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Abstract Background C3H10T1/2 is a mesenchymal cell line capable of differentiating into osteoblasts, adipocytes and chondrocytes. The differentiation of these cells into osteoblasts is modulated by various transcription factors, such as RUNX2. Additionally, several interconnected signaling pathways, including the NOTCH pathway, play a crucial role in modulating their differentiation into mature bone cells. We have investigated the roles of DLK1 and DLK2, two non-canonical inhibitory ligands of NOTCH receptors, in the osteogenic differentiation of C3H10T1/2 cells. Results Our results corroborate existing evidence that DLK1 acts as an inhibitor of osteogenesis. In contrast, we demonstrate for the first time that DLK2 enhances this differentiation process. Additionally, our data suggest that NOTCH2, 3 and 4 receptors may promote osteogenesis, as indicated by their increased expression during this process, whereas NOTCH1 expression, which decreases during cell differentiation, might inhibit osteogenesis. Moreover, treatment with DAPT, a NOTCH signaling inhibitor, impeded osteogenic differentiation. We have confirmed the increase in ERK1/2 MAPK and p38 MAPK phosphorylation in C3H10T1/2 cells induced to differentiate to osteoblasts. Our new findings reveal increased ERK1/2 MAPK phosphorylation in differentiated C3H10T1/2 cells with a decrease in DLK1 expression or an overexpression of DLK2, which is coincident with the behavior of those transfectants where we have detected an increase in osteogenic differentiation. Additionally, p38 MAPK phosphorylation increases in differentiated C3H10T1/2 cells with reduced DLK1 levels. Conclusions Our results suggest that DLK1 may inhibit osteogenesis, while DLK2 may promote it, by modulating NOTCH signaling and the phosphorylation of ERK1/2 and p38 MAPK pathways. Given the established inhibitory effect of DLK proteins on NOTCH signaling, these new insights could pave the way for developing future therapeutic strategies aimed at treating bone diseases.

Dual leucine zipper kinase (DLK) mediates multiple neuronal stress responses, and its expression levels are constantly suppressed to prevent excessive stress signaling. We found that Wallenda (Wnd), the Drosophila ortholog of DLK, is highly enriched in the axon terminals of Drosophila sensory neurons in vivo and that this subcellular localization is necessary for Highwire-mediated Wnd protein turnover under normal conditions. Our structure-function analysis found that Wnd palmitoylation is essential for its axon terminal localization. Palmitoylation-defective Wnd accumulated in neuronal cell bodies, exhibited dramatically increased protein expression levels, and triggered excessive neuronal stress responses. Defective intracellular transport is implicated in neurodegenerative conditions. Comprehensive dominant-negative Rab protein screening identified Rab11 as an essential factor for Wnd localization in axon terminals. Consequently, Rab11 loss-of-function increased the protein levels of Wnd and induced neuronal stress responses. Inhibiting Wnd activity significantly ameliorated neuronal loss and c-Jun N-terminal kinase signaling triggered by Rab11 loss-of-function. Taken together, these suggest that DLK proteins are constantly transported to axon terminals for protein turnover and a failure of such transport can lead to neuronal loss. Our study demonstrates how subcellular protein localization is coupled to protein turnover for neuronal stress signaling.

