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EphrinA4 mimetic peptide targeted to EphA binding site impairs the formation of long-term fear memory in lateral amygdala.

Dines M, Lamprecht R - Transl Psychiatry (2014)

Bottom Line: Acute systemic administration of pep-ephrinA4 1 h after training also impaired long-term fear conditioning memory formation.These results demonstrate that ephrinA4 binding sites in LA are essential for long-term fear memory formation.Moreover, our research shows that ephrinA4 binding sites may serve as a target for pharmacological treatment of fear and anxiety disorders.

View Article: PubMed Central - PubMed

Affiliation: Sagol Department of Neurobiology, University of Haifa, Haifa, Israel.

ABSTRACT
Fear conditioning leads to long-term fear memory formation and is a model for studying fear-related psychopathologies conditions such as phobias and posttraumatic stress disorder. Long-term fear memory formation is believed to involve alterations of synaptic efficacy mediated by changes in synaptic transmission and morphology in lateral amygdala (LA). EphrinA4 and its cognate Eph receptors are intimately involved in regulating neuronal morphogenesis, synaptic transmission and plasticity. To assess possible roles of ephrinA4 in fear memory formation we designed and used a specific inhibitory ephrinA4 mimetic peptide (pep-ephrinA4) targeted to EphA binding site. We show that this peptide, composed of the ephrinA4 binding domain, interacts with EphA4 and inhibits ephrinA4-induced phosphorylation of EphA4. Microinjection of the pep-ephrinA4 into rat LA 30 min before training impaired long- but not short-term fear conditioning memory. Microinjection of a control peptide derived from a nonbinding E helix site of ephrinA4, that does not interact with EphA, had no effect on fear memory formation. Microinjection of pep-ephrinA4 into areas adjacent to the amygdala had no effect on fear memory. Acute systemic administration of pep-ephrinA4 1 h after training also impaired long-term fear conditioning memory formation. These results demonstrate that ephrinA4 binding sites in LA are essential for long-term fear memory formation. Moreover, our research shows that ephrinA4 binding sites may serve as a target for pharmacological treatment of fear and anxiety disorders.

