Eph Receptors and Their Ephrin Ligands in Neural Plasticity

Robert Gerlai Abstract

Eph receptor tyrosine kinases are largely known for their involvement in brain development. But, as these receptors are also expressed in the adult, their possible role in the mature nervous system has begun to be explored. Emerging evidence for the involvement of Eph receptors in synaptic plasticity, learning and memory is discussed in this chapter. It is forecast that the actions of Eph receptors in the adult brain will attract significant attention, and research into their roles will have relevance for the human clinic, particularly in the area of CNS disorders associated with abnormal neural plasticity and memory loss.

Introduction

Tyrosine kinases including the receptors of neurotrophic factors such as NGF (Nerve Growth Factor), BDNF (Brain Derived Neurotrophic Factor) and neurotrophins NT3 and NT/4-5, have enjoyed considerable attention because of their newly discovered roles in neural plasticity and learning and memory. These proteins that were previously thought to function exclusively in brain development, now are known to be key players in synaptic processes thought to underlie LTP and memory formation.62-67 Some argue now that the development of the brain and the development of the memory trace are not fundamentally different in terms of underlying molecular mechanisms.

The focus of the present review is the newest and largest receptor tyrosine kinase family, the Eph tyrosine kinases. Interestingly, the history of research into the function of these kinases is fairly similar to that of the "traditional" nerurotrophic factors and their receptors. The initial functional characterization of Eph tyrosine kinase receptors was also focussed on brain development. Eph receptors were found to mediate the establishment of topographic connections and migration of neuronal cells during ontogenesis. The fact that Eph receptors are expressed in the adult brain escaped attention for several years after the discovery of these receptors. Recently, however, such expression has been clearly demonstrated and the question regarding the possible role these receptors may play in the adult central nervous system has been raised. Here I review the emerging evidence for Eph receptor involvement in neural plasticity, and argue that the actions of Eph kinases in the adult brain will attract much attention and will become a prolific research area, perhaps even more so than in the case of neurotrophins and their tyrosine kinase receptors.

The Promiscuous Family of Eph Receptors

Eph receptors form the largest family of tyrosine kinase receptors with highly conserved amino acid sequence and perhaps function across vertebrate species (for most recent reviews

From Messengers to Molecules: Memories Are Made of These, edited by Gernot Riedel and Bettina Platt. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishers.

see refs. 1, 2). Their ligands, the ephrins, are also a highly abundant class of molecules.1'2 Two main classes of Eph receptors are differentiated, A and B. This classification is based on the homology of the extracellular domains of the receptors and on their ligand preference.3,4 EphA receptors bind ephrinA ligands and EphB receptors bind ephrinB ligands. The ephrin ligands, similarly to their receptors, are characterized by higher sequence homology within a class. The A and B classes of ephrins are also different in the way these ligands are attached to the cell membrane. EphrinA ligands are glycosylphosphatidylinositol (GPI) anchored. EphrinB ligands, however, span the cell membrane as they possess a transmembrane and a cytoplasmic domain. Importantly, the ephrin ligand must be membrane bound in order for it to activate its receptor. Soluble ephrin extracellular domains are inhibitory as they bind to the Eph receptors but are unable to initiate dimerization and autophosphorylation of the receptor. Artificial aggregation of soluble ligands mimics the endogenous physiological conformation of the ligands and can be used to activate the Eph receptor.5 In summary, under physiological conditions receptor-ligand interaction requires cell-cell contact.6

The majority of studies investigating the function of Eph receptors has been largely limited to exploring the developmental role of these receptors.7 Interestingly, however, recently both the receptors and their ligands were found to be expressed in the mature mammalian brain (see e.g., ref. 9 and references therein). This has raised the intriguing possibility that Eph receptors have a role beyond development. Here the first pieces of evidence supporting a role for Eph kinases in the adult nervous system is reviewed. The discussion will be focused on the involvement of Eph receptors in synaptic plasticity and learning and memory. The possible mechanisms of their action will also be outlined.

