Directed robust generation of functional retinal ganglion cells from Müller glia

Abstract

Glaucoma and optic neuropathies cause progressive and irreversible degeneration of retinal ganglion cells (RGCs) and the optic nerve and are currently without any effective treatment. Previous research into cell replacement therapy of these neurodegenerative diseases has been stalled due to the limited capability for grafted RGCs to integrate into the retina and project properly along the long visual pathway to reach their brain targets. In vivo RGC regeneration would be a promising alternative approach but mammalian retinas lack regenerative capacity even though cold-blood vertebrates such as zebrafish have the full capacity to regenerate a damaged retina using Müller glia (MG) as retinal stem cells. Nevertheless, mammalian MG undergo limited neurogenesis when stimulated by retinal injury. Therefore, a fundamental question that remains to be answered is whether MG can be induced to efficiently regenerate functional RGCs for vision restoration in mammals. Here we show that without stimulating proliferation, the transcription factor (TF) Math5 together with a Brn3 TF family member are able to reprogram mature mouse MG into RGCs with exceedingly high efficiency while either alone has no or limited capacity. The reprogrammed RGCs extend long axons that make appropriate intra-retinal and extra-retinal projections through the entire visual pathway including the optic nerve, optic chiasm and optic tract to innervate both image-forming and non-image-forming brain targets. They exhibit typical neuronal electrophysiological properties and improve visual responses in two glaucoma mouse models: Brn3b null mutant mice and mice with the optic nerve crushed (ONC). Together, our data provide evidence that mammalian MG can be reprogrammed by defined TFs to achieve robust 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.

Turning lead into gold: reprogramming retinal cells to cure blindness

“…Second, the newly generated neurons were reported to be morphologically, functionally, and molecularly indistinguishable from wild-type neurons in some studies (6-10) but abnormal and immature in others (3)(4)(5). Third, three studies reported that newly generated RGCs rapidly extended axons through the optic nerve to synapse on central targets and mediate visually evoked behaviors (7,8,10), despite the fact that regeneration of axotomized RGCs is highly inefficient in adult mammalian retina and the very limited number of regenerating axons seldom reach central targets (11).…”

“…Over the past few years, a flurry of studies has built on these findings, manipulating key regulators of gene expression to reprogram adult rodent MG (3-9), as well as amacrine cells (10), resulting in generation of functional retinal neurons. The experimental paradigm is similar in all cases: researchers use adeno-associated viruses (AAVs) as vectors with cell type-specific promoters (7)(8)(9) or Cre-expressing transgenic lines (3)(4)(5)10) to selectively target the cells to be reprogrammed. They then exploit this genetic access to introduce fluorescent reporters that track the cells and their progeny, along with interventions (overexpressed cDNAs, shRNAs, gRNAs, or conditional knockouts) to induce reprogramming (Figure 1).…”

“…Despite this methodological similarity, however, the results differ greatly. Three groups report generation of RGCs from MG (7,8) or interneurons (10) by depletion of Ptbp1 (7) or ectopic expression of Pou4f2 (Brn3b) plus Atoh7 (Math5) (8) or Pou4f2 plus Sox4 (8,10). Two groups generated bipolar and amacrine-like cells by overexpression of Ascl1 (3,4) or conditional deletion of three nuclear factor I (NFI) class transcription factors (5).…”

Advances in Regeneration of Retinal Ganglion Cells and Optic Nerves

“…Furthermore, Neurod1-expressing amacrine and photoreceptor progenitors can be reprogrammed into RGCs when Atoh7 is inserted into the Neurod1 locus [ 64 ]. Xiao et al showed that combining Atho7 with Brn3b was able to reprogram mature mouse MG into RGCs efficiently and the MG-derived RGCs were functional, made appropriate central projections, and improved visual responses [ 119 ]. Ngn2 alone is also sufficient to lineage, reprogramming postnatal mouse MG into RGC-like neurons in vitro and inducing the generation of this neuronal type from late retinal progenitors in vivo [ 63 ].…”

On the Generation and Regeneration of Retinal Ganglion Cells

“…Strategies to reprogram existing cells to generate RGCs have been limited in mammalian retina, highlighting the need for innovative approaches. A preprint from Xiao et al (2019) have described the generation of induced RGCs from Müller glia in mice through the overexpression of Atoh7 and Pou4f2/Brn3b. These cells projected axons to superior targets in the brain and restored the vision in a disease model.…”