Interregulation between fragile X mental retardation protein and methyl CpG binding protein 2 in the mouse posterior cerebral cortex.

Several X-linked neurodevelopmental disorders including Rett syndrome, induced by mutations in the MECP2 gene, and fragile X syndrome (FXS), caused by mutations in the FMR1 gene, share autism-related features. The mRNA coding for methyl CpG binding protein 2 (MeCP2) has previously been identified as a substrate for the mRNA-binding protein, fragile X mental retardation protein (FMRP), which is silenced in FXS. Here, we report a homeostatic relationship between these two key regulators of gene expression in mouse models of FXS (Fmr1 Knockout (KO)) and Rett syndrome (MeCP2 KO). We found that the level of MeCP2 protein in the cerebral cortex was elevated in Fmr1 KO mice, whereas MeCP2 KO mice displayed reduced levels of FMRP, implicating interplay between the activities of MeCP2 and FMRP. Indeed, knockdown of MeCP2 with short hairpin RNAs led to a reduction of FMRP in mouse Neuro2A and in human HEK-293 cells, suggesting a reciprocal coupling in the expression level of these two regulatory proteins. Intra-cerebroventricular injection of an adeno-associated viral vector coding for FMRP led to a concomitant reduction in MeCP2 expression in vivo and partially corrected locomotor hyperactivity. Additionally, the level of MeCP2 in the posterior cortex correlated with the severity of the hyperactive phenotype in Fmr1 KO mice. These results demonstrate that MeCP2 and FMRP operate within a previously undefined homeostatic relationship. Our findings also suggest that MeCP2 overexpression in Fmr1 KO mouse posterior cerebral cortex may contribute to the fragile X locomotor hyperactivity phenotype.

Identification of a molecular locus for normalizing dysregulated GABA release from interneurons in the Fragile X brain.

Principal neurons encode information by varying their firing rate and patterns precisely fine-tuned through GABAergic interneurons. Dysregulation of inhibition can lead to neuropsychiatric disorders, yet little is known about the molecular basis underlying inhibitory control. Here, we find that excessive GABA release from basket cells (BCs) attenuates the firing frequency of Purkinje neurons (PNs) in the cerebellum of Fragile X Mental Retardation 1 (Fmr1) knockout (KO) mice, a model of Fragile X Syndrome (FXS) with abrogated expression of the Fragile X Mental Retardation Protein (FMRP). This over-inhibition originates from increased excitability and Ca2+ transients in the presynaptic terminals, where Kv1.2 potassium channels are downregulated. By paired patch-clamp recordings, we further demonstrate that acutely introducing an N-terminal fragment of FMRP into BCs normalizes GABA release in the Fmr1-KO synapses. Conversely, direct injection of an inhibitory FMRP antibody into BCs, or membrane depolarization of BCs, enhances GABA release in the wild type synapses, leading to abnormal inhibitory transmission comparable to the Fmr1-KO neurons. We discover that the N-terminus of FMRP directly binds to a phosphorylated serine motif on the C-terminus of Kv1.2; and that loss of this interaction in BCs exaggerates GABA release, compromising the firing activity of PNs and thus the output from the cerebellar circuitry. An allosteric Kv1.2 agonist, docosahexaenoic acid, rectifies the dysregulated inhibition in vitro as well as acoustic startle reflex and social interaction in vivo of the Fmr1-KO mice. Our results unravel a novel molecular locus for targeted intervention of FXS and perhaps autism.

FMRP Expression Levels in Mouse Central Nervous System Neurons Determine Behavioral Phenotype.

Fragile X mental retardation protein (FMRP) is absent or highly reduced in Fragile X Syndrome, a genetic disorder causing cognitive impairment and autistic behaviors. Previous proof-of-principle studies have demonstrated that restoring FMRP in the brain using viral vectors can improve pathological abnormalities in mouse models of fragile X. However, unlike small molecule drugs where the dose can readily be adjusted during treatment, viral vector-based biological therapeutic drugs present challenges in terms of achieving optimal dosing and expression levels. The objective of this study was to investigate the consequences of expressing varying levels of FMRP selectively in neurons of Fmr1 knockout and wild-type (WT) mice. A wide range of neuronal FMRP transgene levels was achieved in individual mice after intra-cerebroventricular administration of adeno-associated viral vectors coding for FMRP. In all treated knockout mice, prominent FMRP transgene expression was observed in forebrain structures, whereas lower levels were present in more caudal regions of the brain. Reduced levels of the synaptic protein PSD-95, elevated levels of the transcriptional modulator MeCP2, and abnormal motor activity, anxiety, and acoustic startle responses in Fmr1 knockout mice were fully or partially rescued after expression of FMRP at about 35-115% of WT expression, depending on the brain region examined. In the WT mouse, moderate FMRP over-expression of up to about twofold had little or no effect on PSD-95 and MeCP2 levels or on behavioral endophenotypes. In contrast, excessive over-expression in the Fmr1 knockout mouse forebrain (approximately 2.5-6-fold over WT) induced pathological motor hyperactivity and suppressed the startle response relative to WT mice. These results delineate a range of FMRP expression levels in the central nervous system that confer phenotypic improvement in fragile X mice. Collectively, these findings are pertinent to the development of long-term curative gene therapy strategies for treating Fragile X Syndrome and other neurodevelopmental disorders.

