Ultrasound-Mediated Blood-Brain Barrier Opening Improves Whole Brain Gene Delivery in Mice.

Gene therapy represents a powerful therapeutic tool to treat diseased tissues and provide a durable and effective correction. The central nervous system (CNS) is the target of many gene therapy protocols, but its high complexity makes it one of the most difficult organs to reach, in part due to the blood-brain barrier that protects it from external threats. Focused ultrasound (FUS) coupled with microbubbles appears as a technological breakthrough to deliver therapeutic agents into the CNS. While most studies focus on a specific targeted area of the brain, the present work proposes to permeabilize the entire brain for gene therapy in several pathologies. Our results show that, after i.v. administration and FUS sonication in a raster scan manner, a self-complementary AAV9-CMV-GFP vector strongly and safely infected the whole brain of mice. An increase in vector DNA (19.8 times), GFP mRNA (16.4 times), and GFP protein levels (17.4 times) was measured in whole brain extracts of FUS-treated GFP injected mice compared to non-FUS GFP injected mice. In addition to this increase in GFP levels, on average, a 7.3-fold increase of infected cells in the cortex, hippocampus, and striatum was observed. No side effects were detected in the brain of treated mice. The combining of FUS and AAV-based gene delivery represents a significant improvement in the treatment of neurological genetic diseases.

Analysis of Astroglial Secretomic Profile in the Mecp2-Deficient Male Mouse Model of Rett Syndrome.

Mutations in the X-linked MECP2 gene are responsible for Rett syndrome (RTT), a severe neurological disorder. MECP2 is a transcriptional modulator that finely regulates the expression of many genes, specifically in the central nervous system. Several studies have functionally linked the loss of MECP2 in astrocytes to the appearance and progression of the RTT phenotype in a non-cell autonomous manner and mechanisms are still unknown. Here, we used primary astroglial cells from Mecp2-deficient (KO) pups to identify deregulated secreted proteins. Using a differential quantitative proteomic analysis, twenty-nine proteins have been identified and four were confirmed by Western blotting with new samples as significantly deregulated. To further verify the functional relevance of these proteins in RTT, we tested their effects on the dendritic morphology of primary cortical neurons from Mecp2 KO mice that are known to display shorter dendritic processes. Using Sholl analysis, we found that incubation with Lcn2 or Lgals3 for 48 h was able to significantly increase the dendritic arborization of Mecp2 KO neurons. To our knowledge, this study, through secretomic analysis, is the first to identify astroglial secreted proteins involved in the neuronal RTT phenotype in vitro, which could open new therapeutic avenues for the treatment of Rett syndrome.

Severe offtarget effects following intravenous delivery of AAV9-MECP2 in a female mouse model of Rett syndrome.

Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). RTT is the second most prevalent genetic cause of intellectual disability in girls, and there is currently no cure for the disease. We have previously shown that gene therapy using a self-complementary AAV9 viral vector expressing a codon-optimized Mecp2 version (AAV9-MCO) significantly improved symptoms and increased survival in male Mecp2-deficient mice. Here, we pursued our studies and investigated the safety and efficacy of long-term gene therapy in the genetically relevant RTT mouse model: the heterozygous (HET) Mecp2 deficient female mouse. These mice were injected with the AAV9-MCO vector through the tail vein and an array of behavioral tests was performed. At 16- and 30-weeks post-injection, this treatment was able to rescue apneas and improved the spontaneous locomotor deficits and circadian locomotor activity in Mecp2 HET mice treated with AAV9-MCO at a dose of 5 × 1011 vg/mouse. To examine whether a higher dose of vector could result in increased improvements, we injected Mecp2 HET mice with a higher MCO vector dose (1012 vg/mouse), which resulted in some severe, sometimes lethal, side effects. In order to confirm these effects, a new cohort of Mecp2 HET mice were administered increasing doses of MCO vector (1011, 5 × 1011 and 1012 vg/mouse). Again, two weeks after vector administration, some Mecp2 HET mice were found dead while others displayed severe side effects and had to be euthanized. These deleterious effects were not observed in Mecp2 HET mice injected with a high dose of AAV9-GFP and were directly proportionate to vector dosage (0, 23 or 54% mortality at an AAV9-MCO dose of 1011, 5 × 1011, 1012 vg/mouse, respectively), and no such lethality was observed in wild-type (WT) mice. In the Mecp2 HET mice treated with the high and medium AAV9-MCO doses, blood chemistry analysis and post-mortem histology showed liver damage with drastically elevated levels of liver transaminases and disorganized liver architecture. Apoptosis was confirmed by the presence of TUNEL- and cleaved-caspase 3-positive cells in the Mecp2 HET mice treated with the higher doses of AAV9-MCO. We then studied the involvement of the unfolded protein response (UPR) in triggering apoptosis since it can be activated by AAV vectors. Increased expression of the C/EBP homologous protein (CHOP), one of UPR downstream effectors, was confirmed in Mecp2 HET mice after vector administration. The toxic reaction seen in some treated mice indicates that, although gene therapy for RTT improved breathing deficits observed in Mecp2 HET mice, further studies are needed to better understand the underlying mechanisms and caution must be exercised before similar attempts are undertaken in female Rett patients.

