An orally available compound suppresses glucagon hypersecretion and normalizes hyperglycemia in type 1 diabetes.

Suppression of glucagon hypersecretion can normalize hyperglycemia during type 1 diabetes (T1D). Activating erythropoietin-producing human hepatocellular receptor type-A4 (EphA4) on α cells reduced glucagon hypersecretion from dispersed α cells and T1D islets from both human donor and mouse models. We synthesized a high-affinity small molecule agonist for the EphA4 receptor, WCDD301, which showed robust plasma and liver microsome metabolic stability in both mouse and human preparations. In islets and dispersed islet cells from nondiabetic and T1D human donors, WCDD301 reduced glucagon secretion comparable to the natural EphA4 ligand, Ephrin-A5. In diabetic NOD and streptozotocin-treated mice, once-daily oral administration of WCDD301 formulated with a time-release excipient reduced plasma glucagon and normalized blood glucose for more than 3 months. These results suggest that targeting the α cell EphA4 receptor by sustained release of WCDD301 is a promising pharmacologic pathway for normalizing hyperglycemia in patients with T1D.

Endothelial deletion of EPH receptor A4 alters single-cell profile and Tie2/Akap12 signaling to preserve blood-brain barrier integrity.

Neurobiological consequences of traumatic brain injury (TBI) result from a complex interplay of secondary injury responses and sequela that mediates chronic disability. Endothelial cells are important regulators of the cerebrovascular response to TBI. Our work demonstrates that genetic deletion of endothelial cell (EC)-specific EPH receptor A4 (EphA4) using conditional EphA4f/f/Tie2-Cre and EphA4f/f/VE-Cadherin-CreERT2 knockout (KO) mice promotes blood-brain barrier (BBB) integrity and tissue protection, which correlates with improved motor function and cerebral blood flow recovery following controlled cortical impact (CCI) injury. scRNAseq of capillary-derived KO ECs showed increased differential gene expression of BBB-related junctional and actin cytoskeletal regulators, namely, A-kinase anchor protein 12, Akap12, whose presence at Tie2 clustering domains is enhanced in KO microvessels. Transcript and protein analysis of CCI-injured whole cortical tissue or cortical-derived ECs suggests that EphA4 limits the expression of Cldn5, Akt, and Akap12 and promotes Ang2. Blocking Tie2 using sTie2-Fc attenuated protection and reversed Akap12 mRNA and protein levels cortical-derived ECs. Direct stimulation of Tie2 using Vasculotide, angiopoietin-1 memetic peptide, phenocopied the neuroprotection. Finally, we report a noteworthy rise in soluble Ang2 in the sera of individuals with acute TBI, highlighting its promising role as a vascular biomarker for early detection of BBB disruption. These findings describe a contribution of the axon guidance molecule, EphA4, in mediating TBI microvascular dysfunction through negative regulation of Tie2/Akap12 signaling.

Transcriptional regulation of the mouse EphA4, Ephrin-B2 and Ephrin-A3 genes by the circadian clock machinery.

Circadian rhythms originate from molecular feedback loops. In mammals, the transcription factors CLOCK and BMAL1 act on regulatory elements (i.e. E-boxes) to shape biological functions in a rhythmic manner. The EPHA4 receptor and its ligands Ephrins (EFN) are cell adhesion molecules regulating neurotransmission and neuronal morphology. Previous studies showed the presence of E-boxes in the genes of EphA4 and specific Ephrins, and that EphA4 knockout mice have an altered circadian rhythm of locomotor activity. We thus hypothesized that the core clock machinery regulates the gene expression of EphA4, EfnB2 and EfnA3. CLOCK and BMAL1 (or NPAS2 and BMAL2) were found to have transcriptional activity on distal and proximal regions of EphA4, EfnB2 and EfnA3 putative promoters. A constitutively active form of glycogen synthase kinase 3ß (GSK3ß; a negative regulator of CLOCK and BMAL1) blocked the transcriptional induction. Mutating the E-boxes of EphA4 distal promoter sequence reduced transcriptional induction. EPHA4 and EFNB2 protein levels did not show circadian variations in the mouse suprachiasmatic nucleus or prefrontal cortex. The findings uncover that core circadian transcription factors can regulate the gene expression of elements of the Eph/Ephrin system, which might contribute to circadian rhythmicity in biological processes in the brain or peripheral tissues.

