The Role of Retinal Progenitor microRNAs for Late-stage Progenitor Cell State and Muller Glia Reprogramming

PROJECT SUMMARY Our long-term goal is to prevent blindness, either by interfering with disease progression or by developing cell replacement therapies to restore vision. We believe that microRNAs (miRNAs) are a very powerful and innovative tool to accomplish this, but we first need to identify the set of miRNAs required for retinal cell fate specification and proper cell function in the developing retina. These miRNAs might represent potential therapeutic agents to not only restore imbalances that occur with the onset of retinal disorders, but also to induce specific cell fates for Müller glia (MG) reprogramming. We therefore propose to investigate the role of retinal progenitor cell (RPC)- miRNAs for late-stage RPC state and function and MG reprogramming by Aim 1: identifying the specific miRNAs required in early postnatal development, when rod photoreceptors (PR), bipolar cells (BCs), and MG are generated. Aim 2: testing miRNAs to reprogram MG into functional retinal neurons with focus on BCs and rod PR. We propose to use a mouse line that will not be able to produce miRNAs in their RPCs to better understand the function of miRNAs in retinal development and diseases. We will analyze tissue and cells with regard to morphological and functional alterations and determine which miRNAs are responsible for changes in cellular behavior. If we discover certain cell types that do not form properly without miRNAs, this could mean that specific miRNAs are required for proper development of that cell type. Rescue experiments will show whether disturbed tissue can be restored by miRNA supplementation and would imply potential therapeutic use. These miRNAs might also be new, additional reprogramming factors to regenerate specific cell types from stem cells or MG. My previous work has shown that miRNAs can reprogram MG into neuronal-like cells similar to BCs. However, whether these reprogrammed neurons are functional, and whether other neurons are generated, is still unknown. To address these questions, we will reprogram primary MG from reporter mice to visualize the conversion of MG into RPCs and neuronal-like cells, use patch clamp to measure ion currents, and profile their gene expression. miRNA candidates that successfully converted MG into functional neurons will be tested subsequently in 3D organotypic cultures (intact retinas outside the organism). Explants will be treated with miRNAs and evaluated with regard to cell proliferation and proper differentiation capability. miRNA candidates that can induce MG reprogramming ex vivo will be utilized for in vivo reprogramming approaches. To reveal underlying mechanisms and true miRNAs targets, we will use target prediction and target validation tools to narrow down and test selected candidates via sensors (in vitro proof of miRNA:mRNA prediction) and rescue experiments. This work provides a comprehensive study of miRNAs by combining molecular and cellular analyses with functional testing, in vitro, ex vivo and in vivo and will reveal (1) the set miRNAs required for proper retinal development and cell fate specification of late-born retinal neurons and (2) the set of miRNAs that can reprogram MG into functional neurons.