Ingrid W. Caras

Cloning of AL-1, a ligand for an Eph-related tyrosine kinase receptor involved in axon bundle formation

REK7 is an Eph-related tyrosine kinase receptor expressed exclusively in the nervous system, predominantly in hippocampus and cortex. A soluble REK7-IgG fusion protein, produced to analyze the biological role of REK7, prevents axon bundling in cocultures of cortical neurons with astrocytes, a model of late stage nervous system development and differentiation. Using REK7-IgG as an affinity reagent, we purified and cloned a novel REK7 ligand called AL-1, a GPI-linked protein homologous to other members of an emerging ligand family. Membrane attachment of AL-1 appears necessary for receptor activation, since REK7 on cortical neurons is efficiently activated by transfected cells expressing GPI-linked AL-1, but not by soluble AL-1. Consistent with this, soluble AL-1 blocks axon bundling. Our findings, together with the observation that both molecules are expressed in the brain, suggest a role in the formation of neuronal pathways, a crucial feature of nervous system development and regeneration.

AL‐1‐induced Growth Cone Collapse of Rat Cortical Neurons is Correlated with REK7 Expression and Rearrangement of the Actin Cytoskeleton

Previous experiments identified AL‐1 as a glycosylphosphatidylinositol (GPI)‐linked ligand for the Eph‐related receptor, REK7, and showed that a REK7‐IgG fusion protein blocks axon bundling in co‐cultures of cortical neurons on astrocytes, suggesting a role for REK7 and AL‐1 in axon fasciculation. Subsequent identification of RAGS, the chick homologue of AL‐1, as a repellent axon guidance molecule in the developing chick visual system led to speculation that AL‐1, expressed on astrocytes, provides a repellent stimulus for cortical axons, inducing them to bundle as an avoidance mechanism. Using a growth cone collapse assay to test this hypothesis, we show that a soluble AL‐1‐IgG fusion protein is a potent collapsing factor for embryonic rat cortical neurons. The response is strongly correlated with REK7 expression, implicating REK7 as a receptor mediating AL‐1‐induced collapse. Morphological collapse is preceded by an AL‐1‐IgG‐induced reorganization of the actin cytoskeleton that resembles the effects of cytochalasin D. This suggests a pathway whereby REK7 activation by AL‐1 leads to perturbation of the actin cytoskeleton, possibly by an effect on actin polymerization, followed by growth cone collapse. We further show that AL‐1‐IgG causes collapse of rat hippocampal neurons and rat retinal ganglion cells. These data suggest a role for REK7 and AL‐1 in the patterning of axonal connections in the developing cortex, hippocampus and visual system.

Regulation of Hippocampal Synaptic Plasticity by the Tyrosine Kinase Receptor, REK7/EphA5, and its Ligand, AL-1/Ephrin-A5 ☆

The Eph-related tyrosine kinase receptor, REK7/EphA5, mediates the effects of AL-1/Ephrin-A5 and related ligands and is involved in the guidance of retinal, cortical, and hippocampal axons during development. The continued expression of REK7/EphA5 in the adult brain, in particular in areas associated with a high degree of synaptic plasticity such as the hippocampus, raises the question of its function in the mature nervous system. In this report we examined the role of REK7/EphA5 in synaptic remodeling by asking if agents that either block or activate REK7/EphA5 affect synaptic strength in hippocampal slices from adult mouse brain. We show that a REK7/EphA5 antagonist, soluble REK7/EphA5-IgG, impairs the induction of long-term potentiation (LTP) without affecting other synaptic parameters such as normal synaptic transmission or paired-pulse facilitation. In contrast, perfusion with AL-1/Ephrin-A5-IgG, an activator of REK7/EphA5, induces a sustained increase in normal synaptic transmission that partially mimics LTP. The sustained elevation of normal synaptic transmission could be attributable to a long-lasting binding of the AL-1/Ephrin-A5-IgG to the endogenous REK7/EphA5 receptor, as revealed by immunohistochemistry. Furthermore, maximal electrical induction of LTP occludes the potentiating effects of subsequent treatment with AL-1/Ephrin-A5-IgG. Taken together these results implicate REK7/EphA5 in the regulation of synaptic plasticity in the mature hippocampus and suggest that REK7/EphA5 activation is recruited in the LTP induced by tetanization.

