since the discovery of the fragile x mental retardation 1 ( fmr1 ) gene in 1991 , our knowledge of the molecular mechanisms underlying fragile x syndrome ( fxs ) has increased tremendously .
the disorder is caused by dynamic mutation of a single trinucleotide sequence , cgg , on the x chromosome . in patients , cgg repeat expansion and subsequent
methylation silences the fmr1 gene , preventing transcription of the fragile x mental retardation protein ( fmrp ) .
fmrp is an rna binding protein involved in mrna transport and stability and also in local protein translation .
the protein is estimated to interact with 4% of all brain mrnas and with an unknown number of proteins in the mammalian brain .
absence of fmrp leads to perturbation of the normal cellular function of many mrnas and proteins .
the resulting fragile x phenotype is characterized by intellectual disability , specific physical abnormalities , behavioral problems and epileptic seizures .
elucidating the pathophysiology of fxs is therefore not only important for the identification of potential therapeutic targets , but could also provide further insights into the general mechanisms of cognition and behavior .
various techniques have been used to determine the mrna targets of fmrp , including co - immunoprecipitation followed by microarray analysis , antibody - positioned rna amplification ( apra ) and , most recently , high - throughput sequencing of rnas isolated by crosslinking immunoprecipitation ( hits - clip ) .
each study identified a large number of mrna targets , encoding proteins involved in synaptic signaling pathways , such as glutamate and -aminobutyric acid ( gaba ) , in addition to proteins involved in many other intracellular pathways .
proteomic analysis of murine hippocampal synapses revealed abnormalities due to loss of translational regulation of fmrp . altered expression profiles were found predominantly for proteins in specific functional groups , such as synaptic structure and cytoskeleton organization , neurotransmission and metabolism . a recent study by davidovic and colleagues , published in genome research , provides a new dimension to fragile x research by analyzing the brain metabolome of fmr1-deficient mice . by combining metabonomics data with results from previously published studies and with the knowledge of signaling and metabolic pathways gathered from databases , this work significantly increases our insight into the aberrant signaling pathways in fxs .
metabonomics measures the dynamic multiparametric metabolic response of living systems to pathological stimuli or genetic modification .
the collection of all metabolites ( the intermediate and end products of cellular processes ) in a biological sample is represented by the metabolome , which can reflect the ultimate response of biological systems to genetic or environmental changes .
metabonomics is a rapidly developing field with many potential applications in cancer and neurodegenerative disease diagnostics , monitoring of disease progression , evaluation of drug toxicity and development of therapeutics .
traditionally , genomics and proteomics approaches have been used to study the effect of genetic disorders . however , finding the relationship between altered gene or protein expression and the biological consequences is not always straightforward , especially given that the effect of a single genetic variation is often not limited to one biochemical pathway .
metabonomics can help to identify the metabolites of seemingly unrelated biochemical pathways , and therefore might reveal potential new sites for therapeutic intervention .
the metabolites can be detected by approaches based on mass spectrometry or nuclear magnetic resonance ( nmr ) spectroscopy . in their study , davidovic et al .  
used solid state nmr , specifically h magic - angle - spinning nmr ( h hr - mas nmr ) spectroscopy .
this method has the advantage of being able to analyze small amounts of intact tissues , unlike other methods , which require metabolites to be in solution .
the technique can thus be used even if further analysis of the tissue sample is required .
the outcome of the metabonomic analysis showed 25 metabolites with altered concentrations in the fragile x brain ( figure 1 ) .
these 25 metabolites could be grouped into a small number of affected pathways , involved in one of four categories : neurotransmission , osmoregulation , energy metabolism and oxidative stress response .
some alterations are in line with reported anomalies in fxs , including abnormalities in the glutamatergic and the gabaergic pathways . for instance , a reduced amount of gaba in the cortex and cerebellum is compatible with earlier reports of a compromised gabaergic system in the disorder . increased glutamatergic signaling is another hallmark of the disorder .
it can be hypothesized that the reduced amount of free glutamate observed by davidovic et al .   is a protective reaction of the organism to the overstimulation of the group
there have been isolated reports that the cholinergic system , a second inhibitory system in brain , is also involved in the disorder , mainly on the basis of results from fly studies . the davidovic et al .
study   clearly shows that several components in the cholinergic pathway are compromised in the mouse model .