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EphrinA4 mimetic peptide interacts with EphA4 receptor and inhibits ephrinA4-induced EphA4 phosphorylation. (a) A molecular model of ephrinA4 binding to EphA4 receptor using the Swiss-Model. Upper figure: The ephrinA4 peptide (pep-ephrinA4) is derived from the GH loop binding domain of ephrinA4 (arrow-magenta). The control peptide (pep-control) is derived from E helix of the ephrinA4 (arrow-yellow). Lower figure: A molecular model of pep-ephrinA4 peptide bound to EphA4. (b) Pull-down experiments show that pep-ephrinA4, but not pep-control or agarose beads alone, interacts with EphA4. The analysis of variance (ANOVA) showed a significant effect for group (F(2)=4.513, P<0.05), and post hoc analysis found that more EphA4 was pulled down in the pep-ephrinA4 group (n=4) than in the pep-control (P<0.04; n=3) or beads (P<0.03; n=4). Pep-ephrinA4 did not interact with the EphB2 receptor or elongation factor 2 protein (eEF2) serving as control proteins. (c) Pep-ephrinA4 inhibits ephrinA4-induced EphA4 tyrosine phosphorylation. Brain slices that contain the amygdala were divide to three groups: (1) placed in Ringer's solution, (2) placed in Ringer's solution and stimulated with ephrinA4-Fc for 20 min or (3) placed in Ringer's solution with pep-ephrinA4 for 20 min followed by stimulation with ephrinA4-Fc for 20 min (n=3 each). Protein extracts from the slices were immunoprecpitated with anti-EphA4 antibody and subjected to western blot with anti-EphA4 or anti-phosphotyrosine antibodies. The upper panel shows that pep-ephrinA4 abolished ephrinA4-induced EphA4 receptor tyrosine phosphorylation. The lower panel shows the EphA4 protein level in immunoprecipitates. The ANOVA analysis showed a significant effect for group (F(2)=5.496, P<0.05), and post hoc analysis found significant increase in EphA4 phosphorylation in the ephrinA4 group when compared with the Ringer's solution control (P<0.05) or ephrinA4+pep-ephrinA4 (P<0.03) groups. The phosphorylation of EphA4 in the control Ringer's group is not significantly different from the ephrinA4+pep-ephrinA4 group (P>0.6).
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fig1: EphrinA4 mimetic peptide interacts with EphA4 receptor and inhibits ephrinA4-induced EphA4 phosphorylation. (a) A molecular model of ephrinA4 binding to EphA4 receptor using the Swiss-Model. Upper figure: The ephrinA4 peptide (pep-ephrinA4) is derived from the GH loop binding domain of ephrinA4 (arrow-magenta). The control peptide (pep-control) is derived from E helix of the ephrinA4 (arrow-yellow). Lower figure: A molecular model of pep-ephrinA4 peptide bound to EphA4. (b) Pull-down experiments show that pep-ephrinA4, but not pep-control or agarose beads alone, interacts with EphA4. The analysis of variance (ANOVA) showed a significant effect for group (F(2)=4.513, P<0.05), and post hoc analysis found that more EphA4 was pulled down in the pep-ephrinA4 group (n=4) than in the pep-control (P<0.04; n=3) or beads (P<0.03; n=4). Pep-ephrinA4 did not interact with the EphB2 receptor or elongation factor 2 protein (eEF2) serving as control proteins. (c) Pep-ephrinA4 inhibits ephrinA4-induced EphA4 tyrosine phosphorylation. Brain slices that contain the amygdala were divide to three groups: (1) placed in Ringer's solution, (2) placed in Ringer's solution and stimulated with ephrinA4-Fc for 20 min or (3) placed in Ringer's solution with pep-ephrinA4 for 20 min followed by stimulation with ephrinA4-Fc for 20 min (n=3 each). Protein extracts from the slices were immunoprecpitated with anti-EphA4 antibody and subjected to western blot with anti-EphA4 or anti-phosphotyrosine antibodies. The upper panel shows that pep-ephrinA4 abolished ephrinA4-induced EphA4 receptor tyrosine phosphorylation. The lower panel shows the EphA4 protein level in immunoprecipitates. The ANOVA analysis showed a significant effect for group (F(2)=5.496, P<0.05), and post hoc analysis found significant increase in EphA4 phosphorylation in the ephrinA4 group when compared with the Ringer's solution control (P<0.05) or ephrinA4+pep-ephrinA4 (P<0.03) groups. The phosphorylation of EphA4 in the control Ringer's group is not significantly different from the ephrinA4+pep-ephrinA4 group (P>0.6).

Mentions: To study the roles of ephrinA4 in fear conditioning we designed and used an ephrinA4 peptide targeted to EphA binding site (pep-ephrinA4). The pep-ephrinA4 was designed to mimic the ephrinA4 binding domain (GH loop) according to the structure configuration provided by a molecular model (Figure 1a). A control peptide (pep-control) was derived from a nonbinding site of ephrinA4 (E helix, Figure 1a). Pull-down experiments of rat brain homogenates show that the pep-ephrinA4 binds the EphA4 receptor whereas the pep-control or agarose beads alone do not (Figure 1b). The analysis of variance analysis showed a significant effect for group (F(2)=4.513, P<0.05), and post hoc analysis showed that more EphA4 was pulled down in the pep-ephrinA4 group than in the pep-control (P<0.04) or beads (P<0.03). These results show that the pep-ephrinA4 interacts with EphA4 whereas the pep-control does not. Pep-ephrinA4 did not bind EphB2 receptor and the elongation factor 2 (eEF2) that served as control proteins (Figure 1b).


