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Biomarkers in Fragile X Syndrome (FXS)


Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and monogenic known cause of autism spectrum disorder (ASD). It is a trinucleotide repeat disorder, in which more than 200 CGG repeats in 5’ Untranslated Region (UTR) of FMR1 gene cause the methylation at promoter, results in silencing of the gene and the ultimately loss of product, fragile X mental retardation 1 protein (FMRP). FMRP is RNA binding protein that plays a main role in gene expression and regulates the translation of various mRNAs, many of which are involved in the maintenance and development of neuronal synaptic connections.  FXS patients faces several behavioral challenges, including anxiety, hyperactivity, in addition to seizures, issues with clear speaking and intellectual functioning. Currently, there is no cure or approved medication for the treatment of fragile X Syndrome but with the time our knowledge about proteins that are dysregulated by the loss of FMRP is increasing that leading us to the path of developing validate biomarker for identifying potential targets for therapies. In this paper we reviewed all candidate biomarkers that are identified in mice and now under validation for Human application or already made their way to the clinical trials.

Keywords: Fragile X syndrome; Molecular Biomarkers; FMR1; FMRP; Intellectual disability; Mouse models; Metabotropic Glutamate Receptors; Crebral Protein synthesis; Phosphoinositide 3 kinase; Extracellular- regulated Kinase; Matrix Metalloproteinase-9; Brain Derived Neurotrophic Factor; Mammalian Target of Rapamycin; Substrate p70 Ribosomal S6 Kinase; Amyloid-beta Protein Precursor and Amyloid-beta;

1. Introduction

 A biomarker is “a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention [Biomarker definition working group, 2001].” They are rapid, sensitive, cost-efficient and useful in evaluating the clinical benefits of pharmaceutical interference. Hopes for the cure of several genetic disorders were raised by the identification of genetic reason, elucidating of pathophysiological mechanism and developing of the specific biomarkers for targeted treatment.

Fragile X syndrome (FXS), is the most prevalent inherited cause of X-linked intellectual disability, and the single leading known monogenic cause of autism as 60% of those with full mutation presenting with ASD [Kraan et al., 2018]. The clinical symptoms include anxiety, impairment in cognitive, executive and language performance, hypotonia, flat feet, hyperactivity, impulsivity, insomnia, seizures and hyperextensible joints [Landowska et al., 2018]. FXS is caused by the abnormal expansion of a naturally occurring CGG repeat in the 5’ untranslated region (UTR) of the Fragile X Mental Retardation 1 gene (FMR1) gene to > 200 units which results in hypermethylation and transcriptional silencing of the gene, and the resulting loss or reduction of expression of the gene product, Fragile X Mental Retardation Protein (FMRP) [Verkerk et al., 1991, Oberle et al., 1991, Pieretti et al., 1991]. FMRP is mRNA-binding translational regulator effects synaptic plasticity, morphology, and cellular signaling pathways. Thus the reduced expression of FMRP leads to the abnormalities in neurodevelopmental processes and disturbed neuronal communication [Santos et al., 2014].

The function of FMRP appears to be largely inhibitory as it prevents activity in various biochemical pathways to ensures that neural activation happened in a “controlled” manner [Sunamura et al., 2018]. In a sense, reduced FMRP leads to exaggerated biochemical reactions that adversely affect the neural function. Two decades of research have shown defects in major excitatory glutamatergic and inhibitory GABAergic pathways, and several other neurotransmitter systems [Yang et al., 2018]. Thus, the development of measures that reflect the impact of a drug on FMRP-regulated pathways, including the activity of proteins in the translational activation pathway and proteins whose translation is regulated by FMRP can act as the biomarker for the FXS. For the discovery of these biomarkers, it is important to consider that tissue differences in the interaction of the biomarker with pathways affected by FMRP and there is need of the individualization of a biomarker for agents with various targets.

Development of the FMR1 knockout (KO) mouse model [Bakker et al., 1994], which lacks a functional FMR1 gene and has phenotypes that resemble the human disorder in several ways, including behavior, biochemistry, electrophysiology, and spine morphology provides the way to conduct the pre-clinical studies. These pre-clinical studies in FMR1 KO mice led to successful therapeutic intervention studies that paved the way for clinical trials. Current treatment is symptomatic, including non-pharmacological behavioral and educational therapy, but also extending to pharmacological treatment for aggression, anxiety, and attention deficit hyperactivity disorder (ADHD). However, hope has been tempered by the discovery that it is less straightforward than first assumed to translate encouraging results in the FMR1 KO mouse model into a therapy in the clinical setting. Although they showed the patterns of brain activity, including seizures, similar to those in people with fragile X syndrome but these mice are poor mimics of human behavior. The strain of FMR1 KO mice that often used to test drugs for fragile X syndrome does not show the exact similar cognitive problems like the FXS patients [Berry Kravis E. 2016].

Currently, there is no cure for FXS and recent failure of multiple clinical trials have highlighted the need for the development and validation of new biomarkers to better measure the outcome of these treatments [Scharf et al., 2015, Mullard A., 2016]. The greater understanding of the underlying molecular mechanisms and pathways have led to the development of the specific biomarkers for defining targeted therapeutic strategies that intended to reverse the intellectual and behavioral problems of patients with FXS. In this paper we will review all potential molecular biomarkers for FXS that been identified in FMR1 KO mice as early sign of drug promise and later moved to a larger trial in human being.

2. Variation in FMR1 CGG Repeat and Mosaicism

Variation in FMR1 CGG repeat size is a useful biomarker of various types of risk that could affect people, as it defines differences between full mutation and premutation carriers. The FMR1 expansion leads to the alteration of downstream biochemical processes that indicated the risk in better way by reflecting gene into environment interaction [Belmonte et al., 2006].  FXS incidence is affected not only by premutation carrier frequency, but also by CGG repeat size, since the risk for next-generation expansion to full mutation corresponds to increasing size. Study considered the European population and reported that the Ethnic variability in FMR1 CGG repeat size distribution predicts differences in inter-ethnic fragile X syndrome incidence. Thus Premutation frequency and repeat length must be considered in tabulating risk for fragile X syndrome. This provides a more accurate indication of reproductive risk than reliance on simple carrier frequency [Lazarin et al., 2016].