Eph Receptors Are in the Right Places and at the Right Time

The expression of Eph receptors has been thoroughly investigated in the developing brain. It has been found to be complex, temporally controlled, and tissue specific. Recently, however, continued expression in the adult CNS has been demonstrated by in situ hybridization and immunohistochemical analysis. For example, a strong signal for EphA5, a member of the Eph tyrosine kinase family, was found in all hippocampal neuronal fields, in the cortex, and in the amygdala of the adult rat brain.8 The results were confirmed in two inbred strains of mice (C57BL/6 and DBA/2) by in situ hybridization.8 Strong EphA5 mRNA expression was observed in the hippocampus, and a milder but still clearly detectable message was seen in the cortex, the amygdala, the thalamus and the hypothalamus.9 The presence of EphA5 protein was also revealed.9 It was found in hippocampal tissue in a phosphorylated form, which implies that the Eph kinase was present in an activated form in the adult mouse brain. EphrinA5, a ligand of the EphA5 receptor, was not detected by in situ hybridization in mice.9 Nevertheless, a more sensitive technique, quantitative real time RT-PCR demonstrated the presence of mRNA of this and other ephrin ligands including ephrinA2.9 Other studies using immunostaining revealed the presence of EphA3 and EphA4 receptors and the ephrinA2 ligand in both the adult rat and mouse brains10,11 Clearly, these findings imply a possible functional role for the Eph receptors and their ligands in the adult brain.

The mere presence of these receptors and their ligands in adult brain tissue does not allow one to speculate what role these molecules may play there. However, analysis of their microstructural localization may offer some clues. Eph receptors and ephrinB ligands were found to co-localize with PDZ binding proteins in subcellular fractions (crude synaptosomes, and pre-and post-synaptic membranes) of adult rat cortex, indicating that these molecules may be present at synapses in vivo.12 Moreover, immunohistochemical double labeling for synaptophysin and for Eph receptors or ephrinB ligands has confirmed synaptic localization of these proteins in hippocampal neuronal cultures. 2 Based on these observations a potential role for Eph kinases in the physiology of the synapse has been suggested,12 an idea that has gained considerable support by the results of in vivo and ex vivo analyses of the function of Eph receptors.

Eph Receptors: "New" Players in the Adult Brain

Perhaps the first indication that Eph receptors may function in the adult brain came from a study in which kainate induced excitotoxicity and its effects on Eph gene expression were studied.13 Kainate injection was found to induce the expression of Eph tyrosine kinases, namely EphA4, EphB2 and EphA5. Quantification of the expression levels of these receptors showed significant temporal changes. The results suggested that Eph receptors/ligands might function in neuronal pathfinding after sprouting subsequent to neuronal denervation in the adult, potentially implicating these receptors in such human brain diseases as epilepsy or spinal cord injury. 4 For instance, upon spinal cord injury EphB3 was found to be overexpressed in a rat model of contusive spinal cord trauma suggesting that EphB3 may contribute to the unfavorable environment for axonal regeneration. 8 In another study, ephrinA5 was found to be involved in selective inhibition of spinal cord neurite outgrowth and cell survival14 again suggesting that Eph receptors significantly impair regeneration after injury in the adult CNS. Another interesting recent finding relevant for adult brain injury and repair concerns the expression of EphB1-3 and EphA4 receptors and their ephrinB ligands in the subventricular zone (SVZ) of the lateral ventricles in the adult mammalian brain.69 SVZ, the largest remaining germinal zone of the adult brain contains neuroblast cells migrating rostrally to the olfactory bulb. The Eph receptors were demonstrated to mediate the migration and proliferation of these cells69 raising the intriguing possibility that modulation of Eph receptor function may allow one to develop therapeutic applications by influencing neurogenesis in the adult brain. Finally, in a recent study, investigators using a kindling model found that activation or deactivation of Eph receptors can alter the development of behavioral seizures and change both the extent and the pattern of mossy fiber sprouting.70 In summary, it appears that Eph receptors are involved in processes following injury to the adult brain. But what do they do in the normal brain?

