Identification of new EphA4 inhibitors by virtual screening of FDA-approved drugs.

The receptor tyrosine kinase, erythropoietin-producing hepatocellular A4 (EphA4), was recently identified as a molecular target for Alzheimer's disease (AD). We found that blockade of the interaction of the receptor and its ligands, ephrins, alleviates the disease phenotype in an AD transgenic mouse model, suggesting that targeting EphA4 is a potential approach for developing AD interventions. In this study, we identified five FDA-approved drugs-ergoloid, cyproheptadine, nilotinib, abiraterone, and retapamulin-as potential inhibitors of EphA4 by using an integrated approach combining virtual screening with biochemical and cellular assays. We initially screened a database of FDA-approved drugs using molecular docking against the ligand-binding domain of EphA4. Then, we selected 22 candidate drugs and examined their inhibitory activity towards EphA4. Among them, five drugs inhibited EphA4 clustering induced by ephrin-A in cultured primary neurons. Specifically, nilotinib, a kinase inhibitor, inhibited the binding of EphA4 and ephrin-A at micromolar scale in a dosage-dependent manner. Furthermore, nilotinib inhibited the activation of EphA4 and EphA4-dependent growth cone collapse in cultured hippocampal neurons, demonstrating that the drug exhibits EphA4 inhibitory activity in cellular context. As demonstrated in our combined computational and experimental approaches, repurposing of FDA-approved drugs to inhibit EphA4 may provide an alternative fast-track approach for identifying and developing new treatments for AD.

Thymol has antifungal activity against Candida albicans during infection and maintains the innate immune response required for function of the p38 MAPK signaling pathway in Caenorhabditis elegans.

The Caenorhabditis elegans model can be used to study Candida albicans virulence and host immunity, as well as to identify plant-derived natural products to use against C. albicans. Thymol is a hydrophobic phenol compound from the aromatic plant thyme. In this study, the in vitro data demonstrated concentration-dependent thymol inhibition of both C. albicans growth and biofilm formation during different developmental phases. With the aid of the C. elegans system, we performed in vivo assays, and our results further showed the ability of thymol to increase C. elegans life span during infection, inhibit C. albicans colony formation in the C. elegans intestine, and increase the expression levels of host antimicrobial genes. Moreover, among the genes that encode the p38 MAPK signaling pathway, mutation of the pmk-1 or sek-1 gene decreased the beneficial effects of thymol's antifungal activity against C. albicans and thymol's maintenance of the innate immune response in nematodes. Western blot data showed the level of phosphorylation of pmk-1 was dramatically decreased against C. albicans. In nematodes, treatment with thymol recovered the dysregulation of pmk-1 and sek-1 gene expressions, the phosphorylation level of PMK-1 caused by C. albicans infection. Therefore, thymol may act, at least in part, through the function of the p38 MAPK signaling pathway to protect against C. albicans infection and maintain the host innate immune response to C. albicans. Our results indicate that the p38 MAPK signaling pathway plays a crucial role in regulating the beneficial effects observed after nematodes infected with C. albicans were treated with thymol.

EphA4 has distinct functionality from EphA7 in the corticothalamic system during mouse brain development.

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.

EphA4 is Involved in Sleep Regulation but Not in the Electrophysiological Response to Sleep Deprivation.

STUDY OBJECTIVES: Optimal sleep is ensured by the interaction of circadian and homeostatic processes. Although synaptic plasticity seems to contribute to both processes, the specific players involved are not well understood. The EphA4 tyrosine kinase receptor is a cell adhesion protein regulating synaptic plasticity. We investigated the role of EphA4 in sleep regulation using electrocorticography in mice lacking EphA4 and gene expression measurements. METHODS: EphA4 knockout (KO) mice, Clock(Δ19/Δ19) mutant mice and littermates, C57BL/6J and CD-1 mice, and Sprague-Dawley rats were studied under a 12 h light: 12 h dark cycle, under undisturbed conditions or 6 h sleep deprivation (SLD), and submitted to a 48 h electrophysiological recording and/or brain sampling at different time of day. RESULTS: EphA4 KO mice showed less rapid eye movement sleep (REMS), enhanced duration of individual bouts of wakefulness and nonrapid eye movement sleep (NREMS) during the light period, and a blunted daily rhythm of NREMS sigma activity. The NREMS delta activity response to SLD was unchanged in EphA4 KO mice. However, SLD increased EphA4 expression in the thalamic/hypothalamic region in C57BL/6J mice. We further show the presence of E-boxes in the promoter region of EphA4, a lower expression of EphA4 in Clock mutant mice, a rhythmic expression of EphA4 ligands in several brain areas, expression of EphA4 in the suprachiasmatic nuclei of the hypothalamus (SCN), and finally an unchanged number of cells expressing Vip, Grp and Avp in the SCN of EphA4 KO mice. CONCLUSIONS: Our results suggest that EphA4 is involved in circadian sleep regulation.