Huntingtin phosphorylation governs BDNF homeostasis and improves the phenotype of Mecp2 knockout mice.

Mutations in the X-linked MECP2 gene are responsible for Rett syndrome (RTT), a severe neurological disorder for which there is no treatment. Several studies have linked the loss of MeCP2 function to alterations of brain-derived neurotrophic factor (BDNF) levels, but non-specific overexpression of BDNF only partially improves the phenotype of Mecp2-deficient mice. We and others have previously shown that huntingtin (HTT) scaffolds molecular motor complexes, transports BDNF-containing vesicles, and is under-expressed in Mecp2 knockout brains. Here, we demonstrate that promoting HTT phosphorylation at Ser421, either by a phospho-mimetic mutation or inhibition of the phosphatase calcineurin, restores endogenous BDNF axonal transport in vitro in the corticostriatal pathway, increases striatal BDNF availability and synaptic connectivity in vivo, and improves the phenotype and the survival of Mecp2 knockout mice-even though treatments were initiated only after the mice had already developed symptoms. Stimulation of endogenous cellular pathways may thus be a promising approach for the treatment of RTT patients.

Correcting deregulated Fxyd1 expression rescues deficits in neuronal arborization and potassium homeostasis in MeCP2 deficient male mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the MECP2 gene. In the absence of MeCP2, expression of FXYD domain-containing transport regulator 1 (FXYD1) is deregulated in the frontal cortex (FC) of mice and humans. Because Fxyd1 is a membrane protein that controls cell excitability by modulating Na+, K+-ATPase activity (NKA), an excess of Fxyd1 may reduce NKA activity and contribute to the neuronal phenotype of Mecp2 deficient (KO) mice. To determine if Fxyd1 can rescue these RTT deficits, we studied the male progeny of Fxyd1 null males bred to heterozygous Mecp2 female mice. Maximal NKA enzymatic activity was not altered by the loss of MeCP2, but it increased in mice lacking one Fxyd1 allele, suggesting that NKA activity is under Fxyd1 inhibitory control. Deletion of one Fxyd1 allele also prevented the increased extracellular potassium (K+) accumulation observed in cerebro-cortical neurons from Mecp2 KO animals in response to the NKA inhibitor ouabain, and rescued the loss of dendritic arborization observed in FC neurons of Mecp2 KO mice. These effects were gene-dose dependent, because the absence of Fxyd1 failed to rescue the MeCP2-dependent deficits, and mimicked the effect of MeCP2 deficiency in wild-type animals. These results indicate that excess of Fxyd1 in the absence of MeCP2 results in deregulation of endogenous K+ conductances functionally associated with NKA and leads to stunted neuronal growth.

Rett syndrome from bench to bedside: recent advances.

Rett Syndrome is a severe neurological disorder mainly due to de novo mutations in the methyl-CpG-binding protein 2 gene ( MECP2). Mecp2 is known to play a role in chromatin organization and transcriptional regulation. In this review, we report the latest advances on the molecular function of Mecp2 and the new animal and cellular models developed to better study Rett syndrome. Finally, we present the latest innovative therapeutic approaches, ranging from classical pharmacology to correct symptoms to more innovative approaches intended to cure the pathology.

