Single-Cell Profiles of Retinal Ganglion Cells Differing in Resilience to Injury Reveal Neuroprotective Genes

Abstract

Neuronal types in the central nervous system differ dramatically in their resilience to injury or other insults. Here we studied the selective resilience of mouse retinal ganglion cells (RGCs) following optic nerve crush (ONC), which severs their axons and leads to death of $80% of RGCs within 2 weeks. To identify expression programs associated with differential resilience, we first used single-cell RNA-seq (scRNA-seq) to generate a comprehensive molecular atlas of 46 RGC types in adult retina. We then tracked their survival after ONC; characterized transcriptomic, physiological, and morphological changes that preceded degeneration; and identified genes selectively expressed by each type. Finally, using loss-and gain-of-function assays in vivo, we showed that manipulating some of these genes improved neuronal survival and axon regeneration following ONC. This study provides a systematic framework for parsing type-specific responses to injury and demonstrates that differential gene expression can be used to reveal molecular targets for intervention.

Molecular identification of sixty-three amacrine cell types completes a mouse retinal cell atlas

“…Accordingly, we set out to enrich BCs, RGCs and ACs so we could profile them in sufficient numbers. For BCs and RGCs, we documented the existence of 15 and 46 types, respectively (Shekhar et al, 2016;Tran et al, 2019). These numbers correspond well to those obtained from recent high-throughput electrophysiological, ultrastructural and molecular studies (Baden et al, 2016;Bae et al, 2018;Franke et al, 2017;Greene et al, 2016;Rheaume et al, 2018).…”

“…To sparsely label ACs, we injected mice from Cre-expressing lines listed above with a cre-dependent virus, AAV9-EF1a-BbTagBY (Cai et al, 2013; Addgene #45185-AAV9), AAV9-EF1a-BbChT (Cai et al, 2013; Addgene #45186-AAV9) or AAV9-CAG-tdTomato (Addgene #51503-AAV9). Intravitreal injections were performed as described in Tran et al (2019). Animals were euthanized 3-4 weeks after injection and retinas processed as above.…”

“…Adult amacrine cells were collected and sequenced as part of unrelated projects (Tran et al, 2019;I.B., I.E.W., N. M.T., and J.R.S., unpublished).…”

Laser capture microdissection-based single-cell RNA sequencing reveals optic nerve crush-related early mRNA alterations in retinal ganglion cells

“…We compared our sequencing results with the currently published 10x scRNA-seq results for RGCs [2] and found that there were a large number of ganglion cell markers observed in our sequencing data, such as Pou4, Rbpms and Slc17ab, which meant our sequencing process could obtain ganglion cells accurately and was more economical and thus more suitable for low-throughput PCR to detect specific genes in small samples.…”

“…scRNA-seq can even reveal complex and rare cell populations, uncover regulatory relationships between genes, and track the trajectories of distinct cell lineages during development. Although studies involving 10x scRNA-seq of the retina have been reported recently, [2,3] and their results have been valuable, there are still some shortcomings. This technique does not distinguish cells in advance; therefore, there is a large amount of sequencing data that cannot be referenced to target cells, which is not economical.…”