Figure 14.

KO of MERTK in cultured retina glia (i.e., MG and retinal astrocytes) impairs synaptogenic function. A, Schematic representation of coculture experiments. Purified RGCs were either cultured alone or cocultured with retinal glia. The retinal glia were plated onto transwell inserts, which were pretreated with CRISPR/SaCas9-AAVs 5 d before coculture with neurons (RGCs). For UCM treatment experiments, the control and MERTK KO retinal glia inserts were further treated with UTC conditioned medium prepared from cells transduced by lentiviruses containing control scramble shRNA (CTR UCM) or shRNAs against TSP1, TSP2, and TSP4 (TSP KD UCM) for 3 d before coculture with RGCs. B, Schematic representation of the experimental design. Purified RGCs were treated with either control (SaCas9 only) or MERTK KO (MTK KO) retinal glia conditioned medium (RG CM). After 6 d treatment with conditioned media, the number of synapses is determined by immunocytochemistry. C, Representative images of RGCs stained with antibodies against presynaptic Bassoon (green, pre) and postsynaptic Homer (red, postsynaptic). White arrows indicate the colocalized synaptic puncta. Quantification of fold increase in synapse number of the RGCs that were either (D) cocultured or (E) treated with conditioned medium demonstrates that loss of MERTK in retina glia impairs their synaptogenic activity (n = 30 cells/condition). Data are mean ± SEM. ***p < 0.0001. F, Treatment of MERTK KO retina glia with either scrambled shRNA transduced UCM or TSP1 + TSP2 + TSP4 knockdown shRNA transduced UTC conditioned medium (TSP-KD UCM) rescues the synaptogenic activity (n = 30 cells/condition). Data are mean ± SEM. ***p < 0.0001, *p < 0.05. G, Western blot analyses using the cell lysates from control and MERTK KO retina glia. Represented Western blot images are from an individual experiment with three biological replicates. Quantitative analysis of (H) TSP1, (I) TSP2, (J) GFAP, and (K) GS (n = 3 per condition). L, Left, Western blot analyses using the retina glia conditioned medium. Right, Total protein loaded is visualized by Coomassie blue staining. Quantitative analyses of retina glia-secreted (M) TSP1 and (N) TSP2 (n = 3 per condition). Data are mean ± SEM. *p < 0.05.