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Human Molecular Genetics Advance Access originally published online on February 19, 2007
Human Molecular Genetics 2007 16(6):640-650; doi:10.1093/hmg/ddm007
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

FXYD1 is an MeCP2 target gene overexpressed in the brains of Rett syndrome patients and Mecp2-null mice

Vivianne Deng1,2,{dagger}, Valerie Matagne1,{dagger}, Fatima Banine1, Matthew Frerking3, Patricia Ohliger3, Sarojini Budden4, Jonathan Pevsner5,6, Gregory A. Dissen1, Larry S. Sherman1 and Sergio R. Ojeda1,*

1 Division of Neuroscience, Oregon National Primate Research Center, Beaverton, OR, USA, 2 School of Medicine Graduate Program, Department of Cell and Developmental Biology, 3 Neurological Sciences Institute, 4 Division of Developmental Pediatrics, Oregon Health and Science University, Portland, OR, USA, 5 Department of Neurology, Kennedy Krieger Institute and 6 Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD, USA

* To whom correspondence should be addressed at: Division of Neuroscience, Oregon National Primate Research Center, 505 NW 185th Avenue, Beaverton, OR97006, USA. Tel: +1 5036905303; Fax: +1 5036905384; Email: ojedas{at}ohsu.edu

Received January 16, 2007; Revised January 16, 2007; Accepted January 30, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder linked to heterozygous de novo mutations in the MECP2 gene. MECP2 encodes methyl-CpG-binding protein 2 (MeCP2), which represses gene transcription by binding to 5-methylcytosine residues in symmetrically positioned CpG dinucleotides. Direct MeCP2 targets underlying RTT pathogenesis remain largely unknown. Here, we report that FXYD1, which encodes a transmembrane modulator of Na+,K+-ATPase activity, is elevated in frontal cortex (FC) neurons of RTT patients and Mecp2-null mice. Increasing neuronal FXDY1 expression is sufficient to reduce dendritic arborization and spine formation, hallmarks of RTT neuropathology. Mecp2-null mouse cortical neurons have diminished Na+,K+-ATPase activity, suggesting that aberrant FXYD1 expression contributes to abnormal neuronal activity in RTT. MeCP2 represses Fxyd1 transcription through direct interactions with sequences in the Fxyd1 promoter that are methylated in FC neurons. FXYD1 is therefore a MeCP2 target gene whose de-repression may directly contribute to RTT neuronal pathogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Rett syndrome (RTT) (OMIM #312750) is an X-linked neurodevelopmental disorder that ranks as the second most prevalent cause of mental retardation in girls (1). The disorder is almost exclusively diagnosed in females, because males affected by the disease usually die perinatally due to severe encephalopathy (2). Most cases of RTT are associated with mutations of MeCP2 (3–5), and loss of function of MeCP2 is primarily responsible for the pathology of RTT, although overexpression of MeCP2 can also cause symptoms that resemble RTT (6) (reviewed in 7). Supporting the notion that classical RTT is primarily due to a loss of MeCP2 function, Mecp2-null mice exhibit neurological abnormalities strikingly similar to those of RTT (8–10).

MeCP2 was originally recognized by its ability to bind specifically to methylated CpGs (11), and silence gene expression by recruiting a Sin3A-HDAC1/2 transcription repressor complex to the surrounding chromatin (12,13). Because of these characteristics MeCP2 was considered to be a global gene silencer. However, MeCP2 deficiency does not appear to result in widespread deregulation of gene expression (14), but instead might affect discrete subsets of genes required for mature neuronal functions (14,15). Several genes are now thought to be target genes for MeCP2, including brain-derived neurotrophic factor (BDNF) (16,17), distal-less homeobox 5 (DLX5) (18), the inhibitors of differentiation genes (ID1–ID4) (19), members of the glucocorticoid signaling pathway (20), a component of mitochondrial respiratory complex III (21) and corticotropin releasing hormone (22); however, the involvement of these genes in the classical neuropathology of RTT remains unclear (reviewed in 2,23). Moreover, not all of the proposed target genes for MeCP2 respond to MeCP2 dysfunction as expected: global analysis of gene expression in RTT and mouse Mecp2-null brains have failed to show increased BDNF gene expression (14,24), and measurements of BDNF protein levels in mice have yielded inconsistent results (16,17).

