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.

Retinal ganglion cells (RGCs) convey the visual input from the eye through the optic nerve and optic tract into the brain’s target regions. As typical neurons of the central nervous system (CNS), mammalian RGCs lose most of their capability to regrow injured axons after birth (1, 2), leading to an irreversible functional loss after optic nerve damage. To date, regenerative failure has been mainly attributed to three leading causes: 1) axotomy-induced apoptosis of RGCs, 2) the low intrinsic capacity to regrow axons, and 3) the external inhibitory environment with CNS myelin and glial scar proteins (3, 4).One widely used approach to delay axotomy-induced RGC degeneration and activate the intrinsic regenerative capacity of injured axons is inflammatory stimulation (IS) in the eye induced by a lens injury, intravitreal Pam₃Cys, or zymosan injection (5–7). IS leads to the expression and release of CNTF, LIF, and IL-6 from retinal astrocytes and Müller cells (8–10), which directly interact with RGCs and activate neuroprotective/regenerative signaling such as the JAK/STAT3 pathway (8, 9, 11, 12). IS, therefore, enables moderate axon regeneration beyond the lesion site of the optic nerve. Although combinatorial strategies, together with measures overcoming the inhibitory CNS environment synergistically, further improve IS-mediated optic nerve regeneration (13–17), the overall outcome remains mostly unsatisfactory. Thus, additional unknown mechanisms besides neurodegeneration, low intrinsic capacity, and the inhibitory environment might contribute to optic nerve regeneration failure.The chemokine receptor CXCR4, a seven-transmembrane G protein–coupled receptor, is expressed in embryonic and adult neurons (18–20). We have recently shown that this receptor is also expressed in the somata and axons of adult rat RGCs (18). Next to its role as a coreceptor for HIV entry and cancer-cell migration/proliferation (21, 22), CXCR4 is reportedly involved in neurogenesis and axonal pathfinding during the embryonal development of RGCs (20, 23, 24). CXCR4 regulates different signaling pathways upon binding its ligand CXCL12 (also known as stromal cell–derived factor 1, SDF-1), which is part of the chemokine family of chemotactic cytokines in the immune system involved in the attraction of lymphocytes (25, 26). CXCL12 is also reportedly expressed by some CNS neurons, astrocytes, and microglia (19, 27–30). As the CXCR4/CXCL12 axis is highly conserved between different species (31) and involved in axonal pathfinding during embryonal development of RGCs (20, 32), we speculated that CXCR4 expression in adult RGCs might also play a role in the regenerative processes of mature axons.The current study shows that growth-stimulated axons of RGCs are actively attracted and entrapped at the lesion site of the optic nerve by a CXCL12/CXCR4-dependent mechanism. CXCL12 is expressed in a subpopulation of RGCs and axonally transported, implying its release at the injury site. A different RGC subpopulation expressed CXCR4, causing axons in the distal nerve to return to the injury site. Specific depletion of CXCR4 or CXCL12 in RGCs abolished aberrant growth. It enabled long-distance regeneration in the optic nerve, with some axons reaching the optic chiasm 3 wk after injury. Thus, active CXCL12/CXCR4-mediated entrapment markedly compromises axon extension into the distal optic nerve and contributes to regenerative failure in the optic nerve.