Macrophage recruitment in DRG contributes to sensory axon regeneration after conditioning injury

The central branches of the dorsal root ganglia (DRG) sensory neurons do not spontaneously regenerate. It has been known that the sensory neurons can regenerate if their peripheral branches are severed before SCI (conditioning injury, CI). After peripheral nerve injury, macrophages accumulate in DRGs. Recent studies revealed potential roles of macrophages and accompanying inflammatory reactions in axonal regeneration. However, it has not been addressed whether macrophages in DRG play role in CI-induced axon regeneration. In this thesis research, I examined whether the macrophages in DRGs contribute to the CI-induced sensory axon regeneration. Severance of the sciatic nerve resulted in a time-dependent increase of macrophages, proinflammatory cytokines (such as IL-6, IL-1 β, and TNF-α) and macrophageinduced regenerative factors such as various neurotrophins and oncomodulin, which is known to be a proregenerative molecule produced by macrophages in DGRs. Continuous infusion of macrophage deactivator minocycline to L5 DRG for 7 days after CI significantly reduced the number of macrophages, and almost completely abolished expressions of proinflammatory cytokines, neurotrophic factors, regeneration associated genes (GAP-43) and oncomodulin at 7 days after CI. Furthermore, minocycline infusion reduced CI-induced neurite outgrowth in vitro and sensory axon regeneration after CI in vivo. These data suggest that macrophages play an essential role in the CI-induced axon regeneration. Macrophage conditioned media (CM) treated with cAMP did not exhibit neurite-growing activity. Contrarily, CM from neuron-macrophage co-cultures treated with cAMP significantly promoted neurite outgrowth, highlighting the requirement of neuron-macrophage interactions for the induction of pro-regenerative macrophage phenotype. To elucidate signals mediating neuron-macrophage interactions, I performed a chemokine array in DRG samples taken after CI. To elucidate signals mediating neuron-macrophage interactions, I performed a chemokine array in DRG samples taken after CI. I identified a novel chemokine signaling mediated by CCL2 that links regenerating neurons and macrophage activation with proregenerative phenotypes in the CI model. The expression level of CCL2 was significantly increased in injured DRG neurons, and cAMP significantly upregulated CCL2 expression in DRG neuron culture. Adding neutralizing antibody against CCL2 in neuron-macrophage co-culture profoundly reduced the proregenerative activity of CM. Moreover, intraganglionic CCL2 neutralizing antibody injection abolished CI-induced enhanced neurite outgrowth. I demonstrated that neuronal CCL2, but not CCL2 from macrophages, is responsible for the neuron-macrophage interaction using the cocultures of primary neurons or macrophages from CCL2 knockout mice. Intraganglionic injection of recombinant CCL2 induced increases of macrophage infiltration in the DRGs and enhanced neurite growth of the DRG neurons taken at 7 days after the injection. Injection of either fractalkine or CCL3 failed to enhance neurite outgrowth while both chemokines were sufficient to increase the number of macrophages in the DRGs. The differential effects are probably due to differential macrophage polarization, because CCL2 instructed macrophages to take on M2 phenotype whereas M1 polarization was dominant by the fractalkine and CCL3 in primary macrophage cultures. I also tested whether CCL2 can mimic CI effects on in vivo axon regeneration after spinal injury. Since the enhanced capacity of neurite growth by intraganglionic CCL2 injection was significantly diminished by 4 weeks after the injection. I tested if adeno-associated virus serotype 5 (rAAV5) mediated overexpression of CCL2 can lead to long-lasting enhancement of axon regeneration capacity. Neurite outgrowth of the DRG neurons taken at 4 weeks was significantly increased by the intraganglionic rAAV5-CCL2 injection compared to control rAAV5-GFP injection. Furthermore, overexpression of neuronal CCL2 in DRGs by intraganglionic rAAV5-CCL2 injection enhanced sensory axon regeneration in vivo. This study suggests that chronic elevation of CCL2 level in DRG neurons can enhance long-lasting regenerative capacity. Based on the data obtained from my thesis research, I propose that manipulation of CCL2 signaling and the neuron-macrophage interactions may lead to a novel therapeutic approach to promote axon regeneration after CNS injury.

Author dc:creator

• 권, 민정

Contributors dc:contributor

• 김, 병곤
• 대학원 의생명과학과
• 112416

• 신경세포 재생
• 좌골신경 조건손상
• 대식세포
• CCL2

Language dc:language

en

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000017718
000000017718

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/10875