A fundamental morphological phenotype of RTT is a reduced complexity of the neuronal dendritic tree (25–27), and a loss of dendritic spines in some CNS neurons (28). Surprisingly, these abnormalities are largely restricted to selected subregions of the frontal and motor cortex (25–28), despite the abundance of MeCP2 throughout the brain (29). The mechanisms underlying this phenotypic regionalization remain unknown, but are likely to be related to a region-specific deregulation of genes yet to be identified. Here, we report results suggesting that one of these genes is FXYD domain-containing transport regulator 1 (FXYD1), which encodes phospholemman (PLM, FXYD1), a small, single-spanning membrane protein that controls cell excitability by modulating Na+,K+-ATPase activity (30,31).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
FXYD1 gene expression is increased in the frontal cortex of RTT patients
To identify genes that might be overexpressed in the brain of RTT patients due to MeCP2 mutations, we utilized Affymetrix oligodeoxynucleotide arrays. Because the prefrontal cortex is most distinctly affected by the disease (27), we selected the superior frontal gyrus (SFG) as a representative brain region. Total RNA extracted from SFG samples derived from five RTT and three normal controls of ages spanning the first 10 years of postnatal life was hybridized to a total of six Affymetrix Human U95v2.0 arrays. Twenty-two genes whose expression was elevated in RTT patients at all age groups (2–10 years of age) were identified (Fig. 1A, Supplementary Material, Table S1). Within this cluster, the gene most consistently overexpressed was FXYD1 (Fig. 1A, thicker line). Though not in this cluster, expression of ID1 and ID4 was also increased (1.2–4.9-fold). Notably, BDNF expression was decreased (–2.1- to –1.1-fold) in the RTT brain at all ages examined.


Figure 1
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  		Figure 1. FXYD1 gene expression is consistently upregulated in RTT brains as detected by Affymetrix GeneChip arrays and real-time PCR. (A) Total RNA extracted from the frontal lobe of five RTT patients and three normal controls with ages spanning the first 10 years of postnatal life were hybridized to six Affymetrix U95v2.0 arrays. The cluster (J-Express) shown here represents a group of 22 genes with increased expression in RTT brain when compared with age-matched normal controls across all age groups (Supplementary Material, Table S1). Each line in the cluster represents the expression pattern of a single gene. The thick line represents the FXYD1 gene. The numbers in bold indicate the fold-increase in FXYD1 gene expression detected in RTT brains. (B) Real-time PCR verification of the change in FXYD1 gene expression detected by DNA arrays in RTT patients (R2, R4, R6, R8–1 and R8–2) when compared with normal, age-matched controls (N2, N5 and N10). R2/R4 denotes a pooled RNA sample with equal contribution from R2 and R4. The same RNA samples used for the arrays were reverse-transcribed with random hexamer primers and used for real-time PCR amplification. 18S ribosomal RNA was used as an internal control for normalization purposes. For other details, see Supplementary Material, Note S1. (C) Real-time PCR analysis of FXYD1 mRNA expression in the FC from three additional RTT patients (R10, R25 and R29) and three normal controls (N15, N18 and N19) plus a re-assay of the samples previously measured [shown in (B)]. The upper left panel depicts a representative standard curve constructed with different concentrations (1 fg to 1 ng) of FXYD1 mRNA transcribed from an FXYD1 cDNA isolated from adult human brain and cloned into the vector pGEM-T Easy. The upper right panel shows a representative relative standard curve constructed by serially diluting and PCR amplifying RNA from an experimental sample (R8-1). GAPDH mRNA was used as internal control. GAPDH mRNA content of each sample was estimated by referring each individual value to the relative standard curve. The bottom panel shows FXYD1 mRNA levels detected in all RTT and normal samples. The real-time PCR reactions were carried out as described in Supplementary Material, Note S1.

 
The array results were validated by real-time PCR (Supplementary Material, Note S1) (Fig. 1B). Analysis of three additional RTT patients and three normal controls of older ages demonstrated that FXYD1 mRNA levels were increased in all RTT patients (Fig. 1C), regardless of the Brain Bank (Harvard or Kennedy Krieger, Supplementary Material, Table S2) from which they were obtained.

The FXYD1 protein is expressed in neurons
To begin elucidating the relationship that MeCP2 deficiency may have to increased FXYD1 expression, we used immunohistofluorescence-confocal microscopy (Supplementary Material, Note S2) to determine if the FXYD1 protein has a neuronal or an astroglial localization in the frontal cortex (FC) of wild-type mice. Because an earlier study (31) showed that the FXYD1 protein is specifically localized to the apical membrane of polarized epithelial cells forming the choroid plexus, we used this tissue as a positive control (Fig. 2A). In the FC, FXYD1 immunoreactivity (green color) was associated with cell membranes of neurons (identified by staining for the neuronal marker HuC/D, red color) (Fig. 2B, D and E) but was not detected in HuC/D-negative cells. Not all neurons were FXYD1-positive (arrows in B and D), and the intensity of labeling varied among positive neurons. No FXYD1 staining was seen in astrocytes (identified with GFAP antibodies, red color) (Fig. 2C, F and G) either in the vicinity of FXYD1-positive neurons (Fig. 2C and F, arrowheads) or next to FXYD1-negative cells (Fig. 2G) confirming that FXYD1 immunoreactivity is predominantly localized to neurons.