Function of Eph Receptors in the Normal Brain: Role in Plasticity and Memory

The above question has been difficult to address because of the scarcity of good molecular tools with which one can manipulate Eph function. Specific pharmacological agents are not available for Eph tyrosine kinases. Antisense oligonucleotide knock down approaches have not been attempted. Gene targeting, although successfully employed with a number of Eph receptors and their ligands, has had limited use for the analysis of adult neural function because disruption of a single gene encoding a particular receptor or ligand could be compensated for by the presence of sister molecules. That is, functional redundancy made it difficult for the investigators to analyze the disruption of single members of this large protein family. Another complication in these studies is that these receptors and ligands are involved in CNS development. Thus if their disruption by gene targeting is not compensated for, the effects almost certainly will manifest as significant developmental abnormalities which would make the analysis of their adult neural function complicated. Perhaps, an inducible and cell type restricted knock out approach could adequately address the confounding effects of developmental alterations. But such an approach has not been attempted for these kinases. Furthermore, because of the high redundancy in the Eph family (overlapping expression and high homology between sister receptors or ligands), significant compensation may be expected if a single gene encoding one Eph receptor or ephrin ligand is mutated15 thus double, triple, quadruple, etc. knock outs may be needed. Ultimately, creating all permutations of absence vs. presence of the normal form of certain members of this family may be required, clearly a daunting task that could take decades of experimentation. To solve the above problems an alternative molecular tool, the immunoadhesins16 was utilized.

The immunoadhesins (Fig. 1) employed in the functional analysis of EphA receptors8,9 were comprised of the ligand-binding domain of the EphA5 receptor (EphA5-IgG) or the receptor-binding domain of the ephrin-A5 ligand (ephrinA5-IgG). These immunoadhesins had opposing effects. EphA5-IgG scavenged the endogenous ligand and acted as an antagonist,

Figure 1. Immunoadhesins in the functional characterization of Eph receptors. Immunoadhesins (A) are genetically engineered proteins that consist of the Fc portion of an IgG molecule attached to a cell-surface protein (for review see 16). Immunoadhesins are disulfide-linked homodimers structurally similar to antibodies. They contain an adhesin region derived from a receptor or cell-surface ligand (triangles), the hinge region (white rectangles) and the Fc portion (black rectangles). Immunoadhesins bind to their target (B) with high affinity and specificity because the binding capacity of their adhesin domain is identical to that of the receptor or ligand of interest. For example, the receptor immunoadhesin EphA5—IgG (panel B left side) binds to ephrinA ligands anchored to the cell surface. By scavenging the ligands, it acts as a competitive antagonist of EphA function. The ligand immunoadhesin ephrinA5—IgG (panel B right side) Fc domain, black; receptor-binding domain of ligand attached to the Fc, "claw" shape) binds to EphA receptors (triangle and elliptic shape) and elicits receptor dimerization, which leads to receptor activation and intracellular signaling (but see below).

It is important to stress that these immunoadhesins recognize the ligand or the receptor on the basis of the high-affinity ligand-receptor interaction.16'17 Immunoadhesins therefore may obviate the lack of EphA selective pharmacological agents and, as a result of the unaltered binding sites, immunoadhesins are capable of binding all the relevant proteins that the endogenous Eph receptor and the ephrins would bind. As Eph receptors are promiscuous and interact with several ephrin ligands,3 immunoadhesins allow the manipulation of all functionally relevant ligands and receptors without the confounding effects of compensation by related molecules, as occurs in gene targeting experiments.155958

Several caveats must also be mentioned, however. First, the ability of immunoadhesins to act as agonists may depend on the experimental conditions and the particular target receptor the immunoadhesin is supposed to bind. Eliciting receptor dimerization may require cross linking several immunoadhesins, i.e., the creation of immunoadhesin multimers.16 Second, even the monomer is large enough not to be able to cross the blood brain barrier. Thus the in vivo delivery of the immunoadhesin requires time consuming, delicate, and invasive stereotaxic brain surgery. Third, the immunoadhesin solution may contain endotoxin, a bacterial lipoprotein-polysaccharide complex that may have significant toxic effects in the brain. Fourth, the immunoadhesin, as a foreign protein, may elicit an immune response. Despite these caveats that can complicate the interpretation of immunoadhesin effects, immunoadhesins have been successfully used in the functional analysis of neurotrophic factors and their tyrosine kinase receptors as well as ephrins and their Eph receptors (for a recent review and methods see refs. 60, 61). Figure modified from ref. 60.

whereas ephrinA5-IgG worked as an EphA agonist by dimerizing and initiating the autophosphorylation cycle of the receptor.6'17

Acute administration of EphA5-IgG, the EphA antagonist, resulted in EphA receptor deactivation leading to a significant impairment in long-term potentiation (LTP) in rat hippocam-pal slices.8 Conversely, the agonist immunoadhesin, ephrinA5-IgG, led to synaptic potentiation resembling LTP. These results provided the first direct evidence demonstrating that Eph tyrosine kinases participate in synaptic plasticity in vitro.