FXS occurs in about 1:4000 males but due to its X-linked nature, it is less prevalent in females (1:6000) [Hagerman 2008]. This sex differences in affectedness is the result of the fact that males have a single X chromosome, whereas females have two. Moreover, the process of X inactivation early in embryonic development in females results in the shutting off of one X chromosome in each cell, which effectively reduces the impact of the FMR1 mutation in females relative to males [ Stembalska et al., 2016]. The relative proportions of active and inactive mutation-carrying X chromosomes contribute to differences in affectedness among females, making the activation ratio another useful biomarker especially in the context of vulnerability to parenting stress [Seltzer et al., 2009].

FMR1-related biomarkers relationship with measures of neurocognitive and social-affective functioning and mental health problems have been found. The magnitude of the correlations generally suggests that these biomarkers are accounting for only a rather small proportion of phenotypic variance. Nevertheless, the path from identification of biomarkers of risk in carriers of FMR1expansions to treatment is likely to involve many steps and considerable scientific effort.

3. Metabotropic Glutamate Receptors (mGluRs)

FMRP is involved in transcriptional regulation and transportation of mRNA from the nucleus to the cytoplasm and distal sites both in pre- and post-synaptic terminals. One region of the brain that is significantly impacted by the loss of FMRP is the hippocampus, a structure that plays a critical role in the regulation of mood and cognition [Bostrom et al., 2016]. The “mGluR Theory of FXS” showed that the lowering the level of FMRP leads to the activity in the metabotropic glutamate receptors (mGluRs) (mGluR1 and mGluR5) a major excitatory neurotransmitter in mammalian central nervous system that would be otherwise blocked but now could contribute to the neurological and psychiatric symptoms of FXS [Bear et al., 2004, Dölen et al., 2008, Kim et al., 2012]. It then leads to the long-term depression (LTD), reduced responsiveness to signal in hippocampus and other parts of brain involved in memory and learning [Bear et al., 2004].

In recent past years, our understanding of the molecular pathophysiology of FXS have been substantially advanced by the corroboration of ‘mGlu Theory of FXS’ in a wide range of experiments with number of different mGlu5 inhibitors [ Krueger et al., 2011, Bhakar et al., 2012].  Metabotropic glutamate receptor 5 (mGluR5) exaggerate signaling is account for multiple cognitive and syndromic features of FXS and the reduction of signaling provide evidence regarding the reversal of Fragile X phenotype [Darnel et al., 2013].

To checked the hypothesis that unchecked activation of mGluR5, a metabotropic glutamate receptor results in the psychiatric and neurological symptoms of FXS [Dolen et al., 2007] generated Fmr1 mutant mice with a 50% reduction in mGluR5 expression. They demonstrated that the reduction of mGlu5 expression levels from early embryonic development effectively prevented the onset of a broad range of FXS phenotypes. Similarly, in the relevant study the chronic treatment of Fmr1 KO mice with the long-acting mGlu5 inhibitor 2- chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)- 1H-imidazol-4-yl)ethynyl)pyridine fully corrected numerous phenotypes including the increased synaptic spine density and protein synthesis rate, aberrant synaptic plasticity and learning and memory deficits, and increased body growth rate and sensitivity to audiogenic seizures [Michalon et al., 2012]. Both of the studies shows that the long-term, uninterrupted mGlu5 inhibition likely is important for a successful pharmacological intervention as single dose of the mGlu5 inhibitors is not enough to correct several mouse phenotypes.

A candidate molecular mechanism for mGlu5 dysfunction in FXS is decreased association of mGlu5 with the Homer family of scaffolding proteins. Genetic deletion of H1(activity-inducible isoform of Homer1) restored normal mGlu5-long Homer association in Fmr1 KO mice and corrected much of the mGlu5 dysfunction as well as behavioral phenotypes, such as anxiety and audiogenic seizures [Ronesi et al., 2012]. Alternatively, [Guo et al., 2016] showed that the disruption of mGlu5-Homer in Fmr1 knockin mice leads to phenotypes of FXS including reduced mGlu5 association with the postsynaptic density, enhanced constitutive mGlu5 signaling to protein synthesis, deficits in agonist-induced translational control, protein synthesis-independent LTD, neocortical hyperexcitability, audiogenic seizures, and altered behaviors, including anxiety and sensorimotor gating. The mechanistic link between changes of mGluR5 dynamics and pathological phenotypes of FXS proved that mGluR5 was significantly more mobile at synapses in hippocampal Fmr1 KO mouse neurons, causing an increased synaptic surface co-clustering of mGluR5 and synaptic N-Methyl-D-aspartate receptor (NMDAR). This correlated with a reduced amplitude of synaptic NMDAR currents, a lack of their mGluR5-activated long-term depression, and NMDAR/hippocampus dependent cognitive deficits [Aloisi et al., 2017].

Clinical trials in humans have been conducted to explore the safety, tolerability, and efficacy in FXS patients. Fenobam [Porter et al., 2015], is the first drug evaluated in a single-dose open-label study of 12 male and female adults with FXS (mean age 23.9 years), showed trends of improvement in a prepulse inhibition deficit relative to controls who not received the drug [Berry-Kravais et al., 2009]. An exploratory study with mavoglurant (AFQ056) [Vranesic et al., 2014] reported the efficacy in a small sample of male adult FXS patients with full FMR1 methylation, but not in patients with partial methylation [Jacquemont et al., 2011]. These behavioral effects were not replicated with FXS male and female adolescents and adults either full or partial FMR1 methylation in subsequent 12-week, double-blind mavoglurant studies [Berry-Kravis et al., 2016]. Similarly, a large proof of concept studies has been conducted with basimglurant i.e; a potent and selective mGluR5 NAM [Jaeschke et al., 2015, Lindemann et al., 2015] and mavoglurant in male and female adult suffering from FXS. They ended as no improvement in the clinical phenotype of patients were observed [Roche 2014, Novartis 2014, Mullard A., 2016]. Recently, basimglurant evaluated in a 12-week, double-blind, parallel-group study of 183 adults and adolescents (aged 14–50, mean 23.4 years) with FXS. In this phase 2 clinical trial, basimglurant did not demonstrate improvement over placebo [ Youssef et al., 2018].

The compelling preclinical evidence for the therapeutic potential of mGlu5 inhibitors in rodent disease model did not translate in FXS patients successfully. It is hoped that future research will help unravel the reasons for this lack of translation between preclinical data and clinical outcome, and that the toolbox for the preclinical profiling of novel drug candidates is expanded and refined in an effort to increase the confidence in predicting therapeutic benefits in patients with FXS.