this adds further evidence to the hypothesis that fxs is a consequence of the disturbance of the delicate balance between excitatory and inhibitory signaling in the brain .
the metabolites found to be downregulated ( down - arrows in the left columns ) or upregulated ( up - arrows in the right columns ) in fmr1 knockout mice compared with wild - type mice are shown for each brain region .
co , cortex ; st , striatum ; ce , cerebellum ; hi , hippocampus . to integrate the abnormal metabolite concentrations with the data available from previous studies , the authors   developed a novel integrated metabolome and interactome mapping ( imim ) approach .
this systems biology approach was considered necessary because fmrp does not interact directly with the metabolites themselves and therefore can not directly influence their concentrations .
all the 25 metabolites were linked to fmrp in the network by , on average , just over three intermediate proteins .
the distance within the network represents the mechanistic link between fmrp and each metabolite . within the interaction network , only 12 mrnas and one protein
are linked directly to fmrp . of these , some are known interactors , including the paralogous protein fxr2p and mrnas encoding subunits of the gabaa receptor .
interestingly , a key interactor is the amyloid beta precursor protein ( app ) mrna , which is implicated in alzheimer 's disease .
a previous study indicated that fmrp binds app mrna , but it is striking that app is predicted to be in the center of the metabolite network , implying a pivotal role .
rhoa is a member of the rho gtpase family , and different members of this gene family have opposite roles in neurite outgrowth and retraction .
although it was known that the rho gtpase rac1 , which promotes neural spine formation , was involved in the disorder , this study supports the involvement of the rhoa signaling pathway , which induces spine retraction . in line with this , expression of larg , encoding a protein that activates rhoa , was reported to be compromised in fxs in an earlier study .
these findings emphasize the involvement of rho gtpases in the neuro - anatomical pathophysiology of fxs , more specifically in the aberrant spine morphology associated with the disease .
this story   adds another layer of understanding to the complex phenotype of fxs and it contains both good and bad news for the design of drugs to treat the disorder .
although it confirms a key role of inhibitory and excitatory pathways , the abnormalities in these and other affected systems seem to be specific to particular brain regions and thus may require much more sophisticated drug treatments than currently anticipated .
in addition , the abnormalities were detected in very young animals and an effect of brain development on the disturbances of metabolite concentrations can not be excluded .
the study from davidovic and colleagues   highlights many similarities between the metabolome of fxs and that of other neurological disorders .
examples include the neurodevelopmental disorder rett syndrome and the neurodegenerative disorders batten disease and huntington 's disease , which have been analyzed by other groups using metabonomics .
alterations in the balance between the excitatory ( glutamate ) and inhibitory ( gaba ) neurotransmitters , osmoregulation ( myo - inositol ) , lipid metabolism ( choline ) and energy metabolism ( creatine ) were the most commonly observed metabolic changes across all these diseases .
thus , collectively these findings contribute to our understanding of the mechanisms underlying neurological disorders in general .
app : amyloid beta precursor protein ; fmr1 : fragile x mental retardation 1 gene ; fmrp : fragile x mental retardation protein ; fxs : fragile x syndrome ; gaba : -aminobutyric acid ; larg : leukemia - associated rho guanine nucleotide exchange factor ; nmr : nuclear magnetic resonance ; rhoa : ras homology protein a.
we are supported by the agency for innovation by science and technology in flanders ( iwt ) and the belgian national fund for scientific research - flanders ( fwo ) .
 fragile x syndrome is the most common cause of inherited intellectual disability , but the underlying pathophysiology is complex and effective treatments are lacking . in a recent study of fragile x mental retardation 1 ( fmr1 ) knockout mice , the metabolic profile of the fragile x brain was determined using proton high - resolution magic angle spinning nuclear magnetic resonance spectroscopy . 
 this analysis revealed deficiencies in four metabolic categories : neurotransmission , osmoregulation , energy metabolism and oxidative stress response . 
 abnormalities in the metabolic phenotype were linked to the fragile x mental retardation protein using an integrated metabolome and interactome mapping approach , allowing a global picture of the disorder to emerge . 
The fragile X protein has many interactions
Metabonomics as a tool to study genetic disorders
Defects in key metabolic pathways
The fragile X syndrome profile overlaps with that of other neurological disorders
Abbreviations
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