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Axon integrity is essential for functional connectivity in the nervous system. The degeneration of stressed or damaged axons is a common and sometimes initiating event in neurodegenerative disorders. Stathmin-2 (Stmn2) is an axon maintenance factor that is depleted in amyotrophic lateral sclerosis, and replenishment of Stmn2 can restore neurite outgrowth in diseased neurons. However, mechanisms responsible for Stmn2-mediated axon maintenance in injured neurons are not known. We used primary sensory neurons to interrogate the role of Stmn2 in the degeneration of severed axons. We discover that membrane association of Stmn2 is critical for its axon-protective activity. Structure-function studies revealed that axonal enrichment of Stmn2 is driven by palmitoylation as well as tubulin interaction. Using live imaging, we discover that another Stmn, Stmn3, comigrates with Stmn2-containing vesicles. We also demonstrate that Stmn3 undergoes regulated degradation through dual leucine zipper kinase (DLK)-c-Jun N-terminal kinase signaling. The Stmn2 membrane-targeting domain is both necessary and sufficient for localization to a specific vesicle population and confers sensitivity to DLK-dependent degradation. Our findings reveal a broader role for DLK in tuning the local abundance of palmitoylated Stmns in axon segments. Moreover, palmitoylation is a critical component of Stmn-mediated axon protection, and defining the Stmn2-containing vesicle population will provide important clues toward mechanisms of axon maintenance., Competing Interests: Conflict of interest The authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Maintaining neuronal connectivity in the face of injury and disease is a major challenge for the nervous system. The great length of axons makes them particularly vulnerable to insult with dire consequences for neuronal function. In the peripheral nervous system there is a program of axonal regeneration that can reestablish connectivity. In the mammalian central nervous system, however, injured axons have little or no capacity to regenerate. The molecular mechanisms that promote axon regeneration have begun to be identified and many of the implicated pathways are evolutionarily conserved. Here we discuss Drosophila models of axonal regrowth, describe insights derived from these studies, and highlight future directions in the use of the fly for dissecting the mechanisms of axonal regeneration. [ABSTRACT FROM AUTHOR]

Background and purpose Since tozasertib is neuroprotective for injured optic nerve, this study is intended to test whether tozasertib reduces early brain injury after subarachnoid hemorrhage (SAH) in a rat model. Methods Two hundred sixteen (216) male Sprague-Dawley rats were randomly subjected to endovascular perforation model of SAH and sham group. SAH grade, neurological score, and brain water content were measured at 24 and 72 h after SAH. Dual leucine zipper kinase (DLK) and its downstream factors, JNK-interacting protein 3 (JIP3), MA2K7, p-JNK/JNK (c-Jun N-terminal kinase), and apoptosis related proteins cleaved caspase-3 (CC-3), Bim, Bcl-2, and cleaved caspase-9 (CC-9) were analyzed by western blot at 24 h after SAH. Apoptotic cells were detected by terminal deoxynucleotid transferase-deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL). DLK small interfering RNA (siRNA), JIP3 siRNA and MA2K7 siRNA, the JNK, p38MAPK, and MEK inhibitors SP600125, SB203580, and PD98059 were used for intervention. Results Tozasertib reduced neuronal apoptosis, attenuated brain edema and improved neurobehavioral deficits 24 and 72 h after SAH. At 24 h After SAH, DLK/JIP3/MA2K7/p-JNK/CC-3 expressions were elevated markedly and tozasertib reduced DLK, MA2K7/p-JNK/CC-3 expressions but enhanced JIP3 expression. In the presence of tozasertib, DLK/JIP3/MA2K7 siRNA and SP600125, SB203580 and PD98059 deteriorated the neurobehavioral deficits, brain edema and increased the expression of CC-3. SAH potentiated the expression of Bim, CC-9, and CC-3 but reduced Bcl-2, while tozasertib reduced expression of Bim, CC-9, and CC-3 but enhanced Bcl-2. Conclusions Tozasertib reduced neuronal apoptosis and improved outcome possibly via DLK/JIP3/MA2K7/JNK pathways after SAH. [ABSTRACT FROM AUTHOR]