Figure 2
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  		Figure 2. Neurons, but not astrocytes, of the mouse FC contain FXYD1 protein. (A) As shown by others using a different bleeding of the same rabbit polyclonal antibodies (31), FXYD1 immunoreactive material (green color) is abundant in the apical cell membrane of the polarized epithelium lining the choroid plexus. (B) Low magnification image showing the presence of FXYD1 immunoreactivity in neurons of the FC. The cells were identified as neurons by the detection of HU/CD (red color), a neuron-specific marker. Notice that the apparent abundance of the protein varies from neuron to neuron and that some neurons are FXYD1-negative (arrows). (C) Astrocytes, identified by antibodies to GFAP (red color) are devoid of FXYD1 immunoreactive material. (D and E) Higher magnification view depicting examples of FXYD1-positive neurons. Notice that the FXYD1 immunoreactivity (green) is localized to the periphery of the cells, indicating association to the cell membrane and that some cells are FXYD1-negative [example denoted by arrow in (D)]. (F and G) High magnification view depicting FXYD1-negative astrocytes adjacent to FXYD1-positve cells [arrowheads in (F)] or to FXYD1-negative cells (G). Bars = 50 µm in (A) and (B); 20 µm in (C); 10 µm in (D–G).

 
The FXYD1 gene is overexpressed in the FC of Mecp2-null mice
We measured Fxyd1 mRNA abundance in seven regions of the WT mouse brain to test if Fxyd1 gene expression varies across the brain. Consistent with earlier findings (31), the highest mRNA levels were found in the cerebellum (CB) and the lowest in the FC (Fig. 3A). To determine whether this difference is related to a differential, MeCP2-dependent, regulation of Fxyd1 expression, we compared Fxyd1 mRNA abundance in the FC and CB of 28-day-old WT male mice with that of asymptomatic Mecp2–/y littermates (8,9). We found that Fxyd1mRNA content was increased 2–3-fold in the FC of Mecp2-deficient mice (Fig. 3B), but remained unchanged in the CB (Fig. 3C). In situ hybridization experiments showed a similar increase (Supplementary Material, Fig. S1). Thus, as observed in RTT patients, the Fxyd1 gene is also selectively overexpressed in the FC of Mecp2-deficient mice.


Figure 3
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  		Figure 3. Fxyd1 mRNA and protein content are increased, and Na+,K+-ATPase activity is reduced, in the FC of Mecp2-null mice. (A) Fxyd1 mRNA abundance varies greatly among different brain regions in wild-type mice (n = 5) as assessed by real-time PCR with lowest values detected in the FC and highest in the CB. (B) Fxyd1 mRNA abundance is significantly increased in the FC of Mecp2-null mice when compared with wild-type littermates. (C) No differences in Fxyd1 mRNA between the two groups were detected in the CB. Fxyd1 mRNA levels are expressed as arbitrary units (AU, the mean ratio of each Fxyd1 value over its respective 18S rRNA value). (D) Representative western blots showing that FXYD1 protein content is also elevated in the FC (left panel), but not the CB (right panel) of Mecp2-null mice when compared with wild-type littermates. GAPDH was used as a loading control. (E) Quantification of the western blot results illustrated in (D). The values shown in the graph were calculated by first determining the FXYD1:GADPH ratio in each sample, and then expressing this ratio for each Mecp2-deficient mice as fold-change with regard to the value observed in its respective littermate control. Bars represent group means, vertical lines are SEM and numbers in parenthesis above bars are number of animals per group. Because of the pair-wise nature of these comparisons, the statistical significance of the differences was analyzed using the paired t-test. *P < 0.05 versus wild-type group. (F) Representative experiments showing the effects of ouabain (1 mM) on the holding current of a pyramidal cell from a wild-type (Mecp2+/y) or a Mecp2–/y mouse. Bar indicates the time of ouabain application. In roughly half of the recorded cells, the effects of ouabain slowly reverted over a span of 15–20 min upon washout; however, in the remaining cells, ouabain induced a stable change in current that was followed during the wash by a sharp 1–2 nA inward current, similar to the spreading depression-like depolarization seen during ouabain application in hippocampus (56,57) (data not shown). The induction of this large inward current was not obviously affected by mouse genotype and was not analyzed further. (G) A summary graph showing the ouabain-induced current in wild-type (open circles) and Mecp2-null mice (filled circles). (H) The ouabain-induced current is significantly smaller in cells from the Mecp2-deficient mice (P = 0.02). (I) The initial holding current, measured immediately after obtaining the whole-cell configuration, and prior to the addition of ouabain, was not genotype-dependent (P = 0.94). (J) Likewise, the input resistance of cells was not genotype-dependent (P = 0.95).

