A DLK And JNK Dependent Axon Self- Destruction Program Promotes Wallerian Degeneration

Axons degenerate in a range of neurological disorders including mechanical injury, chemotherapy-induced neuropathy, hereditary neuropathies, glaucoma, diabetes, and neurodegenerative diseases such as Alzheimer’s Disease and Parkinson’s Disease1. Degenerating axons follow a stereotyped pathological progression that was first described in the 1850’s for the breakdown of the distal segments of severed axons and termed Wallerian degeneration 2. This degeneration likely results from an active self-destruction program rather than passive deterioration2 since it is delayed by over-expression of the chimeric Wallerian degeneration slow3 (Wlds) protein and its component nicotinamide mononucleotide adenylyltransferase4, 5, by preventing Ca2+ influx6, by blocking protein degradation7, and by disrupting the phagocytic clearance of axon fragments8. A variety of insults trigger axon degeneration2, 9, so identifying the components of the hypothesized axon self-destruction program could have broad clinical value. To date, however, no loss-of-function mutations have been identified that disrupt the internal axon breakdown machinery, and the internal signaling pathways that orchestrate axon breakdown in injury and disease remain unknown. Candidate components of axon breakdown pathways should be present in axons and activated by diverse cellular insults. One such candidate is DLK, a mitogen-activated protein kinase kinase kinase (MAP3K)10. One of DLK’s downstream targets, the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK), is activated following axonal injury11. We tested the hypothesis that DLK promotes axon degeneration using a well-established Drosophila olfactory receptor neuron (ORN) axotomy model12, 8. We expressed green fluorescent protein (GFP) in ORNs to visualize their axons, which extend from cell bodies in the antennae into the antennal lobes of the brain and across a midline commissure (Fig. 1a). To severe ORN axons and induce degeneration, we removed the antennae from wildtype flies and mutants lacking the Drosophila ortholog of DLK, wallenda (wnd)13. Most wildtype axons degenerated within twenty-four hours (Fig. 1b), while wnd mutant axons were significantly preserved (Fig. 1c). Wnd is therefore required for normal axon degeneration in Drosophila. Fig. 1 Neuronal Wnd promotes axon degeneration in Drosophila Wnd could act within neurons to promote breakdown after injury or within surrounding cells to promote axon clearance. To distinguish between these possibilities, we expressed Wnd in the GFP-expressing subpopulation of ORNs in wnd mutant flies. Such Wnd expression was not sufficient to induce degeneration in the absence of injury. However, we found that the Wnd expressing axons of these otherwise wnd mutant flies were not preserved twenty-four hours after axotomy (Fig. 1d). Thus, Wnd functions in an internal neuronal pathway that promotes injury-induced axon degeneration. Wnd may selectively promote injury induced axon degeneration, as we found no defects in the developmental pruning of mushroom body gamma-lobe axons (data not shown). To determine if DLK promotes Wallerian degeneration in mammals, we used dorsal root ganglion (DRG) cultures from littermate wildtype (Fig. 2a–c) and DLK-deficient (Fig. 2d–f) embryos14 (Supplementary Fig. 1). We severed DRG axons to induce degeneration and evaluated degeneration of the distal axon segment. Twenty-four hours after severing, wildtype axons distal to the transection deteriorated into axon fragments, while DLK-deficient axons remained continuous (Fig. 2b,e). To quantify the extent of axon fragmentation, we measured the fraction of total axonal area occupied by axon fragments (degeneration index, DI), and we found that DLK-deficient axons were significantly preserved (Fig. 2b,e). This delay in fragmentation persisted for forty-eight hours (Supplementary Fig. 2). Since non-neuronal cells are eliminated in this DRG culture system, DLK must operate within mammalian neurons to promote axon breakdown. Neuronal DLK therefore promotes axon fragmentation after injury in flies and mice. Fig. 2 Normal axon degeneration in mice requires DLK in vitro and in vivo To determine if DLK promotes degeneration in response to multiple insults, we assessed the response of DLK-deficient DRG axons to vincristine, a chemotherapeutic drug that induces axon degeneration in vitro, and whose dose-limiting side effects in patients include neuropathy15. We found that DLK-deficient axons were significantly protected from vincristine-induced fragmentation (Fig. 2c,f), suggesting that DLK operates in a general axon breakdown program. To determine if disrupting DLK protects injured axons in vivo, we transected the sciatic nerves of littermate wildtype and DLK-deficient adult mice (Fig. 2g–l). Fifty-two hours post-transection, wildtype axons degenerated, whereas DLK-deficient axons were significantly preserved (Fig. 2h,i). Electron microscopy revealed that preserved axon profiles contain mitochondria and a cytoskeleton (Fig. 2l and Supplementary Fig. 3). Thus, normal Wallerian degeneration in vivo in adult mice requires DLK. DLK is a MAP3K that can activate JNK and p38 via intermediary MAP2Ks10. To determine whether either downstream kinase promotes axon degeneration, we inhibited JNK and p38 in the DRG axotomy model using wildtype cultures. Inhibition of JNK, but not p38, protected transected axons from fragmentation (Fig. 3a–e), and a significant delay in fragmentation persisted for over forty-eight hours (Supplementary Fig. 2). Thus JNK, like DLK, acts within neurons to promote axon degeneration. Fig. 3 JNK promotes Wallerian degeneration and is critical in the first three hours after axotomy Axon degeneration is hypothesized to comprise at least three distinct phases – competence to degenerate, much of which is determined transcriptionally before axotomy; commitment to degenerate, which occurs in the substantial delay period between injury and axon fragmentation; and the execution phase, when axons fragment1. If JNK’s primary role were to promote competence to degenerate, then JNK activity should be required prior to axotomy. This is not the case: applying the JNK inhibitor twenty-four hours prior to axotomy and then removing it just before axotomy was not protective (Fig. 3f). In contrast, JNK inhibition started concurrently with axotomy was protective (Fig. 3g). Thus, JNK promotes axon fragmentation after the competence period and acts within the severed distal axon segment. JNK could commit axons to degenerate during the delay between injury and breakdown, or it could operate during the subsequent execution phase of axon breakdown. To test whether JNK activity is required during the execution phase, we added the JNK inhibitor three hours after axotomy, which is approximately nine hours before the onset of fragmentation. This treatment schedule spans the transition from the proposed commitment phase to the execution phase and the entire execution phase itself. Continuous JNK inhibition beginning three hours post-axotomy did not delay axon fragmentation (Fig. 3h). Therefore, JNK activity is not required during the execution phase of axon fragmentation. Rather, JNK activity is required during the early response to injury that commits the axon to breakdown hours later. Converging lines of evidence suggest that there is a general internal axon self-destruction program, but its molecular components are unknown. We now show that the MAP3K DLK and its downstream MAPK JNK are important elements of such a program. Disrupting this pathway delays axon fragmentation in response to both axotomy and the neurotoxic chemotherapeutic agent vincristine. Thus, a common self-destruction program may promote axon breakdown in response to diverse insults, and so may be targetable in multiple clinical settings.