Dual leucine zipper kinase (DLK) promotes growth cone motility and must be restrained to ensure normal development. PHR (Pam/ Highwire/RPM-1) ubiquitin ligases therefore target DLK for degradation unless axon injury occurs. Overall DLK levels decrease during development, but how DLK levels are regulated within a developing growth cone has not been examined. We analyzed the expression of the fly DLK Wallenda (Wnd) in R7 photoreceptor growth cones as they halt at their targets and become presynaptic boutons. We found that Wnd protein levels are repressed by the PHR protein Highwire (Hiw) during R7 growth cone halting, as has been observed in other systems. However, as R7 growth cones become boutons, Wnd levels are further repressed by a temporally expressed transcription factor, Tramtrack69 (Ttk69). Previously unobserved negative feedback from JNK also contributes to Wnd repression at both time points. We conclude that neurons deploy additional mechanisms to downregulate DLK as they form stable, synaptic connections.We use live imaging to probe the effects of Wnd and Ttk69 on R7 bouton development and conclude that Ttk69 coordinates multiple regulators of this process. [ABSTRACT FROM AUTHOR]

Background: Recent genetic studies in model organisms, such as Drosophila, C. elegans and mice, have highlighted a critical role for dual leucine zipper kinase (DLK) in neural development and axonal responses to injury. However, exactly how DLK fulfills these functions remains to be determined. Using RNA-seq profiling, we evaluated the global changes in gene expression that are caused by shRNA-mediated knockdown of endogenous DLK in differentiated Neuro-2a neuroblastoma cells. Results: Our analysis led to the identification of numerous up- and down-regulated genes, among which several were found to be associated with system development and axon guidance according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, respectively. Because of their importance in axonal growth, pruning and regeneration during development and adult life, we then examined by quantitative RT-PCR the mRNA expression levels of the identified axon guidance genes in DLK-depleted cells. Consistent with the RNA-seq data, our results confirmed that loss of DLK altered expression of the genes encoding neuropilin 1 (Nrp1), plexin A4 (Plxna4), Eph receptor A7 (Epha7), Rho family GTPase 1 (Rnd1) and semaphorin 6B (Sema6b). Interestingly, this regulation of Nrp1 and Plxna4 mRNA expression by DLK in Neuro-2a cells was also reflected at the protein level, implicating DLK in the modulation of the function of these axon guidance molecules. Conclusions: Collectively, these results provide the first evidence that axon guidance genes are downstream targets of the DLK signaling pathway, which through their regulation probably modulates neuronal cell development, structure and function. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is the most prevalent form of neurodegeneration. Despite the well-established link between tau aggregation and clinical progression, the major pathways driven by this protein to intrinsically damage neurons are incompletely understood., Methods: To model AD-relevant neurodegeneration driven by tau, we overexpressed wild-type human tau in primary mouse neurons and characterized the subsequent cellular and molecular changes. RNAseq profiling and functional investigation were performed as well. A direct comparison with a mutant human tau was conducted in detail., Results: We observed substantial axonal degeneration and cell death associated with wild-type tau, a process accompanied by activated caspase 3. Mechanistically, we detected deformation of the nuclear envelope and increased DNA damage response in tau-expressing neurons. Gene profiling analysis further revealed significant alterations in the mitogen-activated protein kinase (MAPK) pathway; moreover, inhibitors of dual leucine zipper kinase (DLK) and c-Jun N-terminal kinase (JNK) were effective in alleviating wild-type human tau-induced neurodegeneration. In contrast, mutant P301L human tau was less toxic to neurons, despite causing comparable DNA damage. Axonal DLK activation induced by wild-type tau potentiated the impact of DNA damage response, resulting in overt neurotoxicity., Conclusions: We have established a cellular tauopathy model highly relevant to AD and identified a functional synergy between DNA damage response and the MAPK-DLK axis in the neuronal degenerative process., Competing Interests: Competing interests Nothing to declare.