 
Consistent with the mRNA results, FXYD1 protein content detected by western blot analysis (Supplementary Material, Note S3) was lower in the FC than in the CB in both WT and Mecp2–/y male mice (Fig. 3D). Further, MeCP2-deficient animals exhibited a 2-fold increase in FXYD1 protein content in the FC and unchanged levels in the CB (Fig. 3D and E). The absence of MeCP2 in the FC of Mecp2-null mice (8) was confirmed by immunohistochemistry (Supplementary Material, Fig. S2).

Na+,K+-ATPase function is altered in the FC of Mecp2-null mice
FXYD1 associates with the catalytic {alpha}-subunit isoforms of the Na+,K+-ATPase to inhibit the pump's activity (reviewed in 32). To determine whether FXYD1 overexpression is accompanied by a reduced Na+,K+-ATPase activity, we made whole-cell voltage clamp recordings from pyramidal neurons in FC slices from Mecp2-null and wild-type mice (Supplementary Material, Note S9). Because the Na+,K+-ATPase is tonically active, this activity can be assessed by monitoring the effects of ATPase inhibition. We found that the Na+,K+-ATPase inhibitor ouabain (1 mM) resulted in a current that was reduced in Mecp2-null cells when compared with wild-type cells (Fig. 3F, G and H). Neither the baseline holding current prior to the addition of ouabain (Fig. 3I) nor the input resistance (Fig. 3J) differed between the two groups, indicating that the basal electrical properties of these cells are not genotype-dependent. Thus, consistent with an increased Fxyd1 expression, Na+,K+-ATPase activity is reduced in FC neurons of Mecp2-null mice.

Neuronal dendritic tree and spine formation are reduced in Fxyd1-overexpressing FC neurons
To selectively increase the FXYD1 levels in FC neurons, we prepared primary cerebro-cortical neuronal cultures and infected them with a lentiviral construct encoding FXYD1 and an enhanced green fluorescence protein (EGFP) (Supplementary Material, Note S4). The cultures were infected 1 h after seeding with a lentivirus encoding FXYD1 (LV-FXYD1) or the lentivirus alone (LV-EGFP) and were processed for morphological evaluation 4 days later. To determine the level of expression attained, we first infected HiB5 hippocampal cells with LV-FXYD1 at different multiplicities of infection and found that 10 viral particles/cell resulted in FXYD1 protein levels similar to those detected in the FC of Mecp2-null mice (Fig. 4A). LV-FXYD1-expressing neurons had a reduced dendritic arborization when compared with LV-EGFP-infected neurons (examples of control neurons in Fig. 4B and C and of FXYD1-overexpressing neurons in Fig. 4D and E). Examination of dendrites at higher magnification demonstrated a striking reduction of spines in neurons overexpressing FXYD1 when compared with LV-EGFP-infected controls (Fig. 4F and G). Sholl analysis (Supplementary Material, Note S5) demonstrated quantitatively the reduction in dendritic tree complexity in FXYD1-overexpressing neurons when compared with controls (Fig. 4H and I). These findings suggest that an increase in FXYD1 expression may contribute to the morphological deficits observed in the FC of RTT patients (25–28).


Figure 4
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  		Figure 4. Increasing FXYD1 expression in neuronal cultures derived from mouse cerebral cortex reduces neuronal dendritic arborization and number of dendritic spines. The cultures were prepared from 9-day-old mice and infected with a lentiviral construct expressing mouse (m)FXYD1a plus EGFP (LV-FXYD1) or the lentivirus alone encoding only an enhanced green fluorescent protein (LV-EGFP) as described in Supplementary Material, Note S6. The cells were cultured for 4 days in the presence of the viruses, before processing them for double immunohistofluorescence and subsequent morphometric analysis. A monoclonal antibody to ß-III Tubulin was used to identify neurons and a polyclonal antibody to EGFP to identify LV- infected neurons. (A) Western blot comparing the FXYD1 protein levels achieved in HiB5 cells infected with different multiplicities of infection with the FXYD1 protein content detected in the FC of Mecp2-null mice. (B and C) Examples of neurons infected with the control virus LV-EGFP. (D and E) Examples of neurons infected with LV-FXYD1. Notice the decreased dendritic arborization of LV-FXYD1-infected cells. (F) Higher magnification view showing the presence of numerous spines (examples denoted by arrowheads) in LV-EGFP-infected cells. (G) Relative absence of spines in LV-FXYD1-infected neurons. (H) Examples of a control neuron subjected to Sholl analysis. (I) Sholl analysis showing the decreased dendritic arborization resulting from LV-mediated increase in FXYD1 expression. *P < 0.01 versus LV-EGFP. Bars = 25 µm in (B–E); 5 µm in (F) and (G).

