CXCR4/CXCL12-mediated entrapment of axons at the injury site compromises optic nerve regeneration

Regenerative failure in the mammalian optic nerve is generally attributed to axotomy-induced retinal ganglion cell (RGC) death, an insufficient intrinsic regenerative capacity, and an extrinsic inhibitory environment. Here, we show that a chemoattractive CXCL12/CXCR4-dependent mechanism prevents the extension of growth-stimulated axons into the distal nerve. The chemokine CXCL12 is chemoattractive toward axonal growth cones in an inhibitory environment, and these effects are entirely abolished by the specific knockout of its receptor, CXCR4 (CXCR4−/−), in cultured regenerating RGCs. Notably, 8% of naïve RGCs express CXCL12 and transport the chemokine along their axons in the nerve. Thus, axotomy causes its release at the injury site. However, most osteopontin-positive α-RGCs, the main neuronal population that survives optic nerve injury, express CXCR4 instead. Thus, CXCL12-mediated attraction prevents growth-stimulated axons from regenerating distally in the nerve, indicated by axons returning to the lesion site. Accordingly, specific depletion of CXCR4 in RGC reduces aberrant axonal growth and enables long-distance regeneration. Likewise, CXCL12 knockout in RGCs fully mimics these CXCR4−/− effects. Thus, active CXCL12/CXCR4-mediated entrapment of regenerating axons to the injury site contributes to regenerative failure in the optic nerve.

Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy

Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or treating wild-type mice with a CCR5 antagonist repressed the effects of CNTF gene therapy. Thus, CCL5 is a previously unrecognized, potent activator of optic nerve regeneration and mediates many of the effects of CNTF gene therapy.