Multitasking: Dual Leucine Zipper-Bearing Kinases in Neuronal Development and Stress Management.

The dual leucine zipper-bearing kinase (DLK) and leucine zipper-bearing kinase (LZK) are evolutionarily conserved MAPKKKs of the mixed-lineage kinase family. Acting upstream of stress-responsive JNK and p38 MAP kinases, DLK and LZK have emerged as central players in neuronal responses to a variety of acute and traumatic injuries. Recent studies also implicate their function in astrocytes, microglia, and other nonneuronal cells, reflecting their expanding roles in the multicellular response to injury and in disease. Of particular note is the potential link of these kinases to neurodegenerative diseases and cancer. It is thus critical to understand the physiological contexts under which these kinases are activated, as well as the signal transduction mechanisms that mediate specific functional outcomes. In this review we first provide a historical overview of the biochemical and functional dissection of these kinases. We then discuss recent findings on regulating their activity to enhance cellular protection following injury and in disease, focusing on but not limited to the nervous system.

The MAP3Ks DLK and LZK Direct Diverse Responses to Axon Damage in Zebrafish Peripheral Neurons.

Mitogen-activated protein kinase kinase kinases (MAP3Ks) dual leucine kinase (DLK) and leucine zipper kinase (LZK) are essential mediators of axon damage responses, but their responses are varied, complex, and incompletely understood. To characterize their functions in axon injury, we generated zebrafish mutants of each gene, labeled motor neurons (MNs) and touch-sensing neurons in live zebrafish, precisely cut their axons with a laser, and assessed the ability of mutant axons to regenerate in larvae, before sex is apparent in zebrafish. DLK and LZK were required redundantly and cell autonomously for axon regeneration in MNs but not in larval Rohon-Beard (RB) or adult dorsal root ganglion (DRG) sensory neurons. Surprisingly, in dlk lzk double mutants, the spared branches of wounded RB axons grew excessively, suggesting that these kinases inhibit regenerative sprouting in damaged axons. Uninjured trigeminal sensory axons also grew excessively in mutants when neighboring neurons were ablated, indicating that these MAP3Ks are general inhibitors of sensory axon growth. These results demonstrate that zebrafish DLK and LZK promote diverse injury responses, depending on the neuronal cell identity and type of axonal injury.SIGNIFICANCE STATEMENT The MAP3Ks DLK and LZK are damage sensors that promote diverse outcomes to neuronal injury, including axon regeneration. Understanding their context-specific functions is a prerequisite to considering these kinases as therapeutic targets. To investigate DLK and LZK cell-type-specific functions, we created zebrafish mutants in each gene. Using mosaic cell labeling and precise laser injury we found that both proteins were required for axon regeneration in motor neurons but, unexpectedly, were not required for axon regeneration in Rohon-Beard or DRG sensory neurons and negatively regulated sprouting in the spared axons of touch-sensing neurons. These findings emphasize that animals have evolved distinct mechanisms to regulate injury site regeneration and collateral sprouting, and identify differential roles for DLK and LZK in these processes.

LZK-dependent stimulation of astrocyte reactivity promotes corticospinal axon sprouting.

Injury to the adult mammalian central nervous system induces compensatory plasticity of spared axons-referred to as collateral axon sprouting-that can facilitate neural recovery. The contribution of reactive astrocytes to axon sprouting remains elusive. Here, we sought to investigate the role of axon degeneration-reactive astrocytes in the regulation of collateral axon sprouting that occurs in the mouse spinal cord after unilateral photothrombotic stroke of the primary motor cortex. We identified astrocytic leucine zipper-bearing kinase (LZK) as a positive regulator of astrocyte reactivity to corticospinal axon degeneration. Remarkably, genetic stimulation of astrocyte reactivity, via LZK overexpression in adult astrocytes, enhanced corticospinal axon sprouting. LZK promoted the production of astrocyte-derived ciliary neurotrophic factor (CNTF) that likely enhanced axon growth in mice with astrocytic LZK overexpression after injury. Our finding that LZK-dependent stimulation of astrocyte reactivity promotes corticospinal axon sprouting highlights the potential of engineering astrocytes to support injury-induced axon plasticity for neural repair.