The question whether similar effects may be seen in vivo has also been addressed.9'18 In these studies, the synaptoplastic and behavioral effects of in vivo chronic (7 day long) bilateral intrahippocampal immunoadhesin infusion were investigated. Although the induction of LTP was found normal in hippocampal slices of C57BL/6 mice previously infused with EphA5-IgG, the potentiated response was shown to decay faster when compared to control slices. The synaptoplastic changes correlated with behavioral alterations. Mice that received bilateral intrahippocampal infusion of EphA5-IgG for a week exhibited impaired T-maze spontaneous alternation (Figs. 2 and 3) as well as disrupted context-dependent fear conditioning performance (Figs. 4 and 5.), behavioral aberrations indicative of hippocampal abnormalities.19'20'21 Thus, inhibition of EphA activity impaired neuronal plasticity, which manifested both in elec-trophysiological as well as behavioral tests. A potential concern could be that the impairment was due to non-specific effects but perhaps general impairment of health or brain function. However, the effects of ephrinA5-IgG induced Eph activation could not be explained by a non-specific action of this immunoadhesin. When infused into the hippocampus of DBA/2

Spontaneous Alternation Maze

Figure 2. The T-maze Continuous Alternation Task (T-CAT). Mice are allowed to alternate between the left and right arms of the T-maze throughout a 15-trial session. Once they have entered a particular arm, a guillotine door is lowered to block entry to the opposite arm (checkered area). The door is removed only after the mice have returned to the start arm, allowing a new alternation trial to be started. Alternation rate is calculated as the ratio between alternating choices and total number of choices (50%, random choice; 100%, alternation at every trial; 0%, no alternation). Time to complete 15 choices is recorded. In addition, several motor and posture patterns are also measured (not shown).

Figure 2. The T-maze Continuous Alternation Task (T-CAT). Mice are allowed to alternate between the left and right arms of the T-maze throughout a 15-trial session. Once they have entered a particular arm, a guillotine door is lowered to block entry to the opposite arm (checkered area). The door is removed only after the mice have returned to the start arm, allowing a new alternation trial to be started. Alternation rate is calculated as the ratio between alternating choices and total number of choices (50%, random choice; 100%, alternation at every trial; 0%, no alternation). Time to complete 15 choices is recorded. In addition, several motor and posture patterns are also measured (not shown).

Figure 3. EphA receptors mediate spontaneous alternation performance in the T-maze. Infusion of EphA5-IgG impairs alternation performance in C57BL/6 mice (A) while ephrinA5-IgG improves alternation performance in DBA/2 mice (C) in the T-maze spontaneous alternation task. The changes are not related to task completion time (B, D) indicating unaltered motor performance or motivation. Mean + standard error are shown. Sample sizes (n) are also indicated.

Figure 3. EphA receptors mediate spontaneous alternation performance in the T-maze. Infusion of EphA5-IgG impairs alternation performance in C57BL/6 mice (A) while ephrinA5-IgG improves alternation performance in DBA/2 mice (C) in the T-maze spontaneous alternation task. The changes are not related to task completion time (B, D) indicating unaltered motor performance or motivation. Mean + standard error are shown. Sample sizes (n) are also indicated.

mice, a strain with impaired hippoca mpal function,21,22,23,24 ephrinA5-IgG led to significantly improved LTP and this improvement correlated with superior performance in both the T-maze alternation task and the context dependent fear conditioning test as compared to control. These results were replicated in another strain (C57BL/6) of mice with the use of modified stimulation and testing protocols9 suggesting that the findings are robust and not unique to a particular inbred mouse strain. Lastly, the involvement of Eph receptors in consolidation of memory has also been demonstrated18 in a ketamine anesthesia induced retrograde amnesia model. In this work, ephrinA5-IgG, infused after ketamine induced disruption of memory consolidation, significantly improved cognitive performance in a hippocampus dependent manner (Fig. 6). In conclusion, the electrophysiological and behavioral observations obtained support a role for Eph receptors in neural plasticity in the adult mammalian brain.

