Article Figures & Data
Figures
- Figure 1.
miR-383-5p is downregulated in proregenerative contexts. A, Schematic of experimental design depicting intravitreal injection of zymosan or PBS vehicle control at the time of ONC injury. B, Representative retinal cross sections stained with HistoGene for LCM (ROI, region of interest; GCL, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer); scale bar, 50 μm. C, qRT-PCR expression of candidate miRNAs in the GCL at 3 DPI (n = 3–4). FCR was calculated based on the 2−ΔΔCT method. D, qRT-PCR miRNA expression summary data for RGCs in response to zymosan intraocular inflammation. Upregulated or downregulated miRNAs in DRG neurons following a sciatic nerve lesion as reported in Strickland et al. (2011). Error bars represent standard errors of the mean (SEM). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
- Figure 2.
MiR-383-5p inhibition promotes neurite outgrowth and optic nerve regeneration. A, C, βIII-tubulin-stained (Tuj1) mouse retinal (A) or cortical (C) neurons treated with LNA-383 or LNA-NT (scale bar, 100 μm). B, D, Quantification of normalized mean neurite outgrowth in retinal (B) or cortical (D) neurons treated with LNA-NT or LNA-383 (n = 4–5; Student's t test). E, Retinal cross sections demonstrating uptake of FAM-labeled LNA into RGCs. GCL, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar, 50 μm. Red box inlet is a zoomed region to demonstrate colocalization of BRN3A-positive RGCs and FAM-labeled LNA. F, Quantification of BRN3A-positive RGCs in retinal whole mounts 7 d following LNA-NT/383 left intravitreal eye injection, normalized to the uninjected contralateral eye (n = 3). G, Experimental design of in vivo LNA-383 regeneration assessment. LNA-NT or LNA-383 was intravitreally injected into the left eye (vitreous chamber) of adult mice at the time of ONC. An additional injection of LNA was conducted 7 d later. At 12 DPI, fluorescently conjugated Ctβ was intravitreally injected, and animals were killed at 14 DPI. H, Maximal projection of z-stacks for whole-mount imaged optic nerves at 14 DPI, cleared and regenerating fibers visualized by Ctβ. Arrowheads mark crush site (scale bar, 300 μm). I, Quantification of axon regeneration (n = 4–7; two-way ANOVA; Šídák's multiple-comparison test). Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 3.
MiR-383-5p overexpression reduces zymosan-induced regeneration. A, Experimental design of in vivo miR-383-5p overexpression and zymosan regeneration assessment. AAV2-GFP or AAV2-383-5p was intravitreally injected into the left eyes of mice 14 d prior to zymosan (Z) injection and ONC. At 14 DPI animals were killed. B, The whole-mount retina expressing AAV2-GFP (scale bar, 500 μm). C, The evaluation of CD68-positive macrophages in whole-mount retinas of mice 2 weeks following PBS or zymosan injection. CD68-positive macrophages are observed in zymosan-injected eyes (white arrows). D, Representative images of regeneration assessment in optic nerve sections stained against GAP43 (scale bar, 250 μm). Red asterisk indicates the site of ONC. E, Quantification of regenerating fibers (n = 3–4; two-way ANOVA; Šídák's multiple-comparison test). Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 4.
MiR-383-5p is regulated by CNTF. A, qRT-PCR expression of miR-383-5p in mouse cortical neurons treated with ACM (n = 4 per condition). B, ELISA demonstrating CNTF concentration in control and ACM (n = 2–3/condition). C, D, MiR-383-5p level in response to mouse cortical neuron treatment with increasing concentrations of CNTF or LIF (D) continuously until 8 DIV (n = 4; one-way ANOVA; Šídák's multiple-comparison test). FCR was calculated based on the 2−ΔΔCT method (n = 3; one-way ANOVA; Dunnet's multiple comparison). *p < 0.05; **p < 0.01.
- Figure 5.