Effects of lncRNA HOXA11-AS on Sevoflurane-Induced Neuronal Apoptosis and Inflammatory Responses by Regulating miR-98-5p/EphA4.

Objective: To explore the molecular mechanism of sevoflurane-induced neurotoxicity and to determine whether lncRNA HOXA11-AS affects sevoflurane-induced neuronal apoptosis and inflammation by regulating miR-98-5p/EphA4. Methods: Morris water maze (MWM) test was used to detect the learning and memory ability of rats, HE staining was used to observe hippocampal pathology, TUNEL staining was used to detect the level of neuronal apoptosis, and RT-qPCR was used to detect the expression of HOXA11-AS, miR-98-5p, IL-6, IL-1ß, and TNF-α. At the same time, the contents of IL-6, IL-1ß, and TNF-α in serum were detected by ELISA. The expressions of apoptosis-related proteins EphA4, Bax, Cleaved caspase 3, and Bcl-2 were detected by Western blot. The dual-luciferase gene reporter verified the targeting relationship between HOXA11-AS and miR-98-5p and the targeting relationship between miR-98-5p and EphA4. Results: The expression of HOXA11-AS was observed in sevoflurane-treated rats or cells and promoted neuronal apoptosis and inflammation. HOXA11-AS was knocked out alone, or miR-98-5p was overexpressed which attenuates neuronal apoptosis and inflammatory inflammation after sevoflurane treatment. Furthermore, knockdown of HOXA11-AS alone was partially restored by knockdown of miR-98-5p or overexpression of EphA4. Conclusion: Inhibition of lncRNA HOXA11-AS attenuates sevoflurane-induced neuronal apoptosis and inflammatory responses via miR-98-5p/EphA4.

EphA4 signaling is involved in the phenotype of well-differentiated oral squamous cell arcinoma with decreased tumor immunity.

Metronomic chemotherapy is defined as a high-frequency low-dose schedule of chemotherapy drug administration. Although metronomic chemotherapy is widely used, the mechanisms underlying resistance to metronomic chemotherapy remain unclear. Therefore, we herein conducted a single institutional phase I/II trial to assess the efficacy and safety of metronomic chemotherapy with bleomycin plus S-1, an oral 5-FU prodrug, in the neoadjuvant setting for patients with oral squamous cell carcinoma (OSCC). The response rate of well-differentiated OSCC to metronomic chemotherapy was significantly lower. We investigated differences in molecular profiles between poorly or moderately differentiated head and neck squamous cell carcinoma (HNSCC) and well-differentiated HNSCC from patients with HNSCC TCGA data. EphA4 expression positively correlated with histological differentiation. An upstream regulator analysis correlated with EphA4 expression identified pathways associated with decreased mTORC1 signaling and T cell activation, including TCR, CD3, CD28, and CD40LG. An EphA4 blocking peptide (KYL) induced mTOR activation in well-differentiated OSCC cell lines. Plasmacytoid dendritic cell and CD8+ T cell numbers were higher in the microenvironment of poorly or moderately differentiated HNSCC than in that of well-differentiated HNSCC. Well-differentiated HNSCC had the characteristics of "cold tumors" (immune-excluded tumors). Moreover, KYL used with chemotherapeutic drugs synergistically increased cancer cell death. Well-differentiated OSCC is depleted of immune cells, which may be partly explained by the receptor tyrosine kinase EphA4.

EphA4/ephrinA3 reverse signaling induced Müller cell gliosis and production of pro-inflammatory cytokines in experimental glaucoma.