A codon-optimized Mecp2 transgene corrects breathing deficits and improves survival in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). RTT is the second most prevalent cause of intellectual disability in girls and there is currently no cure for the disease. The finding that the deficits caused by the loss of Mecp2 are reversible in the mouse has bolstered interest in gene therapy as a cure for RTT. In order to assess the feasibility of gene therapy in a RTT mouse model, and in keeping with translational goals, we investigated the efficacy of a self-complementary AAV9 vector expressing a codon-optimized version of Mecp2 (AAV9-MCO) delivered via a systemic approach in early symptomatic Mecp2-deficient (KO) mice. Our results show that AAV9-MCO administered at a dose of 2×1011 viral genome (vg)/mouse was able to significantly increase survival and weight gain, and delay the occurrence of behavioral deficits. Apneas, which are one of the core RTT breathing deficits, were significantly decreased to WT levels in Mecp2 KO mice after AAV9-MCO administration. Semi-quantitative analysis showed that AAV9-MCO administration in Mecp2 KO mice resulted in 10 to 20% Mecp2 immunopositive cells compared to WT animals, with the highest Mecp2 expression found in midbrain regions known to regulate cardio-respiratory functions. In addition, we also found a cell autonomous increase in tyrosine hydroxylase levels in the A1C1 and A2C2 catecholaminergic Mecp2+ neurons in treated Mecp2 KO mice, which may partly explain the beneficial effect of AAV9-MCO administration on apneas occurrence.

Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression.

In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty.

GABA and glutamate pathways are spatially and developmentally affected in the brain of Mecp2-deficient mice.

Proper brain functioning requires a fine-tuning between excitatory and inhibitory neurotransmission, a balance maintained through the regulation and release of glutamate and GABA. Rett syndrome (RTT) is a rare genetic disorder caused by mutations in the methyl-CpG binding protein 2 (MECP2) gene affecting the postnatal brain development. Dysfunctions in the GABAergic and glutamatergic systems have been implicated in the neuropathology of RTT and a disruption of the balance between excitation and inhibition, together with a perturbation of the electrophysiological properties of GABA and glutamate neurons, were reported in the brain of the Mecp2-deficient mouse. However, to date, the extent and the nature of the GABA/glutamate deficit affecting the Mecp2-deficient mouse brain are unclear. In order to better characterize these deficits, we simultaneously analyzed the GABA and glutamate levels in Mecp2-deficient mice at 2 different ages (P35 and P55) and in several brain areas. We used a multilevel approach including the quantification of GABA and glutamate levels, as well as the quantification of the mRNA and protein expression levels of key genes involved in the GABAergic and glutamatergic pathways. Our results show that Mecp2-deficient mice displayed regional- and age-dependent variations in the GABA pathway and, to a lesser extent, in the glutamate pathway. The implication of the GABA pathway in the RTT neuropathology was further confirmed using an in vivo treatment with a GABA reuptake inhibitor that significantly improved the lifespan of Mecp2-deficient mice. Our results confirm that RTT mouse present a deficit in the GABAergic pathway and suggest that GABAergic modulators could be interesting therapeutic agents for this severe neurological disorder.

Correcting deregulated Fxyd1 expression ameliorates a behavioral impairment in a mouse model of Rett syndrome.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the MECP2. Several genes have been shown to be MECP2 targets. We previously identified FXYD1 (encoding phospholemman; a protein containing the motif phenylalanine-X-tyrosine-aspartate), a gene encoding a transmembrane modulator of the Na, K-ATPase (NKA) enzyme, as one of them. In the absence of MECP2, FXYD1 expression is increased in the frontal cortex (FC) of both RTT patients and Mecp2(Bird) null mice. Here, we show that Fxyd1 mRNA levels are also increased in the FC and hippocampus (HC) of male mice carrying a truncating mutation of the Mecp2 gene (Mecp2(308)). To test the hypothesis that some of the behavioral phenotypes seen in these Mecp2 mutants could be ameliorated by genetically preventing the Fxyd1 response to MECP2 deficiency, we crossed Fxyd1 null male mice with Mecp2(308) heterozygous females and behaviorally tested the adult male offspring. Mecp2(308) mice had impaired HC-dependent novel location recognition, and this impairment was rescued by deletion of both Fxyd1 alleles. No other behavioral or sensorimotor impairments were rescued. These results indicate that reducing FXYD1 levels improves a specific cognitive impairment in MECP2-deficient mice.

Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates.

Other labs have previously reported the ability of adeno-associated virus serotype 9 (AAV9) to cross the blood-brain barrier (BBB). In this report, we carefully characterized variables that might affect AAV9's efficiency for central nervous system (CNS) transduction in adult mice, including dose, vehicle composition, mannitol coadministration, and use of single-stranded versus self-complementary AAV. We report that AAV9 is able to transduce approximately twice as many neurons as astrocytes across the entire extent of the adult rodent CNS at doses of 1.25 × 10¹², 1 × 10¹³, and 8 × 10¹³ vg/kg. Vehicle composition or mannitol coadministration had only modest effects on CNS transduction, suggesting AAV9 crosses the BBB by an active transport mechanism. Self-complementary vectors were greater than tenfold more efficient than single-stranded vectors. When this approach was applied to juvenile nonhuman primates (NHPs) at the middle dose (9-9.5 × 10¹² vg/kg) tested in mice, a reduction in peripheral organ and brain transduction was observed compared to mice, along with a clear shift toward mostly glial transduction. Moreover, the presence of low levels of pre-existing neutralizing antibodies (NAbs) mostly occluded CNS and peripheral transduction using this delivery approach. Our results indicate that high peripheral tropism, limited neuronal transduction in NHPs, and pre-existing NAbs represent significant barriers to human translation of intravascular AAV9 delivery.

Brain region-specific expression of Fxyd1, an Mecp2 target gene, is regulated by epigenetic mechanisms.

Fxyd1 encodes a trans-membrane protein that modulates Na(+) ,K(+) -ATPase activity and is a substrate for multiple protein kinases. Fxyd1 expression is repressed by methyl CpG-binding protein 2 (Mecp2) in the frontal cortex (FC) but not in the cerebellum (CB) of the mouse brain. Consistently with these observations, FXYD1 mRNA abundance is increased in the FC of Rett syndrome (RTT) patients with MECP2 mutations. Because Fxyd1 is implicated in the regulation of neuronal excitability, understanding how Fxyd1 expression is controlled is important. Here we report that basal expression of Fxyd1a and Fxyd1b, the two main alternatively spliced forms of Fxyd1 mRNA, is lower in the FC than in the CB. This difference is accompanied by increased Mecp2 recruitment to the promoter region of these two Fxyd1 mRNA forms. DNA methylation of both promoters is more frequent in the FC than in the CB, and in both cases the most frequently methylated CpG dinucleotides are adjacent to [A/T](4) sequences required for high-affinity Mecp2 binding. Consistently with these features of epigenetic silencing, histone 3 acetylated at lysines 9 and 14 (H3K9/14ac) and histone 3 methylated at lysine 4 (H3K4me3), both activating histone marks, were associated with the Fxyd1 promoter to a lesser degree in the FC than in the CB. These results indicate that differential Fxyd1 expression in these two brain regions is, at least in part, regulated by an epigenetic mechanism involving increased DNA methylation of the two alternative Fxyd1 promoters, enhanced Mecp2 recruitment, and reduced association of activating histones.

The transcriptional control of female puberty.

The initiation of mammalian puberty requires a sustained increase in pulsatile release of gonadotrophin releasing hormone (GnRH) from the hypothalamus. This increase is brought about by coordinated changes in transsynaptic and glial-neuronal communication, consisting of an increase in neuronal and glial stimulatory inputs to the GnRH neuronal network and the loss of transsynaptic inhibitory influences. GnRH secretion is stimulated by transsynaptic inputs provided by excitatory amino acids (glutamate) and at least one peptide (kisspeptin), and by glial inputs provided by growth factors and small bioactive molecules. The inhibitory input to GnRH neurons is mostly transsynaptic and provided by GABAergic and opiatergic neurons; however, GABA has also been shown to directly excite GnRH neurons. There are many genes involved in the control of these cellular networks, and hence in the control of the pubertal process as a whole. Our laboratory has proposed the concept that these genes are arranged in overlapping networks internally organized in a hierarchical fashion. According to this concept, the highest level of intra-network control is provided by transcriptional regulators that, by directing expression of key subordinate genes, impose genetic coordination to the neuronal and glial subsets involved in initiating the pubertal process. More recently, we have begun to explore the concept that a more dynamic and encompassing level of integrative coordination is provided by epigenetic mechanisms.