Neurons remodel their connectivity in response to various insults, including microtubule disruption. How neurons sense microtubule disassembly and mount remodeling responses by altering genetic programs in the soma are not well defined. Here we show that in response to microtubule disassembly, the Caenorhabditis elegans PLM neuron remodels by retracting its synaptic branch and overex-tending the primary neurite. This remodeling required RHGF-1, a PDZ-Rho guanine nucleotide exchange factor (PDZ-RhoGEF) that was associated with and inhibited by microtubules. Independent of the myosin light chain activation, RHGF-1 acted through Rho-depen-dent kinase LET-502/ROCK and activated a conserved, retrograde DLK-1 MAPK (DLK-1/dual leucine zipper kinase) pathway, which triggered synaptic branch retraction and overgrowth of the PLM neurite in a dose-dependent manner. Our data represent a neuronal remodeling paradigm during development that reshapes the neural circuit by the coordinated removal of the dysfunctional synaptic branch compartment and compensatory extension of the primary neurite. [ABSTRACT FROM AUTHOR]

NOTCH receptors regulate cell proliferation and survival in several types of cancer cells. Depending on the cellular context, NOTCH1 can function as an oncogene or as a tumor suppressor gene. DLK1 is also involved in the regulation of cell growth and cancer, but nothing is known about the role of DLK2 in these processes. Recently, the proteins DLK1 and DLK2 have been reported to interact with NOTCH1 and to inhibit NOTCH1 activation and signaling in different cell lines. In this work, we focused on the role of DLK proteins in the control of melanoma cell growth, where NOTCH1 is known to exert an oncogenic effect. We found that human DLK proteins inhibit NOTCH signaling in SK-MEL-2 metastatic melanoma cells. Moreover, the proliferation rate of these cells was dependent upon the level of NOTCH activation and signaling as regulated by DLK proteins. In particular, high levels of NOTCH inhibition resulted in a decrease, whereas lower levels of NOTCH inhibition led to an increase in melanoma cell proliferation rates, both in vitro and in vivo . Finally, our data revealed additive NOTCH-mediated effects of DLK proteins and the γ-secretase inhibitor DAPT on cell proliferation. The data presented in this work suggest that a fine regulation of NOTCH signaling plays an important role in the control of metastatic melanoma cell proliferation. Our results open the way to new research on the role of DLK proteins as potential therapeutic tools for the treatment of human melanoma. [ABSTRACT FROM AUTHOR]

Insulin biosynthesis is an essential β-cell function and inappropriate insulin secretion and biosynthesis contribute to the pathogenesis of diabetes mellitus type 2. Previous studies showed that the dual leucine zipper kinase (DLK) induces β-cell apoptosis. Since β-cell dysfunction precedes β-cell loss, in the present study the effect of DLK on insulin gene transcription was investigated in the HIT-T15 β-cell line. Downregulation of endogenous DLK increased whereas overexpression of DLK decreased human insulin gene transcription. 5′- and 3′-deletion human insulin promoter analyses resulted in the identification of a DLK responsive element that mapped to the DNA binding-site for the β-cell specific transcription factor MafA. Overexpression of DLK wild-type but not its kinase-dead mutant inhibited MafA transcriptional activity conferred by its transactivation domain. Furthermore, in the non-β-cell line JEG DLK inhibited MafA overexpression-induced human insulin promoter activity. Overexpression of MafA and DLK or its kinase-dead mutant into JEG cells revealed that DLK but not its mutant reduced MafA protein content. Inhibition of the down-stream DLK kinase c-Jun N-terminal kinase (JNK) by SP600125 attenuated DLK-induced MafA loss. Furthermore, mutation of the serine 65 to alanine, shown to confer MafA protein stability, increased MafA-dependent insulin gene transcription and prevented DLK-induced MafA loss in JEG cells. These data suggest that DLK by activating JNK triggers the phosphorylation and degradation of MafA thereby attenuating insulin gene transcription. Given the importance of MafA for β-cell function, the inhibition of DLK might preserve β-cell function and ultimately retard the development of diabetes mellitus type 2. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Axons of the mammalian CNS lose the ability to regenerate soon after development due to both an inhibitory CNS environment and the loss of cell-intrinsic factors necessary for regeneration. The complex molecular events required for robust regeneration of mature neurons are not fully understood, particularly in vivo. To identify genes affecting axon regeneration in Caenorhabditis elegans, we performed both an RNAi-based screen for defective motor axon regeneration in unc-70/β-spectrin mutants and a candidate gene screen. From these screens, we identified at least 50 conserved genes with growth-promoting or growth-inhibiting functions. Through our analysis of mutants, we shed new light on certain aspects of regeneration, including the role of β-spectrin and membrane dynamics, the antagonistic activity of MAP kinase signaling pathways, and the role of stress in promoting axon regeneration. Many gene candidates had not previously been associated with axon regeneration and implicate new pathways of interest for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Purpose: To compare the incidence of diffuse lamellar keratitis (DLK) after laser in situ keratomileusis (LASIK) with flap creation using the Femto LDV and IntraLase™ FS60 femtosecond lasers. Methods: A total of 818 consecutive myopic eyes had LASIK performed using either Femto LDV or IntraLase FS60 for flap creation. The same excimer laser, the Allegretto Wave® Eye-Q Laser, was used for correcting refractive errors for all patients. In the preoperative examination, uncorrected distance visual acuity, corrected distance visual acuity, and manifest refraction spherical equivalent were measured. At the postop examination, the same examinations were performed along with a slit-lamp biomicroscopic examination, and patients with DLK were classified into stages. For the statistical analysis of the DLK occurrence rate and the visual and refractive outcomes, the Mann-Whitney's U-test was used. Results: In the Femto LDV group with 514 eyes, 42 (8.17%) had DLK. In the IntraLase FS60 group with 304 eyes, 114 (37.5%) had DLK. There was a statistically significant difference in the DLK incidence rate between these groups (P < 0.0001). Both groups had excellent visual and refractive outcomes. Although low levels of DLK were observed for both groups, they did not affect visual acuity. Conclusion: While there were significantly fewer incidences of low level DLK when using Femto LDV, neither femtosecond laser induced high levels of DLK, and any postoperative DLK cleared up within 1 week. Therefore, both lasers provide excellent results, with no clinical differences, and both excel at flap creation for LASIK. [ABSTRACT FROM AUTHOR]

