Abstract
Purpose :
Retinal diseases are the most common cause of irreversible blindness worldwide. Although most mammals are incapable of regenerating neuronal tissue, other species such as fish/amphibians can regenerate their retina and restore vision post injury. These species employ developmental transcription programs that drive proliferation of support cells, which act as a progenitor pool for subsequent neurogenesis. We previously showed that overexpressing factors Ascl1 and Atoh1 in mouse Müller glia (MG) of acutely injured retinas can lead to neurogenesis. This work interrogates the role of injury mode on the fate of glia-derived neurons by comparing acute versus chronic retinal damage.
Methods :
Two transgenic mouse lines were used with inducible MG-specific expression of Ascl1 (Glast-CreER/LNL-tTA/TetO-Ascl1-GFP) or Ascl1 and Atoh1 (Glast-CreER/LNL-tTA/TetO-Ascl1-GFP/TetO-Atoh1). To model acute retinal injury, mice received an intravitreal injection of NMDA that damages most retinal ganglions. For chronic retinal injury, mice homozygous for the Rpe65 Leu450 were subjected to ~20,000 lux for 6 hours, which led to gradual photoreceptor degeneration. The transgenic mice were injected with tamoxifen to induce the expression of neurogenic factors in MG followed by intravitreal injection of Trichostatin-A. Four weeks later, the number and types of glial-derived neurons were assessed via histology and scRNA-seq.
Results :
Histological analysis of mouse retinas after acute or chronic injury showed no difference in reprogramming efficiency, for both Ascl1 and Ascl1-Atoh1 mediated reprogramming. Ascl1 produced Otx2+ bipolar neurons in both contexts , whereas Ascl1-Atoh1 led to HuC/D+ and Otx2+ neurons, though light-damage produced more Otx2+ cells than NMDA-induced injury. This was further corroborated by scRNA-seq of lineage-traced MG, showing that the classes of neurons obtained following light-damage in the Ascl1-Atoh1 mice were more diverse. Specifically, a new cluster of early RGC-like cells was detected that expressed markers such as Onecut2, Rbpms, Ebf2 and Zic3, some of which were also verified by immunofluorescence.
Conclusions :
This work sets the stage for understanding how reprogramming paradigms are impacted by the mode of injury in the retina. Such insight could predict whether patients with both acute and chronic retinal diseases could benefit equally from a regenerative therapy based on MG reprogramming.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.