New concepts on the control of the onset of puberty.

The initiation of mammalian puberty requires an increased pulsatile release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. This increase is brought about by changes in transsynaptic and glial-neuronal communication. Coordination of these cellular interactions likely requires the participation of sets of genes hierarchically arranged within functionally connected networks. Using high throughput, genetic, molecular and bioinformatics strategies, in combination with a systems biology approach, three transcriptional regulators of the pubertal process have been identified, and the structure of at least one hypothalamic gene network has been proposed. A genomewide analysis of hypothalamic DNA methylation revealed profound changes in methylation patterns associated with the onset of female puberty. Pharmacological disruption of two epigenetic marks associated with gene silencing (DNA methylation and histone deacetylation) resulted in pubertal failure, instead of advancing the onset of puberty, suggesting that disruption of these two silencing mechanisms leads to activation of repressor genes whose expression would normally decrease at puberty. These observations suggest that the genetic underpinnings of puberty are polygenic rather than specified by a single gene, and that epigenetic mechanisms may provide coordination and transcriptional plasticity to this genetic network.

Gene networks and the neuroendocrine regulation of puberty.

A sustained increase in pulsatile release of gonadotrophin releasing hormone (GnRH) from the hypothalamus is an essential, final event that defines the initiation of mammalian puberty. This increase depends on coordinated changes in transsynaptic and glial-neuronal communication, consisting of activating neuronal and glial excitatory inputs to the GnRH neuronal network and the loss of transsynaptic inhibitory tone. It is now clear that the prevalent excitatory systems stimulating GnRH secretion involve a neuronal component consisting of excitatory amino acids (glutamate) and at least one peptide (kisspeptin), and a glial component that uses growth factors and small molecules for cell-cell signaling. GABAergic and opiatergic neurons provide transsynaptic inhibitory control to the system, but GABA neurons also exert direct excitatory effects on GnRH neurons. The molecular mechanisms that provide encompassing coordination to this cellular network are not known, but they appear to involve a host of functionally related genes hierarchically arranged. We envision that, as observed in other gene networks, the highest level of control in this network is provided by transcriptional regulators that, by directing expression of key subordinate genes, impose an integrative level of coordination to the neuronal and glial subsets involved in initiating the pubertal process. The use of high-throughput and gene manipulation approaches coupled to systems biology strategies should provide not only the experimental bases supporting this concept, but also unveil the existence of crucial components of network control not yet identified.

Hypothalamic expression of Eap1 is not directly controlled by ovarian steroids.

A gene termed EAP1 (enhanced at puberty 1) was recently identified as a transcriptional regulator of female neuroendocrine reproductive function. We have now used in vivo and in vitro assays, and the female rat as an animal model, to determine whether Eap1 gene expression is regulated by ovarian steroids. Eap1 mRNA abundance decreases in both the hypothalamus and cerebral cortex during the infantile-juvenile phases of development, but it increases selectively in the hypothalamus at puberty, suggesting that in contrast to the general decline in expression observed in immature animals, the region-specific increase in Eap1 mRNA levels that occurs at puberty might be elicited by ovarian steroids. This is, however, not the case, because hypothalamic Eap1 mRNA levels increase at the expected time of puberty in rats ovariectomized at the beginning of the juvenile period. Although a subpopulation of EAP1-containing cells in the medial basal hypothalamus (MBH) and preoptic area express estrogen receptor-alpha (ERalpha), the 5'-flanking region of the rat Eap1 (rEap1) gene does not contain a complete estrogen-responsive element, and no such estrogen-responsive element is detected within 100 kb of the rEap1 locus. Functional promoter assays showed that neither estradiol (E(2)) alone nor a combination of E(2) plus progesterone increases rEap1 gene transcription. Likewise, E(2) administered to ovariectomized immature rats elicited a robust surge of LH but increased neither preoptic area nor MBH Eap1 mRNA levels. E(2)/progesterone-treated rats showed a massive elevation in plasma LH but only a modest increase in Eap1 mRNA levels, limited to the MBH. These results indicate that hypothalamic Eap1 expression is not directly controlled by ovarian steroids and suggest that Eap1 expression increases at puberty driven by ovary-independent, centrally initiated events.

