EphA4 Induces the Phosphorylation of an Intracellular Adaptor Protein Dab1 via Src Family Kinases.

Dab1 is an intracellular adaptor protein essential for brain formation during development. Tyrosine phosphorylation in Dab1 plays important roles in neuronal migration, dendrite development, and synapse formation by affecting several downstream pathways. Reelin is the best-known extracellular protein that induces Dab1 phosphorylation. However, whether other upstream molecule(s) contribute to Dab1 phosphorylation remains largely unknown. Here, we found that EphA4, a member of the Eph family of receptor-type tyrosine kinases, induced Dab1 phosphorylation when co-expressed in cultured cells. Tyrosine residues phosphorylated by EphA4 were the same as those phosphorylated by Reelin in neurons. The autophosphorylation of EphA4 was necessary for Dab1 phosphorylation. We also found that EphA4-induced Dab1 phosphorylation was mediated by the activation of the Src family tyrosine kinases. Interestingly, Dab1 phosphorylation was not observed when EphA4 was activated by ephrin-A5 in cultured cortical neurons, suggesting that Dab1 is localized in a different compartment in them. EphA4-induced Dab1 phosphorylation may occur under limited and/or pathological conditions in the brain.

Mutually Repulsive EphA7-EfnA5 Organize Region-to-Region Corticopontine Projection by Inhibiting Collateral Extension.

Coordination of skilled movements and motor planning relies on the formation of regionally restricted brain circuits that connect cortex with subcortical areas during embryonic development. Layer 5 neurons that are distributed across most cortical areas innervate the pontine nuclei (basilar pons) by protrusion and extension of collateral branches interstitially along their corticospinal extending axons. Pons-derived chemotropic cues are known to attract extending axons, but molecules that regulate collateral extension to create regionally segregated targeting patterns have not been identified. Here, we discovered that EphA7 and EfnA5 are expressed in the cortex and the basilar pons in a region-specific and mutually exclusive manner, and that their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts in mice. Specifically, EphA7 and EfnA5 forward and reverse inhibitory signals direct collateral extension such that EphA7-positive frontal and occipital cortical areas extend their axon collaterals into the EfnA5-negative rostral part of the basilar pons, whereas EfnA5-positive parietal cortical areas extend their collaterals into the EphA7-negative caudal part of the basilar pons. Together, our results provide a molecular basis that explains how the corticopontine projection connects multimodal cortical outputs to their subcortical targets.SIGNIFICANCE STATEMENT Our findings put forward a model in which region-to-region connections between cortex and subcortical areas are shaped by mutually exclusive molecules to ensure the fidelity of regionally restricted circuitry. This model is distinct from earlier work showing that neuronal circuits within individual cortical modalities form in a topographical manner controlled by a gradient of axon guidance molecules. The principle that a shared molecular program of mutually repulsive signaling instructs regional organization-both within each brain region and between connected brain regions-may well be applicable to other contexts in which information is sorted by converging and diverging neuronal circuits.

Neoadjuvant therapy alters the collagen architecture of pancreatic cancer tissue via Ephrin-A5.

BACKGROUND: The treatment of pancreatic cancer (PDAC) remains clinically challenging, and neoadjuvant therapy (NAT) offers down staging and improved surgical resectability. Abundant fibrous stroma is involved in malignant characteristic of PDAC. We aimed to investigate tissue remodelling, particularly the alteration of the collagen architecture of the PDAC microenvironment by NAT. METHODS: We analysed the alteration of collagen and gene expression profiles in PDAC tissues after NAT. Additionally, we examined the biological role of Ephrin-A5 using primary cultured cancer-associated fibroblasts (CAFs). RESULTS: The expression of type I, III, IV, and V collagen was reduced in PDAC tissues after effective NAT. The bioinformatics approach provided comprehensive insights into NAT-induced matrix remodelling, which showed Ephrin-A signalling as a likely pathway and Ephrin-A5 (encoded by EFNA5) as a crucial ligand. Effective NAT reduced the number of Ephrin-A5+ cells, which were mainly CAFs; this inversely correlated with the clinical tumour shrinkage rate. Experimental exposure to radiation and chemotherapeutic agents suppressed proliferation, EFNA5 expression, and collagen synthesis in CAFs. Forced EFNA5 expression altered CAF collagen gene profiles similar to those found in PDAC tissues after NAT. CONCLUSION: These results suggest that effective NAT changes the extracellular matrix with collagen profiles through CAFs and their Ephrin-A5 expression.

