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Autism-related protein MeCP2 regulates FGF13 expression and emotional behaviors
Journal of Genetics and Genomics xxx (2016) 1e4
Contents lists available at ScienceDirect
Journal of Genetics and Genomics Journal homepage: www.journals.elsevier.com/journal-of-geneticsand-genomics/
Letter to the editor
Autism-related protein MeCP2 regulates FGF13 expression and emotional behaviors
Methyl-CpG binding protein 2 (MeCP2) has a crucial role in tran et al., 2014). scriptional regulation and neural development (Ausio Loss of function mutations of MECP2 in human lead to Rett syndrome (RTT), a severe neurodevelopmental disorders (Amir et al., 1999), whereas individuals with the chromosomal duplications containing the MECP2 locus showed severe autism-like symptoms (Ramocki et al., 2009). MeCP2 is highly expressed in brain during development and adulthood (Shahbazian et al., 2002). For its selective binding to DNA sequences methylated at cytosine in the dinucleotide 50 CpG (Guy et al., 2011), MeCP2 had initially been identified to be a transcriptional repressor (Nan et al., 1997). However, many genes are down-regulated in the brain of Mecp2 null mice, suggesting that MeCP2 may act as an activator for gene regulation (Chahrour et al., 2008). MicroRNAs mediate posttranscriptional gene silencing by controlling the translation of approximately over 60% of protein-coding genes (Esteller, 2011). Recent study reveals that MeCP2 is involved in posttranscriptional gene regulation by regulating nuclear microRNA processing directly (Cheng et al., 2014). Fibroblast growth factor 13 (FGF13), an intracellular member of the FGF homologous factor (FHF) subfamily (Goldfarb, 2005), is widely expressed in the developing and adult nervous systems (Smallwood et al., 1996). Fgf13 is located in the q26 region of the €rjesonX chromosome, deletion of which is responsible for the Bo Forssman-Lehmann syndrome, suggesting Fgf13 as a candidate gene for the X-linked intellectual disabilities (Gecz et al., 1999). FGF13 is required for regulating neural polarization and migration by stabilizing microtubule protein during brain development (Wu et al., 2012). The abnormal behavior observed in Fgf13-deficient mouse suggests the critical role of FGF13 in regulating emotional behaviors and learning memory (Wu et al., 2012; Puranam et al., 2015). Because the dose of MeCP2 protein is critical for neural development and function, we set out to examine whether genes involved in brain disorders may be regulated by MeCP2. In a screen for neuropsychiatric disorders associated genes, we identified that the tumor suppressor gene Phosphatase and tensin homolog (PTEN) was regulated by MeCP2 in a reciprocal manner (Lyu et al., 2016). Interestingly, we found that Fgf13, an intellectual disability (ID)-related gene, was also regulated by MeCP2. We first applied lentivirus harboring short hairpin RNA against the mouse Mecp2 gene and MeCP2 cDNA, respectively, into culture
mouse cortical neurons and examined protein levels of potential targets by Western blotting. We found that the protein level of FGF13 decreased in MeCP2 knockdown neurons and elevated in MeCP2 overexpressed neurons (Fig. S1A). This bi-directional regulation between MeCP2 and FGF13 suggests that the protein level of FGF13 may also be altered in mice models with MeCP2 deletion and duplications. We next examined the protein level of FGF13 in brains of Mecp2 conditional knockout mice (CKO: Nestin-cre, Mecp2flox/y), a mouse model for Rett syndrome (Guy et al., 2001). We found that both isoforms of FGF13, FGF13A and FGF13B, decreased in brains of Mecp2 CKO mice (Fig. 1A), suggesting that the FGF13 protein indeed was regulated in vivo when Mecp2 is manipulated genetically. We then investigated whether FGF13 protein may be regulated in mice with MeCP2 overexpression, the mouse model of MECP2 duplication syndrome (Collins et al., 2004). Consistently, we found that the protein level of FGF13A indeed increased in MeCP2 overexpression (OE) mice (Fig. S1B), indicating the bi-directional regulation of MeCP2 to FGF13 in vivo. The fact that the protein level of FGF13 decreased in Mecp2 CKO mice and increased in MeCP2 OE mice suggested that MeCP2 regulates the expression of FGF13 through post-transcriptional mechanisms, instead of transcriptional repression. We previously found that microRNAs are dramatically altered in Mecp2 KO mice, a process mediated by MeCP2-dependent nuclear microRNA processing (Cheng et al., 2014). We then wonder whether MeCP2 may also regulate the expression of FGF13 through microRNA-mediated mechanisms. Thus we converged microRNAs potentially regulating FGF13 and dys-regulated microRNAs in Mecp2 KO mice, from which we identified two candidates, miR421 and miR539. First, we found