Majid Moshirfar,1– 3 Stephanie Zhang,4 Shreya Pandya,5 Isabella M Stoakes,1,6 Phillip C Hoopes1 1Hoopes Vision Research Center, Hoopes Vision, Draper, UT, USA; 2John A. Moran Eye Center, University of Utah School of Medicine, Salt Lake City, UT, USA; 3Utah Lions Eye Bank, Murray, UT, USA; 4University of Arizona College of Medicine Phoenix, Phoenix, AZ, USA; 5University of Louisville School of Medicine, Louisville, KY, USA; 6Pacific Northwest University of Health Sciences, Yakima, WA, USACorrespondence: Majid Moshirfar, Hoopes Vision Research Center, Hoopes Vision, 11820 S. State St., Ste. 200, Draper, UT, 84020, USA, Tel +1 801-568-0200, Fax +1 801-563-0200, Email cornea2020@me.comPurpose: To investigate the incidence and management of only epithelial-related complications following small incision lenticule extraction (SMILE).Patients and Methods: A retrospective, single-site study analyzed patients who underwent SMILE at Hoopes Vision Clinic in Draper, Utah, from June 2017 to February 2023. Demographic data and preoperative parameters were reviewed. Postoperatively, patients were assessed for visual acuity and complications at different time points. Statistical analyses were conducted between the control and complication groups.Results: Four hundred and thirty-two eyes of 220 patients received SMILE. Postoperative epithelial-related complications were indicated in 68 (15.7%) eyes, including anterior basement membrane (ABM) changes (five [1.2%]) eyes), epithelial ingrowth (nine [2.1%] eyes), erosion (two [0.5%] eyes), rough epithelium (18 [4.2%] eyes), epithelial defect (12 [2.8%] eyes), diffuse lamellar keratitis (DLK) secondary to epitheliopathy (two [0.5%] eyes), microstriae secondary to epitheliopathy (four [0.9%] eyes), interface debris (21 [4.9%] eyes), and incisional fibrosis (one [0.2%] eye). There was a statistically significant difference in age, with older patients more likely to develop epitheliopathy postoperatively (P = 0.001). Additionally, patients with epithelial-related complications were more likely to receive photorefractive keratectomy (PRK) enhancement after SMILE than the control (P = 0.001). However, there was no statistical difference in uncorrected distance visual acuity (UDVA) better than 20/20 and corrected distance visual acuity (CDVA) between the complications group and the control at the last postoperative visit (P = 0.974 and 0.310, respectively). There was no statistically significant difference in the safety and efficacy indices between the complications and control group (P = 0.281 and 0.617, respectively).Conclusion: In our study, epithelial-related complications were more prevalent in older patients and predisposed patients to require PRK enhancements after recovery from SMILE. Despite the incidence of epithelial-related complications, visual prognoses were favorable and achieved through various management strategies.Keywords: ReLEx, epithelial ingrowth, DLK, diffuse lamellar keratitis, microstriae, LASIK

Abstract Axon regeneration in the central nervous system is inefficient. However, the neurons in the peripheral nervous system display robust regeneration after injury, indicating that axonal regeneration is differentially controlled under various conditions. To identify those molecules regulating axon regeneration, comparative analysis from dorsal root ganglion neurons at embryonic or adult stages is utilized, which reveals that PDK1 is functions as a negative regulator of axon regeneration. PDK1 is downregulated in embryonic neurons after axotomy. In contrast, sciatic nerve axotomy upregulated PDK1 at protein levels from adult mice. The knockdown of PDK1 or the chemical inhibition of PDK1 promotes axon regeneration in vitro and in vivo. Here we present PDK1 as a new player to negatively regulate axon regeneration and as a potential target in the development of therapeutic applications.

Protein phosphatase 2A (PP2A) is a major cellular phosphatase with many protein substrates. As expected for a signaling molecule with many targets, inhibition of PP2A disrupts fundamental aspects of cellular physiology including cell division and survival. In post-mitotic neurons, the microtubule associated protein Tau is a particularly well-studied PP2A substrate as hyperphosphorylation of Tau is a hallmark of Alzheimer's disease. Although many cellular targets are likely altered by loss of PP2A, here we find that activation of a single pathway can explain important aspects of the PP2A loss-of-function phenotype in neurons. We demonstrate that PP2A inhibits activation of the neuronal stress kinase DLK and its Drosophila ortholog Wallenda. In the fly, PP2A inhibition activates a DLK/Wallenda-regulated transcriptional program that induces synaptic terminal overgrowth at the neuromuscular junction. In cultured mammalian neurons, PP2A inhibition activates a DLK-dependent apoptotic program that induces cell death. Since hyperphosphorylated Tau is toxic, we wished to test the hypothesis that dephosphorylation of Tau by PP2A is required for neuronal survival. Contrary to expectations, in the absence of Tau PP2A inhibition still activates DLK and induces neuronal cell death, demonstrating that hyperphosphorylated Tau is not required for cell death in this model. Moreover, hyperphosphorylation of Tau following PP2A inhibition does not require DLK. Hence, loss of PP2A function in cortical neurons triggers two independent neuropathologies: 1) Tau hyperphosphorylation and 2) DLK activation and subsequent neuronal cell death. These findings demonstrate that inhibition of the DLK pathway is an essential function of PP2A required for normal Drosophila synaptic terminal development and mammalian cortical neuron survival.

