Figure 4.

AAV-Mediated Delivery of miR-204 Reduces Phagocytic Microglia Activation in RHO-P347S and Aipl1^−/− Mice

(A) Immunofluorescence staining for microglia markers Iba1 (green) and CD68 (red) on PN30 retinal sections of RHO-P347S injected with AAV.CMV.miR204 vector mix (1 × 10⁹ GC AAV.CMV.miR204 and 1 × 10⁸ GC AAV.CMV.EGFP) at PN4. The contralateral eye was injected with the AAV.CMV.EGFP control vector (1.1 × 10⁹ GC). DAPI nuclei counterstaining is shown in blue. Iba1 is a microglia marker, whereas CD68 is a lysosomal marker expressed at high levels in activated/phagocytic microglia. Iba1-positive and CD68-positive, large-bodied ameboid monocytes at the proximity of the photoreceptor outer segments are marked by arrowheads. (B) Microglia marker expression in Aipl1^−/− animals injected with the AAV.CMV.miR204 vector mix (1 × 10⁹ GC AAV.CMV.miR204, 1 × 10⁸ GC AAV.CMV.EGFP) at PN4 and analyzed at PN12, which corresponds to the early occurrence of photoreceptor death in this model. Microglia reactivity is more prominent in the subretinal space (at the proximity of the photoreceptor OS) in control (CMV.EGFP)-treated eyes. In the miR-204 injected eyes, phagocytic microglia are mainly found at the ONL. (C) Luciferase assays assessing the direct binding of miR-204 to the 3′ UTR of Siglec1 and Xaf1. miR-204 (gray bar) and negative control mimics (black bars) were transfected in HeLa cells together with the luciferase vectors bearing either the wild-type 3′ UTR (pTK-LUC-X 3′UTR) or the mutated binding site of the miR-204 seed (pTK-LUC-X 3′UTR mut). Relative luciferase activity is reported as fold change to the negative mimic-transfected cells. Data are represented as mean ± SEM. Statistical significance (two-way ANOVA) is indicated with asterisks (***p < 0.001; n = 6 observations). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: 50 μm.