the seed sequences of miR421 and miR539 located in the 30 UTR of mouse and human Fgf13 gene, suggesting the regulation of FGF13 by miR421 and miR539 (Fig. 1B). We next found that levels of miR421 and miR539 increased in Mecp2 KO mice, suggesting that expression of miR421 and miR539 was repressed by MeCP2 (Fig. S2A and B). Expression levels of miR421 and miR539 also increased in culture neurons with MeCP2 knockdown, whereas miR421 and miR539 decreased in MeCP2 overexpressed neurons (Fig. S2C and D). This evidence indicated the bidirectional regulation of MeCP2 to miR421 and miR539 in vitro and in vivo. We next asked whether miR421 and miR539 may regulate the expression of FGF13 directly. We first transfected miRNA mimics
http://dx.doi.org/10.1016/j.jgg.2016.10.004 1673-8527/Copyright © 2016, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
Please cite this article in press as: Yuan, B., et al.Autism-related protein MeCP2 regulates FGF13 expression and emotional behaviors, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.10.004
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Letter to the editor / Journal of Genetics and Genomics xxx (2016) 1e4
Please cite this article in press as: Yuan, B., et al.Autism-related protein MeCP2 regulates FGF13 expression and emotional behaviors, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.10.004
Letter to the editor / Journal of Genetics and Genomics xxx (2016) 1e4
of miR421, miR539, and Non-specific Control miRNA (NC miRNA) into 293 cells, using co-transfection of Renilla luciferase gene with 30 UTR of Fgf13 gene (~1000 bp, containing targeting sites of miR421 and miR539) as a reporter. We found that miR421 is able to significantly repress Fgf13 30 UTR-mediated gene expression, measured by the luciferase activity of Renilla, suggesting that miR421 may play the major role in regulating FGF13 expression (Fig. S2E). Furthermore, we transfected the miRNA mimics of miR421, miR539, and NC miRNA into Neuro2a cells to examine whether these two miRNAs may regulate the protein level of endogenous FGF13. Consistently, we found that expression of miR421 indeed significantly decreased the protein level of endogenous FGF13 (Fig. 1C). This evidence further suggests that MeCP2 principally regulates expression of FGF13 via miR421. Genetic loss- and gain-of-function of MeCP2 lead to severe behavioral phenotypes including anxiety and emotional behaviors (Goffin et al., 2012, 2014; Samaco et al., 2012). Fgf13 CKO mice also showed defects in various behavioral paradigms (Wu et al., 2012). Thus we would like to examine whether genetic manipulations of MeCP2 and FGF13 may show synergistic or compensatory effects regarding to various behaviors. In order to study whether Fgf13 deletion and MeCP2 overexpression may play synergistic roles in regulating emotional behaviors, we crossed female mice carrying homozygous Emx1-cre, heterozygous Fgf13þ/flox with male MeCP2 overexpression mice (MeCP2Tg), and examined behaviors of offspring mice in following four genotypes, Fgf13 heterozygous deletion (Emx1-cre, Fgf13þ/flox, n ¼ 9), Fgf13 conditional knockout (Emx1-cre, Fgf13flox/Y, n ¼ 11), Fgf13 heterozygous deletion with MeCP2 overexpression (Emx1-cre, Fgf13þ/flox, MeCP2Tg, n ¼ 8), and Fgf13 CKO with MeCP2 overexpression (Emx1-cre, Fgf13flox/Y, MeCP2Tg, n ¼ 9). We first investigated the anxiety-related behaviors of mice in elevated platform tasks. Previously, MeCP2Tg and Fgf13 deletion mice both showed increased anxiety level (Samaco et al., 2012; Wu et al., 2012). In our experiments, we found that mice with both Fgf13 deletion and MeCP2 overexpression showed similar anxiety level comparing to either Fgf13 deletion or MeCP2 overexpression mice measured by duration time spent in open zone, suggesting that MeCP2 and FGF13 may function through parallel pathways to regulate anxiety in mice, as lack of Fgf13 and MeCP2 overexpression did not lead to accumulative defects in anxiety (Fig. 1D and Fig. S3A). We further examined the anxiety level of mice in four genotypes with the light/dark exploration assay with three parameters, including the latency of the first entry to light box, total entries in light box, and duration time spent in light box (Figs. 1E,S3B and C). Consistently, we found that mice with Fgf13 deletion and MeCP2 overexpression exhibited similar anxiety levels comparing to either Fgf13 deletion or MeCP2 overexpression, further confirming that the MeCP2 and FGF13 may regulate anxiety behaviors through parallel pathways. We next examined forced swimming, a depression-like behavior, in these mice. We consistently found that Fgf13 deletion lead to depression-like behaviors, but MeCP2 overexpression did not affect depression-like behaviors, suggesting the specificity of MeCP2 to anxiety behaviors in mice (Fig. S3D).