4. Cerebral protein Synthesis (rCPS)

In learning and memory synaptic strength played a crucial role that being compromised in many neurodevelopmental disorders.  One of the molecular mechanisms that regulating spine shaping is local dendritic protein synthesis that enabling synapses to autonomously alter their function and structure [Jung et al., 2012]. FMRP is crucial in regulating this process and partial or complete lack of FMRP in FXS leads to an increase in protein translation at synapses [Darnell et al., 2011]. The metabotropic glutamate receptor subtype 5 (mGluR5) theory of FXS also indicated that the imbalance of mechanisms involved in protein translation and synaptic shaping are responsible of many of the symptoms observed in FXS patients [Huber et al., 2002]. The application of quantitative autoradiographic l-[1-14C] leucine method to the in vivo determination of regional rates of cerebral protein synthesis (rCPS) showed a substantial elevation rCPS in Fmr1 null mice in adult wild-type (WT) and Fmr1 null mice at 4 and 6 months of age regionally selective elevation in confirmed that FMRP is a suppressor of translation in brain in vivo [Qin et al., 2005].

The fibroblasts from FXS patients also showed the significantly elevated rates of basal protein synthesis along with increased levels of phosphorylated mechanistic target of rapamycin (p‐mTOR), phosphorylated extracellular signal regulated kinase 1/2, and phosphorylated p70 ribosomal S6 kinase 1 (p‐S6K1) [Kumari et al., 2014].  Similarly, [Jacquemont et al., 2018] reported that the level of protein synthesis has been increases in fibroblast of individuals with FXS and Fmr1 KO mice.

FXS subjects under propofol sedation showed a reduced rCPS in whole brain, cerebellum, and cortex compared with sedated controls. Similar results have been observed in most regions examined in seductive KO mice as compared to the WT mice that suggests the changes in synaptic signaling can correct increased rCPS in FXS [Qin et al., 2o13].  [Liu et al., 2012] also showed the efficacy of lithium treatment in animal models and found that the chronic dietary lithium treatment reversed the increased rCPS in Fmr1 KO mice. The treatment of fibroblast cell from FXS patients with small molecules that inhibit S6K1 and a known FMRP target, phosphoinositide 3‐kinase (PI3K) catalytic subunit p110β, lowered the rates of protein synthesis in both control and patient fibroblasts [Kumari et al., 2014]. Number of studies showed that the mechanisms regulating levels of protein synthesis, can be restored by reducing the mGluR5 signaling genetically or with pharmacological treatments [ Dolen et al., 2008, Michalon et al., 2012, Gross et al., 2015, Jacquemont et al., 2015, Lozano et al., 2014, Pop et al., 2014, Osterweil et al., 2013]. Moreover, mGluR5 reduction by haplo-insufficiency, MMP9 reduction, striatal enriched tyrosine phosphatase (STEP) signaling reduction, and S6K signaling reduction can also have restored the synaptic and behavioral phenotypes in FXS mice model [Bhattacharya et al., 2012, Boda et al., 2014, Dolan et al., 2013, Bhattacharya et al., 2016, Dölen et al., 2007, Gross et al., 2010, Hayashi et al., 2007, Tian et al., 2015, Sawicka et al., 2016]. Recently [Pusciuto et al., 2015] showed that mGluR regulated increase of protein synthesis is sustained by the excessive production of soluble amyloid beta precursor protein α (sAPPα) due to the impaired processing of amyloid beta precursor protein (APP). Moreover, treatment of FXS mice with a cell permeable peptide able to modulate ADAM metallopeptidase domain 10 (ADAM10) activity, and APP processing, restores protein synthesis to wild type (WT) levels.

This all preclinical data suggesting the protein synthesis as a potential biomarker and molecular hallmark for FXS but unfortunately, there is not wide success in replicating this optimal translational scenario into reality [ Berry-Kravis et al., 2016]. The extent to which excessive protein synthesis is associated with cognitive and behavioral impairments is also remains unknown. More importantly, none of the human studies have demonstrated yet efficacy in children, adolescents or adults with FXS on the primary outcome measures which were mainly behavioral questionnaires [Jacquemont et al., 2011, Berry Kravis et al., 2012, Berry Kravis et al., 2016]. Despite these setbacks, protein synthesis remains a primary mechanism for neurodevelopmental disorders and molecular mechanisms controlling protein synthesis continue to be the prime targets in FXS and other neurodevelopmental disorders. As FMRP clearly modulates this cellular phenotype but there are fair chances many other molecular factors independent of FMRP (environmental and genetic) contribute to the modulation of homeostasis of molecules involved in synaptic plasticity.

5. Phosphoinositide 3 kinase (PI3K)

 Phosphoinositide 3 kinase (PI3K) is the signaling molecule of many cell surface receptors involved in cell motility, survival, growth and proliferation. Phosphoinositide 3-kinaseclass I catalytic subunits, p110α, p110β, p110γ, and p110δ, have their specific dysregulation in FXS [Gross et al., 2014]. FMRP regulates the synthesis and synaptic localization of p110β, the catalytic subunit of PI3K which is a key signaling molecule downstream of gp1 mGluRs and other membrane receptors. Loss of FMRP in Fmr1 KO mice leads to excess mRNA translation and synaptic protein expression of p110β, that regulates the deregulated protein synthesis [Gross et al., 2010]. The same molecular mechanisms recapitulated in FXS patient cells and showed that the treatment with a p110β-selective antagonist rescues excess protein synthesis in synaptoneurosomes from an FXS mouse model and in patient cells. [Gross et al., 2012].

Catalytic subunit p110β plays an important role in prefrontal cortex (PFC)-dependent cognitive defects in mouse models of Fragile X syndrome (FXS). As PFC-selective knockdown of p110β, an FMRP target that is translationally upregulated in FXS, reverses deficits in higher cognition in Fmr1 knockout mice and normalizes excessive PI3K activity, restores stimulus-induced protein synthesis, and corrects increased dendritic spine density and behavior. [Gross et al., 2015]

PI3K enhancer PIKE that links PI3K catalytic subunits to group 1 metabotropic glutamate receptors (mGlu1/5) and activates PI3K signaling is a key mediator of impaired mGlu1/5-dependent neuronal plasticity in mouse and fly models of the inherited intellectual disability fragile X syndrome (FXS). Normalizing elevated PIKE protein levels in FXS mice reversed deficits in molecular and cellular plasticity and improved behavior [Gross et al., 2015]. This dysregulated protein synthesis and PI3K activity in patient cells might be suitable biomarkers to quantify the efficacy of drugs and could be used for drug screens to refine treatment strategies for individual patients.