Dual leucine zipper kinase (DLK), a mitogen-activated protein kinase kinase kinase, controls axon growth, apoptosis and neuron degeneration during neural development, as well as neurodegeneration after various insults to the adult nervous system. Interestingly, recent studies have also highlighted a role of DLK in promoting axon regeneration in diverse model systems. Invertebrates and vertebrates, cold- and warm-blooded animals, as well as central and peripheral mammalian nervous systems all differ in their ability to regenerate injured axons. Here, we discuss how DLK-dependent signalling regulates apparently contradictory functions during neural development and regeneration in different species. In addition, we outline strategies to fine-tune DLK function, either alone or together with other approaches, to promote axon regeneration in the adult mammalian central nervous system. [ABSTRACT FROM AUTHOR]

Abstract: Originally discovered nearly a century ago, the Notch signaling pathway is critical for virtually all developmental programs and modulates an astounding variety of pathogenic processes. The DSL (Delta, Serrate, LAG-2 family) proteins have long been considered canonical activators of the core Notch pathway. More recently, a wide and expanding network of non-canonical extracellular factors has also been shown to modulate Notch signaling, conferring newly appreciated complexity to this evolutionarily conserved signal transduction system. Here, I review current concepts in Notch signaling, with a focus on work from the last decade elucidating novel extracellular proteins that up- or down-regulate signal potency. [Copyright &y& Elsevier]

Abstract: In developing cerebral cortices, post-mitotic neurons migrate toward the pial surface, elongating their axons concurrently. It has been reported that targeted-deletion of the dual leucine zipper-bearing kinase (DLK)/mitogen-activated protein kinase upstream protein kinase (MUK)/leucine-zipper protein kinase (ZPK) gene, which encodes a MAP kinase kinase kinase (MAPKKK) for c-Jun N-terminal kinase (JNK), leads to a neuronal migration-defect and hypoplasia of axonal fiber tracts including those of the anterior commissure and corpus callosum. However, there is no evidence that DLK directly regulates axonal development, because another possibility, i.e. that the defective axonal development in the DLK mutant might be caused secondary to migration failure cannot be ruled out. In this study, we first examined the distributions of DLK mRNA and its protein in the developing cerebral cortex, and found that major portion of DLK proteins appear to be transported into axons. Using dissociated cortical neurons and PC12 cells, we provide direct evidence that DLK regulates axonal elongation. Furthermore, knock-down of DLK decreased the phosphorylation of JNK and its substrate, microtubule-associated protein 1B (MAP1B), which is known to be involved in axonal elongation. These results suggest that the DLK/MUK/ZPK-JNK pathway directly regulates axonal growth through phosphorylation of MAP1B. [Copyright &y& Elsevier]

