Activity-dependent aberrations in gene expression and PNAS PLUS alternative splicing in a mouse model of Rett syndrome Sivan Osenberga, Ariel Kartena, Jialin Suna, Jin Lib,c, Shaun Charkowicka, Christy A. Felicea, Mary Kritzerd, Minh Vu Chuong Nguyene, Peng Yub,c,1, and Nurit Ballasa,1 aDepartment of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794; bDepartment of Electrical and Computer Engineering, Texas A&M University, College Station, TX 77843; cTEES-AgriLife Center for Bioinformatics and Genomic Systems Engineering, Texas A&M University, College Station, TX 77843; dDepartment of Neurobiology and Behavior, Stony Brook University, Stony Brook, NY 11794; and eGREPI-UGA EA7408, Université Grenoble Alpes, 38400 Saint-Martin-d’Hères, France Edited by Richard H. Goodman, Vollum Institute, Portland, OR, and approved April 25, 2018 (received for review December 27, 2017) Rett syndrome (RTT) is a severe neurodevelopmental disorder that structure/function of the neuronal networks in the maturing as affects about 1 in 10,000 female live births. The underlying cause of well as in the fully matured brain (16–20). Specifically, these RTT is mutations in the X-linked gene, methyl-CpG-binding protein 2 studies have shown that reactivation of MeCP2 in symptomatic (MECP2); however, the molecular mechanism by which these muta- adult RTT mice reverses the RTT phenotype (19), while de- tions mediate the RTT neuropathology remains enigmatic. Specifically, pletion of MeCP2 even at the adult stage results in a severe RTT although MeCP2 is known to act as a transcriptional repressor, anal- phenotype (16–18, 20). In addition, other studies have shown yses of the RTT brain at steady-state conditions detected numerous that there is an excitatory/inhibitory imbalance in the RTT brain, differentially expressed genes, while the changes in transcript levels resulting specifically in hyperexcitability, which likely leads to spe- were mostly subtle. Here we reveal an aberrant global pattern of cific RTT symptoms, including seizures and breathing abnormalities gene expression, characterized predominantly by higher levels of ex- (21–25). Together, these studies suggest that MeCP2 might have a pression of activity-dependent genes, and anomalous alternative key role, specifically during neuronal network activity. splicing events, specifically in response to neuronal activity in a mouse In this study, we tested the hypothesis that MeCP2 function is model for RTT. Notably, the specific splicing modalities of intron re- required for proper regulation of the activity-dependent tran- tention and exon skipping displayed a significant bias toward in- scriptome during neuronal stimulation. We found that neuronal creased retained introns and skipped exons, respectively, in the RTT activity, elicited either in vitro by potassium chloride (KCl) or in brain compared with the WT brain. Furthermore, these aberrations vivo by kainic acid (KA), results in a significantly higher ex- occur in conjunction with higher seizure susceptibility in response to pression of many activity-dependent genes in RTT cortical neurons neuronal activity in RTT mice. Our findings advance the concept that and in the hippocampi of RTT mice, respectively, compared with normal MeCP2 functioning is required for fine-tuning the robust and their WT counterparts. Furthermore, hundreds of genes were dif- immediate changes in gene transcription and for proper regulation of alternative splicing induced in response to neuronal stimulation. ferentially alternatively spliced specifically in response to neuronal activity, of which many of them were alternatively spliced in oppo- Rett syndrome | MeCP2 | neuronal activity | gene expression | site directions (inclusion vs. exclusion) in the hippocampi of RTT alternative splicing Significance ett syndrome (RTT) is a severe postnatal neurodevelopmental Rdisorder characterized by normal development up to 12–18 mo Rett syndrome (RTT) is a severe neurological disease affecting of age, followed by regression in already acquired milestones, such girls in their early childhood. The underlying cause of most RTT as speech and motor skills, as well as by the development of a cases is mutations in the gene methyl-CpG-binding protein 2 MECP2 NEUROSCIENCE myriad of other neurological and behavioral abnormalities, in- ( ). How the loss of MeCP2 function in the brain due to cluding seizures, learning disabilities, and autism (1). The underlying these mutations causes such severe neurological symptoms cause of over 95% of RTT cases is mutations in the X-linked gene remains a mystery. Here, we show widespread aberrations in gene expression and anomalous patterns of alternative splicing, methyl-CpG-binding protein 2 (MECP2) (1, 2), yet how the loss of specifically when neurons of RTT mice are stimulated. Further- MeCP2 function in the brain mediates RTT remains poorly un- more, these aberrations occur in conjunction with higher seizure derstood. Specifically, although MeCP2 was characterized as a susceptibility in response to