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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Caltech Authors 10912 • The Journal of Neuroscience, October 3, 2007 • 27(40):10912–10917 Neurobiology of Disease Brain-Derived Neurotrophic Factor Expression and Respiratory Function Improve after Ampakine Treatment in a Mouse Model of Rett Syndrome Michael Ogier,1 Hong Wang,1 Elizabeth Hong,2 Qifang Wang,1 Michael E. Greenberg,2 and David M. Katz1 1Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, and 2Departments of Neurology and Neurobiology, Harvard Medical School, Boston, Massachusetts 02115 Rett syndrome (RTT) is caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). Although MeCP2 is thought to act as a transcriptional repressor of brain-derived neurotrophic factor (BDNF), Mecp2 null mice, which develop an RTT-like phenotype, exhibit progressive deficits in BDNF expression. These deficits are particularly significant in the brainstem and nodose cranial sensory ganglia (NGs), structures critical for cardiorespiratory homeostasis, and may be linked to the severe respiratory abnormalities characteristic of RTT. Therefore, the present study used Mecp2 null mice to further define the role of MeCP2 in regulation of BDNF expression and neural function, focusing on NG neurons and respiratory control. We find that mutant neurons express significantly lower levels of BDNF than wild-type cells in vitro,asin vivo, under both depolarizing and nondepolarizing conditions. However, BDNF levels in mutant NG cells can be increased by chronic depolarization in vitro or by treatment of Mecp2 null mice with CX546, an ampakine drug that facilitates activation of glutamatergic AMPA receptors. Ampakine-treated Mecp2 null mice also exhibit marked functional improvement, characterized by restoration of normal breathing frequency and minute volume. These data demonstrate that BDNF expression remains plastic in Mecp2 null mice and raise the possibility that ampakine compounds could be of therapeutic value in the treatment of RTT. Key words: Mecp2 null mice; respiratory frequency; minute volume; nodose ganglion; neurotrophin expression; AMPA receptors modulator Introduction works (Viemari et al., 2005), and hyperexcitability in pontine and Rett syndrome (RTT) is an X-linked neurodevelopmental disor- vagal afferent pathways (Stettner et al., 2007). There is no treat- der caused by mutations in the methyl-CpG-binding protein 2 ment currently available for respiratory dysfunction in RTT. gene (MECP2) (Amir et al., 1999). Six to 18 months after birth, Recent studies suggest that alterations in brain-derived neu- RTT patients begin a neurological decline characterized by re- rotrophic factor (BDNF) signaling contribute to RTT pathophys- gression of acquired skills, behavioral disturbances with autistic iology. For example, Mecp2 null mice exhibit progressive deficits features (Hagberg et al., 1983), motor stereotypies, seizures, au- in BDNF levels after birth (Chang et al., 2006; Wang et al., 2006), tonomic dysfunction, and severely disordered breathing (Shah- and genetic restoration of BDNF in the forebrain improves so- bazian and Zoghbi, 2002). Respiratory abnormalities in RTT in- matomotor function and extends lifespan (Chang et al., 2006). clude alternating periods of hyperventilation and breath holds Moreover, neural structures important for cardiorespiratory and forced and apneustic breathing (Weese-Mayer et al., 2006, control, including the nodose cranial sensory ganglia (NGs) and and references therein) and may contribute to up to 26% of brainstem, exhibit the earliest and most significant known defi- deaths in RTT (Kerr et al., 1997). The primary cause of these cits in BDNF expression in the Mecp2 null mouse brain (Wang et breathing alterations is unknown, and current hypotheses in- al., 2006). Because BDNF is required for the development of NG clude cortical dysfunction (Elian and Rudolf, 1991; Marcus et al., and brainstem respiratory neurons, as well as breathing (Katz, 1994), brainstem immaturity (Julu et al., 2001), decreased nor- 2005), we hypothesize that BDNF deficits contribute to the RTT- adrenergic transmission in ponto-medullary respiratory net- like respiratory phenotype of Mecp2 null mice. The fact that Mecp2 null mice exhibit decreased BDNF expression contrasts with the prevailing view that Mecp2 is a transcrip- Received April 24, 2007; revised Aug. 3, 2007; accepted Aug. 20, 2007. This work was supported by grants from the National Heart, Lung, and Blood Institute; the Rett Syndrome tional repressor of Bdnf (Chen et al., 2003). One model proposed Research Foundation; and the National Institutes of Health to D.M.K. and M.E.G. We gratefully acknowledge Dr. to