Knocking Out Non-muscle Myosin II in Retinal Ganglion Cells Promotes Long-Distance Optic Nerve Regeneration

Abstract

In addition to altered gene expression, pathological cytoskeletal dynamics in the axon are another key intrinsic barrier for axon regeneration in the central nervous system (CNS). Here, we show that knocking out myosin IIA and IIB (myosin IIA/B) in retinal ganglion cells alone, either before or after optic nerve crush, induces significant optic nerve regeneration. Combined Lin28a overexpression and myosin IIA/B knockout lead to an additive promoting effect and long-distance axon regeneration. Immunostaining, RNA sequencing, and western blot analyses reveal that myosin II deletion does not affect known axon regeneration signaling pathways or the expression of regeneration-associated genes. Instead, it abolishes the retraction bulb formation and significantly enhances the axon extension efficiency. The study provides clear evidence that directly targeting neuronal cytoskeleton is sufficient to induce significant CNS axon regeneration and that combining altered gene expression in the soma and modified cytoskeletal dynamics in the axon is a promising approach for long-distance CNS axon regeneration.

Keywords: Lin28; axon regeneration; cytoskeleton; growth cone; non-muscle myosin II; optic nerve regeneration; post-injury treatment; retinal ganglion cells; retraction bulb.

Figure 1.. Deletion of Myosin IIA/B in RGCs Induced Significant and Persistent Optic Nerve Regeneration

(A) Top: experimental timeline. Bottom: representative images of optic nerves showing that deletion of myosin IIA/B in RGCs produced significant axon regeneration 2 and 4 weeks after optic nerve crush. The columns on the right display magnified images of the areas in white, dashed boxes on the left, showing axons at 250, 500, 750, 1,250, and 2,000 μm distal to the crush sites. The yellow line indicates the crush sites. Yellow arrows indicate the top 3 longest axons of each nerve. Scale bar, 100 μm (50 mm for the magnified images). (B) Quantification of optic nerve regeneration in (A) (one-way ANOVA followed by Tukey’s multiple comparisons test; p < 0.0001 at 250, 500, 750, 1,000, 1,250, and 1,500 μm; p = 0.0002, 0.0071, 0.3875, and 0.3875 at 1,750, 2,000, 2,250, and 2,500 μm, respectively; n = 10 mice in 4-week wild-type (WT) group, n = 9 mice in other groups). (C) Quantification of the average length of the top 5 longest axons of each nerve in (A) (one-way ANOVA followed by Tukey’s multiple comparisons test; p < 0.0001; n = 10 mice in 4-week WT group, n = 9 mice in other groups). (D) Representative images of flat-mounted retinas showing that deletion of myosin IIA/B had no effect on RGC survival rate 2 weeks after optic nerve crush. Flat- mounted retinas were stained with anti-tubulin b3 antibody (Tuj1, green). Scale bar, 50 μm. (E) Quantification of RGC survival rate in (D) (unpaired t test, p = 0.9092; n = 4 and 3 mice in WT and dKO groups, respectively; 7–8 fields were analyzed for each retina). Data are represented as mean ± SEM; P values of post hoc analyses are illustrated in the figure. n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. dKO, double knockout of myosin IIA/B. See also Figures S1 and S2.

Figure 2.. Myosin IIA/B Deletion and Lin28a Overexpression Had an Additive Effect on Optic Nerve Regeneration