6. Extracellular- regulated Kinase (ERK)

The ERK pathway is a chain of proteins in the cell that communicates a signal from a receptor to the DNA and act as a nodal point for cell signaling cascades. The absence of FMRP in Fmr1 KO mice results in rapid dephosphorylated of ERK upon mGluR1/5 stimulation suggests that over activation of phosphatases in synapses affects the synaptic translation, transcription, and synaptic receptor regulation in FXS [Kim et al., 2008, Bhattacharya et al., 2012, Hou et al., 2006, Michalon et al., 2012]. Delayed early‐phase phosphorylation of extracellular‐signal regulated kinase (ERK), observed in both neurons and thymocytes of Fmr1 KO mice. Likewise, the early‐phase kinetics of ERK activation in lymphocytes from human peripheral blood is also delayed in individuals with FXS, as compared to controls, that suggests it’s potential to serve as a biomarker for FXS [Weng et al., 2008]. In contrast the significantly increased phosphorylation of MEK1/2 and ERK has been reported in human brain tissue and brain tissue from Fmr1 knockout mice. Interestingly, treating Fmr1 knockout mice with the MEK1/2 inhibitor SL327 abrogated audiogenic seizure activity [Wang et al., 2012].

ERK signaling elevation has been observed in the neocortex of fragile X mice. This elevated ERK activity causes over activation of p90-ribosomal S6 kinase (RSK) and hyperphosphorylation of ribosomal protein S6 that facilitates translation, and correlates with neuronal activation. Interestingly RSK inhibitor reduces susceptibility to audiogenic seizures in fragile X mice and identified as a therapeutic target for fragile X syndrome [Sawicka et al., 2016]. [Kravis et al., 2008; Erickson et al., 2011] used lymphocyte-based ERK activation kinetics assay as a biomarker for treatment response and showed that ERK activation rates can be normalized with lithium and riluzole treatment. Despite promising results, this assay has a limited routine applicability since it is difficult to perform and requires specialized apparatuses. Moreover, change in activation rate has not been correlated to clinical response so its significance as an outcome measure remains unclear. Recently, [Pellerin et al., 2016] reported a significant FMRP-dependent increase in FXS platelets and ERK phosphorylation. Moreover, they showed that lovastatin can normalized the aberrant ERK activity partly correlated to clinical response.

7. Gamma-aminobutyric acid (GABA) Receptors.

GABA is the most prominent inhibitory neurotransmitter that acts through two receptors in the brain. GABAA receptors, are ligand-regulated chloride channels that upon activation cause hyperpolarization in mature neurons while GABAB receptors, are heterodimeric G protein-coupled receptors (GPCRs) which expressed mostly presynaptically in brain. GABAB receptor activation reducing glutamatergic signalling at excitatory synapses by dampening presynaptic glutamate release and causesing hyperpolarization of postsynaptic neurons via activation of G protein-activated inward rectifying potassium channels (GIRKs) [Gassmann et al., 2012]. Several alterations including a reduction in the expression of several GABA receptor subunits have been detected in the brains of Fmr1-knockout mice. [d’Hulst et al., 2009] showed that the crucial GABAA family receptors and enzymes expressed at reduced levels in Fmr1-KO mice as compare to control WT mice. Later [ Braat et al., 2015] demonstrates that FMRP directly binds several GABAA receptor mRNAs and rescued the phenotype by introducing a yeast artificial chromosome (YAC) containing the ‘healthy’ human FMR1genomic region into Fmr1-KO mice. Similarly, [Jewett et al., 2018] demonstrated in fmr1 KO mice that chronic elevation of neuronal activity through the inhibition of GABAA receptors draw out the synchronization of neural network activity and homeostatic reduction of the amplitude of spontaneous neural network spikes. Recent electrophysiological study supported the suggestion that δ subunit-containing GABAARs are compromised in the Fmr1 KO mice by reporting the four-fold, decrease in tonic inhibition in the fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2 [Zhang et al., 2017].

The delay in switching from depolarizing to hyperpolarizing GABA has also been observed in the cortex of fragile X mice during development [ Hi et al., 2014]. Moreover, the oxytocin-­mediated, GABA excitation– inhibition shift that occurs in newborn rodents during delivery is absent from the hippocampal neurons of Fmr1-knockout mice [Tyzio et al., 2014].  Promising data from different studies in mice model confirmed that GABA receptors are a suitable target for novel treatment of FXS [Yang et al., 2018, Kang et al., 2017, Ligsay et al., 2016, Wang et al., 2016, Zhao et al., 2015, Fatemi et al., 2015, Braat et al., 2014, Lozano et al., 2014, Olmos et al., 2010, Curia et al., 2008, Centonze et al., 2008]. In a recent study the response of the functional deficit FX neurons that lack the synaptic activity has been investigated to pulse application of the neurotransmitter GABA. The results confirm the that FMRP play a role in the development of the GABAergic synapse during neurogenesis [Telias et al., 2016].  Fmr1­deficient Drosophila showed the reduced specificity in olfactory computations and defective lateral inhibition across projection neurons as response to weaker inhibition from GABAergic interneurons. It provides the direct evidence that deficient inhibition impairs sensory computations and behavior in an in vivo model of FXS [Franco et al., 2017].

Gaboxadol (THIP) a GABAreceptor agonist, can restore principal neuron excitability deficits in the Fmr1 KO amygdala and can improve some specific behavioral characteristics, including hyperactivity and auditory seizures [Olmos et al., 2011]. The treatment of Fmr1 KO mice with bumetanide can restored electrophysiological abnormalities in the mutant offspring as well as hyperactivity and autistic behaviors [Tyzio et al., 2014]. Arbaclofen, a GABAB agonist, improve protein synthesis, the abnormal auditory-evoked gamma oscillations, working memory and anxiety-related behavior in Fmr1 KO mice [Sinclair et al., 2017, Qin et al., 2015, Henderson et al., 2012]. To determine safety and efficacy of arbaclofen for social avoidance in FXS patients two phase 3 placebo-controlled trials were conducted with a flexible dose trial in subjects age 12-50 and a fixed dose trial in subjects age 5-11. Arbaclofen did not meet the primary outcome of improved social avoidance in FXS in either study [Berry Kravis et al., 2017].