explain this apparent discrepancy is that decreased neuronal Diana Kunze for critical review of this manuscript and David T. Hellard for technical help. activity in Mecp2 null mutants (Dani et al., 2005) reduces activity- Correspondence should be addressed to Dr. David M. Katz, Department of Neurosciences, Case Western Reserve dependent BDNF expression, thereby masking any effect of de- University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.1869-07.2007 repression (Chang et al., 2006). To test this hypothesis, we exam- Copyright © 2007 Society for Neuroscience 0270-6474/07/2710912-06$15.00/0 ined BDNF expression in NG neurons cultured under Ogier et al. • Ampakines and Breathing Deficits in Rett Syndrome J. Neurosci., October 3, 2007 • 27(40):10912–10917 • 10913 depolarizing and nondepolarizing conditions. Because the NG com- prises a single neuronal cell type (sensory neurons) and exhibits the Mecp2 null BDNF phenotype in vitro as in vivo (Wang et al., 2006), it provides a simple model for exploring mechanisms that underlie BDNF regulation by MeCP2. Our data indicate that Mecp2 null cells exhibit significantly lower levels of BDNF expression than wild type, under both depolarizing and nondepolarizing conditions. However, BDNF levels in mutant cells can be elevated to wild-type resting levels by depolarizing stimuli in vitro. Similarly, we find that treatment of Mecp2 null mice with the ampakine drug 1-(1,4- benzodioxan-6-yl-carbonyl)piperidine (CX546), which enhances activation of glutamatergic AMPA receptors (Nagarajan et al., 2001), elevates NG BDNF levels in vivo. Moreover, ampakine treatment significantly improves respiratory function in Mecp2 null mice, suggesting that this class of compounds may be of therapeutic Figure1. Mecp2nullmutationisassociatedwithdecreasedexpressionofspecificBdnftran- value in RTT. scriptsinnodoseneurons.BdnftranscriptlevelsinintactNGfromwild-typeandMecp2nullmice were determined using qRT-PCR. The Bdnf gene has a complex structure in which multiple promotersdrivetheexpressionofdifferentmRNAisoformscontainingalternativenoncoding5Ј Materials and Methods exonssplicedtoacommondownstreamcodingexon[exon8;nomenclatureofLiuetal.(2006)]. Animals. Mecp2tm1–1Jae mice (Chen et al., 2001), developed by Dr. R. Total Bdnf mRNA levels (Exon 8), as well as transcripts containing exons 2, 4, and 5 were Jaenisch (Whitehead Institute, Massachusetts Institute of Technology, markedly decreased in mutant NG compared with wild type, whereas transcripts containing Cambridge, MA) and obtained from the Mutant Mouse Regional Re- exon 1 were expressed at levels that were not significantly different from wild type. Results are source Center (University of California Davis, Davis, CA), were main- the mean Ϯ SEM (n ϭ 4). ***p Ͻ 0.001, ANOVA I with post hoc Tukey’s test. tained on a mixed background (129Sv, C57BL/6, BALB/c). Male Mecp2 nulls (Mecp2Ϫ/y) were generated by crossing heterozygous Mecp2tm1–1Jae ϩ knock-out females with Mecp2tm1–1Jae wild-type males (Mecp2 /y). All F5Ј-agcggtgtaggctggaatagactc-3Ј andR5Ј-ggtggaacttctttgcggcttac-3Ј; Bdnf experimental procedures were approved by the Institutional Animal exon 4, F 5Ј-cgccatgcaatttccactatcaataatttaac-3Ј andR5Ј-cgccttcatgcaac- Care and Use Committee at Case Western Reserve University. cgaagtatg-3Ј; Bdnf exon 5, F 5Ј-gatccgagagctttgtgtggac-3Ј andR5Ј-gccttcat- Cell cultures. Wild-type and Mecp2 null mice were killed with CO2 on gcaaccgaagtatg-3Ј; ␤-tubulin III, F 5Ј-cgacaatgaagccctctacgac-3Ј andR5Ј- postnatal day 35 (P35). The NGs were removed, digested in 0.1% colla- atggtggcagacacaaggtggttg-3Ј. genase (Sigma, St. Louis, MO) in Earle’s balanced salt solution (Invitro- BDNF immunoassay. BDNF protein levels in intact NGs or in cultured gen, San Diego, CA) for 70 min at 37°C, triturated in culture medium (see NG cells were measured by ELISA using the BDNF Emax Immunoassay below) containing 0.15% BSA, and plated at a density of one NG per well System (Promega, Madison, WI). Protein extracts from one intact NG or into 96-well flat-bottom ELISA plates coated with poly-D-lysine. Cul- from an equivalent number of cultured cells were used for ELISA. tures were grown for3dinDMEM/F-12 medium supplemented with 5% ␤ MeCP2 and -tubulin III double staining. Mice were killed with CO2 fetal bovine serum (Invitrogen) and 1% penicillin–streptomycin–neo- and perfused with 4% paraformaldehyde, and the head was sectioned at mycin, with or without 40 mM potassium chloride (KCl) or 1.5 ␮M tetro- 10 ␮m with a cryostat. Sections were stained with rabbit polyclonal anti- dotoxin (TTX). MeCP2 (Upstate Biotechnology, Lake Placid, NY) and chicken poly- Ampakine treatment. Beginning on P25,

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