MiR-383-5p targets CNTF receptor components to sensitize neurons to CNTF. A, MiR-383-5p target prediction flowchart. Numerous databases for in silico miRNA target prediction were utilized. Validated targets were added to this list from miRTarBase as well as CLEAR-CLIP whole mouse brain miRNA–mRNA chimeras from M. J. Moore et al. (2015). MiRNA targets were then filtered to include those also expressed in FACS RGCs and E16 cortical neurons. B, Reactome analysis of curated list of miR-383-5p target genes. C, Databases that predict CNTF receptor components targeted by miR-383-5p (Extended Data Table 5-1). D, Quantification of luciferase activity in HEK293 cells cotransfected with M383 and luciferase vectors with 3′UTR of predicted targets (n = 3–4; two-way ANOVA; Šídák's multiple-comparison test). E–H, Retinal cross section presenting the RGC layer of mice intravitreally injected with LNA-NT or LNA-383 and stained with antibodies to LIFR (E) or GP130 (G; scale bar, 25 μm). Quantification of fluorescence intensity of LIFR (F) and GP130 (H) in BRN3A-positive cells (n = 4; two-tailed Student's t test). I, Representative images of adult retinal neuron outgrowth in response to transfection with LNA and culturing in 0 or 200 ng/ml CNTF for 6 DIV (scale bar, 50 μm). J, Quantification of CNTF-dependent neurite outgrowth from adult mouse retinal neurons treated with LNA-NT or LNA-383 and cultured in CNTF (n = 3; two-way ANOVA; Šídák's multiple-comparison test). K, Quantification of retinal neuron survival in culture with LNA-NT/383 and 0/200 ng/ml CNTF. Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
- Figure 6.
Prdx3 is a target of miR-383-5p and is necessary for LNA-383-mediated regeneration. A, Selection of Prdx3 as a prioritized target of miR-383-5p. Filtering of miR-383-5p targets was based on overlap of in silico predicted targets from five or more databases and miRTarBase experimentally validated targets for hsa-miR-383-5p (Extended Data Fig. 1-1B). B, Mean expression values and percentage of RGCs expressing transcripts for Prdx3 and Npat averaged across 46 RGC subtypes. Data were derived from the online RGC expression atlas provided by N. M. Tran et al. (2019). C, Quantification of luciferase activity in HEK293 cells cotransfected with M383 and luciferase vectors with 3′UTR of Prdx3 (n = 3; two-way ANOVA; Šídák's multiple-comparison test). D, E, Western blot and quantification for PRDX3 expression, with mitochondrial marker PDH (n = 4; two-tailed student's t test). F, Retinal cross section focused on the RGC layer of mice intravitreally injected with LNA-NT or LNA-383 and stained to assess PRDX3 expression (scale bar, 25 μm). G, Quantification of fluorescence intensity of PRDX3 in BRN3A-positive cells following injection with LNA-383 or LNA-383 (n = 4; two-tailed Student's t test). H, Western blot analysis of PRDX3 expression from mouse cortical neurons transfected with an siRNA targeting Prdx3 (siPrdx3) or negative control (siNC) for 72 h. I, Neuronal viability in MTT assay in mouse cortical neurons cotransfected with siPrdx and LNA-383 and treated with 10 μM sodium arsenite (n = 4; two-way ANOVA; Dunnett's multiple-comparison test). J, Schematic of experimental workflow for siRNA and LNA combinatorial experiments. K, βIII-tubulin-stained (Tuj1) cortical neurons that were transfected with siRNA 1 DIV, reseeded 72 h later to retract all neuronal processes and then transfected with LNA-NT or LNA-383 for 48 h (scale bar, 100 μm). L, Quantification of neurite outgrowth in cortical neurons treated with siRNA targeting Prdx3 and LNA-383 (n = 7; two-way ANOVA; p < 0.05; Šídák's multiple-comparison test). Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
- Figure 7.
Schematic of miR-383-5p regulation in response to an inflammatory stimulus. Injection of zymosan at the time of ONC stimulates astrocytic release of CNTF. CNTF in turn downregulates miR-383-5p, releasing its break on CNTF receptor components LIFR and GP130 and the mitochondrial antioxidant PRDX3. Inhibition of miR-383-5p relieves multiple proregenerative pathway components to elicit a distributed axon regenerative response following injury.
Extended Data
Table 5-1
Excel file presenting miR-383 targets identified by in silico databases and PANTHER analysis. See figure 5 for more details (Sheet A) In silico miR-383-5p mRNA targets predicted using Diana- microT, microRNA.org, miRDPB, miR-TarBase, RNA22, TargetScan, and TarBase. CNTF receptor components analyzed (Lifr and gp130/Il6st) are highlighted in green. (Sheet B) MiR-383-5p targets predicted in silico by five or more of the seven databases. Experimentally validated miR-383-5p targets were extracted from the miRTarBase database searching for hsa-miR-383-5p. Overlapping genes between these two lists are highlighted in orange. (Sheet C) PANTHER Reactome analysis conducted for targets predicted by two or more of the seven databases to determine statistically overrepresented pathways. Download Table 5-1, XLSX file.
