies. This project will evaluate the impact of AMIGO2 knockdown on RBC morphology and investigate its role in regulating key signaling pathways, such as the Akt pathway. Additionally, single-cell RNA sequencing will profile RBC-specific gene expression in both healthy and diseased retinas, providing insights into molecular mechanisms underlying dendritic remodeling. By combining advanced imaging techniques, gene therapy, and transcriptomic analysis, this research aims to address critical gaps in our understanding of RBC degeneration. The outcomes of this project will inform strategies for optimizing retinal prostheses, improving photoreceptor transplantation, and developing RBC- targeted therapies. Successful completion of these studies will deepen our understanding of RP progression and contribute to transformative therapies that restore vision for millions of affected individuals.