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It was reported that lack of Fgf13 lead to defects in cued fear conditioning, whereas MeCP2 overexpression mice showed elevated levels of fear conditioning responses (Wu et al., 2012). We thus examined the tone-dependent cued fear conditioning test in mice described above. First we found that Fgf13 deletion mice indeed showed defects in cued fear conditioning, however, MeCP2 overexpression seems to overcome the defects of Fgf13 deletion as mice with both Fgf13 deletion and MeCP2 overexpression exhibited elevated cued responses comparing to Fgf13 deletion only (Fig. S4A). We further examined the contextual fear conditioning and found mice with both Fgf13 deletion and MeCP2 overexpression also showed significantly increased freezing time in the contextual fear conditioning test than Fgf13 deletion mice (Fig. 1F). The freezing time of these mice during training session also showed similar trends, indicating the different roles of FGF13 and MeCP2 in regulating fear conditioning (Fig. S4B). These lines of evidence showed that MeCP2 and FGF13 may play convergent roles in regulating fear conditioning, as MeCP2 overexpression seems to compensate the defects of Fgf13 deletion. Therefore, neural circuits affected by MeCP2 overexpression and Fgf13 deletion may converge to common targets, as increased amount of MeCP2 could compensate the defected caused by genetic loss of Fgf13. Various defects in learning and memory paradigms were found in Fgf13 deletion mice (Wu et al., 2012). We then would like to examine whether MeCP2 overexpression may affect the defects in learning and memory of Fgf13 deletion mice. We performed the Morris water maze test on these mice and found that loss of Fgf13 indeed impaired the spatial memory, as the escape latency of Fgf13 heterozygous deletion mice are able to find the escape platform were much sooner than Fgf13 deletion mice (Fig. 1G). Interestingly, MeCP2 overexpression seems to deteriorate the learning ability of Fgf13 heterozygous deletion mice, as mice with MeCP2 overexpression showed compromised spatial learning, regardless the presence of Fgf13 (Fig. 1G). This evidence suggested that MeCP2 was at the downstream position of Fgf13 for regulating spatial memory, as MeCP2 overexpression even impaired spatial memory in Fgf13 heterozygous deletion mice (Fig. 1G). The overall time spent in the platform quadrant was similar between mice across different genotypes (Fig. S5A). The strong inhibition of MeCP2 overexpression on spatial memory could also be found in platform crossing times measure in the water maze test (Fig. 1H). Finally, we found that the MeCP2 overexpression and Fgf13 deletion seems to have strong influence on the swimming speed of mice, suggesting the potential roles of MeCP2 and FGF13 in motor control (Fig. S5B). In this work, we present evidence that FGF13 is regulated by MeCP2 via microRNAs. MeCP2 is a nuclear protein most abundant in brain which occupies a central role in the neurodevelopment (Guy et al., 2011). FGF13 is an auxiliary protein of voltage-gated Naþ channels and critical for synaptic excitatory-inhibitory balance (Puranam et al., 2015). These findings indicate that dys-regulation of FGF13 expression by MeCP2 may have wide-ranging effects on neuronal and synaptic homeostasis and nervous system function. This is the first demonstration of the FGF13 regulating by an
Fig. 1. MeCP2 regulates the expression of FGF13 and anxiety-related behaviors. A: FGF13 expression decreases in MeCP2 conditional knockout mice. Brain tissues from adult mice with different genotypes were collected and analyzed on SDS-PAGE using antibodies indicated. B: Schematic illustration for targeting sites of miR421 and miR539 in the mouse Fgf13 gene 30 UTR. C: Left panel: FGF13 protein level was decreased in Neuro2a cells with the excess of miR421 rather than miR539. Neuro2a cells were transfected by LipofectAMINE 2000 with miRNA mimics of non-specific miRNA control, miR421 or miR539. Right panel: Quantitation of Western blotting experiments. Eight replicates were performed. Error bars denote standard error of mean (SEM), *P < 0.05, one-tail t-test. D: Duration time of mice stay in open zone of elevated plus maze test. Emx1-cre, Fgf13þ/flox, MeCP2Tg and Emx1-cre, Fgf13flox/Y, MeCP2Tg mice were in open zone shorter than Emx1-cre, Fgf13þ/flox and Emx1-cre, Fgf13flox/Y mice. E: Total entries into the light side in the light/dark exploration test. F: MeCP2 and FGF13 play opposite roles in regulating fear conditioning tasks. The percentage of freezing time in contextual memory test. G: Mean escape latencies during hidden platform training of Morris water maze tests. H: Mean values of percentage time spent in quadrant with platform and platform crossing times during day 6 probe trials. D¡H: Error bars denote SEM. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.0001, one-way ANOVA test.
