Laura K.K. Pacey

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 GABAB receptor agonist baclofen to FMR1 mice inhibited seizures, whereas the GABAB 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 GABAB receptor-mediated signaling antagonizes the seizure-promoting effects of the mGluRs in FMR1 knockout mice and point to the potential therapeutic benefit of GABAB agonists for the treatment of fragile X syndrome.

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.

Clustering autism: using neuroanatomical differences in 26 mouse models to gain insight into the heterogeneity

Autism is a heritable disorder, with over 250 associated genes identified to date, yet no single gene accounts for >1–2% of cases. The clinical presentation, behavioural symptoms, imaging and histopathology findings are strikingly heterogeneous. A more complete understanding of autism can be obtained by examining multiple genetic or behavioural mouse models of autism using magnetic resonance imaging (MRI)-based neuroanatomical phenotyping. Twenty-six different mouse models were examined and the consistently found abnormal brain regions across models were parieto-temporal lobe, cerebellar cortex, frontal lobe, hypothalamus and striatum. These models separated into three distinct clusters, two of which can be linked to the under and over-connectivity found in autism. These clusters also identified previously unknown connections between Nrxn1α, En2 and Fmr1; Nlgn3, BTBR and Slc6A4; and also between X monosomy and Mecp2. With no single treatment for autism found, clustering autism using neuroanatomy and identifying these strong connections may prove to be a crucial step in predicting treatment response.

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.

Pathways to Drug Development for Autism Spectrum Disorders

Autism spectrum disorders (ASDs) are neurodevelopmental disorders whose prevalence has risen over the past two decades. Current drug treatments for ASDs and the related disorders—fragile X syndrome (FXS) and Rett syndrome—target specific symptoms but do not address the basic underlying etiologies. However, based partly on an improved understanding of the neurochemical underpinnings of FXS, pharmacotherapy for this syndrome has progressed to the point of clinical trials of several novel drug treatments. By contrast, our overall understanding of the neuropathophysiology of ASDs is still rudimentary. There is hope in the field that knowledge and experience gained in the study of fragile X and Rett syndromes may be applicable to the larger autism patient population. In this review, we discuss how recent advances in our understanding of the biochemistry and neuropathology of these disorders could lead to new more effective treatments for ASDs.

3D visualization of the regional differences.

We examined 26 different mouse models related to autism and identified three groups that shared similar neuroanatomical phenotypes. These three groups and their regional differences are shown here. Anything highlighted in red was determined to be larger in that group (median effect size for group >0.5) and anything highlighted in blue was determined to be smaller in that group (median effect size for group < − 0.5). These differences are shown in 3D in an axial representation either looking from above (a) or below (b) the mouse brain. For more information on this topic, please refer to the article by Ellegood et al on pages 118–125.

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 GABAB receptor (GABABR) 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 GABAB receptor agonists, alone and in combination on receptor protein expression and audiogenic seizures in FMR1 mice. Single doses of MPEP (30 mg/kg), the GABABR orthosteric agonist R-baclofen (1 mg/kg), or the GABABR-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.

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.

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.

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.