In double blind, placebo controlled crossover trial of GABAB agonist baclofen [Berry Kravis et al., 2012] reported the social avoidance improvement in the FXS patients.  Acamprosate, which activates GABAB and GABAA receptors also reported to improveseveral phenotypes in Fmr1-KO mice [Schaefer et al., 2017]. Ganaxolone, a neurosteroid and positive GABAA modulator addresses several phenotypes of Fmr1-KO mice [Braat et al., 2015] has been tested in one the recent a randomized, double-blind, placebo-controlled, crossover trial in children with FXS, aged 6-17 years. It found to be safe and in subgroups of children with FXS, including those with higher anxiety or lower cognitive abilities, might have beneficial effects [Ligsay et al., 2017]. These preclinical and clinical studies strengthen the hypothesis of GABA receptors involvement in the pathology of FXS and as potential biomarker for the targeted therapeutics.

8. Matrix Metalloproteinase-9 (MMP-9)

Matrix metalloproteinase-9 is a Zn2+dependent endopeptidase expressed in both the central- and peripheral nervous systems and acts to cleave components of the extracellular matrix (ECM) as well as cell adhesion molecules, cell surface receptors and other proteases. MMP-9 plays an important role in both establishing synaptic connections during development and in the restructuring of synaptic networks in the adult brain [Reinhard et al., 2015]. In Fragile X syndrome the activity- dependent protein synthesis is dysregulated that results in dendritic spine dysmorphogenesis. MMP-9 mRNA is part of the FMRP complex and colocolizes in dendrites. The translation of MMP-9 is increased at synapses in Fmr1 KO mice and suggest it’s contribution in aberrant dendritic spine morphology observed in the Fmr1 knock-out mice and in FXS patients [ Janusz et al., 2013].

Similarly, [Sidhu et al., 2014] also provided an evidence that matrix metalloproteinase-9 (MMP-9) is necessary to the development of FXS-associated defects in Fmr1 KO mice. They showed that the genetic disruption of MMP-9 rescued key aspects of Fmr1 deficiency, including abnormal mGluR5-dependent LTD and dendritic spine abnormalities. MMP-9 also contributed to event related potential (RRP) habituation and it’s level up regulated in the auditory cortex of adult Fmr1 KO mice. It provides a translation relevant electrophysiological biomarker or sensory deficits in FXS and implicate MMP-9 as a target for drug discovery [Lovelace et al., 2016].

[Bilousova et al., 2008] showed that the antibiotic minocycline decreases MMP-9 in the hippocampus of Fmr1 KO mice and promote the dendritic spine maturation, improved anxiety and strategic exploratory behavior. [AlOlaby et al., 2018] also observed that plasma MMP 9 activity elevated in FXS patients as compared to age matched normal controls while they didn’t observe any changes after sertraline treatment.

9. Brain Derived Neurotrophic Factor (BDNF)

Lost, of FMRP leads to abnormalities in the differentiation of neural progenitor cells (NPCs) and in the development of dendritic spines and neuronal circuits. Brain-Derived Neurotrophic Factor is involved in the regulation of various processes of normal neural circuit function and development. BDNF/TrkB signaling in Fmr1 KO mice showed that the alterations in the BDNF/TrkB signaling modulate brain development and impair synaptic plasticity in FXS [Louhivuori et al., 2011].

[Uutela et al., 2012] examined the effects of reduced BDNF expression on the behavioral phenotype of Fmr1 knockout (KO) mice, crossed with mice carrying a deletion of one copy of the Bdnf gene (Bdnf+/−). They reported age‐dependent alterations in hippocampal BDNF expression of Fmr1 KO mice and showed that the absence of FMRP modifies the diverse effects of BDNF on the FXS phenotype.  While, Aberrances of BDNF signaling in FMRP-deficient Neural Progenitor Cells (NPCs) indicate that alterations of BDNF signaling are involved in perturbations of brain development in FXS at very early stages of development [Castrén et al., 2014].

[Kim et al., 2014] proposed that the activity dependent variation in the sensitivity to BDNF-TrkB signaling may compensate the post synaptic activity by promoting the excessive sponginess and dendritic arborization in FXS. A single-nucleotide polymorphism (SNP) in the human BDNF gene, leads to a Methionine (Met) substitution for Valine (Val) at amino acid 66 interferes with the intercellular trafficking and the activity-dependent secretion of BDNF in cortical neurons. BDNF Met66 allele modulate the epilepsy and associated with epilepsy of finnish FXS male [Louhivuori et al., 2009]. Interestingly [Tondo et al., 2011] didn’t observe any association between Val66Met BDNF polymorphism and epilepsy in group of 77 patients with FXS.

Open-label study of acamprosate showed significant increase in BDNF level after treatment [Erickson et al., 2013]. Similarly, [AlOlaby et al., 2018] also reported increase in BDNF after treatment with sertraline in patients with FXS which shows its significance as pharmacodynamics target.

10. Mammalian Target of Rapamycin (mTOR) and Substrate p70 Ribosomal S6 Kinase (S6K1)

Mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation, autophagy, translation and growth. Component of mTOR signaling cascade present at synapse and influence the synaptic plasticity. mTOR is activated in dendrites by stimulation of group I mGluRs and is required for mGluR-LTD at CA1 synapses [Borrie et al., 2017]. In juvenile Fmr1 knock-out mice that has no functional FMRP the elevated mTOR activity and phosphorylation has been observed in hippocampus. This enhances mTOR signaling associated with increase eukaryotic initiation factor complex F4 (eIF4F) [Sharma et al., 2010]. Followed on Mice studies [Hoeffer et al., 2012] investigated the mTOR signaling in subjects with FXS. They found increased phosphorylation of (mTOR) substrate, p70 ribosomal subunit 6 kinase1 (S6K1) in fibroblast and brain tissues along with high expression of mTOR regulator, the serine/threonine protein kinase (Akt). In addition, they also reported increased phosphorylation of the cap binding protein eukaryotic initiation factor 4E (eIF4E) suggesting that protein synthesis is upregulated in FXS. These very first findings in mice and human provided an evidence of mTOR mis-regulation may act as useful biomarkers for designing targeted treatments in FXS. With the passage of time blood platelets appear to be a promising and appropriate disease model of FXS owing to their close biochemical similarities with neurons. [Pellerin et al., 2018] recapitulate the FXS neuron’s core molecular dysregulations, such as hyperactivity of Akt/mTOR pathways that already been observed in mice and human.

mTOR substrate p70 ribosomal S6 kinase 1 (S6K1), proves as an elongation, and translation initiation regulator, and supports the model that dysregulated protein synthesis is the key causal factor in FXS and that restoration of normal translation can stabilize peripheral and neurological function in FXS. As S6K1 deletion prevented immature dendritic spine morphology and multiple behavioral phenotypes, impaired novel object recognition, and behavioral inflexibility [Bhattacharya et al., 2012]. Two S6K1 inhibitors, PF-4708671 and FS-115, tested to normalize FXS phenotypes exhibited by mice model. It has been found that both inhibitors overlapped in reversing the multiple FXS associated phenotypes instead of differences in pharmacokinetic profiles [Bhattacharya et al., 2016].

