Impact of neuronal CPEB2 on the translation of mRNAs involved in synaptic plasticity

Abstract

Among four members of the CPEB family, much less information is available for CPEB2. Although the expression of CPEB2 has been reported in mouse brain (Theis et al., 2003; Hagele et al., 2009; Turimella et al., in revision), the functional role of CPEB2 was not reported before. Hence, the present study was aimed to elucidate the functional role of CPEB2 in neurons of mouse hippocampus.<br /> In the first part of my work, I have analyzed the expression pattern of all CPEBs (1-4) in different cells (neurons, astrocytes, NG2 glia and microglia) of the mouse brain by single-cell RT-PCR. The incidence of CPEBs expression was higher in neurons compared to the glial subtypes analyzed. Even though CPEBs were described before in mouse brain, no information is available about which isoforms are present in each cell type and across species. The present study gives detailed information of the isoform distribution across cell types and in different species. <br /> In the second part of my work, I have studied the functional role of CPEB2 in mouse hippocampus. For that purpose, I have generated a set of CPEB2/CPEB2∆Zn transgenic mice using tet-OFF system (Lewandoski, 2001). These mice overexpressed either full length or mutant CPEB2 (which lacks the zinc finger essential for RNA binding) in pyramidal neurons of mouse hippocampus. Using immunofluorescence, I have confirmed the transgene expression in pyramidal neurons as well as in dendritic processes where CPEB2 could modulate local protein synthesis. By using this set of overexpressing mice, I have investigated the impact of CPEB2 on the translation of several mRNAs in neurons. CPEB2 was found to repress the basal translation of neuronal mRNAs such as β-catenin, GluR2 and EphA4. By regulating the translation of β-catenin, CPEB2 might regulate dendritic morphogenesis (Yu and Malenka, 2003) and participate in the Wnt signalling cascade (Clevers, 2006). As GluR2 dysfunction is associated with neurodegeneration and epilepsy, CPEB2, by regulating the translation of GluR2, might lead to pathological conditions. CPEB2 also repressed the translation of EphA4, a key player which modulates the glial glutamate transport via interacting with the astrocytic ephrin-A3 ligand (Carmona et al., 2009) and participates in neuron to glia communication. The knockout of EphA4 leads to enhanced glutamate transport (increased GLT-1 and GLAST protein levels) (Filosa et al., 2009), but GLT-1 protein levels were not altered in CPEB2 overexpressing mice. EphA4 protein levels were decreased only by 30% in CPEB2 overexpressing mice. Since a complete knockout of EphA4 lead to enhanced glutamate transport, it could be that only 30% decrease in EphA4 protein levels might have an insufficient impact on GLT-1. But, it was found that with the astrocytic overexpression of another CPEB family member, CPEB3, the protein levels of GLT-1 were decreased (Vangoor et al., unpublished). Since the transcript levels of all the target mRNAs analyzed in the present study were not changed, it can be concluded that CPEB2 regulation happens at the level of translation. <br /> I have also investigated the impact of RNA binding on the translation of CPEB target mRNAs in vitro as well as using CPEB2ΔZn overexpressing mice. By RNA co-immunoprecipitation, I have found that CPEB2 requires the zinc finger for RNA binding since only residual binding was observed with a mutant CPEB2 which lacks the zinc finger. In addition, I have analyzed the protein levels of β-catenin, GluR2 and EphA4 in CPEB2ΔZn overexpressing mice compared to their control littermates. Immunoblot analysis indicated that none of the protein levels were changed, indicating the importance of RNA binding for translational regulation. Since the zinc finger essential for RNA binding is not present in CPEB2ΔZn overexpressing mice, no translational regulation was observed. <br /> In the last part of my work, I have studied the interaction of CPEB2 with other target mRNAs. More than one CPEB can regulate same target mRNA, which means CPEBs have an overlap in the binding specificity of target mRNAs. In the present study, I have investigated the interaction of CPEB2 with CamKIIα (a CPEB1 target) and Cx36 using RNA co-immunoprecipitation or luciferase reporter assays. I found that CPEB2 specifically interacted with CamKIIα, but with less affinity compared to CPEB1. CPEB2 also interacted with Cx36, which could be prevented by knocking down the endogenous as well as overexpressed CPEB2 in HeLa cells. <br /> Taken together, by regulating the translation of several key players in neurons, CPEB2 might participate in synaptic plasticity, learning and memory as well as in neuron to glia communication.