Intercellular communication atlas reveals Oprm1 as a neuroprotective factor for retinal ganglion cells

The neuronal cell death results in neurodegenerative diseases. The extracellular environment plays a critical role in regulating cell viability. In this study, we explore how intercellular communication contributes to the survival of retinal ganglion cells (RGCs) following the optic nerve crush (ONC). By performing single-cell RNA-seq on whole retinal cells, we observed transcriptomic responses in non-RGC retinal cells to the injury, with astrocytes and Müller glia having the most interactions with RGCs. By comparing the RGC subclasses showing distinct resilience cell death, we identified top 47 interactions that are stronger in the high-survival RGCs, likely representing neuroprotective interactions. We performed functional assays on one of the receptors, Mu-opioid receptor (Oprm1). Although Oprm1 is preferentially expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs), its neuroprotective effect could be transferred to multiple RGC subclasses by specific overexpressing Oprm1 in pan-RGCs. Our study provides an atlas of cell-cell interactions in both intact and post-ONC retina and an effective strategy to predict molecular mechanisms in neuroprotection, underlying the principal role played by extracellular environment in supporting neuron survival. Overall design: With 12-hour, 24-hour, 48-hour post-ONC, or sham conditions, the mice were euthanized. Each time point had two biological replicates, except the 12-hour condition had one sample. Retinas were quickly and carefully dissected, and whole retinal cells were dissociated using the Papain enzyme system (LK003150, Worthington). 30mM actinomycin-D was added to the Papain system to prevent post-enucleation transcription. The tissues then underwent manual trituration in neurobasal medium supplemented with 3% BSA, 10 mg/mL Ovomucoid, 1U/uL RNase inhibitor, and 3mM actinomycin D. Cells were filtered through a 40-µm strainer and centrifuged under 500´g. The scRNA-seq libraries were prepared using the 10X Genomics Chromium 3' Single-Cell Gene Expression V3 Kit following the standard protocol by the manufacturer. Approximately 8,000-12,000 cells were loaded onto the platform. Libraries were sequenced with the NovaSeq S2 100 platform.