Previous work showed that ephrinA3/EphA4 forward signaling contributed to retinal ganglion cell (RGC) damage in experimental glaucoma. Since up-regulated patterns of ephrinA3 and EphA4 were observed in Müller cells and RGCs, an EphA4/ephrinA3 reverse signaling may exist in Müller cells of chronic ocular hypertension (COH) retina. We investigated effects of EphA4/ephrinA3 reverse signaling activation on Müller cells in COH retina. Intravitreal injection of the ephrinA3 agonist EphA4-Fc increased glial fibrillary acidic protein (GFAP) levels in normal retinas, suggestive of Müller cell gliosis, which was confirmed in purified cultured Müller cells treated with EphA4-Fc. These effects were mediated by intracellular STAT3 signaling pathway as phosphorylated STAT3 (p-STAT3) levels and ratios of p-STAT3/STAT3 were significantly increased in both COH retinas and EphA4-Fc intravitreally injected retinas, as well as in EphA4-Fc treated purified cultured Müller cells. The increase of GFAP protein levels in EphA4-Fc-injected retinas and EphA4-Fc treated purified cultured Müller cells could be partially eliminated by stattic, a selective STAT3 blocker. Co-immunoprecipitation results testified to the presence of interaction between ephrinA3 and STAT3/p-STAT3. In addition, intravitreal injection of EphA4-Fc or EphA4-Fc treatment of cultured Müller cells significantly up-regulated mRNA and protein contents of pro-inflammatory cytokines. Moreover, intravitreal injection of EphA4-Fc increased the number of apoptotic RGCs, which could be reversed by the tyrosine kinase blocker PP2. Overall, EphA4/ephrinA3 reverse signaling may induce Müller cell gliosis and increases release of pro-inflammatory factors, which could contribute to RGC death in glaucoma. Inhibition of EphA4/ephrinA3 signaling may provide an effective neuroprotection in glaucoma.

EphA4/ephrinA3 reverse signaling mediated downregulation of glutamate transporter GLAST in Müller cells in an experimental glaucoma model.

Deficiency of glutamate transporter GLAST in Müller cells may be culpable for excessive extracellular glutamate, which involves in retinal ganglion cell (RGC) damage in glaucoma. We elucidated how GLAST was regulated in rat chronic ocular hypertension (COH) model. Western blot and whole-cell patch-clamp recordings showed that GLAST proteins and GLAST-mediated current densities in Müller cells were downregulated at the early stages of COH. In normal rats, intravitreal injection of the ephrinA3 activator EphA4-Fc mimicked the changes of GLAST in COH retinas. In purified cultured Müller cells, EphA4-Fc treatment reduced GLAST expression at mRNA and protein levels, which was reversed by the tyrosine kinase inhibitor PP2 or transfection with ephrinA3-siRNA (Si-EFNA3), suggesting that EphA4/ephrinA3 reverse signaling mediated GLAST downregulation. EphA4/ephrinA3 reverse signaling-induced GLAST downregulation was mediated by inhibiting PI3K/Akt/NF-κB pathways since EphA4-Fc treatment of cultured Müller cells reduced the levels of p-Akt/Akt and NF-κB p65, which were reversed by transfecting Si-EFNA3. In Müller cells with ephrinA3 knockdown, the PI3K inhibitor LY294002 still decreased the protein levels of NF-κB p65 in the presence of EphA4-Fc, and the mRNA levels of GLAST were reduced by LY294002 and the NF-κB inhibitor SN50, respectively. Pre-injection of the PI3K/Akt pathway activator 740 Y-P reversed the GLAST downregulation in COH retinas. Western blot and TUNEL staining showed that transfecting of Si-EFNA3 reduced Müller cell gliosis and RGC apoptosis in COH retinas. Our results suggest that activated EphA4/ephrinA3 reverse signaling induces GLAST downregulation in Müller cells via inhibiting PI3K/Akt/NF-κB pathways, thus contributing to RGC damage in glaucoma.

Exosomal miR-532-5p induced by long-term exercise rescues blood-brain barrier function in 5XFAD mice via downregulation of EPHA4.

