Neuronal transmission is regulated by the local circuitry which is composed of principal neurons targeted at different subcellular compartments by a variety of interneurons. However, mechanisms that contribute to the subcellular localisation and maintenance of GABAergic interneuron terminals are poorly understood. Stabilization of GABAergic synapses depends on clustering of the postsynaptic scaffolding protein gephyrin and its interaction with the guanine nucleotide exchange factor collybistin. Lentiviral knockdown experiments in adult rats indicated that the receptor tyrosine kinase EphA7 is required for the stabilisation of basket cell terminals on proximal dendritic and somatic compartments of granular cells of the dentate gyrus. EphA7 deficiency and concomitant destabilisation of GABAergic synapses correlated with impaired long-term potentiation and reduced hippocampal learning. Reduced GABAergic innervation may be explained by an impact of EphA7 on gephyrin clustering. Overexpression or ephrin stimulation of EphA7 induced gephyrin clustering dependent on the mechanistic target of rapamycin (mTOR) which is an interaction partner of gephyrin. Gephyrin interactions with mTOR become released after mTOR activation while enhanced interaction with the guanine nucleotide exchange factor collybistin was observed in parallel. In conclusion, EphA7 regulates gephyrin clustering and the maintenance of inhibitory synaptic connectivity via mTOR signalling.

The shape of a neuron facilitates its functionality within neural circuits. Dendrites integrate incoming signals from axons, receiving excitatory input onto small protrusions called dendritic spines. Therefore, understanding dendritic growth and development is fundamental for discerning neural function. We previously demonstrated that EphA7 receptor signaling during cortical development impacts dendrites in two ways: EphA7 restricts dendritic growth early and promotes dendritic spine formation later. Here, the molecular basis for this shift in EphA7 function is defined. Expression analyses reveal that EphA7 full-length (EphA7-FL) and truncated (EphA7-T1; lacking kinase domain) isoforms are dynamically expressed in the developing cortex. Peak expression of EphA7-FL overlaps with dendritic elaboration around birth, while highest expression of EphA7-T1 coincides with dendritic spine formation in early postnatal life. Overexpression studies in cultured neurons demonstrate that EphA7-FL inhibits both dendritic growth and spine formation, while EphA7-T1 increases spine density. Furthermore, signaling downstream of EphA7 shifts during development, such that in vivo inhibition of mTOR by rapamycin in EphA7-mutant neurons ameliorates dendritic branching, but not dendritic spine phenotypes. Finally, direct interaction between EphA7-FL and EphA7-T1 is demonstrated in cultured cells, which results in reduction of EphA7-FL phosphorylation. In cortex, both isoforms are colocalized to synaptic fractions and both transcripts are expressed together within individual neurons, supporting a model where EphA7-T1 modulates EphA7-FL repulsive signaling during development. Thus, the divergent functions of EphA7 during cortical dendrite development are explained by the presence of two variants of the receptor.

The conversion of proliferating skeletal muscle precursors (myoblasts) to terminally-differentiated myocytes is a critical step in skeletal muscle development and repair. We show that EphA7, a juxtacrine signaling receptor, is expressed on myocytes during embryonic and fetal myogenesis and on nascent myofibers during muscle regeneration in vivo. In EphA7-/- mice, hindlimb muscles possess fewer myofibers at birth, and those myofibers are reduced in size and have fewer myonuclei and reduced overall numbers of precursor cells throughout postnatal life. Adult EphA7-/- mice have reduced numbers of satellite cells and exhibit delayed and protracted muscle regeneration, and satellite cell-derived myogenic cells from EphA7-/- mice are delayed in their expression of differentiation markers in vitro. Exogenous EphA7 extracellular domain will rescue the null phenotype in vitro, and will also enhance commitment to differentiation in WT cells. We propose a model in which EphA7 expression on differentiated myocytes promotes commitment of adjacent myoblasts to terminal differentiation.