 
The two alternative promoters of the FXYD1 gene are differentially methylated in the human brain
To determine whether FXYD1 expression is repressed in a DNA methylation-dependent fashion, we treated two human cell lines with 5'-aza-cytidine (Supplementary Material, Note S6) and found that FXYD1 mRNA levels increase markedly in both cell lines in response to this DNA demethylating agent (Supplementary Material, Fig. S3).

Two transcript variants of human FXYD1 (FXYD1a and FXYD1b) with different 5'-UTR sequences in the same coding region have been described (33). In silico analysis (http://www.Genomatix.de) indicated that FXYD1a and 1b appear to be transcribed from alternative transcription sites using different promoters. Bisulfite conversion of genomic human brain DNA followed by PCR amplification of either the FXYD1b or the FXYD1a promoter (Fig. 5A, bent arrows) (Supplementary Material, Note S7) showed that both promoters contain methylated cytosines. However, only the FXYD1a promoter has a predicted CpG island (http://www.ebi.ac.uk/emboss/cpgplot/) (Fig. 5A). This region also contains the putative binding sites of SP1 and other basal transcription factors such as TBP and TFIID (http://cbil.upenn.edu/tess). The human heart, a tissue in which the FXYD1 gene is highly expressed (33), showed a much lower degree of FXYD1 promoter methylation than the brain (Supplementary Material, Fig. S4). These results suggest that, because of their different degree of methylation, the FXYD1 promoters may be differentially regulated by MeCP2 in the human brain when compared with the heart.


Figure 5
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  		Figure 5. The two alternative FXYD1 promoters are methylated, and MeCP2 physically associates to the methylated regions. (A) Mapping the methylation status of the FXYD1 promoter region in human brain by bisulfite genomic DNA sequencing. Genomic DNA isolated from human brain was subjected to bisulfite treatment. The bisulfite-converted DNA was PCR-amplified with a set of primers favoring bisulfite-converted DNA and targeting 400–500 bp DNA segments in the two putative FXYD1 promoter regions. The PCR products were cloned into the pGEM-T EASY vector and the plasmid DNA from at least 10 individual colonies was purified for subsequent DNA sequencing. The top diagram depicts the 5' flanking region of the hFXYD1 gene with its two alternative transcription start sites (TSS1 and TSS2). Bent arrows show the location of primers used for PCR amplification of bisulfite-converted genomic DNA. Notice that a CpG island was detected only in the region containing the alternative promoter 1a (red box). The positions of CpG sites are numbered relative to the annotated FXYD1b or FXYD1a mRNA start site (+1) for each promoter. Each row represents the individually sequenced PCR products. Methylated-CpG sites are shown as closed circles and unmethylated CpG sites as open circles. (B–G) MeCP2 binds to both the human and the mouse Fxyd1 promoters, and this association does not occur in Mecp2 KO mice. (B) Diagram depicting the two sets of primer pairs (hP1, hP2) used to PCR-amplify DNA segments from the 5' flanking region of the human FXYD1 gene. (C) Each set of primers amplifies DNA fragments from the hFXYD1 promoter immunoprecipitated by MeCP2 antibodies (arrows). Protein–DNA complexes derived from human 293T cells transfected with HA-MeCP2 were cross-linked by incubation with 1% formaldehyde at 37°C. The chromatin was ultrasonically sheared to a length ranging from 500 to 1000 bp. ChIP assays were performed using MeCP2 or HDAC1 polyclonal antibodies, using normal rabbit serum as a negative control (–). (D) Western blot showing that the cells transfected with HA-MeCP2 express the MeCP2 protein, detected as a band identical in size to MeCP2 present in mouse cerebral cortex (CTX). (E) Endogenous HDAC1 also associates to the human FXYD1 promoter region in cells transfected with HA-MeCP2 as HDAC1 antibodies immunoprecipitate protein–DNA complexes containing a DNA segment (hP2) from the hFXYD1 promoter (arrow). (F) Diagram showing the two sets of primers (mP1, mP2) used to PCR-amplify specific DNA segments from the mouse Fxyd1 promoters. The DNA was extracted from the FC of 45-day-old wild-type and Mecp2 KO mice. (G) Protein–DNA complexes immunoprecipitated with MeCP2 antibodies from the FC of wild-type mice contain each of the DNA segments from the mFxyd1 promoter targeted for PCR amplification, indicating that endogenous MeCP2 is associated with the native mFxyd1 promoter under physiological conditions. This association was not present in the FC of Mecp2-null mice. Inp, non-immunoprecipitated input DNA (1/100th of the total sheared DNA); nrs, normal rabbit serum.