Persistent astrocyte activation in the fragile X mouse cerebellum.

BACKGROUND: Fragile X Syndrome, the most common single gene cause of autism, results from loss of the RNA-binding protein FMRP. Although FMRP is highly expressed in neurons, it has also recently been identified in glia. It has been postulated that in the absence of FMRP, abnormal function of non-neuronal cells may contribute to the pathogenesis of the disorder. We previously demonstrated reduced numbers of oligodendrocyte precursor cells and delayed myelination in the cerebellum of fragile X (Fmr1) knockout mice. METHODS: We used quantitative western blotting and immunocytochemistry to examine the status of astrocytes and microglia in the cerebellum of Fmr1 mice during development and in adulthood. RESULTS: We report increased expression of the astrocyte marker GFAP in the cerebellum of Fmr1 mice starting in the second postnatal week and persisting in to adulthood. At 2 weeks postnatal, expression of Tumor Necrosis Factor Receptor 2 (TNFR2) and Leukemia Inhibitory Factor (LIF) were elevated in the Fmr1 KO cerebellum. In adults, expression of TNFR2 and the glial marker S100ß were also elevated in Fmr1 knockouts, but LIF expression was not different from wild-type mice. We found no evidence of microglial activation or neuroinflammation at any age examined. CONCLUSIONS: These findings demonstrate an atypical pattern of astrogliosis in the absence of microglial activation in Fmr1 knockout mouse cerebellum. Enhanced TNFR2 and LIF expression in young mice suggests that changes in the expression of astrocytic proteins may be an attempt to compensate for delayed myelination in the developing cerebellum of Fmr1 mice.

Reduced phenotypic severity following adeno-associated virus-mediated Fmr1 gene delivery in fragile X mice.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by a trinucleotide repeat expansion in the FMR1 gene that codes for fragile X mental retardation protein (FMRP). To determine if FMRP expression in the central nervous system could reverse phenotypic deficits in the Fmr1 knockout (KO) mouse model of FXS, we used a single-stranded adeno-associated viral (AAV) vector with viral capsids from serotype 9 that contained a major isoform of FMRP. FMRP transgene expression was driven by the neuron-selective synapsin-1 promoter. The vector was delivered to the brain via a single bilateral intracerebroventricular injection into neonatal Fmr1 KO mice and transgene expression and behavioral assessments were conducted 22-26 or 50-56 days post injection. Western blotting and immunocytochemical analyses of AAV-FMRP-injected mice revealed FMRP expression in the striatum, hippocampus, retrosplenial cortex, and cingulate cortex. Cellular expression was selective for neurons and reached ∼ 50% of wild-type levels in the hippocampus and cortex at 56 days post injection. The pathologically elevated repetitive behavior and the deficit in social dominance behavior seen in phosphate-buffered saline-injected Fmr1 KO mice were reversed in AAV-FMRP-injected mice. These results provide the first proof of principle that gene therapy can correct specific behavioral abnormalities in the mouse model of FXS.

Delayed myelination in a mouse model of fragile X syndrome.

Fragile X Syndrome is the most common inherited cause of autism. Fragile X mental retardation protein (FMRP), which is absent in fragile X, is an mRNA binding protein that regulates the translation of hundreds of different mRNA transcripts. In the adult brain, FMRP is expressed primarily in the neurons; however, it is also expressed in developing glial cells, where its function is not well understood. Here, we show that fragile X (Fmr1) knockout mice display abnormalities in the myelination of cerebellar axons as early as the first postnatal week, corresponding roughly to the equivalent time in human brain development when symptoms of the syndrome first become apparent (1-3 years of age). At postnatal day (PND) 7, diffusion tensor magnetic resonance imaging showed reduced volume of the Fmr1 cerebellum compared with wild-type mice, concomitant with an 80-85% reduction in the expression of myelin basic protein, fewer myelinated axons and reduced thickness of myelin sheaths, as measured by electron microscopy. Both the expression of the proteoglycan NG2 and the number of PDGFRα+/NG2+ oligodendrocyte precursor cells were reduced in the Fmr1 cerebellum at PND 7. Although myelin proteins were still depressed at PND 15, they regained wild-type levels by PND 30. These findings suggest that impaired maturation or function of oligodendrocyte precursor cells induces delayed myelination in the Fmr1 mouse brain. Our results bolster an emerging recognition that white matter abnormalities in early postnatal brain development represent an underlying neurological deficit in Fragile X syndrome.

