Protein turnover of the Wallenda/DLK kinase regulates a retrograde response to axonal injury

283

427

The cell intrinsic factors that determine whether a neuron regenerates or undergoes apoptosis in response to axonal injury are not well defined. Here we show that the mixed-lineage dual leucine zipper kinase (DLK) is an essential upstream mediator of both of these divergent outcomes in the same cell type. Optic nerve crush injury leads to rapid elevation of DLK protein, first in the axons of retinal ganglion cells (RGCs) and then in their cell bodies. DLK is required for the majority of gene expression changes in RGCs initiated by injury, including induction of both proapoptotic and regeneration-associated genes. Deletion of DLK in retina results in robust and sustained protection of RGCs from degeneration after optic nerve injury. Despite this improved survival, the number of axons that regrow beyond the injury site is substantially reduced, even when the tumor suppressor phosphatase and tensin homolog (PTEN) is deleted to enhance intrinsic growth potential. These findings demonstrate that these seemingly contradictory responses to injury are mechanistically coupled through a DLK-based damage detection mechanism.A xonal damage results in significant neuronal cell death and axon degeneration, often leading to permanent functional deficits. For example, optic nerve crush rapidly induces a stress response in retinal ganglion cells (RGCs) that includes profound alterations in gene expression patterns (1) and ultimately leads to apoptosis of these neurons (2). As axon injury may occur a significant distance from the cell body, it has been proposed that retrograde molecular motors play a critical role in conveying damage signals to the nucleus, allowing the cell to respond to damage (3). Attenuation of this transport mechanism has been shown to reduce degeneration, suggesting that the ability of the nucleus to detect an insult is an essential component of the injury response (4).Recent data suggest that dual leucine zipper kinase (DLK) is an essential component of the neuronal response to axon damage. DLK protein is present in axons, and protein levels are increased in response to axonal injury (5). Loss of DLK has been shown to protect distal axons from Wallerian degeneration (6) and to abrogate stress-induced retrograde c-Jun N-terminal kinase (JNK) signaling through interaction with the scaffolding protein JNK-interacting protein 3 (JIP3) (7-9). In many instances, injury-induced JNK activation in neurons results in apoptosis through phosphorylation of activator protein 1 (AP-1) transcription factors such as c-Jun, which initiates a proapoptotic gene expression program (10, 11). Consistent with this, genetic deletion of JNK2 and/or JNK3 is sufficient to protect neurons from degeneration in a range of CNS injury models, including axotomy (12-14), although the role of DLK in these contexts is not known.In contrast, DLK has been shown to regulate axon regeneration after axonal injury in adult peripheral nerves (9) and invertebrate systems (5, 15). The mechanism underlying the divergence between these apoptotic and reg...

mentioning

confidence: 90%

“…DLK protein is present in axons, and protein levels are increased in response to axonal injury (5). Loss of DLK has been shown to protect distal axons from Wallerian degeneration (6) and to abrogate stress-induced retrograde c-Jun N-terminal kinase (JNK) signaling through interaction with the scaffolding protein JNK-interacting protein 3 (JIP3) (7)(8)(9).…”

mentioning

confidence: 99%

DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury

Watkins¹,

Wang²,

Huntwork‐Rodriguez³

et al. 2013

283

427

“…3A), indicating that increased translation and/or decreased protein turnover must underlie the mechanism of DLK up-regulation. In Drosophila melanogaster, Wallenda/DLK is posttranslationally regulated by the E3 ubiquitin ligase Highwire (36,37). However, mice with a brain-specific conditional knockout of Phr1 (Pam/Highwire/RPM-1 1, the vertebrate Highwire homolog) show no difference in the overall brain levels of DLK protein (38).…”