Please cite this article in press as: Yuan, B., et al.Autism-related protein MeCP2 regulates FGF13 expression and emotional behaviors, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.10.004
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Letter to the editor / Journal of Genetics and Genomics xxx (2016) 1e4
upstream factor. The excess of MeCP2 and the lack of FGF13 play multiple roles in different behaviors. The convergent effects on emotional behaviors and the opposite effects on fear memory indicate that MeCP2 regulates FGF13 in a circuit specific way. The increased MeCP2 level also causes autism like features by affecting neural circuits underlying social behaviors (Samaco et al., 2012). However, we found social behavior abnormalities present on FVB/N and C57BL/6 F1 hybrid mice are absent on C57BL/6 background mice (data not show). These finding suggest that genetic background plays a critical role in determining the social defects appeared in MeCP2 overexpression mice. Thus the social interaction tests were not able to performed for MeCP2 overexpression and Fgf13 deletion mice, due to mixed genetic background. It would be valuable to address whether acutely manipulation of FGF13 in the MeCP2 OE mice may make impacts on the emotional and cognitive behaviors in the future study. Finally, our findings that MeCP2 and FGF13 plays distinct roles in regulating emotional and cognitive behaviors, also crosstalk between MeCP2 and FGF13-related neural circuits provides further insights for molecular controls of emotional and cognitive behaviors in autism spectrum disorders and intellectual disabilities. Acknowledgments We thank Yuefang Zhang for taking care of mouse colony. This work was supported by the Strategic Priority Research Program of Chinese Academy of Sciences (Grant No. XDB02050400), and the National Natural Science Foundation of China (Grant No. #91432111) to Z.Qiu.
Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jgg.2016.10.004. References Amir, R.E., Van den Veyver, I.B., Wan, M., Tran, C.Q., Francke, U., Zoghbi, H.Y., 1999. Rett syndrome is caused by mutations in X-linked MECP2, encoding methylCpG-binding protein 2. Nat. Genet. 23, 185e188. , J., de Paz, A.M., Esteller, M., 2014. MeCP2: the long trip from a chromatin proAusio tein to neurological disorders. Trends Mol. Med. 20, 487e498. Chahrour, M., Jung, S.Y., Shaw, C., Zhou, X., Wong, S.T., Qin, J., Zoghbi, H.Y., 2008. MeCP2, a key contributor to neurological disease, activates and represses transcription. Science 320, 1224e1229. Cheng, T.L., Wang, Z., Liao, Q., Zhu, Y., Zhou, W.H., Xu, W., Qiu, Z., 2014. MeCP2 suppresses nuclear microRNA processing and dendritic growth by regulating the DGCR8/Drosha complex. Dev. Cell 28, 547e560. Collins, A.L., Levenson, J.M., Vilaythong, A.P., Richman, R., Armstrong, D.L., Noebels, J.L., David Sweatt, J., Zoghbi, H.Y., 2004. Mild overexpression of MeCP2 causes a progressive neurological disorder in mice. Hum. Mol. Genet. 13, 2679e2689. Esteller, M., 2011. Non-coding RNAs in human disease. Nat. Rev. Genet. 12, 861e874. Gecz, J., Baker, E., Donnelly, A., Ming, J.E., McDonald-McGinn, D.M., Spinner, N.B., Zackai, E.H., Sutherland, G.R., Mulley, J.C., 1999. Fibroblast growth factor homologous factor 2 (FHF2): gene structure, expression and mapping to the BorjesonForssman-Lehmann syndrome region in Xq26 delineated by a duplication
breakpoint in a BFLS-like patient. Hum. Genet. 104, 56e63. Goffin, D., Allen, M., Zhang, L., Amorim, M., Wang, I.T., Reyes, A.R., MercadoBerton, A., Ong, C., Cohen, S., Hu, L., Blendy, J.A., Carlson, G.C., Siegel, S.J., Greenberg, M.E., Zhou, Z., 2012. Rett syndrome mutation MeCP2 T158A disrupts DNA binding, protein stability and ERP responses. Nat. Neurosci. 15, 274e283. Goffin, D., Brodkin, E.S., Blendy, J.A., Siegel, S.J., Zhou, Z., 2014. Cellular origins of auditory event-related potential deficits in Rett syndrome. Nat. Neurosci. 17, 804e806. Goldfarb, M., 2005. Fibroblast growth factor homologous factors: evolution, structure, and function. Cytokine Growth Factor Rev. 16, 215e220. Guy, J., Cheval, H., Selfridge, J., Bird, A., 2011. The role of MeCP2 in the brain. Annu. Rev. Cell Dev. Biol. 27, 631e652. Guy, J., Hendrich, B., Holmes, M., Martin, J.E., Bird, A., 2001. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nat. Genet. 27, 322e326. Lyu, J.W., Yuan, B., Cheng, T.L., Zhou, W.H., Qiu, Z., 2016. Reciprocal regulation of autism-related genes MeCP2 and PTEN via microRNAs. Sci. Rep. 6, 20392. Nan, X.S., Campoy, F.J., Bird, A., 1997. MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Cell 88, 471e481. Puranam, R.S., He, X.P., Yao, L., Le, T., Jang, W., Rehder, C.W., Lewis, D.V., McNamara, J.O., 2015. Disruption of Fgf13 causes synaptic excitatoryinhibitory imbalance and genetic epilepsy and febrile seizures plus. J. Neurosci. 