Rodent models of nerve injury have increased our understanding of peripheral nerve regeneration, but clinical applications have been scarce, partly because such models do not adequately recapitulate the situation in humans. In human injuries, axons are often required to extend over much longer distances than in mice, and injury leaves distal nerve fibres and target tissues without axonal contact for extended amounts of time. Distal Schwann cells undergo atrophy owing to the lack of contact with proximal neurons, which results in reduced expression of neurotrophic growth factors, changes in the extracellular matrix and loss of Schwann cell basal lamina, all of which hamper axonal extension. Furthermore, atrophy and denervation-related changes in target tissues make good functional recovery difficult to achieve even when axons regenerate all the way to the target tissue. To improve functional outcomes in humans, strategies to increase the speed of axonal growth, maintain Schwann cells in a healthy, repair-capable state and keep target tissues receptive to reinnervation are needed. Use of rodent models of chronic denervation will facilitate our understanding of the molecular mechanisms of peripheral nerve regeneration and create the potential to test therapeutic advances.

Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid and the endocannabinoids, 2-arachidonoylglycerol and arachidonoylethanolamide. Evaluation of a series of COX-2 inhibitors revealed that many weak, competitive inhibitors of arachidonic acid oxygenation are potent inhibitors of endocannabinoid oxygenation. (R)-Enantiomers of ibuprofen, naproxen, and flurbiprofen, which are considered to be inactive as COX-2 inhibitors, are potent “substrate-selective inhibitors” of endocannabinoid oxygenation. Crystal structures of the COX-2-(R)-naproxen and COX-2-(R)-flurbiprofen complexes verified this unexpected binding and defined the orientation of the (R)-enantiomers relative to (S)-enantiomers. (R)-Profens selectively inhibited endocannabinoid oxygenation by lipopolysaccharide-stimulated dorsal root ganglion cells. Substrate-selective inhibition provides novel tools for investigating the role of COX-2 in endocannabinoid oxygenation and a possible explanation for the ability of (R)-profens to maintain endocannabinoid tone in models of neuropathic pain.

During the development of peripheral ganglia 50 % of the neurons generated undergo apoptosis. How the massive numbers of corpses are removed is unknown. We report that satellite glial cell precursors are the primary phagocytic cells for apoptotic corpse removal in developing mouse dorsal root ganglia (DRG). Confocal and electron microscopic analysis revealed that glial precursors, not macrophages, are responsible for clearing most of the dead DRG neurons. Moreover, we identified Jedi-1, a novel engulfment receptor, and MEGF10, a purported engulfment receptor, as homologs of the invertebrate engulfment receptors Draper and CED-1 expressed in the glial precursor cells. Expression of Jedi-1 or MEGF10 in fibroblasts facilitated binding to dead neurons and knocking down either protein in glial cells, or over expressing truncated forms lacking the intracellular domain, inhibited engulfment of apoptotic neurons. Together, these results reveal the cellular and molecular mechanism by which neuronal corpses are culled during DRG development.

During the development of the peripheral nervous system there is extensive apoptosis and these neuronal corpses need to be cleared to prevent an inflammatory response. Recently, Jedi-1 and MEGF10, both expressed in glial precursor cells, were identified in mouse as having an essential role in this phagocytosis (Wu et al., 2009); however, the mechanisms by which they promote engulfment remained unknown. Both Jedi-1 and MEGF10 are homologous to the Drosophila melanogaster receptor Draper, which mediates engulfment through activation of the tyrosine kinase Shark. Here, we identify Syk, the mammalian homolog of Shark, as a signal transducer for both Jedi-1 and MEGF10. Syk interacted with each receptor independently through the Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) in their intracellular domains. The interaction was enhanced by phosphorylation of the tyrosines in the ITAMs by Src family kinases (SFKs). Jedi association with Syk and activation of the kinase was also induced by exposure to dead cells. Expression of either Jedi-1 or MEGF10 in HeLa cells facilitated engulfment of carboxylated microspheres to a similar extent and there was no additive effect when they were co-expressed. Mutation of the ITAM tyrosines of Jedi-1 and MEGF10 prevented engulfment. The SFK inhibitor PP2 or a selective Syk inhibitor (BAY 61-3606) also blocked engulfment. Similarly, in co-cultures of glial precursors and dying sensory neurons from embryonic mice, addition of PP2 or knock down of endogenous Syk decreased the phagocytosis of apoptotic neurons. These results indicate that both Jedi-1 and MEGF10 can mediate phagocytosis independently through the recruitment of Syk.