Expression characteristic, immune signature, and prognosis value of EFNA family identified by multi-omics integrative analysis in pan-cancer.

BACKGROUND: EphrinA (EFNA) are Eph receptor ligands that regulate various disease processes. Nonetheless, the expression characteristics of EFNAs in pan-cancer, their relationship with tumor immune microenvironment, and prognostic value landscape remain unknown. METHODS: A comprehensive landscape of EFNAs was created using various statistical data extracted from 33 cancers. Subsequently, we identified differential expression, genetic variations, potential function enrichment, tumor immune-related analysis, and drug sensitivity. Further, we investigated the clinical features and diagnostic prognostic value of EFNAs. RT-qPCR, western blot and immunohistochemistry (IHC) were used to validate the expression level and significant clinical value of EFNA5 in lung adenocarcinoma cell lines and tissues. RESULTS: EFNAs were highly mutated in various cancers. Genomic and epigenetic alterations of EFNAs were observed in various tumors, where an oncogenic mutation in specific cancer types potentially affected EFNA expression. Moreover, tumor-derived EFNAs were significantly related to the tumor immune microenvironment, suggesting that they are promising therapeutic targets. The majority of EFNA family genes were significantly linked to patient prognosis. Eventually, EFNA5 was an independent prognostic factor in lung adenocarcinoma. CONCLUSION: In summary, EFNAs are crucial in tumor immune regulation, and EFNA5 is a prognostic marker in lung adenocarcinoma. Our findings provide new insights into EFNAs from a bioinformatics standpoint and highlight the significance of EFNAs in cancer diagnosis and treatment.

Spatial organization-dependent EphA2 transcriptional responses revealed by ligand nanocalipers.

Ligand binding induces extensive spatial reorganization and clustering of the EphA2 receptor at the cell membrane. It has previously been shown that the nanoscale spatial distribution of ligands modulates EphA2 receptor reorganization, activation and the invasive properties of cancer cells. However, intracellular signaling downstream of EphA2 receptor activation by nanoscale spatially distributed ligands has not been elucidated. Here, we used DNA origami nanostructures to control the positions of ephrin-A5 ligands at the nanoscale and investigated EphA2 activation and transcriptional responses following ligand binding. Using RNA-seq, we determined the transcriptional profiles of human glioblastoma cells treated with DNA nanocalipers presenting a single ephrin-A5 dimer or two dimers spaced 14, 40 or 100 nm apart. These cells displayed divergent transcriptional responses to the differing ephrin-A5 nano-organization. Specifically, ephrin-A5 dimers spaced 40 or 100 nm apart showed the highest levels of differential expressed genes compared to treatment with nanocalipers that do not present ephrin-A5. These findings show that the nanoscale organization of ephrin-A5 modulates transcriptional responses to EphA2 activation.

Manipulating the Level of Sensorimotor Stimulation during LI-rTMS Can Improve Visual Circuit Reorganisation in Adult Ephrin-A2A5-/- Mice.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique that has the potential to treat a variety of neurologic and psychiatric disorders. The extent of rTMS-induced neuroplasticity may be dependent on a subject's brain state at the time of stimulation. Chronic low intensity rTMS (LI-rTMS) has previously been shown to induce beneficial structural and functional reorganisation within the abnormal visual circuits of ephrin-A2A5-/- mice in ambient lighting. Here, we administered chronic LI-rTMS in adult ephrin-A2A5-/- mice either in a dark environment or concurrently with voluntary locomotion. One day after the last stimulation session, optokinetic responses were assessed and fluorescent tracers were injected to map corticotectal and geniculocortical projections. We found that LI-rTMS in either treatment condition refined the geniculocortical map. Corticotectal projections were improved in locomotion+LI-rTMS subjects, but not in dark + LI-rTMS and sham groups. Visuomotor behaviour was not improved in any condition. Our results suggest that the beneficial reorganisation of abnormal visual circuits by rTMS can be significantly influenced by simultaneous, ambient visual input and is enhanced by concomitant physical exercise. Furthermore, the observed pathway-specific effects suggest that regional molecular changes and/or the relative proximity of terminals to the induced electric fields influence the outcomes of LI-rTMS on abnormal circuitry.