35, 8866e8881. Ramocki, M.B., Peters, S.U., Tavyev, Y.J., Zhang, F., Carvalho, C.M., Schaaf, C.P., Richman, R., Fang, P., Glaze, D.G., Lupski, J.R., Zoghbi, H.Y., 2009. Autism and other neuropsychiatric symptoms are prevalent in individuals with MeCP2 duplication syndrome. Ann. Neurol. 66, 771e782. Samaco, R.C., Mandel-Brehm, C., McGraw, C.M., Shaw, C.A., McGill, B.E., Zoghbi, H.Y., 2012. Crh and Oprm1 mediate anxiety-related behavior and social approach in a mouse model of MECP2 duplication syndrome. Nat. Genet. 44, 206e211. Shahbazian, M.D., Antalffy, B., Armstrong, D.L., Zoghbi, H.Y., 2002. Insight into Rett syndrome: MeCP2 levels display tissue- and cell-specific differences and correlate with neuronal maturation. Hum. Mol. Genet. 11, 115e124. Smallwood, P.M., Munoz-Sanjuan, I., Tong, P., Macke, J.P., Hendry, S.H., Gilbert, D.J., Copeland, N.G., Jenkins, N.A., Nathans, J., 1996. Fibroblast growth factor (FGF) homologous factors: new members of the FGF family implicated in nervous system development. Proc. Natl. Acad. Sci. U. S. A. 93, 9850e9857. Wu, Q.F., Yang, L., Li, S., Wang, Q., Yuan, X.B., Gao, X., Bao, L., Zhang, X., 2012. Fibroblast growth factor 13 is a microtubule-stabilizing protein regulating neuronal polarization and migration. Cell 149, 1549e1564.
Bo Yuan, Tian-lin Cheng Institute of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Kan Yang Institute of Chemistry and Bioengineering, Donghua University, Shanghai 200051, China Xu Zhang, Zilong Qiu* Institute of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Shanghai Clinical Research Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China *
Corresponding author. E-mail address: [email protected] (Z. Qiu). 29 April 2016 Available online xxx
Please cite this article in press as: Yuan, B., et al.Autism-related protein MeCP2 regulates FGF13 expression and emotional behaviors, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.10.004
Contents lists available at ScienceDirect
Journal of Genetics and Genomics Journal homepage: www.journals.elsevier.com/journal-of-geneticsand-genomics/
Letter to the editor
Autism-related protein MeCP2 regulates FGF13 expression and emotional behaviors
Methyl-CpG binding protein 2 (MeCP2) has a crucial role in tran et al., 2014). scriptional regulation and neural development (Ausio Loss of function mutations of MECP2 in human lead to Rett syndrome (RTT), a severe neurodevelopmental disorders (Amir et al., 1999), whereas individuals with the chromosomal duplications containing the MECP2 locus showed severe autism-like symptoms (Ramocki et al., 2009). MeCP2 is highly expressed in brain during development and adulthood (Shahbazian et al., 2002). For its selective binding to DNA sequences methylated at cytosine in the dinucleotide 50 CpG (Guy et al., 2011), MeCP2 had initially been identified to be a transcriptional repressor (Nan et al., 1997). However, many genes are down-regulated in the brain of Mecp2 null mice, suggesting that MeCP2 may act as an activator for gene regulation (Chahrour et al., 2008). MicroRNAs mediate posttranscriptional gene silencing by controlling the translation of approximately over 60% of protein-coding genes (Esteller, 2011). Recent study reveals that MeCP2 is involved in posttranscriptional gene regulation by regulating nuclear microRNA processing directly (Cheng et al., 2014). Fibroblast growth factor 13 (FGF13), an intracellular member of the FGF homologous factor (FHF) subfamily (Goldfarb, 2005), is widely expressed in the developing and adult nervous systems (Smallwood et al., 1996). Fgf13 is located in the q26 region of the €rjesonX chromosome, deletion of which is responsible for the Bo Forssman-Lehmann syndrome, suggesting Fgf13 as a candidate gene for the X-linked intellectual disabilities (Gecz et al., 1999). FGF13 is required for regulating neural polarization and migration by stabilizing microtubule protein during brain development (Wu et al., 2012). The abnormal behavior observed in Fgf13-deficient mouse suggests the critical role of FGF13 in regulating emotional behaviors and learning memory (Wu et al., 2012; Puranam et al., 2015). Because the dose of MeCP2 protein is critical for neural development and function, we set out to examine whether genes involved in brain disorders may be regulated by MeCP2. In a screen for neuropsychiatric disorders associated genes, we identified that the tumor suppressor gene Phosphatase and tensin homolog (PTEN) was regulated by MeCP2 in a reciprocal manner (Lyu et al., 2016). Interestingly, we found that Fgf13, an intellectual disability (ID)-related gene, was also regulated by MeCP2. We first applied lentivirus harboring short hairpin RNA against the mouse Mecp2 gene and MeCP2 cDNA, respectively, into culture
