Sphingosine-1-Phosphate Signaling through Müller Glia Regulates Neuroprotection, Accumulation of Immune Cells, and Neuronal Regeneration in the Rodent Retina

Fluorescence in situ hybridization (FISH) for S1PR1 and SPHK1. Glast-CreER:LNL-tTA:tetO-mAscl1-ires-GFP mice received intraperitoneal injections of tamoxifen or corn oil once daily for 4 consecutive days. Eyes received intravitreal injections with NMDA or saline, and retinas were collected 4 h after NMDA treatment. Retinal sections were labeled with antibodies to Sox2 (cyan), GFP (green), or FISH probes to S1pr1 (red puncta; a, c) or Sphk1 (red puncta; b). Regions of interest (yellow boxes) are enlarged 1.8-fold and displayed in adjacent channels. Solid arrows indicate Sox2⁺/GFP^low MG nuclei, and hollow arrows indicate Sox2⁺/GFP^high MG nuclei. The area occupied by Sox2⁺ (b) or GFP⁺ cells (e), was selected to distinguish FISH puncta labeling within these regions. Small double arrows indicate endothelial cells. Scale bars: a, b, c, and e represent 50 µm. Histograms represent the mean (bar ± SD) number of FISH puncta per MG and each dot represents one biological replicate (d; n = 6 or 7). Significance of difference (p values) was determined by using a paired t test. Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NMDA, N-methyl-D-aspartate; ns, not significant.

Inhibition of S1P signaling enhances Ascl1-mediated reprogramming of MG into neurogenic progenitor cells. Schematic summary of S1P signaling and sites of action for different small molecule agonists and antagonists a, Glast-CreER:LNL-tTA:tetO-mAscl1-ires-GFP mice were intraperitoneally injected with tamoxifen or corn oil once daily for 4 d. Eyes were intravitreally injected NMDA and either a vehicle control or inhibitors to Sphk1 (PF543) and S1pr1/3 (VPC23019). At 24HPI, eyes were treated with vehicle or inhibitors. Eyes were treated with TSA at 48HPI. Retinas were collected 24 d from the first tamoxifen injection. Retinal sections were labeled with DAPI (blue, b, d) or EdU (blue, e, h) and antibodies to GFP (green) and Sox2 (red, b, d), Otx2 (red, e, h), Scgn (red, i), or Lhx4 (red, j). Regions of interest (yellow boxes) are enlarged 1.5 (b, d) or 2-fold (e, h, i, j) and displayed in separate panels. Solid arrows indicate GFP⁺/Sox2^high cells in b and d, and hollow arrowheads indicate GFP⁺/Sox2^low cells. In e and h, solid arrows indicate GFP⁺/Otx2⁺/EdU⁺ cells. Scale bars: b and e represent 50 µm. Histograms represent the mean (bar ± SD) where each dot represents one biological replicate (n = 8), and blue lines connect control and treated eyes from the same individual (c, g, f). Significance of difference (p values) was determined by using a paired t test. Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NMDA, N-methyl-D-aspartate; ns, not significant.

Cross-regulation of S1P/S1pr1 and NFκB signaling in MG. Retinas were obtained from undamaged or NMDA-injured eyes treated with a vehicle control or a small molecule agonist/antagonist a–g, Retinal sections were labeled with DAPI (e) and antibodies to Sox2 (red, a, c, e; pseudocolored cyan, f) and NFκB-GFP (green, a, c, e) or FISH probes to S1pr1 (red puncta; f). Arrows indicate the nuclei of MG. Scale bars: a, c, and f represent 50 µm. Regions of interest (yellow boxes) are enlarged 1.8-fold and displayed in separate panels (f). Histograms illustrate the mean (bar ± SD) number of NFκB-eGFP in MG (b, d) or number of FISH puncta per MG (g), where each dot represents one biological replicate (n = 5–8), and blue lines connect replicates from control and treated retinas from one individual. Significance of difference (p values) was determined by using a paired t test (b, d, g). Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; EdU, 5-ethynyl-2′-deoxyuridine; NMDA, N-methyl-D-aspartate; ns, not significant; UMAP, Uniform Manifold Approximation and Projection.

Immune cell accumulation in S1pr1 KO and Sphk1 KO retinas. Rlbp1-CreERT:S1pr1^fl/fl and Rlbp1-CreERT:Sphk1^fl/fl mice were intraperitoneally injected with tamoxifen or corn oil once daily for 4 d. Eyes were intravitreally injected with NMDA or saline control, and retinas were collected 48HPI. Retinal sections were labeled with antibodies to Iba1 (blue) and CD45 (green; a, b). Solid arrows indicate Iba1⁺/CD45⁺ cells, and hollow arrowheads indicate Iba1⁺/CD45⁻ cells. Scale bars: a and b, represent 50 µm. Histograms represent the mean (bar ± SD) where each dot represents one biological replicate (c, d; n = 5–18). Significance of difference (p values) was determined by using ANOVA with Sidak’s correction. Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NMDA, N-methyl-D-aspartate; ns, not significant.

cKO of S1pr1 from MG is neuroprotective to retinal ganglion cells. Sections of the retina were labeled for fragmented DNA (TUNEL; green; a) and DAPI (blue), or Brn3a (green; d), tdTomato (red) and DAPI (blue), HuC/D (green; f), tdTomato (red) and DAPI (blue), or Pax6 (green, i), tdTomato (red), and DAPI (blue). Arrows indicate TUNEL⁺ nuclei (a) or Brn3a⁺ nuclei (d) or HuC/D⁺ cells (f) and small double arrows indicate GFP⁺ endothelial cells. Scale bars: a, d, and f, represent 50 µm. Histograms represent the mean (bar ± SD) where each dot represents one biological replicate (b, c, e, g, h, j; n = 5–10). Significance of difference (p values) was determined by using ANOVA with Sidak’s correction. Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NMDA, N-methyl-D-aspartate; ns, not significant; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

cKO of Sphk1 from MG is neuroprotective to inner retinal neurons. Sections of the retina were labeled for fragmented DNA (TUNEL; green; a) and DAPI (blue), or Brn3a (green; d), tdTomato (red) and DAPI (blue), or HuC/D (green; f), tdTomato (red) and DAPI (blue), or Pax6 (green; h), tdTomato (red) and DAPI (blue). Single arrows indicate TUNEL⁺ nuclei (a) or Brn3a⁺ nuclei (d) or HuC/D⁺ cells (f), and small double arrows indicate GFP⁺ endothelial cells. Scale bars: a, d, f, and h represent 50 µm. Histograms represent the mean (bar ± SD) where each dot represents one biological replicate (b, c, e, g, i; n = 6–8). Significance of difference (p values) was determined by using ANOVA with Sidak’s correction. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NMDA, N-methyl-D-aspartate; ns, not significant; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.