Abstract
Purpose :
Loss of neurons due to damage or disease is irreversible in humans and most mammals. Although we are incapable of regenerating neuronal tissue, other species such as fish and amphibians can regenerate their retina by employing developmental transcription programs that drive proliferation of support cells, which act as a progenitor pool for subsequent neurogenesis. We previously used transgenic mice to express neurogenic transcription factors in mouse Müller glia (MG) of acutely injured retinas to drive neurogenesis. Now we demonstrate that new lineage-traced neurons can be generated when these factors are delivered with viral vectors in vivo.
Methods :
A mouse line with inducible MG-specific expression of TdTomato was used to lineage trace MG-derived progeny. Mice were intravitreally injected with adeno-associated viral (AAV) vectors with an inducible payload to drive expression of different combinations of TFs: Ascl1, Ascl1-Atoh1 or Ascl1-Atoh7. After a 2-6 week incubation period, the mice were injected with tamoxifen to induce the expression of the vector-borne TFs in MG. From this point on, mice received EdU water ad libitum to label newborn cells. To injure the retina, mice were intravitreally injected with an excitotoxic dose of NMDA, followed by an intravitreal injection of Trichostatin-A. Three weeks later, the number and types of glial-derived neurons were assessed via histology and scRNA-seq.
Results :
The first step towards neurogenesis is the dedifferentiation of MG into a progenitor state that proliferates in response to injury. Histological analysis showed that following injury, MG re-entered the cell-cycle when injected with AAV vectors expressing any of the three TF combinations; untreated MG did not. Furthermore, AAV-borne expression of Ascl1 produced Otx2+/Pcp4+ bipolar neurons, whereas Ascl1-Atoh1/7 led to both HuC/D+ or Otx2+/Pcp4+ neurons. The production of new neurons was verified via lineage tracing MG-derived progeny and EdU incorporation. This was further corroborated by scRNA-seq of lineage-traced MG, showing that the classes of neurons obtained from AAV-treated retinas phenocopied the neurons obtained from glia using only transgenic methods.
Conclusions :
Our results show for the first time that AAV-delivered TFs can stimulate neurogenesis from MG after injury in the adult mouse retina, bringing together AAV-mediated gene therapy and regenerative medicine.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.