The downregulation of receptor tyrosine kinase EphA7 is frequent in epithelial cancers and linked to tumor progression. However, the detailed mechanism of EphA7-mediated prostate tumor progression remains elusive. To test the role of EphA7 receptor in prostate cancer (PCa) progression directly, we generated EphA7 receptor variants that were either lacking the cytoplasmic domain or carrying a point mutation that inhibits its phosphorylation by site-directed mutagenesis. Overexpression of wild-type (WT) EphA7 in PCa cells resulted in decreased tumor volume and increased tumor apoptosis in primary tumors. In addition, ectopic expression of WT EphA7 both can delay PCa cell proliferation and could inhibit PCa cell migration and invasion. This protein can also induce PCa cell apoptosis that correlated with increasing the protein expression levels of Bax, elevating the caspase-3 activities, reducing the protein expression levels of Bcl-2 and facilitating the dephosphorylation of Akt, which is further increased by the stimulation of ephrinA5-Fc. However, expression of these EphA7 mutants in PCa cells has no effect in vivo and in vitro. The expression of EphA7 and ephrinA5 was significantly decreased in PCa specimens compared with BPH tissues or paired normal tissues. Moreover, the phosphorylation of EphA7 was positively related with ephrinA5 expression in human prostate tissues. In sum, receptor phosphorylation of EphA7, at least in part, suppress PCa tumor malignancy through targeting PI3K/Akt signaling pathways.

Prior studies have identified two anatomically and neurochemically distinct cellular compartments within the mammalian striatum, termed striosomes and matrix, which express μ-opioid receptors (μOR) and EphA4, respectively. Here we identify and characterize an additional compartment in the rat striatum composed of neurons that express EphA7. In situ hybridization and immunohistochemical data indicate that neurons expressing EphA7 mRNA and protein are arranged in a banded "matrisome-like" pattern confined to the matrix in the dorsal striatum. Within the ventral striatum, EphA7-positive (+) neurons have a less organized mosaic pattern that partially overlaps areas expressing μOR. Immunolabeling data demonstrate that EphA7+ striatofugal axons form distinct fascicles leaving the striatum. Within the globus pallidus, EphA7+ axons terminate primarily within ventromedial areas of the nucleus and along its striatal border. EphA7+ axons avoid regions containing dopamine neurons within the substantia nigra and preferentially innervate areas near the rostral and caudal margins of the nucleus. Within both nuclei, EphA7+ axons have similar but more restricted terminal fields than the entire population of EphA4+ matrix axons, indicating that EphA7+ axons comprise a subpopulation of matrix axons. Ligand binding data demonstrate that ephrin-A5 selectively binds areas of the striatum, globus pallidus, and substantia nigra containing EphA7+ neurons and axons, but not areas expressing only EphA4. Our findings demonstrate that EphA7 expression identifies a novel "matrisome" compartment within the matrix that binds ephrin-A5 and possesses unique axonal projections. Our findings also suggest that EphA7 and ephrin-A5 may participate in the formation of this matrisome subcompartment and its striatofugal projections.

Zinc finger protein 422 (Zfp422) is a widely expressed zinc finger protein that serves as a transcriptional factor to regulate downstream gene expression, but until now, little is known about its roles in myogenesis. We found here that Zfp422 plays a critical role in skeletal muscle development and regeneration. It highly expresses in mouse skeletal muscle during embryonic development. Specific knockout of Zfp422 in skeletal muscle impaired embryonic muscle formation. Satellite cell-specific Zfp422 deletion severely inhibited muscle regeneration. Myoblast differentiation and myotube formation were suppressed in Zfp422-deleted C2C12 cells, isolated primary myoblasts, and satellite cells. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) revealed that Zfp422 regulated ephrin type-A receptor 7 (EphA7) expression by binding an upstream 169-bp DNA sequence, which was proved to be an enhancer of EphA7. Knocking EphA7 down in C2C12 cells or deleting Zfp422 in myoblasts will inhibit cell apoptosis which is required for myoblast differentiation. These results indicate that Zfp422 is essential for skeletal muscle differentiation and fusion, through regulating EphA7 expression to maintain proper apoptosis.

Deciphering the molecular basis for guiding specific aspects of neocortical development remains a challenge because of the complexity of histogenic events and the vast array of protein interactions mediating these events. The Eph family of receptor tyrosine kinases is implicated in a number of neurodevelopmental activities. Eph receptors have been known to be capable of responding to several ephrin ligands within their subgroups, often eliciting similar downstream effects. However, several recent studies have indicated specificity between receptor-ligand pairs within each subfamily, the functional relevance of which is not defined. Here we show that a receptor of the EphA subfamily, EphA4, has effects distinct from those of its close relative, EphA7, in the developing brain. Both EphA4 and EphA7 interact similarly with corresponding ligands expressed in the developing neocortex. However, only EphA7 shows strong interaction with ligands in the somatosensory thalamic nuclei; EphA4 affects only cortical neuronal migration, with no visible effects on the guidance of corticothalamic (CT) axons, whereas EphA7 affects both cortical neuronal migration and CT axon guidance. Our data provide new evidence that Eph receptors in the same subfamily are not simply interchangeable but are functionally specified through selective interactions with distinct ligands in vivo. J. Comp. Neurol. 524:2080-2092, 2016. © 2015 Wiley Periodicals, Inc.