Figure 4. The fear conditioning paradigm. The paradigm has three phases: a training phase (A), a context dependent test (B), and a cue dependent test (C). For training, mice receive 3 electric foot shocks (1 sec, 0.7 mA, indicated by the thick black bars on the bottom of the cage) each preceded by an 80 dB, 2900 Hz, 20 sec long tone cue (indicated by the black filled circle on the wall). The context test is performed in the training chamber but no shock (thin bars) or tone (empty circle) is delivered. The cue test is carried out in another chamber identical in size but different in visual, olfactory, and tactile cues from those of the training chamber. Tone signals identical to the one used in training are given (black filled circle) but no shock (thin bars) is delivered. Behavior is video-recorded and later quantified using event recording computer programs. Behavior elements correlated with fear, primarily freezing, are measured. The timing of stimulus delivery in each phase of the paradigm is also shown: solid black bars represent the tone, the arrows the shock, and the gray shading the different context.

Figure 4. The fear conditioning paradigm. The paradigm has three phases: a training phase (A), a context dependent test (B), and a cue dependent test (C). For training, mice receive 3 electric foot shocks (1 sec, 0.7 mA, indicated by the thick black bars on the bottom of the cage) each preceded by an 80 dB, 2900 Hz, 20 sec long tone cue (indicated by the black filled circle on the wall). The context test is performed in the training chamber but no shock (thin bars) or tone (empty circle) is delivered. The cue test is carried out in another chamber identical in size but different in visual, olfactory, and tactile cues from those of the training chamber. Tone signals identical to the one used in training are given (black filled circle) but no shock (thin bars) is delivered. Behavior is video-recorded and later quantified using event recording computer programs. Behavior elements correlated with fear, primarily freezing, are measured. The timing of stimulus delivery in each phase of the paradigm is also shown: solid black bars represent the tone, the arrows the shock, and the gray shading the different context.

Mechanisms Mediating Eph Action: The First Working Hypotheses

Admittedly, the potential neurobiological mechanisms underlying the observed behavioral and electrophysiological effects are speculative at this point. The findings obtained so far, however, have led to the emergence of working hypotheses that may be tested in future mechanistic studies. The recent observation showing that Eph receptors and ephrinB ligands contain PDZ recognition motifs and are bound and clustered by PDZ proteins at pre- and postsynaptic sites of neuronal synapses in vitro suggests that Eph receptors are properly positioned to mediate synaptic plasticity.12,25 Moreover, as Eph receptor and ephrin ligand binding interaction requires cell-cell contact (both the ligand and the receptor are membrane bound), Eph receptor mediated signaling can be achieved in a highly localized manner, a crucial prerequisite in the

Figure 5. EphA receptors mediate cognitive performance in a context dependent manner in fear conditioning. The performance of EphA5-IgG infused C57BL/6 mice was significantly impaired compared to control (CD1-IgG infused mice) in the context test (B) but not in other phases of the paradigm (A training, C cue test). The performance of ephrinA5-IgG infused DBA/2 mice after fear-conditioning was significantly improved (increased freezing) compared to the control animals in a context-dependent manner (D training, E context test, F cue test). Note that both the context and the cued tests were carried out 24 hours after the fear conditioning. Mean + standard error are shown. Sample sizes (n) are also indicated. Thin solid lines represent the delivery of tone and the arrows the shocks. (Modified from ref. 9)

Figure 5. EphA receptors mediate cognitive performance in a context dependent manner in fear conditioning. The performance of EphA5-IgG infused C57BL/6 mice was significantly impaired compared to control (CD1-IgG infused mice) in the context test (B) but not in other phases of the paradigm (A training, C cue test). The performance of ephrinA5-IgG infused DBA/2 mice after fear-conditioning was significantly improved (increased freezing) compared to the control animals in a context-dependent manner (D training, E context test, F cue test). Note that both the context and the cued tests were carried out 24 hours after the fear conditioning. Mean + standard error are shown. Sample sizes (n) are also indicated. Thin solid lines represent the delivery of tone and the arrows the shocks. (Modified from ref. 9)

