Identification of a Novel Axon Regeneration Role for Noncanonical Wnt Signaling in the Adult Retina after Injury

Upregulation of endogenous Wnt5a after ONC injury. QPCR quantification of Wnt5a gene expression 3 d after ONC injury showed significant upregulation in retinas from ONC injured mice compared with retinas from uninjured but anesthetized mice (n = 3). Mean ± SD is shown. See Extended Data 1 for detailed statistical analyses.

Wnt5a treatment induced neurite growth, neurite complexity and neurite number in RGC primary cultures. A–D, Representative images of RGC cultures treated with BSA (control), 25, 50, and 100 ng recombinant Wnt5a. Neurites are shown in white, DAPI-stained nuclei are blue. Scale bar: 50 μm. E, Bar diagrams show significantly increased average RGC neurite length, ANC (branch site number) and ANN in different concentrations of Wnt5a-treated cultures compared with BSA-treated cultures. Mean ± SD is shown. See Extended Data 1 for detailed statistical analyses.

Wnt5a protects RGCs after ONC injury. A, Immunohistochemistry on retinal cryosections two weeks after ONC injury showed increased RBPMS-positive RGCs (red) in 20 and 50 ng Wnt5a-injected retinas compared with saline-injected retinas. DAPI-stained nuclei (blue) indicate the retinal layers. Scale bar: 50 μm. B, Quantification of RBPMS immunopositive cells that colocalized with DAPI shows significantly increased RGC density in 20 and 50 ng Wnt5a-injected retinas compared with saline-injected retinas. Mean ± SD is shown. See Extended Data 1 for detailed statistical analyses.

Axonal regeneration two weeks after ONC injury following a single intravitreal injection of Wnt5a. A, Representative images of CTB-labeled axons (white) demonstrating increased axons past the crush region (*) in 50 ng Wnt5a-injected mice compared with saline-injected or 20 ng Wnt5a-injected mice. Scale bar: 100 μm. B, Average axon count for each distance past the crush site (400–1400 μm) demonstrates significantly higher regeneration in 50 ng Wnt5a-injected animals compared with saline and 20 ng Wnt5a-injected animals (mean ± SEM). C, Comparison of the longest axon detected per nerve for each mouse shows significantly longer regrowth in the 50 ng Wnt5a-injected animals compared with saline and 20 ng Wnt5a-injected animals (mean ± SD). S = saline, 20 = 20 ng Wnt5a, 50 = 50 ng Wnt5a; n = 5 or 6 animals per each group. See Extended Data 1 for detailed statistical analyses.

CamKII and JNK signaling is upregulated and PKC signaling is downregulated by Wnt5a. A, Representative Western blot images of phospho and total CamKII, phospho and total JNK, phospho and total PKC, and GAPDH on retinas collected 24 h after Wnt5a or saline injection. B, Normalized phospho protein band intensity with total protein band intensity of CamKII, JNK, and PKC shows significant upregulation of phospho CamKII and JNK in 20 ng Wnt5a-injected retinas compared with saline-injected retinas. In contrast, significant downregulation of phospho PKC was observed in 20 ng Wnt5a-injected retinas compared with the saline-injected retinas (n = 5 in each group). Mean ± SD is shown. See Extended Data 1 for detailed statistical analyses.

CamKII and JNK signaling is upregulated by Wnt5a and PKC signaling is downregulated by Wnt5a. A, Representative Western blot images of phospho and total CamKII, phospho and total JNK, phospho and total PKC, and GAPDH. B, Normalized phospho protein band intensity with total protein band intensity of CamKII, JNK, and PKC shows significant upregulation of phospho CamKII and JNK in 50 ng Wnt5a-injected retinas compared with saline-injected retinas. In contrast, significant downregulation of phospho PKC was observed in 50 ng Wnt5a-injected retinas compared with the saline-injected retinas (n = 5 in each group). Mean ± SD is shown. See Extended Data 1 for detailed statistical analyses.

Increased phosphorylation of CamKII in RGCs 24 h after a Wnt5a injection. Upper panel shows representative images of cross-sections of saline-injected retinas. Lower panel shows representative images of cross-sections of 50 ng Wnt5a-injected retinas. IHC was performed to co-immunostain phospho CamKII (green) and RBPMS (red). Phosphorylation of CamKII was induced in GCL, INL, and OPL of Wnt5a-injected retinas compared with saline-injected retinas (GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer and OS: outer segment layer). Colocalization of CamKII phosphorylation with RBPMS-positive cells is indicated by yellow overlap signal (white arrows). Scale bar: 50 μm.

Increased phosphorylation of JNK in Wnt5a-injected retinas. Upper panel shows representative images of cross-sections of saline-injected retinas. Lower panel shows representative images of cross-sections of 50 ng Wnt5a-injected retinas. IHC was performed to co-immunostain phospho JNK (green) and RBPMS (red). Phosphorylation of JNK was induced in certain cell somas of RGC, IPL, and OPL in Wnt5a-injected retinas compared with saline-injected retinas (GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer and OS: outer segment layer). White arrows indicate induced phosphorylation of JNK that colocalized with RBPMS-positive RGCs. Scale bar: 50 μm.

Increased phosphorylation CREB in Wnt5a-injected retinas. Upper panel shows representative images of cross-sections of saline-injected retinas. Lower panel shows representative images of cross-sections of 50 ng Wnt5a-injected retinas. IHC was performed to co-immunodetect phospho CREB (red) and RBPMS (green). Phosphorylation of CREB was induced in the GCL and other layers of the retina. White arrows indicate induced phosphorylation of CREB in RBPMS-positive RGC cell somas (GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer and OS: outer segment layer). Scale bar: 50 μm.