We have isolated a novel chick Eph-related receptor that corresponds to the EphA7 gene. Within the nervous system, EphA7 expression is restricted to prosomeres 1 and 2 in the diencephalon and all the rhombomeres in the hindbrain during segmentation stages. Later on, a superimposed pattern appears that correlates with the formation of several axonal tracts. In the somitic mesoderm, the expression correlates with segmentation and the guidance of both neural crest and motor axons through the sclerotomes.

Within the first two postnatal weeks, corticostriatal axons from the primary somatosensory cortex (S1) form topographic projections that organize into characteristic bands of axon terminals in the dorsolateral striatum. Molecules regulating the development of these topographically organized projections are currently unknown. Thus, the present study investigated whether EphA receptor tyrosine kinases, which regulate axonal guidance in the visual system via axon repulsion, could participate in the formation of corticostriatal connections during development. Prior studies indicate that EphA7-expressing striatal neurons are organized into banded compartments resembling the matrisome innervation pattern formed by cortical afferents from the S1 cortex and that ephrin-A5, a known EphA7 ligand, is expressed in a medial (high) to lateral (low) gradient in S1. Thus, we hypothesized that the organization of EphA7-expressing striatal neurons in banded domains provides a repulsive barrier preventing corticostriatal axons containing EphA7-ligands from innervating inappropriate regions of the striatum. To evaluate this, we injected the anterograde tracer, biotinylated dextran amine (BDA), into two locations in medial areas of S1 (the anterior and posterior whisker fields), which are reported to express high levels of ephrin-A5 during development. Injections were made in mouse pups on postnatal day 9 (P9) and the animals were processed for immunohistochemistry on P12. Our data demonstrate that projections from both the forelimb/anterior whisker field and the posterior whisker field avoid EphA7-expressing neurons and terminate in a banded pattern in regions with very low EphA7-expression. We also determined that corticothalamic projections from medial S1 also exhibit a restricted distribution in the thalamus and avoid neurons expressing EphA7. Thus, our results support the hypothesis that the anatomical organization of striatal and thalamic neurons expressing EphA7 receptors restricts the topographic distribution of cortical afferents from medial regions of S1 which express high levels of ephrin-A5.

Micro (mi)RNAs serve crucial roles in cancer development although little is known about their cellular mechanisms in the pathogenesis of melanoma. The present study explored the regulatory roles of miR‑18a‑5p in melanoma cell proliferation, apoptosis and autophagy, in addition to its target gene in melanoma cells. miRNA and ephrin receptor A7 (EPHA7) mRNA were analyzed by reverse transcription‑quantitative PCR. Cell Counting Kit‑8 and colony formation assays were performed to examine the cell proliferation rate. Hoechst staining and flow cytometry were performed to investigate cell apoptosis. Western blotting was used to estimate the abundance of proteins. Dual-luciferase reporter assay verified the binding of miRNA with target gene sequences. Melanoma tissues and cell lines exhibited markedly elevated miR‑18a‑5p expression. miR‑18a‑5p inhibitor inhibited proliferation rates, and triggered apoptosis and autophagy marker protein expression in WM266‑4 and A375 cells. It also negatively regulated EPHA7 expression in WM266‑4 and A375 cells by directly binding at the 3'‑untranslated region of EPHA7. miR‑18a‑5p mimics reversed the EPHA7 overexpression‑induced suppression of proliferation, and the EPHA7 overexpression‑induced promotion of apoptosis and autophagy. miR‑18a‑5p triggered proliferation of melanoma cells and inhibited apoptosis and autophagy by directly targeting and inhibiting EPHA7 expression. Thus, the present study aided our understanding of miRNA‑mediated melanoma pathogenesis.