mouse cortical neurons and examined protein levels of potential targets by Western blotting. We found that the protein level of FGF13 decreased in MeCP2 knockdown neurons and elevated in MeCP2 overexpressed neurons (Fig. S1A). This bi-directional regulation between MeCP2 and FGF13 suggests that the protein level of FGF13 may also be altered in mice models with MeCP2 deletion and duplications. We next examined the protein level of FGF13 in brains of Mecp2 conditional knockout mice (CKO: Nestin-cre, Mecp2flox/y), a mouse model for Rett syndrome (Guy et al., 2001). We found that both isoforms of FGF13, FGF13A and FGF13B, decreased in brains of Mecp2 CKO mice (Fig. 1A), suggesting that the FGF13 protein indeed was regulated in vivo when Mecp2 is manipulated genetically. We then investigated whether FGF13 protein may be regulated in mice with MeCP2 overexpression, the mouse model of MECP2 duplication syndrome (Collins et al., 2004). Consistently, we found that the protein level of FGF13A indeed increased in MeCP2 overexpression (OE) mice (Fig. S1B), indicating the bi-directional regulation of MeCP2 to FGF13 in vivo. The fact that the protein level of FGF13 decreased in Mecp2 CKO mice and increased in MeCP2 OE mice suggested that MeCP2 regulates the expression of FGF13 through post-transcriptional mechanisms, instead of transcriptional repression. We previously found that microRNAs are dramatically altered in Mecp2 KO mice, a process mediated by MeCP2-dependent nuclear microRNA processing (Cheng et al., 2014). We then wonder whether MeCP2 may also regulate the expression of FGF13 through microRNA-mediated mechanisms. Thus we converged microRNAs potentially regulating FGF13 and dys-regulated microRNAs in Mecp2 KO mice, from which we identified two candidates, miR421 and miR539. First, we found the seed sequences of miR421 and miR539 located in the 30 UTR of mouse and human Fgf13 gene, suggesting the regulation of FGF13 by miR421 and miR539 (Fig. 1B). We next found that levels of miR421 and miR539 increased in Mecp2 KO mice, suggesting that expression of miR421 and miR539 was repressed by MeCP2 (Fig. S2A and B). Expression levels of miR421 and miR539 also increased in culture neurons with MeCP2 knockdown, whereas miR421 and miR539 decreased in MeCP2 overexpressed neurons (Fig. S2C and D). This evidence indicated the bidirectional regulation of MeCP2 to miR421 and miR539 in vitro and in vivo. We next asked whether miR421 and miR539 may regulate the expression of FGF13 directly. We first transfected miRNA mimics
http://dx.doi.org/10.1016/j.jgg.2016.10.004 1673-8527/Copyright © 2016, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
Please cite this article in press as: Yuan, B., et al.Autism-related protein MeCP2 regulates FGF13 expression and emotional behaviors, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.10.004
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Letter to the editor / Journal of Genetics and Genomics xxx (2016) 1e4
Please cite this article in press as: Yuan, B., et al.Autism-related protein MeCP2 regulates FGF13 expression and emotional behaviors, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.10.004
Letter to the editor / Journal of Genetics and Genomics xxx (2016) 1e4
of miR421, miR539, and Non-specific Control miRNA (NC miRNA) into 293 cells, using co-transfection of Renilla luciferase gene with 30 UTR of Fgf13 gene (~1000 bp, containing targeting sites of miR421 and miR539) as a reporter. We found that miR421 is able to significantly repress Fgf13 30 UTR-mediated gene expression, measured by the luciferase activity of Renilla, suggesting that miR421 may play the major role in regulating FGF13 expression (Fig. S2E). Furthermore, we transfected the miRNA mimics of miR421, miR539, and NC miRNA into Neuro2a cells to examine whether these two miRNAs may regulate the protein level of endogenous FGF13. Consistently, we found that expression of miR421 indeed significantly decreased the protein level of endogenous FGF13 (Fig. 1C). This evidence further suggests that MeCP2 principally regulates expression of FGF13 via miR421. Genetic loss- and gain-of-function of MeCP2 lead to severe behavioral phenotypes including anxiety and emotional behaviors (Goffin et al., 2012, 2014; Samaco et al., 2012). Fgf13 CKO mice also showed defects in various behavioral paradigms (Wu et al., 2012). Thus we would like to examine whether genetic manipulations of MeCP2 and FGF13 may show synergistic or compensatory effects regarding to various behaviors. In order to study whether Fgf13 deletion and MeCP2 overexpression may play synergistic roles in regulating emotional behaviors, we crossed female mice