Gene expression profiling of hypothalamic hamartomas: a search for genes associated with central precocious puberty.

BACKGROUND: Hypothalamic hamartomas (HHs) are congenital lesions composed of neurons and astroglia. Frequently, HHs cause central precocious puberty (CPP) and/or gelastic seizures. Because HHs might express genes similar to those required for the initiation of normal puberty, we used cDNA arrays to compare the gene expression profile of an HH associated with CPP with three HHs not accompanied by sexual precocity. METHODS: Global changes in gene expression were detected using Affymetrix arrays. The results were confirmed by semiquantitative PCR, which also served to examine the expression of selected genes in the hypothalamus of female monkeys undergoing puberty. RESULTS: All HHs were associated with seizures. Ten genes whose expression was increased in the HH with CPP were identified. They encode proteins involved in three key cellular processes: transcriptional regulation, cell-cell signaling, and cell adhesiveness. They include IA-1 and MEF2A, two transcription factors required for neuronal development; mGluR1 and VILIP-1, which encode proteins involved in neuronal communication, and TSG-6 that encodes a protein involved in cell adhesiveness. Of these, expression of mGluR1 also increases in the female monkey hypothalamus at puberty. CONCLUSIONS: Increased expression of these genes in HHs may be relevant to the ability of some HHs to induce sexual precocity.

Oxytocin facilitates female sexual maturation through a glia-to-neuron signaling pathway.

It has been earlier proposed that oxytocin could play a facilitatory role in the preovulatory LH surge in both rats and humans. We here provide evidence that oxytocin also facilitates sexual maturation in female rats. The administration of an oxytocin antagonist for 6 d to immature female rats decreased GnRH pulse frequency ex vivo and delayed the age at vaginal opening and first estrus. The in vitro reduction in GnRH pulse frequency required chronic blockade of oxytocin receptors, because it was not acutely observed after a single injection of the antagonist. Hypothalamic explants exposed to the antagonist in vitro showed a reduced GnRH pulse frequency and failed to respond to oxytocin with GnRH release. Prostaglandin E(2) (PGE(2)) mimicked the stimulatory effect of oxytocin on GnRH pulse frequency, and inhibition of PG synthesis blocked the effect of oxytocin, suggesting that oxytocin accelerates pulsatile GnRH release via PGE(2). The source of PGE(2) appears to be astrocytes, because oxytocin stimulates PGE(2) release from cultured hypothalamic astrocytes. Moreover, astrocytes express oxytocin receptors, whereas GnRH neurons do not. These results suggest that oxytocin facilitates female sexual development and that this effect is mediated by a mechanism involving glial production of PGE(2).

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

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.

Deletion of the Ttf1 gene in differentiated neurons disrupts female reproduction without impairing basal ganglia function.

Thyroid transcription factor 1 (TTF1) [also known as Nkx2.1 (related to the NK-2 class of homeobox genes) and T/ebp (thyroid-specific enhancer-binding protein)], a homeodomain gene required for basal forebrain morphogenesis, remains expressed in the hypothalamus after birth, suggesting a role in neuroendocrine function. Here, we show an involvement of TTF1 in the control of mammalian puberty and adult reproductive function. Gene expression profiling of the nonhuman primate hypothalamus revealed that TTF1 expression increases at puberty. Mice in which the Ttf1 gene was ablated from differentiated neurons grew normally and had normal basal ganglia/hypothalamic morphology but exhibited delayed puberty, reduced reproductive capacity, and a short reproductive span. These defects were associated with reduced hypothalamic expression of genes required for sexual development and deregulation of a gene involved in restraining puberty. No extrapyramidal impairments associated with basal ganglia dysfunction were apparent. Thus, although TTF1 appears to fulfill only a morphogenic function in the ventral telencephalon, once this function is satisfied in the hypothalamus, TTF1 remains active as part of the transcriptional machinery controlling female sexual development.