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. 2023 Mar 31;18(11):2526–2534. doi: 10.4103/1673-5374.371372

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Copyright: © Neural Regeneration Research

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S6K1 activation and inhibition of 4E-BP1 are necessary for caRheb-induced axon regeneration following ONC.

(A) DN-S6K1 decreased p-S6 expression while caS6K1 increased p-S6 expression. Immunofluorescence of retinal sections using p-S6 (red) or Tuj1 (green) antibodies from mice transfected with AAV-DN-S6K1 or AAV-caS6K1. (B) Delivery of 4E-BP1-4A increased 4E-BP1 expression while sh4E-BP1 decreased 4E-BP1 expression. Immunofluorescence analysis for 4E-BP1 levels in RGCs transfected with AAV-4E-BP1-4A or AAV-sh4E-BP1. Cells were stained with antibodies for 4E-BP1 (red) or Tuj1 (green). (C) Mixed injection of AAV was administrated intravitreally to mice 2 weeks before ONC. CTB was intravitreally injected 12 days after ONC and eyes were harvested on days 14 after ONC. (D) DN-S6K1 and 4E-BP1-4A decreased the regenerating axons induced by caRheb. Images of optic nerve sections showing regenerating axons labeled by anterograde transport of CTB on days 14 after ONC from eyes injected with AAV-caRheb, AAV-caRheb + DN-S6K1, AAV-caRheb + caS6K1, AAV-caRheb + 4E-BP1-4A, or AAV-EGFP. Red asterisk indicates crush site. Scale bars: 40 µm (A, B), 100 µm (D). (E) Quantification of regenerating axons 14 days post-crush from eyes injected with AAV-caRheb, AAV-caRheb + DN-S6K1, AAV-caRheb + caS6K1, or AAV-caRheb + 4E-BP1-4A at different distances distal to the lesion site. (F) Quantification of regenerating axons on day 14 post-crush from eyes injected with AAV-EGFP, AAV-caRheb + 4E-BP1-4A, or AAV-caRheb + DN-S6K1 at different distances distal to the lesion site. Data are presented as mean ± SD (n = 8–10). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way analysis of variance with Bonferroni’s post hoc test). 4E-BP1-4A: Non-phosphorylatable 4E-BP1 mutant; AAV: adeno-associated virus; caRheb: constitutively active Ras homolog enriched in brain; caS6K1: constitutively active S6K1; CTB: cholera toxin B subunit; DN-S6K1: dominate negative S6K1; dpc: days post crush; EGFP: enhanced green fluorescent protein; GCL: ganglion cell layer; INL: inner nuclear layer; ONC: optic nerve crush; ONL: outer nuclear layer; p-S6: phospho-S6 ribosomal protein; RGC: retinal ganglion cell; S6K1: mammalian target of rapamycin complex 1 downstream effector; Tuj1: β3-tubulin.