11. Amyloid-beta Protein Precursor and Amyloid-beta

Amyloid precursor protein (APP) facilitates synapse formation in the developing brain, while beta-amyloid (Aβ) accumulation, results in impaired neurotransmission and synaptic loss. FMRP protein binds to the coding region of APP mRNA and results in the increased translation with the help of metabotropic glutamate receptor pathway. The higher steady-state levels of APP have been found in Fmr1KO synaptoneurosomes (SN) and primary cultured Fmr1KO neurons [Westmark et al., 2007]. APP is processed by β- and γ-secretases to produce amyloid-beta (Aβ), which is the prominent peptide found in the case of Alzheimer’s disease (AD). It shows the possible link between AD and FXS and strengthen the hypothesis that APP and Aβ can be developed as biomarkers for FXS disease severity and drug efficacy.

Following Westmark [Liao et al., 2008] indicates an approximately 1.7-fold increase in APP in Fmr1KO versus WT SN by using western blot analysis. In later years [Westmark et al., 2011] shows that the both APP and Aβ are overexpressed in the absence of FMRP (Fmr1 KO mice). The genetic knockdown of one App allele in the Fmr1KO mice rescues FXS phenotypes like anxiety, seizures, mGLuR-LTD, the ratio of mature versus immature dendritic spines. ADAM 10 is a disintegrin and metalloproteinase domain-containing protein 10; aka α-secretase. [Puscioto et al., 2015] reported involvement of APP-ADAM 10 pathway dysregulation in Fragile X patient and significant upregulation of APP expression in whole brain lysates in Fmr1 KO mice. While [Ray B. et al., 2016] finds elevated levels of sAPP, sAPPα, sAPPβ, Aβ1–40, and Aβ1–42 in pediatric FXS plasma as well as elevated sAPP, sAPPα, and Aβ1–40 in FXS brain The Fmr1KO/APPHET slices exhibit complete rescue of UP states in a neocortical hyperexcitability model and demonstrates the role of APP in maintaining a balance in neural circuits [Westmark et al., 2016].

In human blood plasma, the level of Aβ1-42was significantly reduced in full-mutation FXS adult males while APP and Aβ1-40 levels were not altered. These data suggest that Aβ1-42 may be a plausible blood-based biomarker for FXS [Ray et al., 2016]. Later study reported that Aβ1-40, Aβ1-42 and sAPPβ levels are decreased in plasma of youth with severe autism compared to controls whereas sAPPα levels are elevated [Ray et al., 2011].  APP and Aβ evaluated as blood-based biomarkers in a prospective open-label trial of acamprosate in FXS and interestingly acamprosate treatment significantly reduced sAPP and sAPPα in pediatric subjects with FXS-associated autism spectrum disorder [Erisckson et al., 2014].

These studies have issues as blood levels of APP metabolites may not correlate with brain levels, or the anti-coagulant used to collect blood can have large effects on APP metabolites, which may explain the varied results in Aβ levels in plasma [Wetmark et al., 2011]. It also suggests the age-dependent differences in APP expression but in total all these findings support a role for dysregulated APP production and processing. These APP metabolites provides an advantage of protein stability in serum against of measuring the activity of the aforementioned signaling molecules in FXS and indicate that the APP metabolites may be viable therapeutic targets for FXS treatment.

12. Ion Channels (KNa, BKCa, CaVKv, HCN1)

Voltage- gated ion channels are involved in the neural transmission and with some recent past studies showed their involvement in the FXS pathology [Lee et al., 2012]. The sodium-activated potassium channel (KNa) Slack-B is the largest known potassium channel subunit, consisting of an extended cytoplasmic C-terminal. FMRP binds to the C terminus of the Slack sodium-activated potassium channel to activate the channel in mice and loss of FMRP in FXS might result in loss of inactivation of this channel [Brown et al., 2010]. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (CaV) channels and study found that functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP) [Ferron et al., 2014]. FMRP shapes neuron class-specific calcium in developing learning/memory circuitry, and mediates activity-dependent regulation of calcium signaling specifically during the early-use critical period [Doll et al., 2016].

BKCa channels, also known as BK or Maxi-K channels are large-conductance Ca2+-activated K+channels and FMRP regulates the synaptic transmission and neurotransmitter release vis these channels [Deng et al., 2013, Myrick et al., 2015]. BKCa channel opener molecule (BMS-204352) constitutes a promising potential medication for FXS patients correcting a broad spectrum of behavioral impairments (social, emotional and cognitive) and proposed as a new therapeutic target for FXS [Hébert et al., 2014]. Recently, [Deng et al., 2016] showed that upregulation of BK channel activity normalizes multi-level deficits caused by FMRP loss by generating Fmr1/BKβ4 double knockout mice.

FMRP binds mRNAs of several ion channels, numerous encode regulators of ion homeostasis like Kv3.1 and Kv4.2 voltage-dependent K+ channels, nonselective HCN1 channels, and Cav1.3 Ca2+ channels [Darnell et al., 2001, Brown et al., 2001]. The pore-forming subunit of the Cav2.1 channel is less expressed at the plasma membrane of Fmr1 KO neurons and the Caconcentration improves after the KCl-triggered depolarization [Castagnola et al., 2018]. Remarkably, these ion channels can be used as a novel cellular biomarker and is amenable to small molecule screening and identification of new drugs to treat FXS.