 
The FXYD1 promoter is an endogenous Mecp2 target in vivo
MeCP2 selectively binds to a single methylated CpG dinucleotide site and recruits a histone deacetylase (HDAC)-containing complex, resulting in chromatin condensation and gene silencing (34). To determine whether FXYD1 is a MeCP2 target in vivo, we performed chromatin immunoprecipitation (ChIP) assays (Supplementary Material, Note S8) on both the human and mouse FXYD1 promoter. The assays, using human (h) FXYD1 promoter-specific primers (Fig. 5B), revealed that MeCP2 binds to the native hFXYD1 promoter region in human embryonic kidney 293T cells transiently transfected with a pCMV-HA-MeCP2 expressing vector (Fig. 5C). Western blots verified the presence of MeCP2 after HA-MeCP2 transfection (Fig. 5D). In additional ChIP assays, and using a polyclonal antibody against HDAC1, we detected endogenous HDAC1 associated with the hFXYD1 promoter region (Fig. 5E), suggesting that, as typically shown in human cell lines and rodent brain nuclear extracts (34), an HDAC1-containing co-repressor complex is recruited to the hFXYD1 promoter region.

To determine whether the native Fxyd1 promoter is an endogenous MeCP2 target in the mouse FC, we conducted ChIP assays using cortical tissue from wild-type and Mecp2-deficient mice and PCR primers that amplify either the Fxyd1b or Fxyd1a promoter regions (Fig. 5F). MeCP2 was recruited to the Fxyd1b and 1a promoter regions in the FC of wild-type, but not Mecp2-null mice (Fig. 5G), demonstrating that under physiological conditions endogenous MeCP2 associates with the native Fxyd1 promoter in the mouse brain.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
We have identified FXYD1 as a novel MeCP2 target gene that is selectively overexpressed in the FC of both RTT patients and Mecp2-null mice. FXYD1 in the FC is subjected to MeCP2-mediated, DNA methylation-dependent transcriptional repression. Elevating Fxyd1 expression in neuronal cultures is sufficient to recapitulate the loss of dendritic arborization (25–27) and dendritic spines (28) observed in FC neurons of RTT patients, suggesting that derepression of FXYD1 may contribute significantly to the neuropathogenesis of RTT.

FXYD1 has a region-specific distribution in the CNS, with highest levels seen in the choroid plexus and CB and low levels in the rest of the brain (31). In mouse FC, we found that FXYD1 immunoreactivity is selectively present in neurons, suggesting that the wide variation in Fxyd1 mRNA prevalence we observed in different regions of wild-type mouse brains and the increased FXYD1 mRNA abundance detected in the FC of RTT patients and Mecp2-null mice reflect changes in neuronal, but not glial, FXYD1 expression.

MeCP2 likely controls gene expression in a region-dependent manner given the localized neuropathology of RTT (27,35). We found that MeCP2 selectively regulates FXYD1 in the FC, which is most severely affected in RTT (27). In contrast, Mecp2-null mice showed no deregulation of Fxyd1 in the CB. The mechanisms underlying this difference remain to be identified, but they may be related to a differential degree of methylation of the Fxyd1 gene or to a different binding capacity of MeCP2 to the Fxyd1 promoters in these brain regions.

Our results indicate that a reduced neuronal Na+,K+-ATPase activity is a distinct abnormality of FC neurons lacking MeCP2. This finding is in keeping with earlier reports indicating that FXYD1 inhibits Na+,K+-ATPase (reviewed in 32) and with recent electrophysiological evidence showing that FXYD1 overexpression leads to reduced Na+,K+-ATPase activity (36). Although the pump's activity in the heart is reduced in Fxyd1-null mice, this change may be secondary to downregulated ATPase subunit expression (37). Partial inhibition of Na+,K+-ATPase activity can increase neuronal excitability (38,39) and has been implicated as a mechanism underlying epileptogenic seizures (38–40), a condition commonly observed in RTT patients. A relative inability of the neurons to maintain adequate Na+ and K+ gradients across the cell membrane might result in abnormal neuronal excitability when these gradients are stressed. However, the observation that the intrinsic excitability of cortical neurons from Mecp2–/y mice is normal in response to brief current injections (41) suggests that higher levels of activity may be required to stress these gradients sufficiently for the impairment in Na+,K+-ATPase activity to have an effect.