XMAP215 promotes microtubule-F-actin interactions to regulate growth cone microtubules during axon guidance in Xenopuslaevis.

It has long been established that neuronal growth cone navigation depends on changes in microtubule (MT) and F-actin architecture downstream of guidance cues. However, the mechanisms by which MTs and F-actin are dually coordinated remain a fundamentally unresolved question. Here, we report that the well-characterized MT polymerase, XMAP215 (also known as CKAP5), plays an important role in mediating MT-F-actin interaction within the growth cone. We demonstrate that XMAP215 regulates MT-F-actin alignment through its N-terminal TOG 1-5 domains. Additionally, we show that XMAP215 directly binds to F-actin in vitro and co-localizes with F-actin in the growth cone periphery. We also find that XMAP215 is required for regulation of growth cone morphology and response to the guidance cue, Ephrin A5. Our findings provide the first strong evidence that XMAP215 coordinates MT and F-actin interaction in vivo We suggest a model in which XMAP215 regulates MT extension along F-actin bundles into the growth cone periphery and that these interactions may be important to control cytoskeletal dynamics downstream of guidance cues. This article has an associated First Person interview with the first author of the paper.

The Expression of the Cancer-Associated lncRNA Snhg15 Is Modulated by EphrinA5-Induced Signaling.

The Eph receptor tyrosine kinases and their respective ephrin-ligands are an important family of membrane receptors, being involved in developmental processes such as proliferation, migration, and in the formation of brain cancer such as glioma. Intracellular signaling pathways, which are activated by Eph receptor signaling, are well characterized. In contrast, it is unknown so far whether ephrins modulate the expression of lncRNAs, which would enable the transduction of environmental stimuli into our genome through a great gene regulatory spectrum. Applying a combination of functional in vitro assays, RNA sequencing, and qPCR analysis, we found that the proliferation and migration promoting stimulation of mouse cerebellar granule cells (CB) with ephrinA5 diminishes the expression of the cancer-related lncRNA Snhg15. In a human medulloblastoma cell line (DAOY) ephrinA5 stimulation similarly reduced SNHG15 expression. Computational analysis identified triple-helix-mediated DNA-binding sites of Snhg15 in promoters of genes found up-regulated upon ephrinA5 stimulation and known to be involved in tumorigenic processes. Our findings propose a crucial role of Snhg15 downstream of ephrinA5-induced signaling in regulating gene transcription in the nucleus. These findings could be potentially relevant for the regulation of tumorigenic processes in the context of glioma.

Erythropoietin-producing hepatocellular receptor A7 restrains estrogen negative feedback of luteinizing hormone via ephrin A5 in the hypothalamus of female rats.

We have previously shown that systemic injection of erythropoietin-producing hepatocellular receptor A7 (EPHA7)-Fc raises serum luteinizing hormone (LH) levels before ovulation in female rats, indicating the induction of EPHA7 in ovulation. In this study, we aimed to identify the mechanism and hypothalamus-pituitary-ovary (HPO) axis level underlying the promotion of LH secretion by EPHA7. Using an ovariectomized (OVX) rat model, in conjunction with low-dose 17ß-estradiol (E2) treatment, we investigated the association between EPHA7-ephrin (EFN)A5 signaling and E2 negative feedback. Various rat models (OVX, E2-treated OVX, and abarelix treated) were injected with the recombinant EPHA7-Fc protein through the caudal vein to investigate the molecular mechanism underlying the promotion of LH secretion by EPHA7. Efna5 was observed strongly expressed in the arcuate nucleus of the female rat by using RNAscope in situ hybridization. Our results indicated that E2, combined with estrogen receptor (ER)α, but not ERß, inhibited Efna5 and gonadotropin-releasing hormone 1 (Gnrh1) expressions in the hypothalamus. In addition, the systemic administration of EPHA7-Fc restrained the inhibition of Efna5 and Gnrh1 by E2, resulting in increased Efna5 and Gnrh1 expressions in the hypothalamus as well as increased serum LH levels. Collectively, our findings demonstrated the involvement of EPHA7-EFNA5 signaling in the regulation of LH and the E2 negative feedback pathway in the hypothalamus, highlighting the functional role of EPHA7 in female reproduction.

Novel genetic associations with interferon in systemic lupus erythematosus identified by replication and fine-mapping of trait-stratified genome-wide screen.