Abstract: Some data in the literature suggest that serine/threonine phosphorylation is required for activation of the mixed-lineage kinases (MLKs), a subgroup of mitogen-activated protein kinase kinase kinases (MAPKKKs). In this report, we demonstrate that the MLK family member DLK is activated and concurrently tyrosine-phosphorylated in cells exposed to the protein tyrosine phosphatase inhibitor vanadate. Tyrosine phosphorylation appears crucial for activation as incubation of vanadate-activated DLK molecules with a tyrosine phosphatase substantially reduced DLK enzymatic activity. Interestingly, the effects of vanadate on DLK are completely blocked by treatment with a Src family kinase inhibitor, PP2, or the expression of short hairpin RNA (shRNA) directed against Src. DLK also fails to undergo vanadate-stimulated tyrosine phosphorylation and activation in fibroblasts which lack expression of Src, Yes and Fyn, but reintroduction of wild-type Src or Fyn followed by vanadate treatment restores this response. In addition to vanadate, stimulation of cells with platelet-derived growth factor (PDGF) also induces tyrosine phosphorylation and activation of DLK by a Src-dependent mechanism. DLK seems important for PDGF signaling because its depletion by RNA interference substantially reduces PDGF-stimulated ERK and Akt kinase activation. Thus, our findings suggest that Src-dependent tyrosine phosphorylation of DLK may be important for regulation of its activity, and they support a role for DLK in PDGF signaling. [Copyright &y& Elsevier]

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Abstract: Prostate apoptosis response-4 (Par-4) is a 38-kDa protein originally identified as a gene product upregulated in prostate cancer cells undergoing apoptosis. Cell death mediated by Par-4 and its interaction partner DAP like kinase (Dlk) is characterized by dramatic changes of the cytoskeleton. To uncover the role of the cytoskeleton in Par-4/Dlk-mediated apoptosis, we analyzed Par-4 for a direct association with cytoskeletal structures. Confocal fluorescence microscopy revealed that endogenous Par-4 is specifically associated with stress fibers in rat fibroblasts. In vitro cosedimentation analyses and in vivo FRET analyses showed that Par-4 directly binds to F-actin. Actin binding is mediated by the N-terminal 266 amino acids, but does not require the C-terminal region of Par-4 containing the leucine zipper and the death domain. Furthermore, the interaction of Par-4 with actin filaments leads to the formation of actin bundles in vitro and in vivo. In rat fibroblasts, this microfilament association is essential for the pro-apoptotic function of Par-4, since both disruption of the actin cytoskeleton by cytochalasin D treatment and overexpression of Par-4 constructs impaired in actin binding result in a significant decrease of apoptosis induction by Par-4 and Dlk. We propose a model, in which Par-4 recruits Dlk to stress fibers, leading to enhanced phosphorylation of the regulatory light chain of myosin II (MLC) and to the induction of apoptosis. [Copyright &y& Elsevier]

Abstract: The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis. [Copyright &y& Elsevier]

We previously demonstrated that hepatoblasts can be isolated from mouse fetal liver based on the expression of delta-like leucine zipper kinase (Dlk), also known as Pref-1. Each Dlk+ hepatoblast forms a colony containing both albumin+ hepatocytes and cytokeratin 19+ (CK19) cholangiocytic cells on either type IV collagen or laminin. Here we show that extracellular matrices (ECMs) significantly affect the growth of Dlk+ cells. Dlk+ cells vigorously proliferated on type IV collagen-coated dishes in the presence of EGF and HGF during the first 5 days, but their proliferative capability declined thereafter. Dlk+ cells also proliferated on laminin-coated plates and some colonies continued to expand even beyond one month after plating. These hepatic progenitor cells proliferating on laminin (HPPL) efficiently proliferated even after replating. Moreover, they were induced to differentiate into hepatocytes and cholangiocytes by overlaying Engelbreth-Hoim-Swarm sarcoma (EHS) gel and by embedding in type I collagen gel, respectively. IIPPL acquired the metabolic functions of accumulating polysaccharides and detoxifying ammonium ions after hepatic differentiation. Surprisingly, HPPL expressed pancreatic genes such as Pdx1 when dexamethasone was depleted from the culture medium. Therefore, the long-term culture of hepatoblasts on laminin produces multi-potential hepatic progenitors, which possess a strong proliferative cap ability, differentiate into both hepatocytes and cholangiocytes, and potentially give rise to pancreatic cells. [ABSTRACT FROM AUTHOR]

