In vivo Regeneration of Ganglion Cells for Vision Restoration in Mammalian Retinas

Abstract

Glaucoma and other optic neuropathies affect millions of people worldwide, ultimately causing progressive and irreversible degeneration of retinal ganglion cells (RGCs) and blindness. Previous research into cell replacement therapy of these neurodegenerative diseases has been stalled due to the incapability for grafted RGCs to integrate into the retina and project properly along the long visual pathway. In vivo RGC regeneration would be a promising alternative approach but mammalian retinas lack regenerative capacity. It therefore has long been a great challenge to regenerate functional and properly projecting RGCs for vision restoration in mammals. Here we show that the transcription factors (TFs) Math5 and Brn3b together are able to reprogram mature mouse Müller glia (MG) into RGCs. The reprogrammed RGCs extend long axons that make appropriate intra-retinal and extra-retinal projections through the entire visual pathway to innervate both image-forming and non-image-forming brain targets. They exhibit typical neuronal electrophysiological properties and improve visual responses in RGC loss mouse models. Together, our data provide evidence that mammalian MG can be reprogrammed by defined TFs to achieve in vivo regeneration of functional RGCs as well as a promising new therapeutic approach to restore vision to patients with glaucoma and other optic neuropathies.

Critical Examination of Müller Glia-Derived in vivo Neurogenesis in the Mouse Retina

“…During the past decade, many groups have sought to identify key pathways and transcription factors responsible for MG-mediated retinal repair in zebrafish and other species, with the hope of recapitulating the full regenerative processes in mammals. Having built on these findings, several recent high-profile in vivo studies show that after manipulating key regulators in the adult mouse retina, endogenous MG can be reprogrammed to generate multiple types of retinal neurons, including rod photoreceptors ( Yao et al, 2018 ), bipolar or amacrine-like interneurons ( Jorstad et al, 2017 ; Hoang et al, 2020 ), and RGCs ( Zhou et al, 2020 ; Xiao et al, 2021 ).…”

“…The first study by Zhou et al utilized the CRISPR-CasRx system to knock down the expression of Ptbp1 , resulting in MG-to-RGC conversion in the adult mouse retina ( Zhou et al, 2020 ). The second study by Xiao et al reported highly efficient MG-to-RGC conversion after ectopic expression of Math5 and Brn3b, two transcription factors that promote the RGC fate ( Xiao et al, 2021 ). Strikingly, these two studies also reported that newly generated RGC regrew their axons through long-distance travel within the optic nerve all the way to the target regions in the brain, re-establishing the retina-brain connection.…”

“…As a result, MG were exclusively labeled by the AAV-mediated expression system. In another study, MG were marked in three different ways: Firstly by injecting AAV-GFAP-GFP (control) or AAV-GFAP-Math5-Brn3b-GFP into the wild-type mouse retina; Secondly by co-injecting AAV-GFAP-tdTomato-Cre with AAV-CAG-FLEX-GFP (control) or AAV-CAG-FLEX-Math5-Brn3b-GFP into the wild-type mouse retina; thirdly by injecting AAV-CAG-FLEX-GFP (control) or AAV-CAG-FLEX-Math5-Brn3b-GFP into the retinas of tamoxifen-induced Glast-CreER mice ( Xiao et al, 2021 ). Consequently, all three MG labeling approaches exclusively relied on an AAV-mediated expression system.…”

“…Identification of interneuron-like cells and progenitor-like cells, which are normally not present in the mature mouse retina, is a clear indication of MG that undergoe identity changes during the reprogramming process. Although Xiao et al also performed scRNA-seq to capture the intermediate states of MG-to-RGC transition ( Xiao et al, 2021 ), they did not use stringent fate mapping techniques to isolate lineage traced MG. Therefore, genetic-based lineage tracing of MG, independent of the AAV-mediated labeling system, is a prerequisite for scRNA sequence analysis of cellular state changes of reprogrammed MG.…”

“…However, their results only showed a growing number of MG-derived RGCs, without providing authentic morphological changes during the process of MG-to-RGC conversion ( Zhou et al, 2020 ). Xiao et al generated a Brn3b-GFP reporter mouse line, in which GFP was simultaneously expressed with Brn3b to monitor the progress of MG-to-RGC conversion ( Xiao et al, 2021 ). However, two prominent morphological changes essential for MG-to-RGC conversion were missing in these studies: first, the migration path of MG somas from the middle of INL crossing the inner plexiform layer before reaching the ganglion cell layer; second, the trajectory of axon growth path from the newly generated RGC somas going towards the optic nerve head to exit the retina followed by extension of the axons in the optic nerve before the RGC axons reach their brain targets.…”