carrying homozygous Emx1-cre, heterozygous Fgf13þ/flox with male MeCP2 overexpression mice (MeCP2Tg), and examined behaviors of offspring mice in following four genotypes, Fgf13 heterozygous deletion (Emx1-cre, Fgf13þ/flox, n ¼ 9), Fgf13 conditional knockout (Emx1-cre, Fgf13flox/Y, n ¼ 11), Fgf13 heterozygous deletion with MeCP2 overexpression (Emx1-cre, Fgf13þ/flox, MeCP2Tg, n ¼ 8), and Fgf13 CKO with MeCP2 overexpression (Emx1-cre, Fgf13flox/Y, MeCP2Tg, n ¼ 9). We first investigated the anxiety-related behaviors of mice in elevated platform tasks. Previously, MeCP2Tg and Fgf13 deletion mice both showed increased anxiety level (Samaco et al., 2012; Wu et al., 2012). In our experiments, we found that mice with both Fgf13 deletion and MeCP2 overexpression showed similar anxiety level comparing to either Fgf13 deletion or MeCP2 overexpression mice measured by duration time spent in open zone, suggesting that MeCP2 and FGF13 may function through parallel pathways to regulate anxiety in mice, as lack of Fgf13 and MeCP2 overexpression did not lead to accumulative defects in anxiety (Fig. 1D and Fig. S3A). We further examined the anxiety level of mice in four genotypes with the light/dark exploration assay with three parameters, including the latency of the first entry to light box, total entries in light box, and duration time spent in light box (Figs. 1E,S3B and C). Consistently, we found that mice with Fgf13 deletion and MeCP2 overexpression exhibited similar anxiety levels comparing to either Fgf13 deletion or MeCP2 overexpression, further confirming that the MeCP2 and FGF13 may regulate anxiety behaviors through parallel pathways. We next examined forced swimming, a depression-like behavior, in these mice. We consistently found that Fgf13 deletion lead to depression-like behaviors, but MeCP2 overexpression did not affect depression-like behaviors, suggesting the specificity of MeCP2 to anxiety behaviors in mice (Fig. S3D).
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It was reported that lack of Fgf13 lead to defects in cued fear conditioning, whereas MeCP2 overexpression mice showed elevated levels of fear conditioning responses (Wu et al., 2012). We thus examined the tone-dependent cued fear conditioning test in mice described above. First we found that Fgf13 deletion mice indeed showed defects in cued fear conditioning, however, MeCP2 overexpression seems to overcome the defects of Fgf13 deletion as mice with both Fgf13 deletion and MeCP2 overexpression exhibited elevated cued responses comparing to Fgf13 deletion only (Fig. S4A). We further examined the contextual fear conditioning and found mice with both Fgf13 deletion and MeCP2 overexpression also showed significantly increased freezing time in the contextual fear conditioning test than Fgf13 deletion mice (Fig. 1F). The freezing time of these mice during training session also showed similar trends, indicating the different roles of FGF13 and MeCP2 in regulating fear conditioning (Fig. S4B). These lines of evidence showed that MeCP2 and FGF13 may play convergent roles in regulating fear conditioning, as MeCP2 overexpression seems to compensate the defects of Fgf13 deletion. Therefore, neural circuits affected by MeCP2 overexpression and Fgf13 deletion may converge to common targets, as increased amount of MeCP2 could compensate the defected caused by genetic loss of Fgf13. Various defects in learning and memory paradigms were found in Fgf13 deletion mice (Wu et al., 2012). We then would like to examine whether MeCP2 overexpression may affect the defects in learning and memory of Fgf13 deletion mice. We performed the Morris water maze test on these mice and found that loss of Fgf13 indeed impaired the spatial memory, as the escape latency of Fgf13 heterozygous deletion mice are able to find the escape platform were much sooner than Fgf13 deletion mice (Fig. 1G). Interestingly, MeCP2 overexpression seems to deteriorate the learning ability of Fgf13 heterozygous deletion mice, as mice with MeCP2 overexpression showed compromised spatial learning, regardless the presence of Fgf13 (Fig. 1G). This evidence suggested that MeCP2 was at the downstream position of Fgf13 for regulating spatial memory, as MeCP2 overexpression even impaired spatial memory in Fgf13 heterozygous deletion mice (Fig. 1G). The overall time spent in the platform quadrant was similar between mice across different genotypes (Fig. S5A). The strong inhibition of MeCP2 overexpression on spatial memory could also be found in platform crossing times measure in the water maze test (Fig. 1H). Finally, we found that the MeCP2 overexpression and Fgf13 deletion seems to have strong influence on the swimming speed of mice, suggesting the potential roles of MeCP2 and FGF13 in motor control (Fig. S5B). In this work, we present evidence that FGF13 is regulated by MeCP2 via microRNAs. MeCP2 is a nuclear protein most abundant in brain which occupies a central role in the neurodevelopment (Guy et al., 2011). FGF13 is an auxiliary protein of voltage-gated Naþ channels and critical for synaptic excitatory-inhibitory balance (Puranam et al., 2015). These findings indicate that dys-regulation of FGF13 expression by MeCP2 may have wide-ranging effects on neuronal and synaptic homeostasis and nervous system function. This is the first demonstration of the FGF13 regulating by an