BACKGROUND/PURPOSE: High serum interferon alpha (IFN-α) is an important heritable phenotype in systemic lupus erythematosus (SLE) which is involved in primary disease pathogenesis. High vs. low levels of IFN-α are associated with disease severity and account for some of the biological heterogeneity between SLE patients. The aim of the study was to replicate and fine-map previously detected genetic associations with serum IFN-α in SLE. METHODS: We previously undertook a case-case genome-wide association study of SLE patients stratified by ancestry and extremes of phenotype in serum IFN-α. Single nucleotide polymorphisms (SNPs) in seven loci identified in this screen were selected for follow up in a large independent cohort of 1370 SLE patients (703 European-ancestry, 432 African ancestry, and 235 Amerindian ancestry). Each ancestral background was analyzed separately, and ancestry-informative markers were used to control for ancestry and admixture. RESULTS: We find a rare haplotype spanning the promoter region of EFNA5 that is strongly associated with serum IFN-α in both African-American and European-American SLE patients (OR = 3.0, p = 3.7 × 10-6). We also find SNPs in the PPM1H, PTPRM, and NRGN regions associated with IFN-α levels in European-American, Amerindian, and African-American SLE patients respectively. Many of these associations are within regulatory regions of the gene, suggesting an impact on transcription. CONCLUSION: This study demonstrates the power of molecular sub-phenotypes to reveal genetic factors involved in complex autoimmune disease. The distinct associations observed in different ancestral backgrounds emphasize the heterogeneity of molecular pathogenesis in SLE.

Mapping of breakpoints in balanced chromosomal translocations by shallow whole-genome sequencing points to EFNA5, BAHD1 and PPP2R5E as novel candidates for genes causing human Mendelian disorders.

BACKGROUND: Mapping the breakpoints in de novo balanced chromosomal translocations (BCT) in symptomatic individuals provides a unique opportunity to identify in an unbiased way the likely causative genetic defect and thus find novel human disease candidate genes. Our aim was to fine-map breakpoints of de novo BCTs in a case series of nine patients. METHODS: Shallow whole-genome mate pair sequencing (SGMPS) together with long-range PCR and Sanger sequencing. In one case (BCT disrupting BAHD1 and RET) cDNA analysis was used to verify expression of a fusion transcript in cultured fibroblasts. RESULTS: In all nine probands 11 disrupted genes were found, that is, EFNA5, EBF3, LARGE, PPP2R5E, TXNDC5, ZNF423, NIPBL, BAHD1, RET, TRPS1 and SLC4A10. Five subjects had translocations that disrupted genes with so far unknown (EFNA5, BAHD1, PPP2R5E, TXNDC5) or poorly delineated impact on the phenotype (SLC4A10, two previous reports of BCT disrupting the gene). The four genes with no previous disease associations (EFNA5, BAHD1, PPP2R5E, TXNDC5), when compared with all human genes by a bootstrap test, had significantly higher pLI (p<0.017) and DOMINO (p<0.02) scores indicating enrichment in genes likely to be intolerant to single copy damage. Inspection of individual pLI and DOMINO scores, and local topologically associating domain structure suggested that EFNA5, BAHD1 and PPP2R5E were particularly good candidates for novel disease loci. The pathomechanism for BAHD1 may involve deregulation of expression due to fusion with RET promoter. CONCLUSION: SGMPS in symptomatic carriers of BCTs is a powerful approach to delineate novel human gene-disease associations.

Recent advances in the development and function of type II spiral ganglion neurons in the mammalian inner ear.

In hearing, mechanically sensitive hair cells (HCs) in the cochlea release glutamate onto spiral ganglion neurons (SGNs) to relay auditory information to the central nervous system (CNS). There are two main SGN subtypes, which differ in morphology, number, synaptic targets, innervation patterns and firing properties. About 90-95% of SGNs are the type I SGNs, which make a single bouton connection with inner hair cells (IHCs) and have been well described in the canonical auditory pathway for sound detection. However, less attention has been given to the type II SGNs, which exclusively innervate outer hair cells (OHCs). In this review, we emphasize recent advances in the molecular mechanisms that control how type II SGNs develop and form connections with OHCs, and exciting new insights into the function of type II SGNs.

Proper closure of the optic fissure requires ephrin A5-EphB2-JNK signaling.