Development of the thalamo-dorsal ventricular ridge tract in the Chinese soft-shelled turtle, Pelodiscus sinensis.

With the exception of that from the olfactory system, the vertebrate sensory information is relayed by the dorsal thalamus (dTh) to be carried to the telencephalon via the thalamo-telencephalic tract. Although the trajectory of the tract from the dTh to the basal telencephalon seems to be highly conserved among amniotes, the axonal terminals vary in each group. In mammals, thalamic axons project onto the neocortex, whereas they project onto the dorsal pallium and the dorsal ventricular ridge (DVR) in reptiles and birds. To ascertain the evolutionary development of the thalamo-telencephalic connection in amniotes, we focused on reptiles. Using the Chinese soft-shelled turtle (Pelodiscus sinensis), we studied the developmental course of the thalamic axons projecting onto the DVR. We found, during the developmental period when the thalamo-DVR connection forms, that transcripts of axon guidance molecules, including EphA4 and Slit2, were expressed in the diencephalon, similar to the mouse embryo. These results suggest that the basic mechanisms responsible for the formation of the thalamo-telencephalic tract are shared across amniote lineages. Conversely, there was a characteristic difference in the expression patterns of Slit2, Netrin1, and EphrinA5 in the telencephalon between synapsid (mammalian) and diapsid (reptilian and avian) lineages. This indicates that changes in the expression domains of axon guidance molecules may modify the thalamic axon projection and lead to the diversity of neuronal circuits in amniotes.

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 effects of Eph-ephrin mutations on pre-pulse inhibition in mice.

Eph-ephrin signaling is known to be important in directing topographic projections in the afferent auditory pathway, including connections to various subdivisions of the inferior colliculus (IC). The acoustic startle-response (ASR) is a reliable reflexive behavioral response in mammals elicited by an unexpected intense acoustic startle-eliciting stimulus (ES). It is mediated by a sub-cortical pathway that includes the IC. The ASR amplitude can be measured with an accelerometer under the subject and can be decreased in amplitude by presenting a less intense, non-startling stimulus 5-300ms before the ES. This reflexive decrement in ASR is called pre-pulse inhibition (PPI) and indicates that the relatively soft pre-pulse was heard. PPI is a general trait among mammals. Mice have been used recently to study this response and to reveal how genetic mutations affect neural circuits and hence the ASR and PPI. In this experiment, we measured the effect of Eph-ephrin mutations using control mice (C57BL/6J), mice with compromised EphA4 signaling (EphA4(lacZ/+), EphA4(lacZ/lacZ)), and knockout ephrin-B3 mice (ephrin-B3 (+/-, -/-)). Control and EphA4(lacZ/+s)trains showed robust PPI (up to 75% decrement in ASR) to an offset of a 70dB SPL background noise at 50ms before the ES. Ephrin-B3 knockout mice and EphA4 homozygous mutants were only marginally significant in PPI (<25% decrement and <33% decrement, respectively) to the same conditions. This decrement in PPI highlights the importance of ephrin-B3 and EphA4 interactions in ordering auditory behavioral circuits. Thus, different mutations in certain members of the signaling family produce a full range of changes in PPI, from minimal to nearly maximal. This technique can be easily adapted to study other aspects of hearing in a wider range of mutations. Along with ongoing neuroanatomical studies, this allows careful quantification of how the auditory anatomical, physiological and now behavioral phenotype is affected by changes in Eph-ephrin expression and functionality.

Zic2-dependent axon midline avoidance controls the formation of major ipsilateral tracts in the CNS.