Hepatoblasts give rise to both mature hepatocytes and cholangiocytes. While Notch signaling has been implicated in the formation of bile ducts composed of cholangiocytes, little is known about the mechanism of lineage commitment of hepatoblasts. Here we describe the role of the Notch pathway in hepatoblast differentiation. Immunohistochemical analysis showed that Jagged1 was expressed in the cells surrounding the portal veins and Notch2 was expressed in most hepatic cells at mid gestation when ductal plates are formed surrounding the portal veins. Interestingly, the Jagged1+ cells were adjacent to ductal plates, suggesting that the Notch signaling is activated in hepatoblasts that undergo differentiation into cholangiocytes. In fact, expression of the Notch intracellular domain in Dlk+ hepatoblasts inhibited hepatic differentiation and significantly reduced the expression of albumin, a marker of both hepatoblasts and hepatocytes. Furthermore, the addition of Matrigel to the hepatoblast culture upregulated the expression of cytokeratin 7 and 19, integrin β4, and HNF1β, which are known to be expressed in cholangiocytes. By contrast, down regulation of the Notch signaling by siRNA specific for Notch2 mRNA as well as by the γ-secretase inhibitor L-685,458 promoted the hepatic differentiation. Consistent with the previous finding that mature cholangiocytes strongly express HNF1β, but barely express HNF1α, HNF4, and C/EBPα, activation of the Notch signaling upregulated HNF1β expression, whereas it down regulated the expression of HNF1α, HNF4, and C/EBPα. These results suggest that the Notch signaling contributes to form a network of these transcription factors suitable for cholangiocyte differentiation. [ABSTRACT FROM AUTHOR]

: ObjectivecDNA microarray technology identifies genes that are differentially expressed between tissues. Our previous study identified several genes that might contribute to the fibroid phenotype. We therefore sought to confirm genes involved in three distinct signal transduction pathways.: DesignEvaluation of differential mRNA and protein expression of Dlk, Frizzled-2, and CD-24 in fibroids compared with adjacent myometrium.: SettingUniversity hospital.: Patient(s)Five women undergoing medically indicated hysterectomy for symptomatic fibroids.: Intervention(s)Microarray analysis of up to 33,000 genes, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time RT-PCR, Western blot, and immunohistochemistry.: Main outcome measure(s)Expression of mRNA transcripts and protein in fibroid compared with myometrium.: Result(s)A more extensive microarray confirmed differential expression of Frizzled-2 and CD-24 but did not confirm Dlk overexpression. RT-PCR and real-time PCR demonstrated equivalent Dlk mRNA expression between fibroid and myometrium (ratio, 1.02), a slight Frizzled-2 overexpression (ratio, 2.09), and robust CD-24 overexpression in fibroids (ratio, 12.35). Western blot and immunohistochemistry confirmed Frizzled-2 overexpression, but did not confirm Dlk overexpression.: Conclusion(s)Microarray technology is the first phase of tissue evaluation, but changes in gene expression must be confirmed. Confirmed genes can then be used to generate hypotheses testing their involvement in fibroid development. [Copyright &y& Elsevier]

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Objective: To use microarray analysis as an unbiased approach to identify genes involved in the induction and growth of uterine leiomyomata.Design: Screen by arrays for up to 12,000 genes in leiomyoma (L) and control myometrium (M) from nine patients.Setting: University research laboratories.Patient(s): Nine patients in the follicular and luteal phases of the menstrual cycle.Intervention(s): mRNA from L and M was converted to biotin-labeled cRNA and hybridized to cDNA oligonucleotide sequences on the arrays.Main Outcome Measure(s): Greater than two-fold change in gene expression between leiomyoma and matched myometrium.Result(s): Prominent among the 67 genes overexpressed in L relative to M were dlk or Pref-1, doublecortin, JM27, ionotropic glutamate receptor subunit 2, apolipoprotein E3, IGF2, semaphorin F, myelin proteolipid protein, MEST, frizzled, CRABP II, stromelysin-3, and TGFbeta3. The genes dlk, IGF2, and MEST are paternally expressed imprinted genes, and the others are involved in tissue differentiation and growth. Prominent among the 78 genes down-regulated in L relative to M were alcohol dehydrogenases 1alpha-gamma, tryptase, dermatopontin, thrombospondin, coxsackievirus receptor, nur77, and c-kit.Conclusion(s): Arrays offer large-scale screening of mRNA expression, which will help us differentiate between the genes and metabolic pathways necessary for leiomyoma growth and those regulating myometrial contractions. [ABSTRACT FROM AUTHOR]

