Expression of MUK/DLK/ZPK, an activator of the JNK pathway, in the nervous systems of the developing mouse embryo

doi:10.1016/j.modgep.2004.12.002

Gene Expression Patterns

Volume 5, Issue 4, April 2005, Pages 517-523

https://doi.org/10.1016/j.modgep.2004.12.002 Get rights and content

Abstract

C-Jun N-terminal kinase (JNK) is implicated in regulating the various cellular events during neural development that include differentiation, apoptosis and migration. MUK/DLK/ZPK is a MAP kinase kinase kinase (MAPKKK) enzyme that activates JNK via MAP kinase kinases (MAPKK) such as MKK7. We show here that the expression of MUK/DLK/ZPK protein in the developing mouse embryo is almost totally specific for the neural tissues, including central, peripheral, and autonomic nervous systems. The only obvious exception is the liver, in which the protein is temporally expressed at around E11. The expression becomes obvious in the neurons of the brain and neural crest tissues at embryonic day 10 (E10) after neuron production is initiated. By E14, MUK/DLK/ZPK proteins are found in various neural tissues including the brain, spinal cord, sensory ganglia (such as trigeminal and dorsal root ganglia), and the sympathetic and visceral nerves. The localization of MUK/DLK/ZPK protein in neural cells almost consistently overlapped that of βIII-tubulin, a neuron specific tubulin isoform, and both proteins were more concentrated in axons than in cell bodies and dendrites. The intensely overlapping localization of βIII-tubulin and MUK/DLK/ZPK indicated that this protein kinase is tightly associated with the microtubules of neurons.

Section snippets

Results and discussion

Originally identified as a subgroup of MAP kinases that are activated by cellular stress, and which regulate cell differentiation and apoptosis (Kyriakis and Avruch, 2001), JNK is now recognized as a key component of the signal transduction pathways involved in the regulation of planner cell polarity and cell migration, both of which are essential for ontogeny (Hirai et al., 2002, Weston and Davis, 2002, Xia and Karin, 2004). The multiplicity of cellular events regulated by JNK indicates that

Experimental procedures

Mouse embryos at embryonic day 10–16 were fixed overnight in 4% PFA at 4 °C and then embedded in paraffin wax. Paraffin sections (6 μm thick) were hydrolyzed, heated at 120 °C for 20 min in 10 mM sodium citrate, pH 6.0 and then immunohistochemically stained using a standard protocol. Briefly, sections were sequentially incubated with primary affinity-purified anti-MUK rabbit antibody raised against the C-terminal part of MUK (Hirai et al., 2002), anti-MAP2 monoclonal antibody (Sigma), rabbit