The breakdown of the blood-brain barrier, which develops early in Alzheimer's disease (AD), contributes to cognitive impairment. Exercise not only reduces the risk factors for AD but also confers direct protection against cognitive decline. However, the exact molecular mechanisms remain elusive, particularly whether exercise can liberate the function of the blood-brain barrier. Here, we demonstrate that long-term exercise promotes the clearance of brain amyloid-ß by improving the function of the blood-brain barrier in 5XFAD mice. Significantly, treating primary brain pericytes or endothelial cells with exosomes isolated from the brain of exercised 5XFAD mice improves cell proliferation and upregulates PDGFRß, ZO-1, and claudin-5. Moreover, exosomes isolated from exercised mice exhibit significant changes in miR-532-5p. Administration or transfection of miR-532-5p to sedentary mice or primary brain pericytes and endothelial cells reproduces the improvement of blood-brain barrier function. Exosomal miR-532-5p targets EPHA4, and accordingly, expression of EphA4 is decreased in exercised mice and miR-532-5p overexpressed mice. A specific siRNA targeting EPHA4 recapitulates the effects on blood-brain barrier-associated cells observed in exercised 5XFAD mice. Overall, our findings suggest that exosomes released by the brain contain a specific miRNA that is altered by exercise and has an impact on blood-brain barrier function in AD.

EphA4/EphrinB2 signaling mediates pericyte-induced transient glia limitans formation as a secondary protective barrier after subarachnoid hemorrhage in mice.

BACKGROUND: Most patients with subarachnoid hemorrhage (SAH) do not exhibit brain parenchymal injury upon imaging but present significant blood-brain barrier (BBB) disruption and secondary neurological deficits. The aim of this study was to investigate whether stressed astrocytes act as a secondary barrier to exert a protective effect after SAH and to investigate the mechanism of glial limitan formation. METHODS: A total of 204 adult male C57BL/6 mice and an endovascular perforation SAH model were employed. The spatiotemporal characteristics of glial limitan formation after SAH were determined by immunofluorescence staining and transmission electron microscopy. The molecular mechanisms by which pericytes regulate glia limitans formation were analyzed using polymerase chain reaction, Western blotting, immunofluorescence staining and ELISA in a pericyte-astrocyte contact coculture system. The findings were validated ex vivo and in vivo using lentiviruses and inhibitors. Finally, pericytes were targeted to regulate glial limitan formation, and the effect of the glia limitans on secondary brain injury after SAH was evaluated by flow cytometry and analysis of neurological function. RESULTS: Stress-induced glial limitan formation occurred 1 day after SAH and markedly subsided 3 days after ictus. Pericytes regulated astrocyte glia limitan formation via EphA4/EphrinB2 signaling, inhibited inflammatory cell infiltration and altered neurological function. CONCLUSIONS: Astrocyte-derived glia limitans serve as a secondary protective barrier following BBB disruption after SAH in mice, and pericytes can regulate glial limitan formation and alter neurological function via EphA4/EphrinB2 signaling. Strategies for maintaining this secondary protective barrier may be novel treatment approaches for alleviating early brain injury after SAH.

Lipidation and PEGylation Strategies to Prolong the in Vivo Half-Life of a Nanomolar EphA4 Receptor Antagonist.

The EphA4 receptor tyrosine kinase plays a role in neurodegenerative diseases, inhibition of nerve regeneration, cancer progression and other diseases. Therefore, EphA4 inhibition has potential therapeutic value. Selective EphA4 kinase inhibitors are not available, but we identified peptide antagonists that inhibit ephrin ligand binding to EphA4 with high specificity. One of these peptides is the cyclic APY-d3 (ßAPYCVYRßASWSC-NH2), which inhibits ephrin-A5 ligand binding to EphA4 with low nanomolar binding affinity and is highly protease resistant. Here we describe modifications of APY-d3 that yield two different key derivatives with greatly increased half-lives in the mouse circulation, the lipidated APY-d3-laur8 and the PEGylated APY-d3-PEG4. These two derivatives inhibit ligand induced EphA4 activation in cells with sub-micromolar potency. Since they retain high potency and specificity for EphA4, lipidated and PEGylated APY-d3 derivatives represent new tools for discriminating EphA4 activities in vivo and for preclinical testing of EphA4 inhibition in animal disease models.