Fig. 1. MeCP2 regulates the expression of FGF13 and anxiety-related behaviors. A: FGF13 expression decreases in MeCP2 conditional knockout mice. Brain tissues from adult mice with different genotypes were collected and analyzed on SDS-PAGE using antibodies indicated. B: Schematic illustration for targeting sites of miR421 and miR539 in the mouse Fgf13 gene 30 UTR. C: Left panel: FGF13 protein level was decreased in Neuro2a cells with the excess of miR421 rather than miR539. Neuro2a cells were transfected by LipofectAMINE 2000 with miRNA mimics of non-specific miRNA control, miR421 or miR539. Right panel: Quantitation of Western blotting experiments. Eight replicates were performed. Error bars denote standard error of mean (SEM), *P < 0.05, one-tail t-test. D: Duration time of mice stay in open zone of elevated plus maze test. Emx1-cre, Fgf13þ/flox, MeCP2Tg and Emx1-cre, Fgf13flox/Y, MeCP2Tg mice were in open zone shorter than Emx1-cre, Fgf13þ/flox and Emx1-cre, Fgf13flox/Y mice. E: Total entries into the light side in the light/dark exploration test. F: MeCP2 and FGF13 play opposite roles in regulating fear conditioning tasks. The percentage of freezing time in contextual memory test. G: Mean escape latencies during hidden platform training of Morris water maze tests. H: Mean values of percentage time spent in quadrant with platform and platform crossing times during day 6 probe trials. D¡H: Error bars denote SEM. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.0001, one-way ANOVA test.
Please cite this article in press as: Yuan, B., et al.Autism-related protein MeCP2 regulates FGF13 expression and emotional behaviors, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.10.004
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Letter to the editor / Journal of Genetics and Genomics xxx (2016) 1e4
upstream factor. The excess of MeCP2 and the lack of FGF13 play multiple roles in different behaviors. The convergent effects on emotional behaviors and the opposite effects on fear memory indicate that MeCP2 regulates FGF13 in a circuit specific way. The increased MeCP2 level also causes autism like features by affecting neural circuits underlying social behaviors (Samaco et al., 2012). However, we found social behavior abnormalities present on FVB/N and C57BL/6 F1 hybrid mice are absent on C57BL/6 background mice (data not show). These finding suggest that genetic background plays a critical role in determining the social defects appeared in MeCP2 overexpression mice. Thus the social interaction tests were not able to performed for MeCP2 overexpression and Fgf13 deletion mice, due to mixed genetic background. It would be valuable to address whether acutely manipulation of FGF13 in the MeCP2 OE mice may make impacts on the emotional and cognitive behaviors in the future study. Finally, our findings that MeCP2 and FGF13 plays distinct roles in regulating emotional and cognitive behaviors, also crosstalk between MeCP2 and FGF13-related neural circuits provides further insights for molecular controls of emotional and cognitive behaviors in autism spectrum disorders and intellectual disabilities. Acknowledgments We thank Yuefang Zhang for taking care of mouse colony. This work was supported by the Strategic Priority Research Program of Chinese Academy of Sciences (Grant No. XDB02050400), and the National Natural Science Foundation of China (Grant No. #91432111) to Z.Qiu.
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Bo Yuan, Tian-lin Cheng Institute of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Kan Yang Institute of Chemistry and Bioengineering, Donghua University, Shanghai 200051, China Xu Zhang, Zilong Qiu* Institute of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Shanghai Clinical Research Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China *
Corresponding author. E-mail address: [email protected] (Z. Qiu). 29 April 2016 Available online xxx
Please cite this article in press as: Yuan, B., et al.Autism-related protein MeCP2 regulates FGF13 expression and emotional behaviors, Journal of Genetics and Genomics (2016), http://dx.doi.org/10.1016/j.jgg.2016.10.004