Erythropoietin producing hepatocellular (Eph) forms the largest family of receptor tyrosine kinases (RTK). As a family, Eph regulates physiological events such as cell-cell interaction, cell migration, and adhesion. The Kinase domain is the catalytic core of the Eph receptor and is highly conserved sequentially. EphA7 has been recently regarded as a cancer driver gene and comprises several clinically important mutations. Three of the EphA7 mutations Gly656Glu, Gly656Arg, and Asp751His, present in the kinase domain, are predicted to be highly pathogenic. Furthermore, Gly656Glu and Gly656Arg are reported to be hotspot mutations. Considering the importance of mutations, crystals structure of EphA7 Gly656Glu, Gly656Arg, and Asp751His mutants has been explored. Changes in folding pattern and intramolecular interactions were observed in mutant structures. Secondary structural changes were observed in the hinge region of EphA7 Gly656Arg and Asp751His structure, affecting the transition of kinase domain between open and closed conformations. EphA7 Asp751His mutant structure shows a distorted nucleotide-binding groove. Differences were observed in hydrogen bonding and hydrophobic interactions between the catalytic and highly conserved DFG motif in the EphA7 mutants, which may influence the catalytic activity of kinase domain.

Members of the Eph/ephrin gene families act as key regulators of cerebellar development during embryogenesis. Aberrant signaling of Eph family of receptor tyrosine kinases and their ephrin ligands has also been implicated in human cancers. Medulloblastoma is an aggressive primitive neuroectodermal tumor that originates from granule neuron precursors in the cerebellum. Previous studies have suggested a role for the ephrin-A5 ligand and its receptors, EphA4 and EphA7, in granule cell-precursor formation and in guiding cell migration. In the present study, we investigated the effects of genetic loss of ephrin-A5, EphA4, and EphA7 on the spatiotemporal development of medulloblastoma tumors in the context of the smoothened transgenic mouse model system.

Patients with osteosarcoma (OS) usually have poor overall survival because of frequent metastasis. Long non-coding RNAs (lncRNAs) have been reported to be associated with tumorigenesis and metastasis. In this study, we investigated the expression and roles of lncRNA human histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) in OS, aiming to provide a novel molecular mechanism for OS. HCP5 was up-regulated both in OS tissues and cell lines and high expression of HCP5 was associated to low survival in OS patients. Down-regulation of HCP5 inhibited cell proliferation, migration, and invasion, suggesting its carcinogenic role in OS. miR-101 was targeted by HCP5 and its expression was decreased in OS. The inhibitor of miR-101 reversed the impact of HCP5 down-regulation on cell proliferation, apoptosis, and metastasis in OS. Ephrin receptor 7 (EPHA7) was proved to be a target of miR-101 and had ability to recover the effects of miR-101 inhibitor in OS. In conclusion, lncRNA HCP5 knockdown suppressed cell proliferation, migration, and invasion, and induced apoptosis through depleting the expression of EPHA7 by binding to miR-101, providing a potential therapeutic strategy of HCP5 in OS.

Insights into cancer genetics can lead to therapeutic opportunities. By cross-referencing chromosomal changes with an unbiased genetic screen we identify the ephrin receptor A7 (EPHA7) as a tumor suppressor in follicular lymphoma (FL). EPHA7 is a target of 6q deletions and inactivated in 72% of FLs. Knockdown of EPHA7 drives lymphoma development in a murine FL model. In analogy to its physiological function in brain development, a soluble splice variant of EPHA7 (EPHA7(TR)) interferes with another Eph-receptor and blocks oncogenic signals in lymphoma cells. Consistent with this drug-like activity, administration of the purified EPHA7(TR) protein produces antitumor effects against xenografted human lymphomas. Further, by fusing EPHA7(TR) to the anti-CD20 antibody (rituximab) we can directly target this tumor suppressor to lymphomas in vivo. Our study attests to the power of combining descriptive tumor genomics with functional screens and reveals EPHA7(TR) as tumor suppressor with immediate therapeutic potential.

The role of secreted molecules in cellular reprogramming has been poorly understood. Here we identify a truncated form of ephrin receptor A7 (EPHA7) as a key regulator of reprogramming. Truncated EPHA7 is prominently upregulated and secreted during reprogramming. EPHA7 expression is directly regulated by OCT3/4. EphA7 knockdown results in marked reduction of reprogramming efficiency, and the addition of truncated EPHA7 is able to restore it. ERK activity is markedly reduced during reprogramming, and the secreted, truncated EPHA7 is responsible for ERK activity reduction. Remarkably, treatment of EphA7-knockdown MEFs with the ERK pathway inhibitor restores reprogramming efficiency. Analyses show that truncated EPHA7-induced ERK activity reduction plays an important role in the middle phase of reprogramming. Thus, our findings uncover the importance of secreted EPHA7-induced ERK activity reduction in reprogramming.