EphrinA4 mimetic peptide targeted to EphA binding site impairs the formation of long-term fear memory in lateral amygdala.

Dines M, Lamprecht R - Transl Psychiatry (2014)

EphrinA4 mimetic peptide interacts with EphA4 receptor and inhibits ephrinA4-induced EphA4 phosphorylation. (a) A molecular model of ephrinA4 binding to EphA4 receptor using the Swiss-Model. Upper figure: The ephrinA4 peptide (pep-ephrinA4) is derived from the GH loop binding domain of ephrinA4 (arrow-magenta). The control peptide (pep-control) is derived from E helix of the ephrinA4 (arrow-yellow). Lower figure: A molecular model of pep-ephrinA4 peptide bound to EphA4. (b) Pull-down experiments show that pep-ephrinA4, but not pep-control or agarose beads alone, interacts with EphA4. The analysis of variance (ANOVA) showed a significant effect for group (F(2)=4.513, P<0.05), and post hoc analysis found that more EphA4 was pulled down in the pep-ephrinA4 group (n=4) than in the pep-control (P<0.04; n=3) or beads (P<0.03; n=4). Pep-ephrinA4 did not interact with the EphB2 receptor or elongation factor 2 protein (eEF2) serving as control proteins. (c) Pep-ephrinA4 inhibits ephrinA4-induced EphA4 tyrosine phosphorylation. Brain slices that contain the amygdala were divide to three groups: (1) placed in Ringer's solution, (2) placed in Ringer's solution and stimulated with ephrinA4-Fc for 20 min or (3) placed in Ringer's solution with pep-ephrinA4 for 20 min followed by stimulation with ephrinA4-Fc for 20 min (n=3 each). Protein extracts from the slices were immunoprecpitated with anti-EphA4 antibody and subjected to western blot with anti-EphA4 or anti-phosphotyrosine antibodies. The upper panel shows that pep-ephrinA4 abolished ephrinA4-induced EphA4 receptor tyrosine phosphorylation. The lower panel shows the EphA4 protein level in immunoprecipitates. The ANOVA analysis showed a significant effect for group (F(2)=5.496, P<0.05), and post hoc analysis found significant increase in EphA4 phosphorylation in the ephrinA4 group when compared with the Ringer's solution control (P<0.05) or ephrinA4+pep-ephrinA4 (P<0.03) groups. The phosphorylation of EphA4 in the control Ringer's group is not significantly different from the ephrinA4+pep-ephrinA4 group (P>0.6).
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fig1: EphrinA4 mimetic peptide interacts with EphA4 receptor and inhibits ephrinA4-induced EphA4 phosphorylation. (a) A molecular model of ephrinA4 binding to EphA4 receptor using the Swiss-Model. Upper figure: The ephrinA4 peptide (pep-ephrinA4) is derived from the GH loop binding domain of ephrinA4 (arrow-magenta). The control peptide (pep-control) is derived from E helix of the ephrinA4 (arrow-yellow). Lower figure: A molecular model of pep-ephrinA4 peptide bound to EphA4. (b) Pull-down experiments show that pep-ephrinA4, but not pep-control or agarose beads alone, interacts with EphA4. The analysis of variance (ANOVA) showed a significant effect for group (F(2)=4.513, P<0.05), and post hoc analysis found that more EphA4 was pulled down in the pep-ephrinA4 group (n=4) than in the pep-control (P<0.04; n=3) or beads (P<0.03; n=4). Pep-ephrinA4 did not interact with the EphB2 receptor or elongation factor 2 protein (eEF2) serving as control proteins. (c) Pep-ephrinA4 inhibits ephrinA4-induced EphA4 tyrosine phosphorylation. Brain slices that contain the amygdala were divide to three groups: (1) placed in Ringer's solution, (2) placed in Ringer's solution and stimulated with ephrinA4-Fc for 20 min or (3) placed in Ringer's solution with pep-ephrinA4 for 20 min followed by stimulation with ephrinA4-Fc for 20 min (n=3 each). Protein extracts from the slices were immunoprecpitated with anti-EphA4 antibody and subjected to western blot with anti-EphA4 or anti-phosphotyrosine antibodies. The upper panel shows that pep-ephrinA4 abolished ephrinA4-induced EphA4 receptor tyrosine phosphorylation. The lower panel shows the EphA4 protein level in immunoprecipitates. The ANOVA analysis showed a significant effect for group (F(2)=5.496, P<0.05), and post hoc analysis found significant increase in EphA4 phosphorylation in the ephrinA4 group when compared with the Ringer's solution control (P<0.05) or ephrinA4+pep-ephrinA4 (P<0.03) groups. The phosphorylation of EphA4 in the control Ringer's group is not significantly different from the ephrinA4+pep-ephrinA4 group (P>0.6).
Mentions: To study the roles of ephrinA4 in fear conditioning we designed and used an ephrinA4 peptide targeted to EphA binding site (pep-ephrinA4). The pep-ephrinA4 was designed to mimic the ephrinA4 binding domain (GH loop) according to the structure configuration provided by a molecular model (Figure 1a). A control peptide (pep-control) was derived from a nonbinding site of ephrinA4 (E helix, Figure 1a). Pull-down experiments of rat brain homogenates show that the pep-ephrinA4 binds the EphA4 receptor whereas the pep-control or agarose beads alone do not (Figure 1b). The analysis of variance analysis showed a significant effect for group (F(2)=4.513, P<0.05), and post hoc analysis showed that more EphA4 was pulled down in the pep-ephrinA4 group than in the pep-control (P<0.04) or beads (P<0.03). These results show that the pep-ephrinA4 interacts with EphA4 whereas the pep-control does not. Pep-ephrinA4 did not bind EphB2 receptor and the elongation factor 2 (eEF2) that served as control proteins (Figure 1b).