13. Striatal-enriched Protein Tyrosine Phosphatase (STEP)

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific tyrosine phosphatase that plays a major role in the development of synaptic plasticity [Goebel et al., 2012a, Goebel et al., 2012b]. Regulation of STEP is complex, and recent work has shown the involvement of STEP dysregulation in the pathophysiology of several neuropsychiatric disorders [Johnson et al., 2012]. The absence of FMRP upregulates the translation of some of the important mRNAs including of STEP (Striatal-Enriched protein tyrosine Phosphatase) [Darnell et al., 2011, Goebel et al., 2012b, Goebel et al., 2012c]. It results in increase level of STEP protein in hippocampus of Fmr1 KO mice and genetically reducing STEP significantly diminishes seizures and restores select social and nonsocial anxiety‐related behaviors in Fmr1KO mice suggested it as a target for drug discovery in FXS.  [Goebel et al., 2012c]. Benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153) is newly discovered STEP inhibitor [Xu et al., 2014]. In recent study [Chatterjee et al., 2018] reported the targeting STEP inhibitor (TC-2153) with reduced seizure incidences and hyperactivity, normal anxiety state and improved sociability, reverses electrophysiological deficits in acute brain slices, and improves spine morphology in Fmr1 KO mice.

It suggested the role and potential of STEP inhibition in future FXS therapeutics.

14. Endocannabinoid System (eCS)

The endocannabinoid system (eCS) consists of receptors located in the brain as a key modulator of synaptic plasticity, cognitive performance, anxiety, nociception, seizure susceptibility and the endogenous cannabinoid ligands anandamide (AEA) and 2­arachidonoylglycerol (2­AG) [kano et al., 2009]. The endocannabinoids modulate the synaptic activity by binding to the G protein-coupled receptors CB1 and CB2 [Mouslech et al., 2009, Pacher et al., 2006]. Rimonabant (5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide, SR141716A), was the first selective CB1 receptor antagonist [Rinaldi e al., 1994].  [Gomis et al., 2016] showed that the treatment with rimonabant normalizes anomalous synaptic plasticity in the hippocampus of Fmr1 KO mice. Moreover, Fmr1 deletion affects the endocannabinoid mediated depolarization induced suppression of excitation (DSE) in autaptic hippocampal neurons cultured from Fmr1 KO mice [Straiker et al., 2013].  2AG is produced in the brain as response to activation of metabotropic receptors in dendrites. In the Fmr1 KO mice the absence of FMRP leads to 2­AG-dependent and mGluRI-dependent synaptic plasticity abnormalities, such as enhanced LTD (long-lasting decrease in synaptic strength to below the normal baseline level after prolonged, low frequency stimulation

) at inhibitory synapses [Zhang et al.,2010, Maccarrone et al., 2010, Busquets et al., 2013] and decreased LTD at excitatory synapses [Jung et al., 2012]. The inhibiting of 2 AG degradation results in the improvement of several phenotypes of the FXS mouse [Myrick et al., 2015]. [Qin et al., 2015] indicated with the number of different expariments that endocannabinoid system is involved in FXS and suggest that this system is a promising target for treatment of FXS.

15. MicroRNA’s (miRNA’s)

MicroRNAs (miRNAs) are known as a class of small RNA molecules (19-23 nucleotides) that regulates almost 30% of genes at post transcriptional level in eukaryotic organisms [Lin et al., 2015]. The pathological cause of the Fragile X syndrome likely resides in interactions between r(CGG) over-expansion and FMR1 promoter hypermethylation and the r(CGG)-derived miRNAs are involved in gene silencing [Kelley et al., 2012]. [Lin et al., 2006, Chang et al., 2008] provided the first evidence of miRNA involvement in pathogenesis of FXS by identifying and isolating several r(CGG)-derived miRNAs including miR-fmr1-27 and miR-fmr1-42 in zebrafish FXS model. Among identified miRNA’s on further testing miR-fmr1-affected FXS neurons in FXS zebrafish brains exhibit long stripe dendrites and disconnected synapses exact similar to those found in the human FXS hippocampal–neocortical junction [Lin et al., 2008]. The microarray analyses of miRNA’s associated with FMRP in FMR1 KO mice brains identifies miR-125a, 125b, and 132 have known for involvement in the pathogenesis of FXS and confirm that the lack of FMRP disrupts the regulating the microRNA miR-125- and miR-132-mediated protein translation results in premature neural development and synaptic physiology [Muddashetty et al., 2011, Edbauer et al., 2010].

Following it another microarray study conducted by [Liu et al., 2015] in FMR1 KO mice which showed the interaction of miR-34b, miR-340, and miR-148a with the Met 3′ UTR of FMR1 gene and depicts that the miRNA expression alterations resulted from the absence of FMRP and contribute to molecular pathology of FXS. [Fazlei et al., 2018] reported the association of FMR1 CGG expansion with the enhanced expression of miR-510 in full mutation female carriers. They suggested VHL and PPP2R5E genes as potential new targets for future therapy options of FXS. In addition to FMR1 gene mRNA’s also found to be regulated the expression of FXR1 (Fragile X related gene 1), one of homologous genes of FMR1 and involved in the pathogenesis of FXS [Xu et al., 2011, Siew et al.,2013]. [Cheever et al., 2010] found that the miR-367 could significantly decrease the expression of endogenous FXR1P in human HeLa cell lines. In addition, [Gessert et al., 2010] reported that the loss of FMR1 or FXR1 changed the expression level of miR-130a, miR-200b, miR-96, miR-196a and other miRNAs, resulting in abnormal eye development as well as defects in cranial cartilage in Xenopus laevis. In recent study [Ma et al., 2016] showed that miR-19b-3p plays an important role in the molecular pathology of FXS by interacting with FXR1 and influencing the growth of SH-SY5Y cells.

16. Glycogen Synthase Kinase 3 (GSK3)

Glycogen synthase kinase-3 (GSK-3) is a protein kinase that phosphorylated and inhibited glycogen synthase and regulates a variety of developmental processes, such as cell migration, cell morphology, neurogenesis, gliogenesis via interaction with a variety of signaling pathways [Castano et al., 2010]. FMRP is known to play a critical role in adult hippocampal neurogenesis and regulates adult neural stem cell (NSC) fate by modulating the translation of glycogen synthase kinase-β (GSK-3β) [ Beurel et al., 2009]. GSK-3β inhibition increased hippocampal neurogenesis and better the performance in hippocampal-dependent learning tasks. The lack of FMRP results in an abnormal increase in glycogen synthase kinase 3β (GSK3β) mRNA and protein levels results in the decrease of hippocampal neurogenesis that might contribute to the pathogenies of FXS [Portis et al., 2012].