The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders. Therefore, the aim of our study was to generate potent and selective Nanobodies against the ligand-binding domain of the human EphA4 receptor. We identified two Nanobodies, Nb 39 and Nb 53, that bind EphA4 with affinities in the nanomolar range. These Nanobodies were most selective for EphA4, with residual binding to EphA7 only. Using Alphascreen technology, we found that both Nanobodies displaced all known EphA4-binding ephrins from the receptor. Furthermore, Nb 39 and Nb 53 inhibited ephrin-induced phosphorylation of the EphA4 protein in a cell-based assay. Finally, in a cortical neuron primary culture, both Nanobodies were able to inhibit endogenous EphA4-mediated growth-cone collapse induced by ephrin-B3. Our results demonstrate the potential of Nanobodies to target the ligand-binding domain of EphA4. These Nanobodies may deserve further evaluation as potential therapeutics in disorders in which EphA4-mediated signaling plays a role.

The striatum integrates limbic and neocortical inputs to regulate sensorimotor and psychomotor behaviors. This function is dependent on the segregation of striatal projection neurons into anatomical and functional components, such as the striosome and matrix compartments. In the present study the association of ephrin-A cell surface ligands and EphA receptor tyrosine kinases (RTKs) with the organization of these compartments was determined in postnatal rats. Ephrin-A1 and ephrin-A4 selectively bind to EphA receptors on neurons restricted to the matrix compartment. Binding is absent from the striosomes, which were identified by mu-opioid receptor immunostaining. In contrast, ephrin-A2, ephrin-A3, and ephrin-A5 exhibit a different mosaic binding pattern that appears to define a subset of matrix neurons. In situ hybridization for EphA RTKs reveals that the two different ligand binding patterns strictly match the mRNA expression patterns of EphA4 and EphA7. Ligand-receptor binding assays indicate that ephrin-A1 and ephrin-A4 selectively bind EphA4 but not EphA7 in the lysates of striatal tissue. Conversely, ephrin-A2, ephrin-A3, and ephrin-A5 bind EphA7 but not EphA4. These observations implicate selective interactions between ephrin-A molecules and EphA RTKs as potential mechanisms for regulating the compartmental organization of the striatum.

Much cell-to-cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we apply three complementary methods to map the molecular context and substrate profiles of RTKs. We use affinity purification coupled to mass spectrometry (AP-MS) to characterize stable binding partners and RTK-protein complexes, proximity-dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also use kinase-deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well-studied RTKs, offers insights into the functions of less well-studied RTKs, and highlights RTK-RTK interactions and shared signaling pathways.

We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role in vitro, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only MYC, DNAH5, CSFR1, EPHA7, ARID1B, and KMT2C preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of MYC, KMT2C, and EPHA7 best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25 (p<0.0001), and early relapse during the adjuvant hormonal treatment. The presence of the D348N mutation in KMT2C and/or the T666I mutation in the kinase domain of EPHA7 conferred hormonal resistance in vitro. Novel inactivating mutations in KMT2C and EPHA7, which confer hormonal resistance, are linked to adverse clinical course in HRPBC.

ASD-associated genes are enriched for synaptic proteins and epigenetic regulators. How those chromatin modulators establish ASD traits have remained unknown. We find haploinsufficiency of Ash1l causally induces anxiety and autistic-like behavior, including repetitive behavior, and alters social behavior. Specific depletion of Ash1l in forebrain induces similar ASD-associated behavioral defects. While the learning ability remains intact, the discrimination ability of Ash1l mutant mice is reduced. Mechanistically, deletion of Ash1l in neurons induces excessive synapses due to the synapse pruning deficits, especially during the post-learning period. Dysregulation of synaptic genes is detected in Ash1l mutant brain. Specifically, Eph receptor A7 is downregulated in Ash1l+/- mice through accumulating EZH2-mediated H3K27me3 in its gene body. Importantly, increasing activation of EphA7 in Ash1l+/- mice by supplying its ligand, ephrin-A5, strongly promotes synapse pruning and rescues discrimination deficits. Our results suggest that Ash1l haploinsufficiency is a highly penetrant risk factor for ASD, resulting from synapse pruning deficits.