Subchronic administration and combination metabotropic glutamate and GABAB receptor drug therapy in fragile X syndrome.

The most common cause of inherited mental retardation, fragile X syndrome, results from a triplet repeat expansion in the FMR1 gene and loss of the mRNA binding protein, fragile X mental retardation protein (FMRP). In the absence of FMRP, signaling through group I metabotropic glutamate receptors (mGluRs) is enhanced. We previously proposed a mechanism whereby the audiogenic seizures exhibited by FMR1 null mice result from an imbalance in excitatory mGluR and inhibitory GABA(B) receptor (GABA(B)R) signaling (Mol Pharmacol 76:18-24, 2009). Here, we tested the mGluR5-positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), the mGluR5 inverse agonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), and GABA(B) receptor agonists, alone and in combination on receptor protein expression and audiogenic seizures in FMR1 mice. Single doses of MPEP (30 mg/kg), the GABA(B)R orthosteric agonist R-baclofen (1 mg/kg), or the GABA(B)R-positive allosteric modulator N,N'-dicyclopentyl-2-(methylthio)-5-nitro-4,6-pyrimidine diamine (GS-39783) (30 mg/kg), reduced the incidence of seizures. However, when administered subchronically (daily injections for 6 days), MPEP retained its anticonvulsant activity, whereas R-baclofen and GS-39783 did not. When administered at lower doses that had no effect when given alone, a single injection of MPEP plus R-baclofen also reduced seizures, but the effect was lost after subchronic administration. We were surprised to find that subchronic treatment with R-baclofen also induced tolerance to a single high dose of MPEP. These data demonstrate that tolerance develops rapidly to the antiseizure properties of R-baclofen alone and R-baclofen coadministered with MPEP, but not with MPEP alone. Our findings suggest that cross-talk between the G-protein signaling pathways of these receptors affects drug efficacy after repeated treatment.

The neurochemical basis for the treatment of autism spectrum disorders and Fragile X Syndrome.

Autism spectrum disorders (ASD) and Fragile X Syndrome (FXS) are neurodevelopmental disorders that share overlapping behavioral characteristics. While FXS is known to result from a specific genetic mutation, the causes of the majority of cases of ASD are unknown. Animal models of FXS have revealed new insight into the cellular and biochemical changes that occur in the central nervous system in this disorder, while human genetic studies on individuals with autism have identified sets of genes that may increase susceptibility to the disorder. Together these discoveries suggest overlapping biochemical characteristics and reveal new directions for the potential development of pharmacological therapies that might prove useful in the treatment of both FXS and ASD. In particular, delayed synaptic maturation, abnormal synaptic structure and/or function and alterations in intracellular signaling pathways have been linked to the pathogenesis of FXS and ASD. Aberrations in GABA(A) receptor ion channels and the G-protein coupled metabotropic glutamate and GABA(B) transmitter systems are also linked to both disorders and these receptors are currently at the forefront of preclinical and clinical research into treatments for both autism and Fragile X Syndrome.

Genetic deletion of regulator of G-protein signaling 4 (RGS4) rescues a subset of fragile X related phenotypes in the FMR1 knockout mouse.

Fragile X syndrome (FXS), the most common cause of inherited mental retardation, is caused by the loss of the mRNA binding protein, FMRP. Persons with FXS also display epileptic seizures, social anxiety, hyperactivity, and autistic behaviors. The metabotropic glutamate receptor theory of FXS postulates that in the absence of FMRP, enhanced signaling though G-protein coupled group I metabotropic glutamate receptors in the brain contributes to many of the abnormalities observed in the disorder. However, recent evidence suggests that alterations in cellular signaling through additional G-protein coupled receptors may also be involved in the pathogenesis of FXS, thus providing impetus for examining downstream molecules. One group of signaling molecules situated downstream of the receptors is the regulator of G-protein signaling (RGS) proteins. Notably, RGS4 is highly expressed in brain and has been shown to negatively regulate signaling through Group I mGluRs and GABA(B) receptors. To examine the potential role for RGS4 in the pathogenesis of FXS, we generated FXS/RGS4 double knockout mice. Characterization of these mice revealed that a subset of FXS related phenotypes, including increased body weight, altered synaptic protein expression, and abnormal social behaviors, were rescued in the double knockout mice. Other phenotypes, such as hyperactivity and macroorchidism, were not affected by the loss of RGS4. These findings suggest that tissue and cell-type specific differences in GPCR signaling and RGS function may contribute to the spectrum of phenotypic differences observed in FXS.