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.

Abstract Background To report a first case of late-onset diffuse lamellar keratitis (DLK) occurring 4 years after femtosecond laser-assisted small incision lenticule extraction (SMILE). Case presentation A 41-year-old man who underwent SMILE 4 years prior developed DLK in the right eye 1 day after he was struck in the eye by a finger while playing with his son. Slim-lamp microscopy and anterior segment optical coherence tomography (AS-OCT) were used to evaluate the cornea of the right eye. Slit-lamp examination of the right eye revealed epithelial exfoliation and stage 3 DLK with diffuse, dot-like, granular haze in the interface between the cap and stromal bed. After intensive treatment with topical corticosteroids, the DLK resolved and corneal transparency was achieved. Conclusions This case indicates that DLK can occur several years after SMILE. Ocular trauma may be a risk factor for the development of DLK. The prognosis is usually favorable with early diagnosis and treatment with topical corticosteroids.

Objective: Dual leucine zipper kinase (DLK/MA3K12) has been reported involved in apoptosis and neuronal degeneration during neural development and traumatic brain injury. This study was designed to investigate the role of DLK with its adaptor protein JNK interacting protein-3 (JIP3), and its downstream MA2K7/JNK signaling pathway in early brain injury (EBI) after subarachnoid hemorrhage (SAH) in a rat model. Design: Controlled in vivo laboratory study. Setting: Animal research laboratory. Subjects: Two hundred and twenty-three adult male Sprague-Dawley rats weighing 280–320 g. Interventions: SAH was induced by endovascular perforation in rats. The SAH grade, neurological score, and brain water content were measured at 24 and 72 h after SAH. Immunofluorescence staining was used to detect the cells that expressed DLK. The terminal deoxynucleotid transferase-deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) was used to detect the neuronal apoptosis. In mechanism research, the expression of DLK, JIP3, phosphorylated-JNK (p-JNK)/JNK, and cleaved caspase-3 (CC-3) were analyzed by western blot at 24 h after SAH. The DLK small interfering RNA (siRNA), JIP3 siRNA, MA2K7 siRNA and recombinant DLK protein which injected intracerebroventricularly were given as the interventions. Measurements and main results: The DLK expression was increased in the left cortex neurons and peaked at 24 h after SAH. DLK siRNA attenuated brain edema, reduced neuronal apoptosis, and improved the neurobehavioral functions after SAH, but the recombinant DLK protein deteriorated neurobehavioral functions and brain edema. DLK siRNA decreased and recombinant DLK protein increased the expression of MA2K7/p-JNK/CC-3 at 24 h after SAH. The JIP3 siRNA reduced the level of JIP3/MA2K7/p-JNK/CC-3, combined DLK siRNA and JIP3 siRNA further decreased the expression of DLK/MA2K7/p-JNK/CC-3, and MA2K7 siRNA lowered the amount of MA2K7/p-JNK/CC-3 at 24 h after SAH. Conclusions: As a negative role, DLK was involved in EBI after SAH, possibly mediated by its adaptor protein JIP3 and MA2K7/JNK signaling pathways. To reduce the level of DLK may be a new target as intervention for SAH.

Nerve injury can lead to axonal regeneration, axonal degeneration, and/or neuronal cell death. Remarkably, the MAP3K dual leucine zipper kinase, DLK, promotes each of these responses, suggesting that DLK is a sensor of axon injury. In Drosophila, mutations in proteins that stabilize the actin and microtubule cytoskeletons activate the DLK pathway, suggesting that DLK may be activated by cytoskeletal disruption. Here we test this model in mammalian sensory neurons. We find that pharmacological agents designed to disrupt either the actin or microtubule cytoskeleton activate the DLK pathway, and that activation is independent of calcium influx or induction of the axon degeneration program. Moreover, activation of the DLK pathway by targeting the cytoskeleton induces a pro-regenerative state, enhancing axon regeneration in response to a subsequent injury in a process akin to preconditioning. This highlights the potential utility of activating the DLK pathway as a method to improve axon regeneration. Moreover, DLK is required for these responses to cytoskeletal perturbations, suggesting that DLK functions as a key neuronal sensor of cytoskeletal damage.