Figure 6. EphA receptors are involved in consolidation of memory. The performance of C57BL/6 mice were significantly disrupted by surgical anesthesia (ketamine) delivered 90 min after completion of training (A). The retrograde amnesia is robust in the context test (B), and almost completely absent in the cue test (C). EphrinA5-IgG infusion significantly ameliorates surgical anesthesia induced retrograde amnesia (D training, E context test, F cue test) in C57BL/6 mice. Mean + standard error are shown. Sample sizes (n) are also indicated. Thin solid lines represent the delivery of tone and the arrows the shocks. (Modified from).18

Figure 6. EphA receptors are involved in consolidation of memory. The performance of C57BL/6 mice were significantly disrupted by surgical anesthesia (ketamine) delivered 90 min after completion of training (A). The retrograde amnesia is robust in the context test (B), and almost completely absent in the cue test (C). EphrinA5-IgG infusion significantly ameliorates surgical anesthesia induced retrograde amnesia (D training, E context test, F cue test) in C57BL/6 mice. Mean + standard error are shown. Sample sizes (n) are also indicated. Thin solid lines represent the delivery of tone and the arrows the shocks. (Modified from).18

activation/deactivation of single synapses essential for proper stimulus processing. Eph receptors may interact with a number of proteins through their PDZ binding domains that mediate cytoskeletal processes12 and thus potentially affect a range of subcellular mechanisms influencing synaptic transmission and/or plasticity. Such mechanisms may include, for example, the trafficking and docking of presynaptic vesicles,26 the clustering of neurotransmitter receptors, e.g., AMPA-R and NMDA-R,27 and the formation of "perforated" synapses associated with LTP28,29,30 and perhaps with memory formation. Interestingly, a member of the Eph family, the EphA5 receptor, has been shown to mediate actin polymerization, and its activation by administration of ephrinA5-IgG leads to actin depolymerization and axonal growth cone collapse in neuronal cell cultures and cortical explants.6 Depolymerization of actin, a component of the scaffolding of the synapse, may allow the synapse to undergo plastic structural modification. Indeed, actin has been found to be a crucial component of the cytoskeleton present in presynaptic as well as postsynaptic terminals31,32,33 and has been shown to be associated with structural changes underlying synaptic plasticity34,31,35,32 affecting both presynaptic and postsynapric mechanisms including paired pulse facilitation, and LTP36 Remarkably, it has been demonstrated that application of the EphA agonist ephrinA5-IgG, which destabilizes actin filaments6 improves LTP. Therefore, the assumption that EphA receptor activation mobilizes the synapse by destabilizing actin filaments thus allowing the synapse to undergo structural modifications necessary for plastic changes to take place is not far fetched. Perhaps this hypothesis may be tested by detailed electron- or confocal microscopy analyses coupled with electrophysiological manipulation and monitoring of the synapse.

The possibility that Eph receptors play roles in cytostructural processes is consistent with the changes that were observed in the expression of the tubulin and MAP2 (microtubule associated protein 2) genes in response to EphA5-IgG or ephrinA5-IgG treatment.9 Tubulin and MAP2 were overexpressed as a result of EphA receptor inactivation and were underexpressed due to receptor activation in the adult mouse hippocampus. First, these findings are compatible with the known arresting effects of ephrinA ligands on axonal and dendritic growth during CNS development.17,6,15 Second, they are also consistent with the suggested cytostructural role of the Eph receptors in neural plasticity: removal of the structural components tubulin and MAP2 may be a prerequisite of plastic changes of the synapse. In the adult brain, where major developmental alterations do not take place, transcriptional regulation of tubulin, and perhaps other genes of cytoskeletal proteins, may subserve the development of new or altered synaptic connections, i.e., neural plasticity as previously assumed.37,3 ,39

Although the above hypotheses are plausible, they are not the only possible ones. Eph receptors may also influence synaptic mechanisms via mediating adhesion processes. For example, phosphorylation of L1, a transmembrane adhesion molecule, was demonstrated following EphB2 activation,40 and disruption of L1 function by anti-L1 antibody application was shown to impair synaptic plasticity.41 EphA receptor induced signaling via ephrinA ligands (e.g., ephrinA5) should also be mentioned here as it was shown to increase the attachment of neuronal cells to the extracellular matrix,42 a process that may influence synaptic plasticity.43 Furthermore, Eph receptors contain a cytoplasmic sequence motif, YEPD, that mediates binding src non-receptor tyrosine kinases, including src and fyn.44 fyn is involved in the phosphorylation of NMDA-R,45 a key player in LTP,46 and fyn null mutant mice exhibit impaired spatial learning and blunted hippocampal LTP.47 src also modulates NMDA-R function48 and plays a crucial role in LTP.49 LTP, and NMDA-R itself, has been implicated in acquisition and consolidation of memory .50,46,51,52,19,53,54,55 Thus, src kinase mediated synaptic plasticity may be a potential substrate of Eph action. Lastly, EphB receptors have been shown to directly interact with NMDA receptors, a process that may influence synapse formation and function.56