Bottom Line: Acute systemic administration of pep-ephrinA4 1 h after training also impaired long-term fear conditioning memory formation.These results demonstrate that ephrinA4 binding sites in LA are essential for long-term fear memory formation.Moreover, our research shows that ephrinA4 binding sites may serve as a target for pharmacological treatment of fear and anxiety disorders.

View Article: PubMed Central - PubMed

Affiliation: Sagol Department of Neurobiology, University of Haifa, Haifa, Israel.

ABSTRACT
Fear conditioning leads to long-term fear memory formation and is a model for studying fear-related psychopathologies conditions such as phobias and posttraumatic stress disorder. Long-term fear memory formation is believed to involve alterations of synaptic efficacy mediated by changes in synaptic transmission and morphology in lateral amygdala (LA). EphrinA4 and its cognate Eph receptors are intimately involved in regulating neuronal morphogenesis, synaptic transmission and plasticity. To assess possible roles of ephrinA4 in fear memory formation we designed and used a specific inhibitory ephrinA4 mimetic peptide (pep-ephrinA4) targeted to EphA binding site. We show that this peptide, composed of the ephrinA4 binding domain, interacts with EphA4 and inhibits ephrinA4-induced phosphorylation of EphA4. Microinjection of the pep-ephrinA4 into rat LA 30 min before training impaired long- but not short-term fear conditioning memory. Microinjection of a control peptide derived from a nonbinding E helix site of ephrinA4, that does not interact with EphA, had no effect on fear memory formation. Microinjection of pep-ephrinA4 into areas adjacent to the amygdala had no effect on fear memory. Acute systemic administration of pep-ephrinA4 1 h after training also impaired long-term fear conditioning memory formation. These results demonstrate that ephrinA4 binding sites in LA are essential for long-term fear memory formation. Moreover, our research shows that ephrinA4 binding sites may serve as a target for pharmacological treatment of fear and anxiety disorders.

Show MeSH
Related in: MedlinePlus