(A) Top: experimental timeline. Bottom: representative images of optic nerves showing that combining myosin IIA/B deletion with Lin28a overexpression in RGCs produced much stronger axon regeneration 2 weeks after the optic nerve crush. The columns on the right display magnified images of the areas in white, dashed boxes on the left, showing axons at 250, 750, 1,000, 1,500, 2,000, and 3,000 μm distal to the crush sites. The yellow line indicates the crush sites. Yellow arrows indicate the top 3 longest axons of each nerve. Scale bar, 100 μm (50 μm for the magnified images). (B) Quantification of optic nerve regeneration in (A) (one-way ANOVA followed by Tukey’s multiple comparisons test; p < 0.0001 at 250, 500, 750, 1,000, 1,250, 1,500, 1,750, and 2,000 μm; p = 0.0004, 0.0206, 0.0092, 0.0042, 0.0026, and 0.0844 at 2,250, 2,500, 2,750, 3,000, 3,250, and 3,500 μm, respectively; n = 9 mice in Lin28a overexpression [O/E] group, n = 8 mice in other groups). (C) Quantification of the average length of the top 5 longest axons of each nerve in (A) (one-way ANOVA followed by Tukey’s multiple comparisons test, p < 0.0001; n = 9 mice in Lin28a O/E group, n = 8 mice in other groups). (D) Representative images of flat-mounted retinas showing that neither myosin IIA/B deletion nor combination of myosin IIA/B deletion and Lin28a overexpression had any effect on RGC survival rate 2 weeks after optic nerve crush. Flat-mounted retinas were stained with anti-tubulin β3 antibody (Tuj1, green). Scale bar, 50 μm. (E) Quantification of RGC survival rate in (D) (one-way ANOVA followed by Tukey’s multiple comparisons test, p = 0.0672; n = 3 mice in dKO group, n = 4 mice in other groups; 7–8 fields were analyzed for each retina). Note that the WT and dKO groups are identical to those in Figure 1E. Data are represented as mean ± SEM; P values of post hoc analyses are illustrated in the figure. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S3.

Figure 3.. Myosin-IIA/B-Deletion-Induced Optic Nerve Regeneration Was Not Mediated by mTOR or GSK3β Pathway

(A) Representative images of retinal sections showing that the deletion of myosin IIA/B did not activate mTOR (marked by pS6) in RGCs, whereas Lin28a overexpression markedly activated mTOR in RGCs 2 weeks after optic nerve crush. The two columns on the right display magnified images of the RGCs marked in white, dashed boxes on the left. Retinal sections were stained with anti-pS6 (green) and anti-tubulin β3 (magenta) antibodies. Scale bar, 50 μm (12.5 mm for the magnified images). (B) Quantification of the percentage of pS6⁺ RGCs in (A) (one-way ANOVA followed by Tukey’s multiple comparisons test, p < 0.0001; n = 3 mice in WT group, n = 5 mice in dKO group, n = 4 mice in Lin28a O/E group; at least 363 RGCs from at least 7 non-adjacent retinal sections were analyzed for each mouse). (C) Quantification of average fluorescence intensity of pS6 in all RGCs (one-way ANOVA followed by Tukey’s multiple comparisons test, p < 0.0001; n = 20 retinal sections with identical imaging configurations from at least 2 mice were analyzed for each group). (D) Quantification of average fluorescence intensity of pS6 in pS6⁺ RGCs (one-way ANOVA followed by Tukey’s multiple comparisons test, p < 0.0001; n = 42, 49, and 53 RGCs with identical imaging configurations from at least 2 mice were analyzed for WT, dKO, and Lin28a O/E groups, respectively). (E) Representative images of retinal sections showing that the deletion of myosin IIA/B did not inactivate GSK3 β (marked by pGSK3β) in RGCs, whereas Lin28a overexpression markedly inactivated GSK3 β in RGCs 2 weeks after optic nerve crush. The right two columns display magnified images of the RGCs marked in white, dashed boxes on the left. Retinal sections were stained with anti-pGSK3 β (green) and anti-tubulin b3 (magenta) antibodies. Scale bar, 50 μm (12.5 mm for the magnified images). (F) Quantification of the percentage of pGSK3 β⁺ RGCs in (E) (one-way ANOVA followed by Tukey’s multiple comparisons test, p < 0.0001; n = 3 mice in WT group, n = 4 mice in other groups; at least 434 RGCs from at least 7 non-adjacent retinal sections were analyzed for each mouse). (G) Quantification of average fluorescence intensity of pGSK3 β in all RGCs (one-way ANOVA followed by Tukey’s multiple comparisons test, p < 0.0001; n = 20 retinal sections with identical imaging configurations from at least 2 mice were analyzed for each group). (H) Quantification of average fluorescence intensity of pGSK3 β in pGSK3 β⁺ RGCs (one-way ANOVA followed by Tukey’s multiple comparisons test, p < 0.0001; n = 54, 40, and 65 RGCs with identical imaging configurations from at least 2 mice were analyzed for WT, dKO, and Lin28a O/E groups, respectively). Data are represented as mean ± SEM; P values of post hoc analyses are illustrated in the figure. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S4.