EphA4 regulates white matter remyelination after ischemic stroke through Ephexin-1/RhoA/ROCK signaling pathway.

Ischemic stroke, which accounts for nearly 80% of all strokes, leads to white matter injury and neurobehavioral dysfunction, but relevant therapies to inhibit demyelination or promote remyelination after white matter injury are still unavailable. In this study, the middle cerebral artery occlusion/reperfusion (MCAO/R) in vivo and oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro were used to establish the ischemic models. We found that Eph receptor A4 (EphA4) had no effect on the apoptosis of oligodendrocytes using TUNEL staining. In contrast, EphA4 promoted proliferation of oligodendrocyte precursor cells (OPCs), but reduced the numbers of mature oligodendrocytes and the levels of myelin-associated proteins (MAG, MOG, and MBP) in the process of remyelination in ischemic models in vivo and in vitro as determined using PDGFRα-EphA4-shRNA and LV-EphA4 treatments. Notably, conditional knockout of EphA4 in OPCs (EphA4fl/fl + AAV-PDGFRα-Cre) improved the levels of myelin-associated proteins and functional recovery following ischemic stroke. In addition, regulation of remyelination by EphA4 was mediated by the Ephexin-1/RhoA/ROCK signaling pathway. Therefore, EphA4 did not affect oligodendrocyte (OL) apoptosis but regulated white matter remyelination after ischemic stroke through the Ephexin-1/RhoA/ROCK signaling pathway. EphA4 may provide a novel and effective therapeutic target in clinical practice of ischemic stroke.

Vitronectin, a Novel Urinary Proteomic Biomarker, Promotes Cell Pyroptosis in Juvenile Systemic Lupus Erythematosus.

Objective: Identifying new markers of juvenile systemic lupus erythematosus (JSLE) is critical event to predict patient stratification and prognosis. The aim of the present study is to analyze alteration of urinary protein expression and screen potential valuable biomarkers in juvenile systemic lupus erythematosus (JSLE). Methods: The urine was collected from the patients with or without JSLE and detected by mass spectrometry to analyze proteomic changes. ELISA was used to verify the Vitronectin (VTN) changes in a new set of patients. The clinical correlation was performed to analyze between VTN and clinical pathological parameters. WB and ELISA were used to analyze VTN-mediated cell pyroptosis. Results: Herein, we have identified a group of 105 differentially expressed proteins with ≥1.3-fold upregulation or ≤0.77-fold downregulation in JSLE patients. These proteins were involved in several important biological processes, including acute phase inflammatory responses, complement activation, hemostasis, and immune system regulation through Gene Ontology and functional enrichment analysis. Interestingly, urinary ephrin type-A receptor 4 (EPHA4) and VTN were significantly reduced in both inactive and active JSLE patients, and VTN treatment in THP-1 derived macrophages led to a significant increased cell pyroptosis by activation of Nod-like receptor family protein 3 (NLRP3) inflammasomes, resulting in caspase-1 activation, cleaved gasdermin D (GSDMD), and IL-18 secretion. Most importantly, the urinary VTN was also linearly correlated with clinical characteristics of JSLE, implying that VTN could be a specific diagnostic biomarker to distinguish inactive and active JSLE. Conclusion: This study provided a novel role of VTN in pyroptosis in JSLE through the urinary proteomic profile for JSLE, which could be a nonintrusive monitoring strategy in clinical diagnosis.

The Eph receptor A4 plays a role in demyelination and depression-related behavior.

Proper myelination of axons is crucial for normal sensory, motor, and cognitive function. Abnormal myelination is seen in brain disorders such as major depressive disorder (MDD), but the molecular mechanisms connecting demyelination with the pathobiology remain largely unknown. We observed demyelination and synaptic deficits in mice exposed to either chronic, unpredictable mild stress (CUMS) or LPS, 2 paradigms for inducing depression-like states. Pharmacological restoration of myelination normalized both synaptic deficits and depression-related behaviors. Furthermore, we found increased ephrin A4 receptor (EphA4) expression in the excitatory neurons of mice subjected to CUMS, and shRNA knockdown of EphA4 prevented demyelination and depression-like behaviors. These animal data are consistent with the decrease in myelin basic protein and the increase in EphA4 levels we observed in postmortem brain samples from patients with MDD. Our results provide insights into the etiology of depressive symptoms in some patients and suggest that inhibition of EphA4 or the promotion of myelination could be a promising strategy for treating depression.