In bilaterally symmetric organisms, interhemispheric communication is essential for sensory processing and motor coordination. The mechanisms that govern axon midline crossing during development have been well studied, particularly at the spinal cord. However, the molecular program that determines axonal ipsilaterality remains poorly understood. Here, we demonstrate that ipsilateral neurons whose axons grow in close proximity to the midline, such as the ascending dorsospinal tracts and the rostromedial thalamocortical projection, avoid midline crossing because they transiently activate the transcription factor Zic2. In contrast, uncrossed neurons whose axons never approach the midline control axonal laterality by Zic2-independent mechanisms. Zic2 induces EphA4 expression in dorsospinal neurons to prevent midline crossing while Robo3 is downregulated to ensure that axons enter the dorsal tracts instead of growing ventrally. Together with previous reports, our data reveal a critical role for Zic2 as a determinant of axon midline avoidance in the CNS across species and pathways.

A network of genetic repression and derepression specifies projection fates in the developing neocortex.

Neurons within each layer in the mammalian cortex have stereotypic projections. Four genes-Fezf2, Ctip2, Tbr1, and Satb2-regulate these projection identities. These genes also interact with each other, and it is unclear how these interactions shape the final projection identity. Here we show, by generating double mutants of Fezf2, Ctip2, and Satb2, that cortical neurons deploy a complex genetic switch that uses mutual repression to produce subcortical or callosal projections. We discovered that Tbr1, EphA4, and Unc5H3 are critical downstream targets of Satb2 in callosal fate specification. This represents a unique role for Tbr1, implicated previously in specifying corticothalamic projections. We further show that Tbr1 expression is dually regulated by Satb2 and Ctip2 in layers 2-5. Finally, we show that Satb2 and Fezf2 regulate two disease-related genes, Auts2 (Autistic Susceptibility Gene2) and Bhlhb5 (mutated in Hereditary Spastic Paraplegia), providing a molecular handle to investigate circuit disorders in neurodevelopmental diseases.

Inhibition of Eph receptor-ephrin ligand interaction by tea polyphenols.

Tea contains a variety of bioactive chemicals, such as catechins and other polyphenols. These compounds are thought to be responsible for the health benefits of tea consumption by affecting the function of many cellular targets, not all of which have been identified. In a high-throughput screen for small molecule antagonists of the EphA4 receptor tyrosine kinase, we identified five tea polyphenols that substantially inhibit EphA4 binding to a synthetic peptide ligand. Further characterization of theaflavin monogallates from black tea and epigallocatechin-3,5-digallate from green tea revealed that these compounds at low micromolar concentrations also inhibit binding of the natural ephrin ligands to EphA4 and several other Eph receptors in in vitro assays. The compounds behave as competitive EphA4 antagonists, and their inhibitory activity is affected by amino acid mutations within the ephrin binding pocket of EphA4. In contrast, the major green tea catechin, epigallocatechin-3-gallate (EGCG), does not appear to be an effective Eph receptor antagonist. In cell culture assays, theaflavin monogallates and epigallocatechin-3,5-digallate inhibit ephrin-induced tyrosine phosphorylation (activation) of Eph receptors and endothelial capillary-like tube formation. However, the wider spectrum of Eph receptors affected by the tea derivatives in cells suggests additional mechanisms of inhibition besides interfering with ephrin binding. These results show that tea polyphenols derived from both black and green tea can suppress the biological activities of Eph receptors. Thus, the Eph receptor tyrosine kinase family represents an important class of targets for tea-derived phytochemicals.

Hypoxia disruption of vertebrate CNS pathfinding through ephrinB2 Is rescued by magnesium.

The mechanisms of hypoxic injury to the developing human brain are poorly understood, despite being a major cause of chronic neurodevelopmental impairments. Recent work in the invertebrate Caenorhabditis elegans has shown that hypoxia causes discrete axon pathfinding errors in certain interneurons and motorneurons. However, it is unknown whether developmental hypoxia would have similar effects in a vertebrate nervous system. We have found that developmental hypoxic injury disrupts pathfinding of forebrain neurons in zebrafish (Danio rerio), leading to errors in which commissural axons fail to cross the midline. The pathfinding defects result from activation of the hypoxia-inducible transcription factor (hif1) pathway and are mimicked by chemical inducers of the hif1 pathway or by expression of constitutively active hif1α. Further, we found that blocking transcriptional activation by hif1α helped prevent the guidance defects. We identified ephrinB2a as a target of hif1 pathway activation, showed that knock-down of ephrinB2a rescued the guidance errors, and showed that the receptor ephA4a is expressed in a pattern complementary to the misrouting axons. By targeting a constitutively active form of ephrinB2a to specific neurons, we found that ephrinB2a mediates the pathfinding errors via a reverse-signaling mechanism. Finally, magnesium sulfate, used to improve neurodevelopmental outcomes in preterm births, protects against pathfinding errors by preventing upregulation of ephrinB2a. These results demonstrate that evolutionarily conserved genetic pathways regulate connectivity changes in the CNS in response to hypoxia, and they support a potential neuroprotective role for magnesium.

