Recovery is distressingly poor following acute (e.g., stroke or traumatic injury) or chronic (e.g., neurodegenerative disease) insults to the human central nervous system (CNS). Many neurons die and those that survive are generally incapable of growing new axons and re-establishing synaptic connections with their partners. Over the past few decades, however, some of the factors that limit neuronal survival and regeneration have been identified, and these discoveries have led to the development of interventions aimed at promoting recovery. Nonetheless, few if any are sufficient to restore useful function, so further advances are needed. In seeking them, a useful strategy has been to compare adult CNS neurons, which regenerate poorly, with neurons that grow or regenerate well ?for example, those in the developing mammalian CNS, the adult mammalian peripheral nervous system, or the CNS of lower vertebrates. Here, we propose a complementary approach: comparing vulnerable and resilient cell types within a single tissue. We will use the mouse optic nerve as a model. The optic nerve carries the axons of retinal ganglion cells (RGCs), the sole retinal output neurons, to the brain. There are ?40 RGC types in mice; all are similar in most ways, but they are tuned to distinct visual features and exhibit limited molecular differences. Following optic nerve crush (ONC) to sever all RGC axons, ~80% of the RGCs die within 2 weeks. Virtually none of the survivors regenerate axons spontaneously, but limited regeneration can be provoked in several ways. Recently, we used molecular markers to assess the fate of a small set of RGC types following ONC. We found dramatic differences in their ability to survive following ONC, and in the ability of survivors to regenerate following treatment. We also found a gene selectively expressed by a resilient type that can promote regeneration when overexpressed. These findings led us to believe that a deeper understanding of type-specific differences in response to injury could lead to new strategies for improving recovery. To identify these differences, we will use a high- throughput single cell RNA sequencing (scRNAseq) method called Drop-seq that we helped pioneer. We have now used Drop-seq to profile >80,000 cells from control retina, generating molecular profiles for over half of all retinal types and optimizing a scalable pipeline for further studies. Using Drop-seq and customized computational approaches, we will now profile RGCs in control retina and 2 weeks after ONC to comprehensively map the vulnerability of all types. We will then profile RGCs at 3, 12 and 48 hours after ONC, to find transcriptomic differences that correlate with, and could contribute to their differential vulnerability. Our results will provide a foundation for (a) identifying the cell types on which available interventions act, (b) elucidating their mechanisms of action, so they can be improved, and (c) discovering new ways to transform vulnerable populations into resilient ones.
Injuries to the human central nervous system (CNS) are devastating: many neurons die and those that survive generally do not form new connections, which are required to restore function. It turns out, however, that CNS neurons differ in their reactions to injury, and learning what makes some types resilient could lead to interventions that would improve the lot of the more vulnerable. This project therefore maps the vulnerability of many types of retinal neurons, and then seeks genes that correlate with, and may contribute to resilience.