FXS mice model showed the high activity of Glycogen synthase kinase-3 (GSK3) in several regions of the brain [Min et al., 2009]. [McBride et al., 2005] reported first time in the Drosophila model of FXS that lithium treatment ameliorated impairments in behavioral phenotypes. A later study confirmed that lithium treatment rescued the FXS-associated impairments in the Drosophila model of FXS and demonstrated that this rescue was sustainable throughout the aging process [Choi et al., 2010]. Later [Yuskaitis et al., 2010a] provide the direct evidence of GSK3 involvement in the pathology of FXS by reducing audiogenic seizure activity, and improved performance on open field, elevated plus maze, and passive avoidance tests in Fmr1 knockout (KO) mice with GSK3 inhibitors and lithium treatment. Followed this study [Mines et al., 2010] reported the improvement in the social defects of the Fmr1 KO mice with lithium treatment. Additionally, reactive astrocytes have been found in many brain regions of Fmr1 knockout mice. The attenuation of this phenotype with lithium treatment, providing further evidence of the involvement of GSK-3 in FXS [Yuskaitis et al., 2010b]. These all findings raise the possibility that GSK3 is a fundamental and central component of FXS pathology, with a substantial treatment potential [Mines et al., 2011].

Guo et al., 2011] strengthen this idea by reversing the hippocampus-dependent learning deficits in Fmr1 Ko mice with GSK3β inhibitor, SB216763. [Franklin et al., 2014] reported the improvement in cognitive deficits in Fmr1 KO mice after treatment with GSK3 inhibitors. [Telias et al., 2015] first time test the GSK3β theory of FXS in FX-human embryonic stem cells (FX-hESCs), derived from FX human blastocysts. Interestingly, they didn’t find any evidence for a pathological increase in GSK3β protein levels upon cellular loss of FMRP, in contrast to what was found in the brain of Fmr1 knockout. One of the provided justification that GSK3β involvement in FXS neuropathology is mediated through the canonical Wnt/β-catenin signaling pathway i.e. a critical signaling pathway for embryonic neural development as well as for adult neurogenesis.

17. Other Potential Biomarkers

Bone Morphogenetic Protein Receptor Type 2 (BMPR2)

Bone morphogenetic protein receptor type 2 (BMPR2) is a binding target of FMRP and involved in dendrite formation. Depletion of FMRP protein in FXS results in increase BMPR2 expression and activation of BMP signaling to changing dendritic spine morphology and improve the actin polymerization. The high level of BMPR2 and a marker of LIM domain kinase 1 (LIMK1) observed in prefrontal cortex of patients with FXS as compared to post-­mortem prefrontal cortex tissue from healthy individuals, suggesting that increased BMPR2 signal transduction is associated with FXS and might be a putative therapeutic target for FXS. To test this hypothesis Fmr1 KO mice and Drosophila treated with an LIMK1 inhibitor and it reversed the abnormal dendritic spine morphology of neuron to the wild-type phenotype [Kashmina et al., 2016]. Later the high throughput drug screening confirms the role of LIMK1 inhibitors in improving nerological and behavioral phenotypes of FXS fly model but also reduced the hyperactivity in a mouse model [Kashmina et al., 2017]. These initial studies showed the potential of BMPR2 as putative target for FXS treatment but there is need for more evidences.


Plasma Cytokines and Chemokines

Cytokines are most important mediators of cell-cell communication in the human immune system. They perform the variety of functions like modulation of central nervous system (CNS), brain functioning and responses to infections or injury. [Ashwood et al., 2010] reported the significant differences in plasma cytokine and chemokines levels. They found the high level of IL-1alpha, and the chemokines; RANTES and IP-10, in FXS patients as compared to controls. It is currently unknown if the changes in the cytokine and chemokines are determinant in development of FXS there is need of longitudinal studies to investigate the changes over the period of lifetime of FXS patients.

Diacylglycerol Kinase Kappa (Dgkκ)

Diacylglycerol kinase kappa (Dgkκ), is a master regulator that controls two key signaling pathways involve in protein synthesis. The absence of FMRP in Fmr1 KO mice neurons results in loss of Dgkκ expression along with group 1 metabotropic glutamate receptor-dependent DGK activity. This reduction of Dgkκ in neurons caused the synaptic plasticity alterations, dendritic spine abnormalities and behavior disorders. The overexpression of Dgkκ in neurons is able to rescue the dendritic spine defects of the Fmr1 KO mice neurons. It shows the involvement of Dgkκ deregulation in FXS pathology and account for many symptoms associated with FXS. It suggests that targeting DGKκ signalling might provide new therapeutic approaches for FXS [Tabet et al.,2016].


Fragile X syndrome is a challenging disorder in term of clinical implementation and drug development. There is splendid work has been done in animal models that suggested the way to improve the behavior and cognitive ability but there are few factors (complex clinical phenotype, the genetic variability, a gender difference, simultaneous use of multiple medications, and a limited utility of tools) which might contribute to the lack of translation from the preclinical to the clinical situation. Nevertheless, when looking at the design of the preclinical studies to date, one can see some limitations: 1) Currently, there is no preclinical studies investigating combinations of drugs as it could help in understanding whether these medications could modulate or even inhibit a therapeutic effect. 2) Almost most of the FXS research in mammalian model systems is limited to one single disease model, the Fmr1 KO mouse line. A central issue in use of this model is variability and small effect size of the mouse phenotype in the area of cognitive defects. Moreover, overlapping phenotypes in the Fmr1 KO mice indicate the over prediction of the therapeutic potential of novel drug treatments.

On the other side, the molecular biomarkers are easy and minimally invasive diagnostic tools that can offer help in clinical trials. However, up to this point there is not reproducible molecular biomarkers available for FXS. The current research on molecular biomarkers in FXS suffers from the following limitations: 1) FXS is neurological disorder but there is inaccessibility of the brain as sample tissue, therefore biomarkers must be developed in a tissue that can be biopsied easily like blood, fibroblasts, etc., 2) no single consistent molecule or modification state (ie, phosphorylation or acetylation) has been reported to be differentially regulated in FXS patients versus controls across multiple testing sites, 3) no one substrate’s activity/expression is dynamic enough in the span of few weeks to accurately represent changes in disease modifications in a trial setting, 4) no clinical history for any marker is available, and 5) lengthy and expensive processing is required to generate test substrates such as primary fibroblasts (and/or induced pluripotent stem cells).

In summary there is a great need of novel and stable biomarkers in FXS. The development of non-rodent disease models which may be closer to the human pathophysiology helps in making assessments that are translatable to the clinical outcome measures. In last, clinical and preclinical scientists will need to work more collaboratively to make sure the translation of animal findings to human trials and also translation of key findings in human studies for development of new phenotypic measures in mice.

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