Abstract
During brain development, growth cones respond to attractive and repulsive axon guidance cues. How growth cones integrate guidance instructions is poorly understood. Here, we demonstrate a link between BDNF (brain derived neurotrophic factor), promoting axonal branching and ephrin-A5, mediating axonal repulsion via Eph receptor tyrosine kinase activation. BDNF enhanced growth cone filopodial dynamics and neurite branching of primary neurons. We show that ephrin-A5 antagonized this BDNF-evoked neuronal motility. BDNF increased ERK phosphorylation (P-ERK) and nuclear ERK entry. Ephrin-A5 suppressed BDNF-induced ERK activity and might sequester P-ERK in the cytoplasm. Neurotrophins are well established stimulators of a neuronal immediate early gene (IEG) response. This is confirmed in this study by e.g. c-fos, Egr1 and Arc upregulation upon BDNF application. This BDNF-evoked IEG response required the transcription factor SRF (serum response factor). Notably, ephrin-A5 suppressed a BDNF-evoked neuronal IEG response, suggesting a role of Eph receptors in modulating gene expression. In opposite to IEGs, long-term ephrin-A5 application induced cytoskeletal gene expression of tropomyosin and actinin. To uncover specific Eph receptors mediating ephrin-As impact on neurotrophin signaling, EphA7 deficient mice were analyzed. In EphA7 deficient neurons alterations in growth cone morphology were observed. However, ephrin-A5 still counteracted neurotrophin signaling suggesting that EphA7 is not required for ephrin and BDNF crosstalk. In sum, our data suggest an interaction of ephrin-As and neurotrophin signaling pathways converging at ERK signaling and nuclear gene activity. As ephrins are involved in development and function of many organs, such modulation of receptor tyrosine kinase signaling and gene expression by Ephs might not be limited to the nervous system.
Highlights
During brain development, axons encounter attractive and repulsive guidance cues, whose interplay instructs growth cones with directional information, thereby ensuring target recognition
To study an interplay between ephrin-A and BDNF stimulated neuronal signaling, we investigated growth cone morphology (Fig. 1) and neurite branching (Fig. S1) of primary hippocampal neurons
We investigated whether modulation of a BDNF-mediated immediate early gene (IEG) induction by ephrin-A5 is a general feature of repulsive guidance cues
Summary
Axons encounter attractive and repulsive guidance cues, whose interplay instructs growth cones with directional information, thereby ensuring target recognition. In EphA forward signaling, ephrin-A ligands can activate multiple Eph receptor tyrosine kinase receptors (EphA1-EphA8 and e.g. EphB2; see below) in a highly promiscuous manner. This usually results in contactmediated repulsion, e.g. growth cone collapse [9,10,11]. In Eph reverse signaling, membrane-bound ephrin-As are ‘‘receptors’’ activated by EphA ‘‘ligands’’. This results in attractive [14] and repulsive [15,16,17] axon guidance responses, depending on e.g. axonal subtype investigated. Ephrin-A5 activates EphA and potentially EphB2 forward signaling (in this study summarized as Eph forward signaling)
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