The EGF-like membrane protein dlk plays a crucial role in the control of cell differentiation. Overexpression of the protein prevents, whereas inhibition of its expression increases, adipocyte differentiation of 3T3-L1 cells in response to Insulin-like Growth Factor I (IGF-1) or insulin. We have investigated whether dlk modulates the signaling pathways known to control this process. We found that the levels of dlk expression modulated signaling through the IGF-1 receptor, causing changes in the activation levels and kinetics of Extracellular-Regulated Kinase/Mitogen-Activated Protein Kinase (ERK/MAPK) that correlated with differentiation outcome. These changes occurred in response to IGF-1 or insulin but not in response to Epidermal Growth Factor. However, the levels of expression of IGF-1 receptor, or the activation of Insulin Receptor Substrate-1 in response to IGF-1, were not affected by the levels of dlk expression. Therefore, dlk appears to modulate ERK/MAPK signaling in response to specific differentiation signals. [Copyright &y& Elsevier]

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Levels of dlk, an EGF-like homeotic protein, are critical for several differentiation processes. Because growth and differentiation are, in general, exclusive of each other, and increasing evidence indicates that Dlk1 expression changes in tumorigenic processes, we studied whether dlk could also affect cell growth. We found that, in response to glucocorticoids, Balb/c 3T3 cells with diminished levels of dlk expression develop foci-like cells that have lost contact inhibition, display altered morphology, and grow faster than control cell lines. Balb/c 3T3 cells spontaneously growing more rapidly are also dlk-negative cells. Moreover, screening by the yeast two-hybrid system, using Dlk1 constructs as baits, resulted in the isolation of GAS1 and acrogranin cDNAs. Interestingly, these proteins are cysteine-rich molecules involved in the control of cell growth. Taken together, these observations suggest that dlk may participate in a network of interactions controlling how the cells respond to growth or differentiation signals. [Copyright &y& Elsevier]

The dual leucine zipper kinase (DLK) is a key regulator of axon regeneration and degeneration in response to neuronal injury; however, regulatory mechanisms of the DLK function via its interacting proteins are largely unknown. To better understand the molecular mechanism of DLK function, we performed yeast two-hybrid screening analysis and identified FK506-binding protein-like (FKBPL, also known as WAF-1/CIP1 stabilizing protein 39) as a DLK-binding protein. FKBPL binds to the kinase domain of DLK and inhibits its kinase activity. In addition, FKBPL induces DLK protein degradation through ubiquitin-dependent pathways. We further assessed other members in the FKBP protein family and found that FK506-binding protein 8 (FKBP8) also induced DLK degradation. We identified the lysine 271 residue in the kinase domain as a major site of DLK ubiquitination and SUMO3 conjugation and was thus responsible for regulating FKBP8-mediated proteasomal degradation that was inhibited by the substitution of the lysine 271 to arginine. FKBP8-mediated degradation of DLK is mediated by autophagy pathway because knockdown of Atg5 inhibited DLK destabilization. We show that in vivo overexpression of FKBP8 delayed the progression of axon degeneration and suppressed neuronal death after axotomy in sciatic and optic nerves. Taken together, this study identified FKBPL and FKBP8 as novel DLK-interacting proteins that regulate DLK stability via the ubiquitin-proteasome and lysosomal protein degradation pathways., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Hyperglycemia enhancing the intracellular levels of reactive oxygen species (ROS) contributes to dysfunction and progressive loss of beta cells and thereby to diabetes mellitus. The oxidation sensitive calcium/calmodulin dependent phosphatase calcineurin promotes pancreatic beta cell function and survival whereas the dual leucine zipper kinase (DLK) induces apoptosis. Therefore, it was studied whether calcineurin interferes with DLK action. In a beta cell line similar concentrations of H 2 O 2 decreased calcineurin activity and activated DLK. DLK interacted via its φLxVP motif (aa 362–365) with the interface of the calcineurin subunits A and B. Mutation of the Val prevented this protein protein interaction, hinting at a distinct φLxVP motif. Indeed, mutational analysis revealed an ordered structure of DLK's φLxVP motif whereby Val mediates the interaction with calcineurin and Leu maintains an enzymatically active conformation. Overexpression of DLK wild-type but not the DLK mutant unable to bind calcineurin diminished calcineurin-induced nuclear localisation of the nuclear factor of activated T-cells (NFAT), suggesting that both, DLK and NFAT compete for the substrate binding site of calcineurin. The calcineurin binding-deficient DLK mutant exhibited increased DLK activity measured as phosphorylation of the downstream c-Jun N-terminal kinase, inhibition of CRE-dependent gene transcription and induction of apoptosis. These findings show that calcineurin interacts with DLK; and inhibition of calcineurin increases DLK activity. Hence, this study demonstrates a novel mechanism regulating DLK action. These findings suggest that ROS through inhibition of calcineurin enhance DLK activity and thereby lead to beta cell dysfunction and loss and ultimately diabetes mellitus. [Display omitted] • Inhibition of beta cell protective calcineurin activates beta cell damaging DLK. • DLK interacts with calcineurin through its φLxVP motif. • The calcineurin binding-deficient DLK mutant is more active than DLK wild-type. [ABSTRACT FROM AUTHOR]

