Transcriptomic profiling of retinal cells reveals a subpopulation of microglia/macrophages expressing Rbpms marker of retinal ganglion cells (RGCs) that confound identification of RGCs

Analysis of retinal ganglion cells (RGCs) by scRNA-seq is emerging as a state-of-the-art method for studying RGC biology and subtypes, as well as for studying the mechanisms of neuroprotection and axon regeneration in the central nervous system (CNS). Rbpms has been established as a pan-RGC marker, and Spp1 has been established as an aRGC type and macrophage marker. Here, we analyzed by scRNA-seq retinal microglia and macrophages, and found Rbpms+ subpopulations of retinal microglia/macrophages, which pose a potential pitfall in scRNA-seq studies involving RGCs. We performed comparative analysis of cellular identity of the presumed RGC cells isolated in recent scRNA-seq studies, and found that Rbpms+ microglia/macrophages confounded identification of RGCs. We also showed using immunohistological analysis that, Rbpms protein localizes to stress granules in a subpopulation of retinal microglia after optic nerve injury, which was further supported by bioinformatics analysis identifying stress granule-associated genes enriched in the Rbpms+ microglia/macrophages. Our findings suggest that the identification of Rbpms+ RGCs by immunostaining after optic nerve injury should exclude cells in which Rbpms signal is restricted to a subcellular granule, and include only those cells in which the Rbpms signal is labeling cell soma diffusely. Finally, we provide solutions for circumventing this potential pitfall of Rbpm-expressing microglia/macrophages in scRNA-seq studies, by including in RGC and aRGC selection criteria other pan-RGC and aRGC markers. Overall design: We generated 10x Genomics 3'-droplet based scRNA-seq dataset of macrophage/microglia from adult (10 weeks old) uninjured and injured (2 weeks after ONC) retinas/optic nerves from mice of both sexes using methods we previously described (Rheaume et al 2018; Xing et al, 2023). ONC injury was performed at 8 weeks. Cells from were loaded for capture onto the Chromium System using the v2 single cell reagent kit (10X Genomics). Following capture and lysis, cDNA was synthesized and amplified (12 cycles) as per manufacturer's protocol (10X Genomics). After demultiplexing and aligning to the mouse genome and transcriptome (mm10) using the CellRanger pipeline and merging/integration in Rstuduio, 1168 macrophage/microglia passed quality control (including filters of a minimum of 200 genes and a maximum of 6500 genes). Batches were merged, normalized and scaled using Seurat. Batch correction/integration of datasets was performed using the R package harmony.