neuronal stimulation in these RTT transcriptional repressor, transcriptional profiling studies on the mice. Our findings suggest that MeCP2 is required for adjusting wholeRTTmousebrainandevenspecificareasofthebrain the robust changes in gene transcription and for proper regulation revealed numerous genes that are differentially expressed, while the of alternative splicing during neuronal stimulation. changes in transcript levels were mostly subtle (3–5). This conundrum has led to other studies, some of which have suggested that MeCP2 is Author contributions: S.O., A.K., J.S., S.C., C.A.F., and N.B. designed research; S.O., A.K., not a gene-specific transcriptional repressor but rather a global re- J.S., S.C., C.A.F., and M.K. performed research; S.O., A.K., J.S., J.L., S.C., C.A.F., M.V.C.N., pressor that abundantly binds methylated DNA and dampens tran- P.Y., and N.B. analyzed data; S.O., J.L., S.C., and P.Y. performed computational analysis; and S.O., A.K., J.S., M.K., P.Y., and N.B. wrote the paper. scriptional noise genomewide (6), that MeCP2 acts as both a repressor and an activator (5, 7, 8), that MeCP2 functions in a cell- The authors declare no conflict of interest. type-specific manner (9, 10), and that MeCP2 is a long-gene-specific This article is a PNAS Direct Submission. repressor (4). Other studies have suggested that MeCP2 functions Published under the PNAS license. posttranscriptionally as a regulator of alternative splicing (11, 12), or Data deposition: The data reported in this paper have been deposited in the Gene Ex- – pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. as a regulator of microRNA expression (13 15). Although these GSE113477). studies advanced our understanding of the function of MeCP2, they 1To whom correspondence may be addressed. Email: [email protected] or also clearly emphasize that the molecular mechanism which mediates [email protected]. RTT is likely complex and has yet to be deciphered. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Several recent studies, including our own, support the notion 1073/pnas.1722546115/-/DCSupplemental. that MeCP2 function is required for maintaining the proper Published online May 16, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1722546115 PNAS | vol. 115 | no. 23 | E5363–E5372 Downloaded by guest on October 2, 2021 mice. Thus, we propose that MeCP2 is required to regulate the lack of bias for specific cell types between the WT and Mecp2-null robust changes in the transcriptome that occur in response to cultures (SI Appendix,Fig.S1A and B). We further validated by neuronal activity. quantitative RT-PCR that the WT and the Mecp2-null cortical cultures expressed similar levels of neuronal and glial specific Results markers (SI Appendix, Fig. S1C). We induced membrane de- KCl-Induced Membrane Depolarization Results in Differential Expression polarization in neurons by treating the cultures with 30 mM KCl of Many Activity-Dependent Genes in Mecp2-Null Compared with WT for 3 h (without presilencing of the spontaneous neuronal activity), Neuron-Enriched Cortical Cultures. Because RTT is associated with performed RNA sequencing (RNA-Seq) analysis, and compared hyperexcitability of the cortex and hippocampus (21–23), we rea- the mRNA profiles of untreated and KCl-treated, WT and mutant soned that analysis of the gene expression profile in response to neuron-enriched cortical cultures. Our analysis shows that at neuronal activity, where the immediate and robust changes in gene steady-state conditions (untreated), only a small number of genes expression should be tightly regulated, could provide a new per- displayed differential gene expression between mutant and WT spective and important insight into the function of MeCP2. This cultures. Specifically, of the ∼14,000 expressed genes, only approach also allowed us to examine mainly the primary, rather 19 genes were down-regulated and eight genes were up-regulated than the secondary, effect of the loss of MeCP2 on the induced gene in mutant compared with the WT (Dataset S1). In contrast, expression program. analysis of the KCl-induced activity-dependent genes, as defined We first isolated cortical neurons from WT and Mecp2-null by up- or down-regulation of at least twofold (q-value < 0.05) in littermate male mice at postnatal day 0–1 and cultured them for the WT and/or mutant, showed that hundreds of genes were 10 d. We analyzed by immunostaining the composition of the differentially expressed between the mutant and WT neuron- cultures, using several cell-type-specific markers, and verified the enriched cultures (Fig. 1 A and B and Datasets S2 and S3). In- enrichment of neurons (65%) compared with glia (∼33% as- terestingly, most of the up-regulated genes showed a propensity trocytes, ∼2% oligodendrocytes, and <0.2% microglia), and the toward a higher increase in gene expression in mutant compared Fig. 1. Membrane depolarization induced by KCl results in greater changes in expression level of activity-dependent genes in Mecp2-null cortical cul- tures.