Eph tyrosine kinase receptor EphA4 is required for the topographic mapping of the corticospinal tract.

Fine movement in the body is controlled by the motor cortex, which signals in a topographically specific manner to neurons in the spinal cord by means of the corticospinal tract (CST). How the correct topography of the CST is established is unknown. To investigate the possibility that the Eph tyrosine kinase receptor EphA4 is involved in this process, we have traced CST axons in mice in which the EphA4 gene has been deleted. The forelimb subpopulation of CST axons is unaffected in the EphA4-/- mice, but the hindlimb subpopulation branches too early within the cord, both temporally and spatially. EphA4 shows a dynamic expression pattern in the environment of the developing CST in the spinal cord: high at the time of forelimb branching and down-regulated before hindlimb branching. To examine whether the fore- and hindlimb subpopulations of CST axons respond differently to EphA4 in their environment, neurons from fore- and hindlimb motor cortex were cultured on a substrate containing EphA4. Neurons from the hindlimb cortex showed reduced branching on the EphA4 substrate compared with their forelimb counterparts. Neurons from the hindlimb cortex express ephrinA5, a high-affinity ligand for EphA4, at higher levels compared with forelimb cortex neurons, and this expression is down-regulated before hindlimb branching. Together, these findings suggest that EphA4 regulates topographic mapping of the CST by controlling the branching of CST axons in the spinal cord.

Hoxa2- and rhombomere-dependent development of the mouse facial somatosensory map.

In the mouse trigeminal pathway, sensory inputs from distinct facial structures, such as whiskers or lower jaw and lip, are topographically mapped onto the somatosensory cortex through relay stations in the thalamus and hindbrain. In the developing hindbrain, the mechanisms generating such maps remain elusive. We found that in the principal sensory nucleus, the whisker-related map is contributed by rhombomere 3-derived neurons, whereas the rhombomere 2-derived progeny supply the lower jaw and lip representation. Moreover, early Hoxa2 expression in neuroepithelium prevents the trigeminal nerve from ectopically projecting to the cerebellum, whereas late expression in the principal sensory nucleus promotes selective arborization of whisker-related afferents and topographic connectivity to the thalamus. Hoxa2 inactivation further results in the absence of whisker-related maps in the postnatal brain. Thus, Hoxa2- and rhombomere 3-dependent cues determine the whisker area map and are required for the assembly of the whisker-to-barrel somatosensory circuit.

EphA family gene expression in the developing mouse neocortex: regional patterns reveal intrinsic programs and extrinsic influence.

Parcellation of the mammalian cerebral cortex into distinct areas is essential for proper cortical function; however, the developmental program that results in the genesis of distinct areas is not fully understood. We examined the expression of members of the EphA family-the EphA receptor tyrosine kinases and the ephrin-A ligands-within the developing mouse cerebral cortex, with the aim of characterizing this component of the molecular landscape during cortical parcellation. We found that specific embryonic zones, such as the ventricular, subventricular, intermediate, subplate, and marginal zones, as well as the cortical plate, were positive for particular EphA genes early in corticogenesis (E12-E15). Along with this zone-selective expression, several genes (EphA3, EphA4, EphA5) were evenly expressed along the axes of the developing cortex, whereas one family member (EphA7) was expressed in a distinct anteroposterior pattern. Later in corticogenesis (E16-E18), other EphA family members became selectively expressed, but only within the cortical plate: EphA6 was present posteriorly, and ephrin-A5 was expressed within a middle region. At birth, patterning of EphA gene expression was striking. Thus, we found that the expression of a single EphA gene or a combination of family members can define distinct embryonic zones and anteroposterior regions of the neocortex during development. To examine whether cellular context affects the patterning of EphA expression, we examined gene expression in embryonic cortical cells grown in vitro, such that all cellular contacts are lacking, and in Mash-1 mutant mice, in which thalamocortical connections do not form. We found that the expression patterns of most EphA family members remained stable in these scenarios, whereas the pattern of ephrin-A5 was altered. Taken together, this work provides a comprehensive picture of EphA family expression during mouse corticogenesis and demonstrates that most EphA expression profiles are cell intrinsically based, whereas ephrin-A5 is plastically regulated.