Involvement of Eph receptors in adult neural plasticity implies that Eph receptor function must be modulated in a precise time and location specific manner. At this point, however, it is unclear how this is achieved. Ephrin ligands, compared to their receptors, are expressed at low levels in the adult brain9 implying that perhaps a considerable proportion of Eph receptors is not activated under basal conditions. It is plausible that localized induction of expression of the ligands is the primary process that leads to receptor activation at the appropriate synaptic sites, however, this has not been investigated. Perhaps sensitive single cell PCR techniques or expression profiling using gene arrays will be able to address this question. It is also possible that proper clustering of the GPI anchored membrane bound or transmembrane ephrin ligands underlies receptor activation, as at least two ligand molecules need to be in close proximity to induce receptor dimerization and initiate the autophosphorylation process.16 Although no direct evidence has been obtained to confirm the validity of this suggestion, ephrinA5 ligands have been found in specialized membrane rafts, called caveolae, which perhaps facilitate clustering of EphA receptors42 and eprhinB ligands.12 Activity dependent induction of EphA and EphB receptors (e.g., EphA4, EphA5, EphB2) at the mRNA level has been demonstrated in the hippocampus13 suggesting that transcriptional regulation of the receptors may be possible. Alternatively, or additionally, modulation of Eph receptor signaling may be achieved through the tyrosine phosphorylation sites identified at the juxtamembrane, SAP, and kinase domains of the Eph receptor (reviewed in refs. 1, 2). But again, the molecular components involved in such processes are not well understood. Similarly, the downstream elements of Eph signalling are not yet elucidated. Nevertheless, based on the binding domains identified on the Eph receptor, downstream molecular interactions could involve numerous signaling pathways acting through src family cytoplasmic tyrosine kinases, the RasGAP pathway, the LMW-PTP phophotyrosine phosphatase, PI3 kinase, the Grb2, Grb10 and SLAP adaptor proteins, and several PDZ domain containing proteins including GRIP (reviewed in refs. 1, 2). Finally, signal transduction via ephrin ligands must also be mentioned. EphrinB ligands possess a cytoplasmic domain and have been clearly shown to transduce signals (reviewed in ref. 57) and ephrinA ligands (ephrinA5), as already mentioned, may also be involved in signal transduction (for review see refs. 1, 2).

Concluding Remarks

The molecular cascade of events in which Eph receptors are involved, including both the upstream and downstream elements, are far from understood. The potential neurobiological mechanisms associated with Eph action are also highly speculative. Nevertheless, the gross anatomical localization of Eph receptors and ephrin ligands in the adult brain, and the localization of some of these proteins at the synapse, suggest that this receptor system is involved not only in development of the brain but also in adult neural function. This conclusion is now supported by the findings demonstrating that significant changes occur in synaptic plasticity following acute or chronic modulation of Eph function in hippocampal slices and that significant changes are also observed in learning and memory after chronic modulation of Eph function in vivo.

This is a promising start by all means, but much needs to be done before the exact role of Eph receptors in adult neural function can be understood. Characterization of the signaling pathways upstream and downstream of the Eph receptor will be a complex task given the multitude of potential molecular interactions in which these receptors and ligands participate. It is also not clear whether different members of the Eph receptor tyrosine kinase family have spatially and/or temporally distinct roles in the adult brain. Inducible and cell type restricted gene targeting or the use of immunoadhesins and perhaps novel small molecules, specific pharmacological tools to be developed for particular Eph receptors, will advance our understanding of the actions of the Eph receptors. Ultimately, these techniques will enable us to address the intriguing question whether the development of our brain and the development of our memories share common molecular mechanisms.

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