Phosphorylation of guanosine monophosphate reductase triggers a GTP-dependent switch from pro- to anti-oncogenic function of EPHA4.

Signal transduction pathways post-translationally regulating nucleotide metabolism remain largely unknown. Guanosine monophosphate reductase (GMPR) is a nucleotide metabolism enzyme that decreases GTP pools by converting GMP to IMP. We observed that phosphorylation of GMPR at Tyr267 is critical for its activity and found that this phosphorylation by ephrin receptor tyrosine kinase EPHA4 decreases GTP pools in cell protrusions and levels of GTP-bound RAC1. EPHs possess oncogenic and tumor-suppressor activities, although the mechanisms underlying switches between these two modes are poorly understood. We demonstrated that GMPR plays a key role in EPHA4-mediated RAC1 suppression. This supersedes GMPR-independent activation of RAC1 by EPHA4, resulting in a negative overall effect on melanoma cell invasion and tumorigenicity. Accordingly, EPHA4 levels increase during melanoma progression and inversely correlate with GMPR levels in individual melanoma tumors. Therefore, phosphorylation of GMPR at Tyr267 is a metabolic signal transduction switch controlling GTP biosynthesis and transformed phenotypes.

Role of EphA4 in Mediating Motor Neuron Death in MND.

Motor neuron disease (MND) comprises a group of fatal neurodegenerative diseases with no effective cure. As progressive motor neuron cell death is one of pathological characteristics of MND, molecules which protect these cells are attractive therapeutic targets. Accumulating evidence indicates that EphA4 activation is involved in MND pathogenesis, and inhibition of EphA4 improves functional outcomes. However, the underlying mechanism of EphA4's function in MND is unclear. In this review, we first present results to demonstrate that EphA4 signalling acts directly on motor neurons to cause cell death. We then review the three most likely mechanisms underlying this effect.

NMR-Guided Design of Potent and Selective EphA4 Agonistic Ligands.

In this paper, we applied an innovative nuclear magnetic resonance (NMR)-guided screening and ligand design approach, named focused high-throughput screening by NMR (fHTS by NMR), to derive potent, low-molecular-weight ligands capable of mimicking interactions elicited by ephrin ligands on the receptor tyrosine kinase EphA4. The agents bind with nanomolar affinity, trigger receptor activation in cellular assays with motor neurons, and provide remarkable motor neuron protection from amyotrophic lateral sclerosis (ALS) patient-derived astrocytes. Structural studies on the complex between EphA4 ligand-binding domain and a most active agent provide insights into the mechanism of the agents at a molecular level. Together with preliminary in vivo pharmacology studies, the data form a strong foundation for the translation of these agents for the treatment of ALS and potentially other human diseases.

Dissecting indirect genetic effects from peers in laboratory mice.

BACKGROUND: The phenotype of an individual can be affected not only by the individual's own genotypes, known as direct genetic effects (DGE), but also by genotypes of interacting partners, indirect genetic effects (IGE). IGE have been detected using polygenic models in multiple species, including laboratory mice and humans. However, the underlying mechanisms remain largely unknown. Genome-wide association studies of IGE (igeGWAS) can point to IGE genes, but have not yet been applied to non-familial IGE arising from "peers" and affecting biomedical phenotypes. In addition, the extent to which igeGWAS will identify loci not identified by dgeGWAS remains an open question. Finally, findings from igeGWAS have not been confirmed by experimental manipulation. RESULTS: We leverage a dataset of 170 behavioral, physiological, and morphological phenotypes measured in 1812 genetically heterogeneous laboratory mice to study IGE arising between same-sex, adult, unrelated mice housed in the same cage. We develop and apply methods for igeGWAS in this context and identify 24 significant IGE loci for 17 phenotypes (FDR < 10%). We observe no overlap between IGE loci and DGE loci for the same phenotype, which is consistent with the moderate genetic correlations between DGE and IGE for the same phenotype estimated using polygenic models. Finally, we fine-map seven significant IGE loci to individual genes and find supportive evidence in an experiment with a knockout model that Epha4 gives rise to IGE on stress-coping strategy and wound healing. CONCLUSIONS: Our results demonstrate the potential for igeGWAS to identify IGE genes and shed light into the mechanisms of peer influence.