Dual leucine zipper kinase (DLK, Map3k12) is an axonal protein that governs the balance between degeneration and regeneration through its downstream effectors c-jun N-terminal kinase (JNK) and phosphorylated c-jun (p-c-Jun). In peripheral nerves DLK is generally inactive until induced by injury, after which it transmits signals to the nucleus via retrograde transport. Here we report that in contrast to this mode of regulation, in the uninjured adult mouse cerebellum, DLK constitutively drives nuclear p-c-Jun in cerebellar granule neurons, whereas in the forebrain, DLK is similarly expressed and active, but nuclear p-c-Jun is undetectable. When neurodegeneration results from mutant human tau in the rTg4510 mouse model, p-c-Jun then accumulates in neuronal nuclei in a DLK-dependent manner, and the extent of p-c-Jun correlates with markers of synaptic loss and gliosis. This regional difference in DLK-dependent nuclear p-c-Jun accumulation could relate to differing levels of JNK scaffolding proteins, as the cerebellum preferentially expresses JNK-interacting protein-1 (JIP-1), whereas the forebrain contains more JIP-3 and plenty of SH3 (POSH). To characterize the functional differences between constitutive- versus injury-induced DLK signaling, RNA sequencing was performed after DLK inhibition in the cerebellum and in the non-transgenic and rTg4510 forebrain. In all contexts, DLK inhibition reduced a core set of transcripts that are associated with the JNK pathway. Non-transgenic forebrain showed almost no other transcriptional changes in response to DLK inhibition, whereas the rTg4510 forebrain and the cerebellum exhibited distinct differentially expressed gene signatures. In the cerebellum, but not the rTg4510 forebrain, pathway analysis indicated that DLK regulates insulin growth factor-1 (IGF1) signaling through the transcriptional induction of IGF1 binding protein-5 (IGFBP5), which was confirmed and found to be functionally relevant by measuring signaling through the IGF1 receptor. Together these data illuminate the complex multi-functional nature of DLK signaling in the central nervous system (CNS) and demonstrate its role in homeostasis as well as tau-mediated neurodegeneration. [ABSTRACT FROM AUTHOR]

The mRNA expression level of the gene delta-like (Dlk), coding for a signal transducer related to the Delta-Notch family, was measured using real-time PCR methodology in a large series of control biopsies (n=61) and bone marrow trephines from patients with myelodysplastic syndrome (MDS) (n=93) and related myeloid disorders (AML, n=16 and chronic myeloproliferative disease (CMPD), n=38). It turned out that dlk is strongly overexpressed in a subset of MDS (16%) cases, however with higher frequency in blast rich MDS cases. Therefore, the quantitative detection of dlk mRNA can support the morphological diagnosis of MDS in selected cases. [Copyright &y& Elsevier]