Early developmental alterations in GABAergic protein expression in fragile X knockout mice.

Fragile X syndrome is the most common heritable form of mental retardation. It is caused by silencing of the Fmr1 gene and the absence of the encoded protein. The purpose of this study was to examine global protein expression levels of GABA(A) and GABA(B) receptors, and GABAergic enzymes and trafficking proteins in fragile X knockout mice during brain maturation. Quantitative western blotting of homogenates of forebrain revealed that the levels of GABA(A) beta1 and beta3, GABA(B)-R1, NKCC1, KCC2, gephyrin and ubiquilin were not significantly different from wild-type mice at any of the postnatal time points examined. In contrast, the GABA(A) receptor alpha1, beta2, and delta subunits, and the GABA enzymes GABA transaminase and succinic semialdehyde dehydrogenase were down-regulated during postnatal development, while GAD65 was up-regulated in the adult knockout mouse brain. The GABA(A) receptor alpha1 and beta2 subunits displayed a divergent pattern of developmental expression whereby alpha1 was reduced in the immature brain but regained a level of expression similar to wild-type mice by adulthood, while the expression of beta2 was similar to wild-types at postnatal day 5 but reduced at day 12 and in the adult brain. The GABA(A) receptor delta subunit and the GABA catabolic enzymes GABA transaminase and succinic semialdehyde dehydrogenase were simultaneously but transiently decreased only at postnatal day 12. Our results demonstrate that GABA(A) receptor subunits and GABA enzymes display complex patterns of changes during brain development suggesting that dynamic interactions may occur between GABA transmitter levels and GABA receptors in fragile X syndrome.

Anatomical phenotyping in a mouse model of fragile X syndrome with magnetic resonance imaging.

Fragile X Syndrome (FXS) is the most common single gene cause of inherited mental impairment, and cognitive deficits can range from simple learning disabilities to mental retardation. Human FXS is caused by a loss of the Fragile X Mental Retardation Protein (FMRP). The fragile X knockout (FX KO) mouse also shows a loss of FMRP, as well as many of the physical and behavioural characteristics of human FXS. This work aims to characterize the anatomical changes between the FX KO and a corresponding wild type mouse. Significant volume decreases were found in two regions within the deep cerebellar nuclei, namely the nucleus interpositus and the fastigial nucleus, which may be caused by a loss of neurons as indicated by histological analysis. Well-known links between these nuclei and previously established behavioural and physical characteristics of FXS are discussed. The loss of FMRP has a significant effect on these two nuclei, and future studies of FXS should evaluate the biochemical, physiological, and behavioral consequences of alterations in these key nuclei.

Increased GABA(B) receptor-mediated signaling reduces the susceptibility of fragile X knockout mice to audiogenic seizures.

Mice lacking the gene encoding fragile X mental retardation protein (FMR1) are susceptible to audiogenic seizures, and antagonists of the group I metabotropic glutamate receptors (mGluRs) have been shown to block seizures in FMR1 knockout mice. We investigated whether the G-protein-inhibitory activity of the regulator of G-protein signaling protein, RGS4, could also alter the susceptibility to audiogenic seizures in FMR1 mice. We were surprised to find that male FMR1/RGS4 double-knockout mice showed reduced susceptibility to audiogenic seizures compared with age-matched FMR1 mice. These data raised the intriguing possibility that loss of RGS4 increased signaling through another G-protein pathway that reduces seizure susceptibility in FMR1 mice. Indeed, administration of the GABA(B) receptor agonist baclofen to FMR1 mice inhibited seizures, whereas the GABA(B) receptor antagonist (3-aminopropyl)(cyclohexylmethyl)phosphinic acid (CGP 46381) increased seizure incidence in double-knockout mice but not in wild-type mice. Finally, audiogenic seizures could be induced in wild-type mice by coadministering CGP 46381 and the mGluR5-positive allosteric modulator 3-cyano-N-(1,2 diphenyl-1H-pyrazol-5-yl) benzamide. These data show for the first time that GABA(B) receptor-mediated signaling antagonizes the seizure-promoting effects of the mGluRs in FMR1 knockout mice and point to the potential therapeutic benefit of GABA(B) agonists for the treatment of fragile X syndrome.