A cancer mutation promotes EphA4 oligomerization and signaling by altering the conformation of the SAM domain.

The Eph receptor tyrosine kinases and their ephrin ligands regulate many physiological and pathological processes. EphA4 plays important roles in nervous system development and adult homeostasis, while aberrant EphA4 signaling has been implicated in neurodegeneration. EphA4 may also affect cancer malignancy, but the regulation and effects of EphA4 signaling in cancer are poorly understood. A correlation between decreased patient survival and high EphA4 mRNA expression in melanoma tumors that also highly express ephrinA ligands suggests that enhanced EphA4 signaling may contribute to melanoma progression. A search for EphA4 gain-of-function mutations in melanoma uncovered a mutation of the highly conserved leucine 920 in the EphA4 sterile alpha motif (SAM) domain. We found that mutation of L920 to phenylalanine (L920F) potentiates EphA4 autophosphorylation and signaling, making it the first documented EphA4 cancer mutation that increases kinase activity. Quantitative Föster resonance energy transfer and fluorescence intensity fluctuation (FIF) analyses revealed that the L920F mutation induces a switch in EphA4 oligomer size, from a dimer to a trimer. We propose this switch in oligomer size as a novel mechanism underlying EphA4-linked tumorigenesis. Molecular dynamics simulations suggest that the L920F mutation alters EphA4 SAM domain conformation, leading to the formation of EphA4 trimers that assemble through two aberrant SAM domain interfaces. Accordingly, EphA4 wild-type and the L920F mutant are affected differently by the SAM domain and are differentially regulated by ephrin ligand stimulation. The increased EphA4 activation induced by the L920F mutation, through the novel mechanism we uncovered, supports a functional role for EphA4 in promoting pathogenesis.

Human urine-derived stem cell-derived exosomal miR-21-5p promotes neurogenesis to attenuate Rett syndrome via the EPha4/TEK axis.

Rett syndrome (RTT) is a rare neurodevelopmental disorder that results in multiple disabilities. Exosomal microRNA (miRs) from urine-derived stem cells (USCs) have been shown to induce neurogenesis and aid in functional recovery from brain ischemia. In the present study, we sought to determine whether that exosomal miR-21-5p from USCs could promote early neural formation in a model of RTT. USCs were isolated and evaluated by flow cytometry. Exosomes were analyzed by transmission electron microscopy, tunable resistive pulse sensing (TRPS), and western blotting. PKH26 fluorescent dyes were used to observe intake of exosomes in vivo and in vitro. An RTT mouse model was treated with exosomes for behavioral studies. Dual-luciferase report gene assays were conducted to evaluate the relationship between miR-21-5p and Eph receptor A4 (EphA4). In vitro, treatment with exosomes from human urine-derived stem cells (USC-Exos) increased the percentage of neuron-specific class III beta-tubulin (Tuj1)+ nerve cells as well as the transcription levels of ß-III tubulin and doublecortin (DCX). A higher level of miR-21-5p was observed in USC-Exos, which promoted differentiation in NSCs by targeting the EPha4/TEK axis. In vivo, exosomal miR-21-5p improved the behavior, motor coordination, and cognitive ability of mice, facilitated the differentiation of NSCs in the subventricular zone of the lateral ventricle and promoted a marked rise in the number of DCX+ cells. Our data provide evidence that exosomal miR-21-5p from human USCs facilitate early nerve formation by regulating the EPha4/TEK axis.