The development of complex organs such as the eye requires a delicate and coordinated balance of cell division and cell death. Although apoptosis is prevalent in the proximoventral optic cup, the precise role it plays in eye development needs to be investigated further. In this study, we show that reduced apoptosis in the proximoventral optic cup prevents closure of the optic fissure. We also show that expression of ephrin A5 (Efna5) partially overlaps with Eph receptor B2 (Ephb2) expression in the proximoventral optic cup and that binding of EphB2 to ephrin A5 induces a sustained activation of JNK. This prolonged JNK signal promotes apoptosis and prevents cell proliferation. Thus, we propose that the unique cross-subclass interaction of EphB2 with ephrin A5 has evolved to function upstream of JNK signaling for the purpose of maintaining an adequate pool of progenitor cells to ensure proper closure of the optic fissure.

Epha2 and Efna5 participate in lens cell pattern-formation.

Ephrin type-A receptor 2 (EPHA2) and one of its ligands, ephrin-A5 (EFNA5), have been associated with loss of eye lens transparency, or cataract, - an important cause of visual impairment. Here we show that mice functionally lacking EPHA2 (Epha2-null), EFNA5 (Efna5-null), or both receptor and ligand (Epha2/Efna5-null) consistently develop mostly transparent lenses with an internal refractive disturbance and a grossly disturbed cellular architecture. In situ hybridization localized Epha2 and Efna5 transcripts to lens epithelial cells and nascent fiber cells at the lens equator. In vivo labeling of Epha2-null lenses with a thymidine analog detected a significant decrease in lens epithelial cell proliferation within the germinative zone resulting in impaired early lens growth. Ex vivo imaging of Epha2-null, Efna5-null, and Epha2/Efna5-null lenses labelled in vivo with a membrane-targeted red fluorescent protein revealed misalignment of elongating fiber cells at the lens equator and loss of Y-suture pattern formation near the anterior and posterior poles of the lens. Immuno-fluorescent labeling of lens major intrinsic protein or aquaporin-0 (MIP/AQP0) showed that the precise, radial column patterning of hexagonal fiber cells throughout the cortex region was disrupted in Epha2-null, Efna5-null and Epha2/Efna5-null lenses. Collectively, these data suggest that Epha2 and Efna5 participate in the complex, global patterning of lens fiber cells that is necessary for maximal optical quality.

PTP-PEST controls EphA3 activation and ephrin-induced cytoskeletal remodelling.

Eph receptors and their corresponding membrane-bound ephrin ligands regulate cell positioning and establish tissue patterns during embryonic and oncogenic development. Emerging evidence suggests that assembly of polymeric Eph signalling clusters relies on cytoskeletal reorganisation and underlies regulation by protein tyrosine phosphatases (PTPs). PTP-PEST (also known as PTPN12) is a central regulator of actin cytoskeletal dynamics. Here, we demonstrate that an N-terminal fragment of PTP-PEST, generated through an ephrinA5-triggered and spatially confined cleavage mediated by caspase-3, attenuates EphA3 receptor activation and its internalisation. Isolation of EphA3 receptor signalling clusters within intact plasma membrane fragments obtained by detergent-free cell fractionation reveals that stimulation of cells with ephrin triggers effective recruitment of this catalytically active truncated form of PTP-PEST together with key cytoskeletal and focal adhesion proteins. Importantly, modulation of actin polymerisation using pharmacological and dominant-negative approaches affects EphA3 phosphorylation in a similar manner to overexpression of PTP-PEST. We conclude that PTP-PEST regulates EphA3 activation both by affecting cytoskeletal remodelling and through its direct action as a PTP controlling EphA3 phosphorylation, indicating its multifaceted regulation of Eph signalling.

Thalamic afferents influence cortical progenitors via ephrin A5-EphA4 interactions.

The phenotype of excitatory cerebral cortex neurons is specified at the progenitor level, orchestrated by various intrinsic and extrinsic factors. Here, we provide evidence for a subcortical contribution to cortical progenitor regulation by thalamic axons via ephrin A5-EphA4 interactions. Ephrin A5 is expressed by thalamic axons and represents a high-affinity ligand for EphA4 receptors detected in cortical precursors. Recombinant ephrin A5-Fc protein, as well as ephrin A ligand-expressing, thalamic axons affect the output of cortical progenitor division in vitro. Ephrin A5-deficient mice show an altered division mode of radial glial cells (RGCs) accompanied by increased numbers of intermediate progenitor cells (IPCs) and an elevated neuronal production for the deep cortical layers at E13.5. In turn, at E16.5 the pool of IPCs is diminished, accompanied by reduced rates of generated neurons destined for the upper cortical layers. This correlates with extended infragranular layers at the expense of superficial cortical layers in adult ephrin A5-deficient and EphA4-deficient mice. We suggest that ephrin A5 ligands imported by invading thalamic axons interact with EphA4-expressing RGCs, thereby contributing to the fine-tuning of IPC generation and thus the proper neuronal output for cortical layers.