Acknowledgements

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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MKK4 and MKK7 have yet to be tested independently or together in a model of ocular hypertension. Upstream of JNK and the MAP2Ks are the MAP3Ks, most notably the dual leucine zipper kinase (DLK) and leucine-zipper bearing kinase (LZK), that are expressed in axons and contribute to axonal and somal degeneration after glaucomatous related injuries (Farley and Watkins, 2018; Fernandes et al., 2014; Gerdts et al., 2016; Hirai et al., 2005; Tedeschi and Bradke, 2013; Valakh et al., 2015; Watkins et al., 2013; Welsbie et al., 2013, 2017). The MAP3Ks often control the activation of different physiological and pathological pools of JNK (Chen et al., 2002; Craig et al., 2008).
Axonal degeneration is an active, highly controlled process that contributes to beneficial processes, such as developmental pruning, but also to neurodegeneration. In glaucoma, ocular hypertension leads to vision loss by killing the output neurons of the retina, the retinal ganglion cells (RGCs). Multiple processes have been proposed to contribute to and/or mediate axonal injury in glaucoma, including: neuroinflammation, loss of neurotrophic factors, dysregulation of the neurovascular unit, and disruption of the axonal cytoskeleton. While the inciting injury to RGCs in glaucoma is complex and potentially heterogeneous, axonal injury is ultimately thought to be the key insult that drives glaucomatous neurodegeneration. Glaucomatous neurodegeneration is a complex process, with multiple molecular signals contributing to RGC somal loss and axonal degeneration. Furthermore, the propagation of the axonal injury signal is complex, with injury triggering programs of degeneration in both the somal and axonal compartment. Further complicating this process is the involvement of multiple cell types that are known to participate in the process of axonal and neuronal degeneration after glaucomatous injury. Here, we review the axonal signaling that occurs after injury and the molecular signaling programs currently known to be important for somal and axonal degeneration after glaucoma-relevant axonal injuries.
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Both, TNFα and IL-1β stimulate MLK3 kinase activity at least in part dependent on the activation of Rac/Cdc42 and the interaction with the Rac/Cdc42 interaction site (CRIB) within MLK3 [10–12]. DLK is expressed in many and diverse tissues like brain and the peripheral nervous system, β-cells and primary islets [8,13,14]. Mice lacking DLK die perinatally, exhibiting impaired axon growth and neuronal radial migration, absence of the anterior commissure and reduced apoptosis in multiple neuronal populations during development [15–17].
The dual leucine zipper kinase DLK induces β-cell apoptosis by inhibiting the transcriptional activity conferred by the β-cell protective transcription factor cAMP response element binding protein CREB. This action might contribute to β-cell loss and ultimately diabetes. Within its kinase domain DLK shares high homology with the mixed lineage kinase (MLK) 3, which is activated by tumor necrosis factor (TNF) α and interleukin (IL)-1β, known prediabetic signals. In the present study, the regulation of DLK in β-cells by these cytokines was investigated. Both, TNFα and IL-1β induced the nuclear translocation of DLK. Mutations within a putative nuclear localization signal (NLS) prevented basal and cytokine-induced nuclear localization of DLK and binding to the importin receptor importin α, thereby demonstrating a functional NLS within DLK. DLK NLS mutants were catalytically active as they phosphorylated their down-stream kinase c-Jun N-terminal kinase to the same extent as DLK wild-type but did neither inhibit CREB-dependent gene transcription nor transcription conferred by the promoter of the anti-apoptotic protein BCL-xL. In addition, the β-cell apoptosis-inducing effect of DLK was severely diminished by mutation of its NLS. In a murine model of prediabetes, enhanced nuclear DLK was found. These data demonstrate that DLK exerts distinct functions, depending on its subcellular localization and thus provide a novel level of regulating DLK action. Furthermore, the prevention of the nuclear localization of DLK as induced by prediabetic signals with consecutive suppression of β-cell apoptosis might constitute a novel target in the therapy of diabetes mellitus.
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Viral reactivation could be completed when these signaling pathways reach full threshold. As DLK and JIP-3 are expressed almost exclusively in neurons (Hirai et al., 2005; Kelkar et al., 2000), targeting them would be an effective mechanism to prevent HSV reactivation in response to multiple triggers. We have further defined phase I of reactivation as being dependent upon JNK signaling but independent of histone demethylase activity.
Herpes simplex virus (HSV) reactivation from latent neuronal infection requires stimulation of lytic gene expression from promoters associated with repressive heterochromatin. Various neuronal stresses trigger reactivation, but how these stimuli activate silenced promoters remains unknown. We show that a neuronal pathway involving activation of c-Jun N-terminal kinase (JNK), common to many stress responses, is essential for initial HSV gene expression during reactivation. This JNK activation in neurons is mediated by dual leucine zipper kinase (DLK) and JNK-interacting protein 3 (JIP3), which direct JNK toward stress responses instead of other cellular functions. Surprisingly, JNK-mediated viral gene induction occurs independently of histone demethylases that remove repressive lysine modifications. Rather, JNK signaling results in a histone methyl/phospho switch on HSV lytic promoters, a mechanism permitting gene expression in the presence of repressive lysine methylation. JNK is present on viral promoters during reactivation, thereby linking a neuronal-specific stress pathway and HSV reactivation from latency.
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Five days after axonal injury pJNK fills RGC somas in wildtype retinas, but was not present in RGCs of Dlk−/− mice (Fig. 3A). These data were surprising since DLK is known to be expressed in axons and regulate axonal injury signaling (Eto et al., 2010; Hirai et al., 2005; Xiong et al., 2010). However, it is possible that there is some level of pJNK activation that is controlled by DLK in the proximal axon that was not observed by immunohistochemistry.
Injury to retinal ganglion cell (RGC) axons triggers rapid activation of Jun N-terminal kinase (JNK) signaling, a major prodeath pathway in injured RGCs. Of the multiple kinases that can activate JNK, dual leucine kinase (Dlk) is known to regulate both apoptosis and Wallerian degeneration triggered by axonal insult. Here we tested the importance of Dlk in regulating somal and axonal degeneration of RGCs following axonal injury. Removal of DLK from the developing optic cup did not grossly affect developmental RGC death or inner plexiform layer organization. In the adult, Dlk deficiency significantly delayed axonal-injury induced RGC death. The activation of JUN was also attenuated in Dlk deficient retinas. Dlk deficiency attenuated the activation of the somal pool of JNK but did not prevent activation of the axonal pool of JNK after axonal injury, indicating that JNK activation in different cellular compartments of an RGC following axonal injury is regulated by distinct upstream kinases. In contrast to its robust influence on somal degeneration, Dlk deficiency did not alter RGC axonal degeneration after axonal injury as assessed using physiological readouts of optic nerve function.

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Expression of MUK/DLK/ZPK, an activator of the JNK pathway, in the nervous systems of the developing mouse embryo

Abstract

Section snippets

Results and discussion

Experimental procedures

Acknowledgements

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Curr. Opin. Genet. Dev.

Trends Cell Biol.

Cell-specific expression of the ZPK gene in adult mouse tissues

DNA Cell Biol.