Several previous studies have reported enhanced glutamate release and/or impaired long-term plasticity in the hippocampus in both the Mecp2-null mice used here and in Mecp2308/Y truncation mutants (42–44). Surprisingly, results from the FC suggest that basal glutamatergic transmission is impaired in Mecp2-null mice rather than enhanced (41), suggesting that synaptic defects may not be identical across different brain regions. Regardless, the defects in synaptic plasticity and transmission that have been described so far are likely to be fundamentally distinct from the electrophysiological defects on cellular excitability reported here, because the defects we observed were detected in the presence of the voltage-gated Ca2+ channel blocker cadmium to block depolarization-evoked synaptic transmission.

A loss of dendritic arborization and dendritic spines in subpopulations of neurons, especially in the FC (10,25,27), are hallmarks of RTT neuropathology. Our results showing loss of dendritic arborization and spine formation following overexpression of FXYD1 suggest that deregulation of FXYD1 is one of the factors underlying this neuropathology. Dendritic arborization is not affected in Mecp2308/Y mutant mice (42), but is reduced in Mecp2 null mice (10). Moreover, knockdown of MeCP2 using shRNA leads to a reduction in the complexity of the dendritic arbor (45), similar to the results seen in the null mice. The reason for the discrepancy between the Mecp2308/Y mice and both null mice and RTT patients remains unclear. Because FXYD1 is overexpressed in the FC of RTT patients and Fxyd1 overexpression leads to alterations in dendritic arborization, our results raise the possibility that FXYD1 overexpression may be a cause of the dendritic abnormalities observed in RTT patients, even if these abnormalities are not expressed in Mecp2308/Y mice for an as-yet unknown reason. It remains unclear how FXYD1 causes the dendritic abnormalities seen here, but it is tempting to speculate that morphological defects may follow the initial loss of ionic neuronal homeostasis caused by a chronic FXYD1-dependent reduction in Na+,K+-ATPase activity.

In an earlier publication, Colantuoni et al. (24). used membrane-based, low-density cDNA arrays to analyze the gene expression profile of brain tissue from RTT patients and matched controls and reached the conclusion that the RTT brain had increased expression of a number of glial transcripts and decreased expression of neuron-specific mRNAs. This decrease was particularly evident in transcripts encoding presynaptic proteins. Our results are not directly comparable to those of Colantuoni et al., because we used a different brain region for analysis (SFG) in our study versus the Broadman areas 1–5 (junction between the frontal and parietal cortex) in their analysis. Despite this difference, we consider that our studies complement and expand those earlier findings (24) by showing that along with the increased abundance of gene transcripts expressed in glia, such as S100ß, the excitatory amino acid co-transporter EAAT2, glypican and FGFR3; there is also an increased expression of several neuronal genes involved in neural development and mature function (Supplementary Material, Table S1). FXYD1 is one such gene.

Collectively, our data indicate that FXYD1 is important for maintaining normal neuronal activity and morphology. Given that mutations in Na+,K+-ATPase subunits have been linked to several genetic disorders that share clinical features and neuronal abnormalities with RTT (46–48), FXYD1 may contribute to the pathogenesis of other brain disorders linked to aberrant MeCP2 expression or activity including schizophrenia and age-related neurodegenerative disease (49,50). Our finding that FXYD1 overexpression is sufficient to promote key features of RTT pathogenesis raises the possibility of using drugs blocking FXYD1 function to ameliorate RTT symptoms that may be caused by an excess of FXYD1 production.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Tissue acquisition and RNA isolation
Human brain tissue from the FC of RTT patients and age-matched normal controls were acquired from two sources: the Kennedy Krieger Research Institute and the Harvard Brain Tissue Resource Center (Supplementary Material, Table S2). The Mecp2-null mice and their littermate wild-type controls were obtained by breeding heterozygous Mecp2-deficient female mice (JAX: strain B6.129P2c-Mecp2tm1.1Bird/J) with wild-type C57BL/6 males. RNA was extracted as described in Supplementary Material, Note S1.

Affymetrix GeneChip oligonucleotide array analysis
Total RNA from five RTT patients was pooled to generate samples representing three age groups (2–4, 5–6 and 8–10 years of age) and these samples were compared with their respective age-matched normal controls. Total RNA was labeled and hybridized to six Affymetrix GeneChip oligonucleotide arrays at the Gene Microarray Shared Resource (Oregon Health and Science University, Portland, OR, USA). To ensure the quality of hybridization, labeled RNA samples were first hybridized to a test array containing 345 control probe sets in total, including 37 human probe sets. Quality-tested samples were then hybridized to the HU_U95Av2 array containing 12 625 human probes. Results were processed with Affymetrix Microarray Suite 4.0 (MAS 4.0, Santa Clara, CA, USA). Comparative analysis was performed for each paired sample (i.e. RTT patients and age-matched normal controls) and the difference in hybridization intensity between each pair of samples was calculated and expressed as a fold-change (RTT/normal control). Genes showing an absence call on all six arrays and genes consistently showing less than 2-fold change in either direction between RTT and normal controls in any age group were removed from the analysis. Genes sharing a similar expression pattern in all age groups were clustered and visualized via a K-means clustering algorithm using J-Express 2.0 software (De Nova, Vancouver, BC, Canada).