Isostructural fluorescent and radioactive probes for monitoring neural stem and progenitor cell transplants.

A construct for tagging neurospheres and monitoring cell transplantations was developed using a new technology for producing luminescent and radiolabeled probes that have identical structures. The HIV1-Tat basic domain derivatives NAcGRKKRRQRRR(SAACQ)G (SAACQ-1) and [NAcGRKKRRQRRR(Re(CO)3SAACQ)G]+ (ReSAACQ-1) were prepared in excellent yields using the single amino acid chelate-quinoline (SAACQ) ligand and its Re(I) complex and conventional automated peptide synthesis methods. The distribution of the luminescent Re probe, using epifluorescence microscopy, showed that it localized primarily in the cell nucleus with a significant degree of association on the nuclear envelope. A smaller amount was found to be dispersed in the cytoplasm. The 99m Tc analogue was then prepared in 43+/-7% (n=12) yield and very high effective specific activity. Following incubation, average uptake of the probe in neurospheres ranged between 10 and 20 Bq/cell. As determined by colorimetric assays, viability for cells labeled with high effective specific activity 99m TcSAACQ-1 was 97+/-4% at 2 h postlabeling and 85+/-25% at 24 h postlabeling for incubation activities ranging from 245 to 8900 Bq/cell. DNA analysis showed that at these levels, there was no significant difference between the extent of DNA damage in the treated cells versus control cells. A series of preliminary SPECT/CT studies of transplants in mice were performed, which showed that the strategy is convenient and feasible and that it is possible to routinely assess procedures noninvasively and determine the number of cells transplanted.

Developmental expression of FMRP in the astrocyte lineage: implications for fragile X syndrome.

One of the most common causes of mental retardation in humans, Fragile X syndrome, results from the absence of FMRP, the protein product of the FMR1 gene. In the nervous system, expression of FMRP has been thought to be confined mainly to neurons as little research has examined FMRP expression in non-neuronal lineages. We present evidence that, in addition to neuronal expression, FMRP is expressed in developing CNS glial cells in vitro and in vivo. The neurosphere assay was used to establish cultures of stem and progenitor cells from the brains of wildtype and FMRP knockout (B6.129.FMR1/FvBn) mouse pups. When the neurospheres were differentiated in vitro, approximately 50% of the FMRP positive cells also expressed GFAP. Immunocytochemical studies of the embryonic and postnatal mouse brain revealed coexpression of FMRP and GFAP in the developing hippocampus. Prominent coexpression was also observed in ependymal cells surrounding the third ventricle and astrocytes of the glia limitans. No double-labeled cells were evident in the brains of young adult mice. Cells coexpressing FMRP and the oligodendrocyte precursor marker NG2 were also identified in the hippocampus and corpus callosum of the early postnatal brain. Our results suggest that FMRP is expressed in cells of non-neuronal lineage(s) during development. This represents potential involvement of glial cells in the neural development of fragile X syndrome.

Neural stem cells from protein tyrosine phosphatase sigma knockout mice generate an altered neuronal phenotype in culture.

BACKGROUND: The LAR family Protein Tyrosine Phosphatase sigma (PTPsigma) has been implicated in neuroendocrine and neuronal development, and shows strong expression in specific regions within the CNS, including the subventricular zone (SVZ). We established neural stem cell cultures, grown as neurospheres, from the SVZ of PTPsigma knockout mice and sibling controls to determine if PTPsigma influences the generation and the phenotype of the neuronal, astrocyte and oligodendrocyte cell lineages. RESULTS: The neurospheres from the knockout mice acquired heterogeneous developmental characteristics and they showed similar morphological characteristics to the age matched siblings. Although Ptprs expression decreases as a function of developmental age in vivo, it remains high with the continual renewal and passage of the neurospheres. Stem cells, progenitors and differentiated neurons, astrocytes and oligodendrocytes all express the gene. While no apparent differences were observed in developing neurospheres or in the astrocytes and oligodendrocytes from the PTPsigma knockout mice, the neuronal migration patterns and neurites were altered when studied in culture. In particular, neurons migrated farther from the neurosphere centers and the neurite outgrowth exceeded the length of the neuronal processes from age matched sibling controls. CONCLUSION: Our results imply a specific role for PTPsigma in the neuronal lineage, particularly in the form of inhibitory influences on neurite outgrowth, and demonstrate a role for tyrosine phosphatases in neuronal stem cell differentiation.