A transgenic line that labels the RGCs in the retina (lateral clusters) and the axons in the optic nerve leading up to the optic tectum.
By Rebecca Salowe
Scheie Vision Annual Report 2021
Michael Granato, PhD, Professor of Cell and Developmental Biology, is conducting groundbreaking research on spontaneous regeneration of the optic nerve. A vision scientist at the University of Pennsylvania, he has received two R01 grants from the National Eye Institute to investigate this poorly understood topic.
In mammals, the central nervous system (CNS) has a minimal capacity for regeneration. The CNS includes the retina and the optic nerve, which is made up of axons from retinal ganglion cells (RGCs). These axons extend from the retina to the brain and associated glia. When the optic nerve becomes damaged in diseases such as glaucoma, the injury can lead to irreversible vision loss and blindness due to the limited capacity of the optic nerve for regeneration.
Amphibians and fish, on the other hand, have a remarkable capacity for optic nerve regeneration following injury. As a result, these animals are often used as model systems to study injury to the CNS. “Zebrafish in particular are a productive model for spontaneous spinal cord regeneration, as well as optic nerve regeneration,” said Dr. Granato.
There are many unanswered questions about regeneration. Models such as zebrafish can be used to understand how optic nerve axons and surrounding glia interact during optic nerve regeneration. For example, how are immune and glia cells summoned to the injury site, and how do they provide guidance to regenerate RGCs?
Prior research in this area has uncovered several intrinsic neural signaling pathways that boost axonal growth of injured RGCs and suppress cell death. However, this growth is frequently characterized by axonal misguidance and limited functional regeneration.
This limitation spurred Dr. Granato’s interest in studying how guidance cues and underlying cellular mechanisms play a role in optic nerve regeneration.
“Which extrinsic cues and guidance pathways ensure correct RGC guidance during regeneration is unclear,” said Dr. Granato. “Identifying these cues, and the underlying cellular mechanisms, appears paramount to achieving functional optic nerve regeneration.”
A large reason for these gaps in knowledge is due to challenges in live cell imaging in mammals. Dr. Granato sought to fill this void by investigating the cellular and molecular pathways of spontaneous optic nerve regeneration in larval zebrafish. He received an R01 grant from the National Eye Institute in 2014 for this project, which was recently renewed for a second five-year period, extending to 2024.
Dr. Granato specifically investigates larval zebrafish, as they have a functional visual system yet still maintain optical transparency. His laboratory was among the first to develop larval zebrafish as a model for optic nerve regeneration. The research team established a powerful assay to transect the optic nerve in larval zebrafish and monitor the regeneration of axons.
“We found that RGC axonal regeneration is rapid, as re-growing axons enter the optic tectum by 48 hours post transection, independent of RGC cell death or proliferation,” said Dr. Granato.
The team also performed a candidate gene screen and a small molecule screen to investigate which genes are involved in this spontaneous regeneration. The genetic screen identified mutants in two genes that are critical for guidance of injured RGC axons.
“Combined, our preliminary data support the hypothesis that these two genes participate in a molecular pathway that selectively provides extrinsic guidance to regenerating RGC axons, likely by surrounding glia,” explained Dr. Granato.
The renewal grant will use live cell imaging to elucidate the cellular interplay between regenerating RGC axons and glia cells. This will be the first time that these interactions are observed in their native environment in a vertebrate system.
“This proposal will also determine the cellular and molecular mechanisms by which the two genes we identified ensure proper regenerative guidance in vivo,” said Dr. Granato. “Additionally, mutations in the human counterpart of one of these genes leads to glaucoma and irreversible optic nerve degeneration. Thus, the proposed experiments will identify the mode of action of a glaucoma-causing disease gene.”
This research has implications for many human diseases that damage the optic nerve, such as hereditary optic neuropathies and glaucoma. It will allow for specific hypotheses of optic nerve regeneration that can be applied to restoration of human vision in the future.
“The results from this proposal on spontaneous optic nerve regeneration will provide answers to fundamental questions of optic nerve regeneration across the board, including in mammals,” said Dr. Granato.