Real-time and semi-quantitative PCR
We used real-time PCR to verify and expand the results of the DNA arrays, and to measure Fxyd1 mRNA levels in the brain of Mecp2-null mice and their respective controls. The procedure (Supplementary Material, Note S1) has been described in detail elsewhere (51).

Immunohistofluorescence and in situ hybridization
To identify the cell types containing FXYD1 immunoreactive protein in the mouse FC, we used double immunofluorescence-confocal microscopy as previously described (Supplementary Material, Note S2). FXYD1 was identified with rabbit polyclonal antibodies (C2, generously provided by Dr Randall Moorman, University of Virginia, Charlottesville, VA, USA), and previously characterized by others (36). Neurons were identified by staining with a monoclonal antibody to HuC/HuD, a neuronal-specific protein; astrocytes were recognized with a monoclonal antibody to GFAP. To localize Fxyd1 mRNA in the mouse brain, we utilized a 274 nt cRNA probe transcribed from a cDNA template generated by PCR amplification of mouse cerebral cortex RNA. The procedure used is briefly described in Supplementary Material, Note S2.

Western blots
FXYD1 and MeCP2 were detected in immunoblots using previously characterized polyclonal antibodies (9,31), and following a procedure described in Supplementary Material, Note S3.

Preparation of lentiviral constructs, neuronal cultures and morphological analysis
The coding sequence of mFxyd1a (NM_019503 [GenBank] ) was PCR-amplified from total RNA extracted from mouse cerebral cortex and was cloned into a lentiviral vector that also expresses EGFP (Supplementary Material, Note S4). Viral particles encoding Fxyd1a plus EGFP or only EGFP were used to infect primary cultures of mouse cerebro-cortical neurons. The neurons were cultured as recommended (52), with the modifications described in Supplementary Material, Note S4. Neuronal dendritic arborization and spine formation were analyzed as described in Supplementary Material, Note S5.

Plasmid constructs, transient transfections and 5-aza-cytidine treatment
The pCMV-HA-MeCP2 construct was the generous gift from A. Bird (University of Edinburgh, UK). Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA). When reaching 70% confluence, the cells were transfected with 1 µg of pCMV-HA-MeCP2 using Lipofectamine 2000 (Invitrogen) according to manufacturer's protocol. After 48 h, the cells were subjected to ChIP assay. To determine whether FXYD1 expression is normally repressed in human cells in a methylation-dependent manner, human cells lines were treated with the demethylating agent 5-aza-cytidine and FXYD1 mRNA were measured by semi-quantitative PCR, as outlined in Supplementary Material, Note S6.

Bisulfite PCR sequencing of human and mouse genomic DNA
Genomic DNA from adult human brain and heart were purchased from BioChain, Inc. (Hayward, CA, USA). Genomic DNA from the mouse FC and CB was extracted as described (53). The presence of 5'-methylcytosines was determined by bisulfite genomic sequencing. Bisulfite conversion of DNA was performed as described (54), with some modifications. This procedure and that used to PCR amplify bisulfite-treated DNA are described in Supplementary Material, Note S7. The primers used are listed in Supplementary Material, Table S3.

ChIP assay
The ChIP assay was carried out using commercial ChIP Assay Kits, as described in Supplementary Material, Note S8. The PCR primers used for amplification are listed in Supplementary Material, Table S3.

Electrophysiology
Slice preparation and recording techniques were similar to those described previously (55) and are described in Supplementary Material, Note S9.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
These studies were supported by the Northwest Rett Syndrome Foundation, in addition to NIH grants HD25123, U-54 HD18185 and RR00163 (SRO), NS045101 (M.F.) and a grant from the Christopher Reeves Paralysis Foundation (L.S.S.). V.D. presented material contained in this paper in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Oregon Health and Sciences University School of Medicine Graduate Program in the Department of Cell and Developmental Biology, Portland, OR. V.M. was supported by the Belgian American Educational Foundation, Francqui and Coleen Fellowship.

Conflict of Interest statement. None declared.

The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo) and are accessible through GEO Series accession number GSE6965 [NCBI GEO] .


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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