Lerk2 (Ephrin-B1) Is a Collapsing Factor for a Subset of Cortical Growth Cones and Acts by a Mechanism Different from AL-1 (Ephrin-A5)

The transmembrane (TM) subfamily of Eph ligands and their receptors have been implicated in axon pathfinding and in pattern formation during embryogenesis. These functions are thought to involve repulsive interactions but this has not been demonstrated directly. In this study we used a growth cone collapse assay to determine if the TM ligands Lerk2 and HtkL have repellant guidance activity. We show that Lerk2, but not HtkL, is a collapsing factor for a subset of embryonic cortical neurons. Analysis of the effects of Lerk2 on both the morphology and the cytoskeleton of cortical neurons suggests a mechanism of action different from that of AL-1, a GPI-linked Eph ligand having similar repellant activity. Treatment with Lerk2 disrupts the organization of both the actin cytoskeleton and the microtubules and induces the formation of swellings in the center of the growth cone and along the axon. Measurement of the relative F-actin concentrations in the neurites and soma indicated that F-actin levels in the neurites decrease while those in the soma increase, with the net F-actin content of the neuron remaining unchanged. In contrast, we show that prolonged treatment with AL-1 leads to a net loss of F-actin, consistent with the hypothesis that AL-1 acts by perturbing actin polymerization. These results provide evidence that the ectodomain of Lerk2 functions as a repellant guidance cue and show that, despite overlapping specificities in vitro, the biological activities of related ligands are not necessarily overlapping. Further, TM and GPI-linked Eph ligands appear to exert repellant activity by different mechanisms, opening up the possibility that they may have different effects on growth cones in vivo.

A link between axon guidance and axon fasciculation suggested by studies of the tyrosine kinase receptor EphA5/REK7 and its ligand Ephrin-A5/AL-1

Abstract. The possible roles of the tyrosine kinase receptor EphA5 and its ligand Ephrin-A5 in axon guidance and axon fasciculation are reviewed.

Csk and BatK show opposite temporal expression in the rat CNS: consistent with its late expression in development, BatK induces differentiation of PC12 cells.

BatK is a second member of the Csk family of regulatory kinases that phosphorylate a key inhibitory tyrosine on Src family kinases, leading to down‐regulation. To investigate the roles of BatK and Csk, both of which are expressed in the brain, we compared their temporal expression patterns during development of the central nervous system (CNS) in rats. BatK mRNA is undetectable at embryonic day 12 (E12), appears in the developing nervous system at approximately E15, and its expression progressively increases up to the time of birth, thereafter remaining high throughout the adult brain. In striking contrast, Csk is highly expressed throughout embryonic development and remains high in the CNS until birth. It is then dramatically down‐regulated in the adult brain except in the olfactory bulb. BatK and Csk thus exhibit complementary temporal expression patterns. Since BatK expression correlates with late‐stage development and terminal differentiation, we speculated that it might be involved in regulating neuronal differentiation. Using PC12 cells as a model system, we show that overexpression of BatK is sufficient to induce neurite outgrowth in the absence of nerve growth factor. Further, overexpression of BatK activates the mitogen‐activated protein kinase cascade. We propose a model suggesting that, despite overlapping in vifro activities, BatK and Csk regulate different targets in vivo and have different functions during and after neuronal development, BatK being the dominant regulator of Src kinases in the fully differentiated adult brain.

Protein targeting in the analysis of learning and memory: a potential alternative to gene targeting

Abstract Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins – cell-adhesion molecules – and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location and timing of delivery are controlled. Here, we present methodological details of this novel approach and argue that infusion of immunoadhesins will be useful for studying the role particular receptors play in behavioral and neural plasticity.