The Neural Cell Adhesion Molecule (NCAM) Promotes Clustering and Activation of EphA3 Receptors in GABAergic Interneurons to Induce Ras Homolog Gene Family, Member A (RhoA)/Rho-associated protein kinase (ROCK)-mediated Growth Cone Collapse.

Establishment of a proper balance of excitatory and inhibitory connectivity is achieved during development of cortical networks and adjusted through synaptic plasticity. The neural cell adhesion molecule (NCAM) and the receptor tyrosine kinase EphA3 regulate the perisomatic synapse density of inhibitory GABAergic interneurons in the mouse frontal cortex through ephrin-A5-induced growth cone collapse. In this study, it was demonstrated that binding of NCAM and EphA3 occurred between the NCAM Ig2 domain and EphA3 cysteine-rich domain (CRD). The binding interface was further refined through molecular modeling and mutagenesis and shown to be comprised of complementary charged residues in the NCAM Ig2 domain (Arg-156 and Lys-162) and the EphA3 CRD (Glu-248 and Glu-264). Ephrin-A5 induced co-clustering of surface-bound NCAM and EphA3 in GABAergic cortical interneurons in culture. Receptor clustering was impaired by a charge reversal mutation that disrupted NCAM/EphA3 association, emphasizing the importance of the NCAM/EphA3 binding interface for cluster formation. NCAM enhanced ephrin-A5-induced EphA3 autophosphorylation and activation of RhoA GTPase, indicating a role for NCAM in activating EphA3 signaling through clustering. NCAM-mediated clustering of EphA3 was essential for ephrin-A5-induced growth cone collapse in cortical GABAergic interneurons, and RhoA and a principal effector, Rho-associated protein kinase, mediated the collapse response. This study delineates a mechanism in which NCAM promotes ephrin-A5-dependent clustering of EphA3 through interaction of the NCAM Ig2 domain and the EphA3 CRD, stimulating EphA3 autophosphorylation and RhoA signaling necessary for growth cone repulsion in GABAergic interneurons in vitro, which may extend to remodeling of axonal terminals of interneurons in vivo.

EphA2 and ephrin-A5 are not a receptor-ligand pair in the ocular lens.

Eph-ephrin bidirectional signaling is essential for eye lens transparency in humans and mice. Our previous studies in mouse lenses demonstrate that ephrin-A5 is mainly expressed in the anterior epithelium, where it is required for maintaining the anterior epithelial monolayer. In contrast, EphA2 is localized in equatorial epithelial and fiber cells where it is essential for equatorial epithelial and fiber cell organization and hexagonal cell shape. Immunostaining of lens epithelial and fiber cells reveals that EphA2 and ephrin-A5 are also co-expressed in anterior fiber cell tips, equatorial epithelial cells and newly formed lens fibers, although they are not precisely colocalized. Due to this complex expression pattern and the promiscuous interactions between Eph receptors and ephrin ligands, as well as their complex bidirectional signaling pathways, cataracts in ephrin-A5(-/-) or EphA2(-/-) lenses may arise from loss of function or abnormal signaling mechanisms. To test whether abnormal signaling mechanisms may play a role in cataractogenesis in ephrin-A5(-/-) or EphA2(-/-) lenses, we generated EphA2 and ephrin-A5 double knockout (DKO) mice. We compared the phenotypes of EphA2(-/-) and ephrin-A5(-/-) lenses to that of DKO lenses. DKO lenses displayed an additive lens phenotype that was not significantly different from the two single KO lens phenotypes. Similar to ephrin-A5(-/-) lenses, DKO lenses had abnormal anterior epithelial cells leading to a large mass of epithelial cells that invade into the underlying fiber cell layer, directly resulting in anterior cataracts in ephrin-A5(-/-) and DKO lenses. Yet, similar to EphA2(-/-) lenses, DKO lenses also had abnormal packing of equatorial epithelial cells with disorganized meridional rows, lack of a lens fulcrum and disrupted fiber cells. The DKO lens phenotype rules out abnormal signaling by EphA2 in ephrin-A5(-/-) lenses or by ephrin-A5 in EphA2(-/-) lenses as possible cataract mechanisms. Thus, these results indicate that EphA2 and ephrin-A5 do not form a lens receptor-ligand pair, and that EphA2 and ephrin-A5 have other binding partners in the lens to help align differentiating equatorial epithelial cells or maintain the anterior epithelium, respectively.