Section: Axonal Injury Up-regulates Dlk Expression Through a Posttranmentioning

confidence: 99%

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death

Welsbie

Yang

et al. 2013

Self Cite

230

307

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.G laucoma is the leading cause of irreversible blindness worldwide (1). It is a neurodegenerative disease in which vision loss is caused by the axonal injury and death of retinal ganglion cells (RGCs) (2), the projection neurons that process and transmit vision from the retina to the brain. Current therapies (i.e., surgery, laser, and eye drops) all act by lowering intraocular pressure (IOP). However, pressure reduction can be difficult to achieve, and even with significant pressure lowering, RGC loss can continue. Efforts have therefore been made to develop neuroprotective agents that would complement IOP-lowering therapies by directly inhibiting the RGC cell death process (3, 4). However, no neuroprotective agent has yet been approved for clinical use.Protein kinases provide attractive targets for the development of neuroprotective agents. A number of kinases, including cyclindependent kinases, death-associated protein kinases, JNK1-3, MAPKs, and glycogen synthase kinase-3β, are involved in neuronal cell death (5-12). An additional attraction is that protein kinases are readily druggable. The pharmacology and medicinal chemistry of kinase inhibitors are well-developed, with kinases now being the most important class of drug targets after G protein-coupled receptors (13). Although the primary clinical use of kinase inhibitors continues to be as antineoplastic agents, increasing attention is being paid to their use in other areas (14,15).To identify, in a comprehensive and unbiased manner, kinases that could serve as targets for neuroprotective glaucoma therapy, we screened the entire mouse kinome for kinases whose inhibition promotes RGC survival. For this screen, we develope...

“…JNK signaling is implicated in axon injury response in many systems (6,(14)(15)(16)(17). We therefore hypothesized that JNK signaling would be required for increased microtubule dynamics and for dendrite stabilization.…”

Section: Increased Microtubule Dynamics and Dendrite Stabilization Afmentioning

confidence: 99%

“…We therefore hypothesized that JNK signaling would be required for increased microtubule dynamics and for dendrite stabilization. To test this hypothesis, we compared both responses in control neurons and neurons that expressed a dominant-negative form of the Drosophila JNK protein bsk (bskDN), which blocks axon injury signaling in Drosophila motor neurons (17). Expression of bskDN dampened the microtubule number increase after axon injury (Fig.…”

Section: Increased Microtubule Dynamics and Dendrite Stabilization Afmentioning

confidence: 99%

Axon injury and stress trigger a microtubule-based neuroprotective pathway

Chen

Stone

Tao

et al. 2012

102

Axon injury elicits profound cellular changes, including axon regeneration. However, the full range of neuronal injury responses remains to be elucidated. Surprisingly, after axons of Drosophila dendritic arborization neurons were severed, dendrites were more resistant to injury-induced degeneration. Concomitant with stabilization, microtubule dynamics in dendrites increased. Moreover, dendrite stabilization was suppressed when microtubule dynamics was dampened, which was achieved by lowering levels of the microtubule nucleation protein γ-tubulin. Increased microtubule dynamics and global neuronal stabilization were also activated by expression of expanded polyglutamine (poly-Q) proteins SCA1, SCA3, and huntingtin. In all cases, dynamics were increased through microtubule nucleation and depended on JNK signaling, indicating that acute axon injury and long-term neuronal stress activate a common cytoskeleton-based stabilization program. Reducing levels of γ-tubulin exacerbated long-term degeneration induced by SCA3 in branched sensory neurons and in a well established Drosophila eye model of poly-Q-induced neurodegeneration. Thus, increased microtubule dynamics can delay short-term injury-induced degeneration, and, in the case of poly-Q proteins, can counteract progressive longer-term degeneration. We conclude that axon injury or stress triggers a microtubulebased neuroprotective pathway that stabilizes neurons against degeneration.M any animals generate a single set of neurons that must function for the entire life of the individual. Each neuron typically has a single axon that transmits signals to other neurons or output cells such as muscle. As axons can extend long distances, they are at risk for injury, and, if the single axon is damaged, the cell can no longer function. Many neurons thus mount major responses to axon injury. The best characterized of these responses is axon regeneration, the process in which a neuron extends the stump of the existing axon or grows a new axon from a dendrite (1-3).In addition to the regenerative response, axon injury can cause other less well-understood changes. For example, in mammalian dorsal root ganglion cells, injury of the peripheral axon causes a transcriptional response that increases the capacity of the central axon to regenerate if it is subsequently injured (4, 5). In Drosophila sensory neurons, axon injury causes cytoskeletal changes in the entire dendrite arbor, specifically the number of growing microtubules is up-regulated (6). In this study, we investigated the functional significance of the cytoskeletal changes in the dendrite arbor. We present results that suggest the altered microtubule dynamics in dendrites acts to stabilize them, and thus axon injury seems to trigger a neuroprotective pathway that acts on the rest of the cell. However, this neuroprotective pathway is turned on only transiently after axon injury and subsides as axon regeneration initiates.Axon injury is a very acute neuronal stress. Neurons are also subject to a variety of long-term stress...