The role of CXCL12, its receptors and signaling pathways in axonal regeneration in the central nervous system
Molecular Biology and Physiology of Neurons and Glial Cells
Final Report Abstract
1st part: Mature retinal ganglion cells (RGCs) do not normally regenerate injured axons but undergo apoptosis soon after axotomy. Besides the insufficient intrinsic capability of mature neurons to regrow axons inhibitory molecules located in myelin of the central nervous system as well as the glial scar forming at the site of injury strongly limit axon regeneration. Nevertheless, RGCs can be transformed into a regenerative state upon inflammatory stimulation (IS), enabling these neurons to grow axons into the injured optic nerve. The outcome of IS stimulated regeneration was, however, still limited by the inhibitory extracellular environment. We found that the chemokine CXCL12/SDF-1 can moderately stimulate neurite growth of mature RGCs on laminin in culture and, in contrast to CNTF, exerts potent disinhibitory effects towards myelin. Consistently, co-treatment of RGCs with CXCL12 facilitated CNTF stimulated neurite growth of RGCs on myelin. Mature RGCs express CXCR4, the cognate CXCL12 receptor. Furthermore, the neurite growth promoting and disinhibitory effects of CXCL12 were abrogated by a specific CXCR4 antagonist and by inhibition of the PI3K/AKT/mTOR-, but not the JAK/STAT3-pathway. In vivo, intravitreal application of CXCL12 sustained mTOR activity in RGCs upon optic nerve injury and moderately stimulated axon regeneration in the optic nerve without affecting the survival of RGCs. Moreover, intravitreal application of CXCL12 also significantly increased IS triggered axon regeneration in vivo. 2nd part: We next investigated in mice to what extent the CXCR4 receptor in RGCs was involved in these processes using a genetic knockdown strategy. Surprisingly, we found that growth-stimulated axons (LI) are actively contained at the lesion site by a chemoattractive, CXCL12/CXCR4-dependent mechanism thereby preventing axon extension into the distal optic nerve. We showed that disinhibitory CXCL12 attracted axons of growth-stimulated adult RGCs on inhibitory myelin, and specific knockout of CXCR4 (CXCR4-/-), in these neurons fully abolished this effect. Notably, 8% of naïve RGCs expressed and axonally transported CXCL12, implying its release at the injury site. Other, RGCs, including αRGCs, expressed or even upregulated CXCR4 upon axotomy, causing aberrant fibers to return to the injury site. Accordingly, specific depletion of CXCR4 in RGC axons reduced aberrant growth and markedly enhanced IS-mediated nerve regeneration at long distances. Also, CXCL12 knockout in RGCs fully mimicked these CXCR4-/-effects. Thus, active, CXCL12/CXCR4-mediated attraction to the injury site compromises axon extension and thereby limits CNS axon regeneration. 3rd Part: To test the contribution of CXCL12 in microglia at the lesion site we also performed an extra study investigating the effect of the depletion of microglia on optic nerve regeneration. We found that treatment with the pharmacological colony stimulating factor 1 receptor inhibitor PLX5622 specifically eliminated microglia in murine retinae and optic nerves with high efficiency. Interestingly, time course and extent of retinal ganglion cell (RGC) degeneration after optic nerve crush remained unaffected upon microglia depletion, although remnants of prelabeled apoptotic RGCs were not cleared from the retina in these animals. In addition, microglia depletion neither affected the induction of regeneration associated genes upon optic nerve injury nor the increased regenerative potential of RGCs upon lens injury (LI). However, although the repopulation of the optic nerve lesion site by astrocytes was significantly delayed upon microglia depletion, spontaneous and LI-induced axon regeneration were unaffected by PLX5622 treatment or peripheral macrophage depletion by clodronate liposome treatment. Only concurrent double depletion of microglia and infiltrated macrophages slightly, but significantly, compromised optic nerve regeneration. Therefore, microglia or CXCL12 expressed in microglia are not essentially involved in RGC degeneration or axonal regeneration after acute CNS injury.
Publications
- (2013) CXCL12/SDF-1 facilitates optic nerve regeneration Neurobiology of Disease 55; 76-86
Heskamp A, Leibinger M, Andreadaki A, Gobrecht P, Diekmann H, Fischer D
(See online at https://doi.org/10.1016/j.nbd.2013.04.001) - (2017) Microglia are irrelevant for neuronal degeneration and axon regeneration after acute injury Journal of Neuroscience 37(25): 6124-6133
Hilla A, Diekmann H, Fischer D
(See online at https://doi.org/10.1523/JNEUROSCI.0584-17.2017) - (2018) Studying the Role of Microglia in Neurodegeneration and Axonal Regeneration in the Murine Visual System BIO-PROTOCOL 8 (16)
Hilla A and Fischer D
(See online at https://doi.org/10.21769/BioProtoc.2979)