EphA7 signaling guides cortical dendritic development and spine maturation.

The process by which excitatory neurons are generated and mature during the development of the cerebral cortex occurs in a stereotyped manner; coordinated neuronal birth, migration, and differentiation during embryonic and early postnatal life are prerequisites for selective synaptic connections that mediate meaningful neurotransmission in maturity. Normal cortical function depends upon the proper elaboration of neurons, including the initial extension of cellular processes that lead to the formation of axons and dendrites and the subsequent maturation of synapses. Here, we examine the role of cell-based signaling via the receptor tyrosine kinase EphA7 in guiding the extension and maturation of cortical dendrites. EphA7, localized to dendritic shafts and spines of pyramidal cells, is uniquely expressed during cortical neuronal development. On patterned substrates, EphA7 signaling restricts dendritic extent, with Src and Tsc1 serving as downstream mediators. Perturbation of EphA7 signaling in vitro and in vivo alters dendritic elaboration: Dendrites are longer and more complex when EphA7 is absent and are shorter and simpler when EphA7 is ectopically expressed. Later in neuronal maturation, EphA7 influences protrusions from dendritic shafts and the assembling of synaptic components. Indeed, synaptic function relies on EphA7; the electrophysiological maturation of pyramidal neurons is delayed in cultures lacking EphA7, indicating that EphA7 enhances synaptic function. These results provide evidence of roles for Eph signaling, first in limiting the elaboration of cortical neuronal dendrites and then in coordinating the maturation and function of synapses.

[Changes in the expression of EphA5/ephrinA5 in the CA3 region of the hippocampus in rats with epilepsy and their role in the pathogenesis of temporal lobe epilepsy].

OBJECTIVE: To investigate the changes in the expression of EphA5 and its ligand ephrinA5 in the hippocampus of rats with epilepsy and their role in the pathogenesis of temporal lobe epilepsy (TLE). METHODS: A total of 240 Sprague-Dawley rats were randomly divided into control group and TLE group, with 120 rats in each group. A rat model of lithium-pilocarpine TLE was established, and then the rats were divided into subgroups at 12 and 24 hours and 7, 15, 30, and 60 days after epilepsy was induced. In-situ hybridization was used to measure the mRNA expression of ephrinA5 in the CA3 region and the dentate gyrus of the hippocampus in 9 rats; immunohistochemistry was used to measure the protein expression of EphA5 in the CA3 region and the dentate gyrus of the hippocampus in 9 rats; Neo-Timm silver staining was used to observe mossy fiber sprouting in the CA3 region of the hippocampus in 2 rats. RESULTS: In-situ hybridization showed mRNA expression of ephrinA5 in the CA3 region of the hippocampus, but this was not found in the dentate gyrus. Compared with the control group at the same time point, the TLE group had a significant reduction in the mRNA expression of ephrinA5 in the CA3 region of the hippocampus at 7 and 15 days after epilepsy was induced (P<0.05); at 30 and 60 days after epilepsy was induced, the TLE group had a gradual increase in the mRNA expression of ephrinA5 in the CA3 region of the hippocampus, and there was no significant difference between the TLE and control groups (P>0.05). Immunohistochemistry showed that EphA5 protein was expressed in the CA3 region and the dentate gyrus of the hippocampus and had a similar trend of change as ephrinA5 mRNA. Neo-Timm silver staining showed that the TLE group developed marked mossy fiber sprouting in the CA3 region of the hippocampus at 7 and 15 days after epilepsy was induced. CONCLUSIONS: Downregulation of ephrinA5 and EphA5 in the CA3 region of the hippocampus may participate in the mechanism of mossy fiber sprouting and is closely associated with the development and progression of epilepsy.