Activation of MAP3K DLK and LZK in Purkinje cells causes rapid and slow degeneration depending on signaling strength.

The conserved MAP3K Dual-Leucine-Zipper Kinase (DLK) and Leucine-Zipper-bearing Kinase (LZK) can activate JNK via MKK4 or MKK7. These two MAP3Ks share similar biochemical activities and undergo auto-activation upon increased expression. Depending on cell-type and nature of insults DLK and LZK can induce pro-regenerative, pro-apoptotic or pro-degenerative responses, although the mechanistic basis of their action is not well understood. Here, we investigated these two MAP3Ks in cerebellar Purkinje cells using loss- and gain-of function mouse models. While loss of each or both kinases does not cause discernible defects in Purkinje cells, activating DLK causes rapid death and activating LZK leads to slow degeneration. Each kinase induces JNK activation and caspase-mediated apoptosis independent of each other. Significantly, deleting CELF2, which regulates alternative splicing of Map2k7, strongly attenuates Purkinje cell degeneration induced by LZK, but not DLK. Thus, controlling the activity levels of DLK and LZK is critical for neuronal survival and health.

Targeted disruption of dual leucine zipper kinase and leucine zipper kinase promotes neuronal survival in a model of diffuse traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a major cause of CNS neurodegeneration and has no disease-altering therapies. It is commonly associated with a specific type of biomechanical disruption of the axon called traumatic axonal injury (TAI), which often leads to axonal and sometimes perikaryal degeneration of CNS neurons. We have previously used genome-scale, arrayed RNA interference-based screens in primary mouse retinal ganglion cells (RGCs) to identify a pair of related kinases, dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) that are key mediators of cell death in response to simple axotomy. Moreover, we showed that DLK and LZK are the major upstream triggers for JUN N-terminal kinase (JNK) signaling following total axonal transection. However, the degree to which DLK/LZK are involved in TAI/TBI is unknown. METHODS: Here we used the impact acceleration (IA) model of diffuse TBI, which produces TAI in the visual system, and complementary genetic and pharmacologic approaches to disrupt DLK and LZK, and explored whether DLK and LZK play a role in RGC perikaryal and axonal degeneration in response to TAI. RESULTS: Our findings show that the IA model activates DLK/JNK/JUN signaling but, in contrast to axotomy, many RGCs are able to recover from the injury and terminate the activation of the pathway. Moreover, while DLK disruption is sufficient to suppress JUN phosphorylation, combined DLK and LZK inhibition is required to prevent RGC cell death. Finally, we show that the FDA-approved protein kinase inhibitor, sunitinib, which has activity against DLK and LZK, is able to produce similar increases in RGC survival. CONCLUSION: The mitogen-activated kinase kinase kinases (MAP3Ks), DLK and LZK, participate in cell death signaling of CNS neurons in response to TBI. Moreover, sustained pharmacologic inhibition of DLK is neuroprotective, an effect creating an opportunity to potentially translate these findings to patients with TBI.

Leucine Zipper-Bearing Kinase Is a Critical Regulator of Astrocyte Reactivity in the Adult Mammalian CNS.

Reactive astrocytes influence post-injury recovery, repair, and pathogenesis of the mammalian CNS. Much of the regulation of astrocyte reactivity, however, remains to be understood. Using genetic loss and gain-of-function analyses in vivo, we show that the conserved MAP3K13 (also known as leucine zipper-bearing kinase [LZK]) promotes astrocyte reactivity and glial scar formation after CNS injury. Inducible LZK gene deletion in astrocytes of adult mice reduced astrogliosis and impaired glial scar formation, resulting in increased lesion size after spinal cord injury. Conversely, LZK overexpression in astrocytes enhanced astrogliosis and reduced lesion size. Remarkably, in the absence of injury, LZK overexpression alone induced widespread astrogliosis in the CNS and upregulated astrogliosis activators pSTAT3 and SOX9. The identification of LZK as a critical cell-intrinsic regulator of astrocyte reactivity expands our understanding of the multicellular response to CNS injury and disease, with broad translational implications for neural repair.

Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons.

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.