Bradley R. Miller

A dual leucine kinase–dependent axon self-destruction program promotes Wallerian degeneration

Axon degeneration underlies many common neurological disorders, but the signaling pathways that orchestrate axon degeneration are unknown. We found that dual leucine kinase (DLK) promoted degeneration of severed axons in Drosophila and mice, and that its target, c-Jun N-terminal kinase, promoted degeneration locally in axons as they committed to degenerate. This pathway also promoted degeneration after chemotherapy exposure and may be a component of a general axon self-destruction program.

Amyloid Precursor Protein Cleavage-Dependent and -Independent Axonal Degeneration Programs Share a Common Nicotinamide Mononucleotide Adenylyltransferase 1-Sensitive Pathway

Axonal degeneration is a hallmark of many debilitating neurological disorders and is thought to be regulated by mechanisms distinct from those governing cell body death. Recently, caspase 6 activation via amyloid precursor protein (APP) cleavage and activation of DR6 was discovered to induce axon degeneration after NGF withdrawal. We tested whether this pathway is involved in axonal degeneration caused by withdrawal of other trophic support, axotomy or vincristine exposure. Neurturin deprivation, like NGF withdrawal activated this APP/DR6/caspase 6 pathway and resulted in axonal degeneration, however, APP cleavage and caspase 6 activation were not involved in axonal degeneration induced by mechanical or toxic insults. However, loss of surface APP (sAPP) and caspase 6 activation were observed during axonal degeneration induced by dynactin 1(Dctn1) dysfunction, which disrupts axonal transport. Mutations in Dctn1 are associated with motor neuron disease and frontal temporal dementia, thus suggesting that the APP/caspase 6 pathway could be important in specific types of disease-associated axonal degeneration. The NGF deprivation paradigm, with its defined molecular pathway, was used to examine the context of Nmnat-mediated axonal protection. We found that although Nmnat blocks axonal degeneration after trophic factor withdrawal, it did not prevent loss of axon sAPP or caspase 6 activation within the axon, suggesting it acts downstream of caspase 6. These results indicate that diverse insults induce axonal degeneration via multiple pathways and that these degeneration signals converge on a common, Nmnat-sensitive program that is uniquely involved in axonal, but not cell body, degeneration.

SCG10 is a JNK target in the axonal degeneration pathway

Axons actively self-destruct following genetic, mechanical, metabolic, and toxic insults, but the mechanism of axonal degeneration is poorly understood. The JNK pathway promotes axonal degeneration shortly after axonal injury, hours before irreversible axon fragmentation ensues. Inhibition of JNK activity during this period delays axonal degeneration, but critical JNK substrates that facilitate axon degeneration are unknown. Here we show that superior cervical ganglion 10 (SCG10), an axonal JNK substrate, is lost rapidly from mouse dorsal root ganglion axons following axotomy. SCG10 loss precedes axon fragmentation and occurs selectively in the axon segments distal to transection that are destined to degenerate. Rapid SCG10 loss after injury requires JNK activity. The JNK phosphorylation sites on SCG10 are required for its rapid degradation, suggesting that direct JNK phosphorylation targets SCG10 for degradation. We present a mechanism for the selective loss of SCG10 distal to the injury site. In healthy axons, SCG10 undergoes rapid JNK-dependent degradation and is replenished by fast axonal transport. Injury blocks axonal transport and the delivery of SCG10, leading to the selective loss of the labile SCG10 distal to the injury site. SCG10 loss is functionally important: Knocking down SCG10 accelerates axon fragmentation, whereas experimentally maintaining SCG10 after injury promotes mitochondrial movement and delays axonal degeneration. Taken together, these data support the model that SCG10 is an axonal-maintenance factor whose loss is permissive for execution of the injury-induced axonal degeneration program.

Increased vesicular glutamate transporter expression causes excitotoxic neurodegeneration.

Increases in vesicular glutamate transporter (VGLUT) levels are observed after a variety of insults including hypoxic injury, stress, methamphetamine treatment, and in genetic seizure models. Such overexpression can cause an increase in the amount of glutamate released from each vesicle, but it is unknown whether this is sufficient to induce excitotoxic neurodegeneration. Here we show that overexpression of the Drosophila vesicular glutamate transporter (DVGLUT) leads to excess glutamate release, with some vesicles releasing several times the normal amount of glutamate. Increased DVGLUT expression also leads to an age-dependent loss of motor function and shortened lifespan, accompanied by a progressive neurodegeneration in the postsynaptic targets of the DVGLUT-overexpressing neurons. The early onset lethality, behavioral deficits, and neuronal pathology require overexpression of a functional DVGLUT transgene. Thus overexpression of DVGLUT is sufficient to generate excitotoxic neuropathological phenotypes and therefore reducing VGLUT levels after nervous system injury or stress may mitigate further damage.

A tyrosine-based motif localizes a Drosophila vesicular transporter to synaptic vesicles in vivo.

Vesicular neurotransmitter transporters must localize to synaptic vesicles (SVs) to allow regulated neurotransmitter release at the synapse. However, the signals required to localize vesicular proteins to SVs in vivo remain unclear. To address this question we have tested the effects of mutating proposed trafficking domains in Drosophila orthologs of the vesicular monoamine and glutamate transporters, DVMAT-A and DVGLUT. We show that a tyrosine-based motif (YXXY) is important both for DVMAT-A internalization from the cell surface in vitro, and localization to SVs in vivo. In contrast, DVGLUT deletion mutants that lack a putative C-terminal trafficking domain show more modest defects in both internalization in vitro and trafficking to SVs in vivo. Our data show for the first time that mutation of a specific trafficking motif can disrupt localization to SVs in vivo and suggest possible differences in the sorting of VMATs versus VGLUTs to SVs at the synapse.

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.

Vesicular neurotransmitter transporter trafficking in vivo: Moving from cells to flies

During exocytosis, classical and amino acid neurotransmitters are released from the lumen of synaptic vesicles to allow signaling at the synapse. The storage of neurotransmitters in synaptic vesicles and other types of secretory vesicles requires the activity of specific vesicular transporters. Glutamate and monoamines such as dopamine are packaged by VGLUTs and VMATs respectively. Changes in the localization of either protein have the potential to up- or down regulate neurotransmitter release, and some of the mechanisms for sorting these proteins to secretory vesicles have been investigated in cultured cells in vitro. We have used Drosophila molecular genetic techniques to study vesicular transporter trafficking in an intact organism and have identified a motif required for localizing Drosophila VMAT (DVMAT) to synaptic vesicles in vivo. In contrast to DVMAT, large deletions of Drosophila VGLUT (DVGLUT) show relatively modest deficits in localizing to synaptic vesicles, suggesting that DVMAT and DVGLUT may undergo different modes of trafficking at the synapse. Further in vivo studies of DVMAT trafficking mutants will allow us to determine how changes in the localization of vesicular transporters affect the nervous system as a whole and complex behaviors mediated by aminergic circuits.