Figure 4.. Myosin IIA/B Deletion Did Not Significantly Affect General Gene Transcription in RGCs

(A) Principal-component analysis of RNA-seq libraries of purified RGCs showing the high degree of similarity in gene transcription between WT and myosin-IIA/B-deleted RGCs. (B) Hierarchical clustering of RNA-seq libraries showing the similarity in transcriptome between WT and myosin-IIA/B-deleted RGCs. The value in each grid represents the Euclidean distance between two libraries. (C) Pairwise correlations of RNA-seq libraries showing that the myosin IIA/B deletion had little impact on gene transcription of RGCs. The lower left shows the scattered plots of normalized counts between pairwise libraries. The upper right shows the Pearson correlation coefficient between pairwise libraries. (D) Gene Ontology analysis of differentially expressed genes between WT and myosin-IIA/B-deleted RGCs showing that myosin IIA/B deletion did not affect axon regeneration related gene transcription in RGCs. See also Figure S5 and Table S1.

Figure 5.. Myosin IIA/B Deletion Modified RGC Axonal Morphology after Optic Nerve Injury

(A) Representative images of optic nerves showing that the deletion of myosin IIA/B in RGCs markedly reduced the formation of retraction bulbs in optic nerves 2 and 4 weeks after optic nerve crush. Yellow arrows indicate retraction bulbs. Scale bar, 50 μm. (B) Quantification of retraction bulbs in (A) (Fisher’s exact test, p = 0.0004 and 0.0322 for 2 weeks and 4 weeks after optic nerve crush, respectively; n = 120 and 100 axonal tips from 12 nerves in 2-week WT and 10 nerves in dKO groups, respectively; n = 60 axonal tips from 6 nerves in each 4-week group). (C) Representative images of retraction bulbs and growth cones found in different optic nerves. Scale bar, 5 μm. (D) Quantification of axon extension efficiency in (F) (unpaired t test, p = 0.0032; n = 6 mice in each group; at least 35 axons were analyzed for each mouse). (E) Quantification of U-turn rate in Figure S7 (unpaired t test, p = 0.0210; n = 6 mice in each group; top 15 longest axons were analyzed for each mouse). (F) Left: representative images of optic nerves showing that the deletion of myosin IIA/B in RGCs improved axon extension efficiency 4 weeks after optic nerve crush. Middle: sketches of all axon traces in the left column. Right: detailed trajectories of a few axons (each color represents a single axon) in the left column. As illustrated, the extension efficiency of each nerve was calculated by dividing the summed displacement by the summed length of all traced axons. Scale bar, 50 μm. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S6 and S7 and Videos S1 and S2.

Figure 6.. Post-injury Deletion of Myosin IIA/B Could Also Induce Optic Nerve Regeneration

(A) Top: experimental timeline. Bottom: representative images of optic nerves showing that post-injury deletion of myosin IIA/B in RGCs induced axon regeneration 3 weeks after optic nerve crush. The columns on the right display magnified images of the areas in white, dashed boxes; on the left, showing axons at 250, 500, 750, and 1,000 μm distal to the crush sites. The yellow line indicates the crush sites. Yellow arrows indicate the top 3 longest axons of each nerve. Scale bar, 100 μm (50 μm for the magnified images). (B) Quantification of optic nerve regeneration in (A) (unpaired t test; p = 0.0017 at 250 μm; p < 0.0001 at 500, 750, and 1,000 μm; p = 0.0009, 0.0085, 0.1396, and 0.3343 at 1,250, 1,500, 1,750, and 2,000 μm, respectively; n = 8 mice in each group). Data are represented as mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.