The role of microRNAs in Müller glia reprogramming into neurons in the postnatal murine retina: cellular analysis in vitro and in vivo

• Applicant: Dr. Stefanie Wohl
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells ; Ophthalmology ; Evolutionary Cell and Developmental Biology (Zoology)
• Term: from 2014 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 259990820

MicroRNAs (miRNAs) are small, non-coding RNA molecules that act as translational repressors and play important roles in the development and function of different tissues. Recent studies have shown that miRNAs regulate several key steps in retinal development and photoreceptor function. In addition, studies of regenerating zebrafish retina have implicated miRNAs in regeneration. This process occurs via a specialized type of retinal glia, the Müller glia. In the fish retina, Müller glia cells can dedifferentiate into progenitor-like cells and regenerate damaged neurons after injury. This phenomenon has been variously called transdifferentiation or regulated reprogramming, but the latter term is more appropriate since transdifferentiation implies direct conversion of one cell type into another. Key regulators in this regulated reprogramming are miRNAs. In mammals, the potential for regeneration of neurons from Müller glia appears to be repressed, although the underlying mechanisms for this repression are still unknown. Until now, there are no reports analyzing miRNAs in mammalian Müller glia. Therefore, the overall goal of this proposal is to reveal the set of miRNAs expressed by murine Müller glia cells in order to 1. Determine if specific miRNA repress the reprogramming potential of murine Müller glia cells and 2. Analyze if overexpression of miRNAs, which are required to induce neurogenesis, can reprogram murine Müller glia into retinal progenitor cells and/or mature neurons. Gain and loss of function experiments of promising candidates which have been described for other cell types, i.e. the miRNAs let-7, as repressor of regeneration, and miR-124, as activator of neurogenesis, will be performed by means of lentiviral gene transfer and RNA transfection in Müller glia primary cell cultures of postnatal day (P) 12 mice as well as in vivo after N-methyl-D-aspartate (NMDA) damage in adult mice. Cellular analysis will be conducted by RT-qPCR, Western Blot, and immunofluorescent stainings with subsequent confocal microscopy. In order to reveal the entire set of murine Müller glia miRNAs, and find new potential candidates for Müller glia reprogramming, next generation RNA-Sequencing will be performed, an innovative method to uncover presence and quantity of RNA at a certain time point. These proposed experiments will provide insight into the molecular regulation of the Müller glia cell state and whether or not miRNAs can be used as a tool to successfully reprogram murine Müller glia cells to restore retinal tissue following damage.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Thomas A. Reh, Ph.D.