(limit: 30 lines of text) 2 As we age, diseases such as glaucoma, diabetic retinopathy, and age-related macular degeneration (AMD) 3 result in irreversible loss of retinal neurons and thus sight, which leads to a reduced quality of life and a 4 significant clinical burden. While several therapeutic approaches are being pursued, recent advances in gene 5 therapy show promise for treating genetic disorders leading to retinal degeneration. It would also be 6 advantageous if a similar approach could be employed in retinal regenerative strategies for diseases such as 7 AMD, which has no obvious genetic cause. One potential source of retinal regeneration may be found within 8 mammalian Mller glial cells (MGs). Unlike mammalian counterparts, zebrafish MGs have the ability to 9 generate multipotent cells that can differentiate into new retinal neurons capable of restoring vision. Our lab is 10 currently investigating the cellular and molecular mechanisms that potentially block mammalian MG-cell 11 mediated retinal regeneration and we have uncovered such a role for the Hippo signaling pathway. Therefore, 12 my proposal aims to use Adeno-associated virus (AAV)-mediated gene delivery to manipulate the Hippo 13 pathway within mouse MGs and assess their regenerative potential. 14 The Hippo signaling pathway has been shown to restrain cardiomyocyte proliferation and disruption of 15 Hippo signaling significantly promotes regeneration of the mammalian heart. In my first co-first author 16 publication, by using transgenic expression of a constitutively active form of the transcription cofactor YAP 17 (YAP5SA), I was able to bypass the negative regulation of Hippo signaling. These Hippo non-responsive MGs 18 spontaneously entered the cell cycle and reprogrammed to an identity more reminiscent of multipotent retinal 19 progenitor cells (RPCs). Recently, it was shown that overexpression of the Wnt pathway effector -CATENIN 20 also reprogrammed MGs into a RPC-like state, which could then be re-differentiated into functional rods to 21 restore vision. In other systems, YAP and -CATENIN have been shown to functionally cooperate in a 22 transcription factor complex. Taken together, these data have led me to hypothesize that YAP and -CATENIN 23 cooperatively drive MG reprogramming to a multipotent state, capable of differentiating into retinal neurons. 24 The main objective of this project is to determine whether YAP and -CATENIN similarly reprogram 25 MGs, resulting in the same neurogenic potential. First, I will determine the physical interaction between YAP 26 and -CATENIN in MGs. Then, using AAV-mediated gene delivery to -Catenin mutant MGs, I will determine 27 whether YAP5SA-mediated MG reprogramming requires active -CATENIN. Finally, I will determine whether 28 YAP5SA-reprogrammed MGs are indeed multipotential and thereby capable of differentiation into several 29 retinal neuron identities. Successful completion of this proposal will likely lead to future development of AAV 30 approaches to manipulate both the Hippo and Wnt pathways for bona fide retinal regeneration.
Unlike the mammalian retina, Mller glial cells (MGs) of zebrafish retina have the remarkable ability to regenerate lost neurons and restore vision. In preliminary studies, we have found that the Hippo signaling pathway serves as an intrinsic blocking mechanism that prevents mouse MGs from proliferating and reprogramming to a progenitor-like state during damage. This proposal aims to determine whether bypass of Hippo signaling results in MG-mediated regeneration of the mouse retina akin to zebrafish.
